Endocrine Reviews 22 (5): 565-604
Copyright © 2001 by The Endocrine Society
Mechanisms and Physiological Significance of the Cholinergic Control of Pancreatic ß-Cell Function
Patrick Gilon and
Jean-Claude Henquin
Unité d’Endocrinologie et Métabolisme, University of
Louvain Faculty of Medicine, B-1200 Brussels, Belgium
Correspondence: Address all correspondence and requests for reprints to: Dr. Patrick Gilon, Unité d’Endocrinolgie et Métabolisme, UCL 55.30, Avenue Hippocrate 55, B-1200 Brussels, Belgium. E-mail:
gilon{at}endo.ucl.ac.be
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Abstract
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Acetylcholine (ACh), the major parasympathetic neurotransmitter, is
released by intrapancreatic nerve endings during the preabsorptive and
absorptive phases of feeding. In ß-cells, ACh binds to muscarinic
M3 receptors and exerts complex effects, which culminate in
an increase of glucose (nutrient)-induced insulin secretion. Activation
of PLC generates diacylglycerol. Activation of PLA2
produces arachidonic acid and lysophosphatidylcholine. These
phospholipid-derived messengers, particularly diacylglycerol,
activate PKC, thereby increasing the efficiency of free cytosolic
Ca2+ concentration ([Ca2+]c) on
exocytosis of insulin granules. IP3, also produced by PLC,
causes a rapid elevation of [Ca2+]c by
mobilizing Ca2+ from the endoplasmic reticulum; the
resulting fall in Ca2+ in the organelle produces a
small capacitative Ca2+ entry. ACh also depolarizes the
plasma membrane of ß-cells by a Na+- dependent mechanism.
When the plasma membrane is already depolarized by secretagogues such
as glucose, this additional depolarization induces a sustained increase
in [Ca2+]c. Surprisingly, ACh can also
inhibit voltage-dependent Ca2+ channels and stimulate
Ca2+ efflux when [Ca2+]c is
elevated. However, under physiological conditions, the net effect of
ACh on [Ca2+]c is always positive. The
insulinotropic effect of ACh results from two mechanisms: one involves
a rise in [Ca2+]c and the other involves a
marked, PKC-mediated increase in the efficiency of Ca2+ on
exocytosis. The paper also discusses the mechanisms explaining the
glucose dependence of the effects of ACh on insulin release.
I. Introduction
II. The Innervation of the Endocrine Pancreas
A. General anatomical considerations
B. The parasympathetic innervation
C. The sympathetic innervation
D. Sensory fibers
E. Other types of nerves
III. Physiological Role of the Parasympathetic Control of ß-Cells
A. Difficulties and pitfalls of in vivo studies
B. Physiological situations
C. Pathophysiological situations: hyperinsulinemia, obesity, and insulin
resistance
IV. General Characteristics of Acetylcholine (ACh) Effects on Insulin
Secretion in Vitro
V. Effects of ACh on ß-Cell Phospholipases
A. Activation of PLC
B. Activation of PLA2
C. Activation of PLD
VI. Effects of ACh on the Membrane Potential of ß-Cells
A. Dependence on the electrical resistance of the plasma membrane
B. Mechanisms of the depolarization
C. Paradoxical hyperpolarization by ACh
VII. Other Effects of ACh in Islet Cells
A. Effects on glucose metabolism
B. Effects on cyclic nucleotides
C. Effects on cytoplasmic pH
VIII. ACh Controls Free Cytosolic Ca2+ Concentration
([Ca2+]c) in
ß-Cells
A. Mechanisms by which ACh increases
[Ca2+]c
B. Mechanisms by which ACh decreases
[Ca2+]c
IX. Mechanisms of the Stimulation of Insulin Secretion by ACh
A. The rise in [Ca2+]c by
ACh triggers exocytosis
B. ACh increases the efficacy of Ca2+ on
exocytosis
C. Delayed effects of ACh on insulin secretion
D. Muscarinic responses are often abnormal in insulin-secreting cell
lines
X. Nature of the Muscarinic Receptor Activated by ACh
A. Pharmacological studies
B. Binding studies
C. Molecular identification of the receptor subtypes
D. One or several receptor subtypes for several transduction pathways?
XI. Summary and Conclusions
A. The physiological role of ACh
B. The mechanisms of action of ACh in ß-cells
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I. Introduction
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DESPITE THE ALTERNATION of fasting and feeding periods, the
concentration of plasma glucose is maintained within a narrow range by
a finely tuned balance between insulin, the only hypoglycemic hormone,
and glucagon, epinephrine, corticosteroids, and GH, the major
hyperglycemic hormones. The secretion of insulin by ß-cells of the
endocrine pancreas is regulated by glucose and other circulating
nutrients. It is also modulated by several hormones and
neurotransmitters, among which acetylcholine (ACh) plays a prominent
role.
The complex neural control of hormone secretion by the endocrine
pancreas has been the subject of other reviews (1, 2, 3). It
will be addressed only briefly in our contribution, which focuses on
the cholinergic control of the ß-cell function. After an overview of
the in vivo data demonstrating the role of the
parasympathetic system in the regulation of glycemia, we analyze and
synthesize the in vitro experiments that have elucidated the
cellular mechanisms by which ACh influences ß-cells. Particular
attention is paid to the effects of ACh on phospholipid metabolism,
membrane potential, free cytosolic Ca2+
concentration ([Ca2+]c),
and insulin secretion. This article updates and extends other reviews
on the subject (4, 5, 6, 7).
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II. The Innervation of the Endocrine Pancreas
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A. General anatomical considerations
The endocrine pancreas is organized in small organs, the
pancreatic islets or islets of Langerhans, that are dispersed in the
exocrine parenchyma. The islets are composed of a few hundred to
several thousands of cells, of which 65–80% are insulin-secreting
ß-cells. These cells are mainly located in the center of the islet
and are surrounded by a mantel of three other cell types,
i.e., glucagon-secreting 
-cells,
somatostatin-secreting 
-cells, and pancreatic polypeptide-secreting
cells (PP-cells).
The endocrine pancreas is richly innervated, but the abundance and
organization of this innervation are highly variable between species
(8). Most of the nerve fibers enter the pancreas along the
arteries (9, 10). Unmyelinated nerve fibers are found in
the neighborhood of all islet cell types at the periphery and within
the islet. At some distance from the islets, glial Schwann cells often
form a thin sheet around nerve fibers on their travel toward and within
the islet. In the vicinity of islet cells, however, it is not rare to
see some nerve fibers lacking this glial protection and coming close to
or ending blindly 20–30 nm from the endocrine cells (8, 11, 12, 13, 14, 15, 16, 17). Well differentiated synapses with islet cells have
rarely been observed (18, 19, 20). Interestingly, the
innervation of the islet is very plastic, as suggested by the
observation that islets transplanted in the portal vein of diabetic
rats became reinnervated by hepatic nerves (21).
The autonomic innervation of the endocrine pancreas has several origins
(for review, see Refs. 2 and 3). Classically,
the autonomic nervous system uses two interconnected neurons to control
effector functions and is divided into two systems, the sympathetic and
the parasympathetic nervous systems, according to the location of the
preganglionic cell bodies. However, there are indications suggesting
that these two systems are not always independent of each other, but
display anatomical interactions (22) or share similar
neurotransmitters (23, 24, 25). The endocrine pancreas also
receives other types of nerves, the anatomical origin and the function
(motor efferent or sensory afferent) of which are not clearly known.
These nerves are of peptidergic and nonpeptidergic nature (2, 3).
B. The parasympathetic innervation
The preganglionic fibers of the parasympathetic limb originate
from perikarya located in the dorsal motor nucleus of the vagus
(26, 27, 28, 29, 30, 31, 32, 33) and possibly also in the nucleus ambiguus
(26, 34, 35, 36, 37), which are both under the control of the
hypothalamus. They are organized in well separated branches traveling
within the vagus nerves (cranial nerve X), and through the hepatic,
gastric (31, 38), and possibly celiac branches of the
vagus (39), they reach intrapancreatic ganglia that are
dispersed in the exocrine tissue. These ganglia send unmyelinated
postganglionic fibers toward the islets (9, 10, 38, 40).
Preganglionic vagal fibers release ACh that binds to nicotinic
receptors on intraganglionic neurons. Postganglionic vagal fibers
release several neurotransmitters: ACh, VIP, gastrin-releasing peptide
(GRP), nitric oxide (NO), and pituitary adenylate cyclase-activating
polypeptide (PACAP) (3, 27, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51). Cholinergic terminals
are found in the neighborhood of all islet cell types at the periphery
and within the islet (50, 52, 53, 54, 55, 56). The importance of the
cholinergic innervation of the endocrine pancreas is attested by the
presence of a 10-fold higher activity of choline acetyltransferase and
acetylcholinesterase (the enzymes involved, respectively, in the
synthesis and the degradation of ACh) in the islets than in the
surrounding exocrine tissue (57). Cholinergic synapses
with endocrine cells have been observed in some species (58, 59).
Understanding the organization of the pancreatic innervation
permits correct interpretation of some experiments using different
cholinergic antagonists. The stimulation of insulin release occurring
upon electrical stimulation of vagal nerves in the dog is abolished by
both nicotinic and muscarinic antagonists (60). In the
perfused rat pancreas, nicotine produces an increase of insulin
secretion that is blocked by atropine (10). These
observations can be explained by the presence of nicotinic receptors on
pancreatic ganglia and nerves (61, 62, 63, 64) and muscarinic
receptors on ß-cells (see Section X).
The overall effect of a parasympathetic stimulation is an increase of
insulin secretion (see Section III). Because
postganglionic fibers contain various neurotransmitters in addition to
the classic neurotransmitter ACh, it is important to keep in mind that
parasympathetic neurotransmission is the sum of various biological
effects. VIP and PACAP stimulate insulin secretion by increasing cAMP
levels (3). GRP and its amphibian homolog, bombesin
(3), are also insulinotropic (3, 42, 65, 66, 67, 68). They act on the same family of receptors
(69) and exert their action by two mechanisms, directly by
stimulating ß-cells through the PLC-PKC pathway (3), and
indirectly by activating intrapancreatic postganglionic nerves that
stimulate insulin secretion (68). NO synthase has been
detected in nerves in several organs wherein NO is considered a
neurotransmitter (70, 71), and in pancreatic nitrergic
nerves (45, 48, 49, 67). Various effects of NO on
ß-cells have been reported (72, 73, 74, 75), but it is unclear
whether NO is implicated in the parasympathetic modulation of insulin
secretion.
The parasympathetic system also controls the secretion of the other
islet hormones. Vagal nerve stimulation increases glucagon (31, 41, 60, 76, 77, 78) and PP secretion (41). The effect
of vagal stimulation on -cells is less clear, as it was reported to
stimulate (79) or inhibit somatostatin secretion
(78, 80). In vitro and in vivo
experiments using various cholinergic agents have shown that ACh
stimulates glucagon and PP secretion through atropine-sensitive
mechanisms (81, 82, 83, 84). The effects of cholinergic agonists
on in vitro somatostatin secretion are again controversial
(2, 80, 82, 85), although this might reflect species
differences.
C. The sympathetic innervation
The sympathetic innervation of the pancreas originates from
preganglionic perikarya located in the thoracic and upper lumbar
segments of the spinal cord (86). The myelinated axons of
these cells traverse the ventral roots to form the white communicating
rami of the thoracic and lumbar nerves that reach the paravertebral
sympathetic chain (87). Preganglionic fibers either
communicate with a nest of ganglion cells within the paravertebral
sympathetic chain or pass through the sympathetic chain, travel through
the splanchnic nerves, and reach the celiac (2, 3, 35, 86, 88) and mesenteric ganglia (86). Ganglia within the
paravertebral sympathetic chain, and the celiac and mesenteric ganglia,
give off postganglionic fibers that eventually reach the pancreas. The
existence of intrapancreatic sympathetic ganglia has also been reported
(25, 26, 37). The preganglionic fibers release ACh that
acts on nicotinic receptors on intraganglionic neurons, whereas the
postganglionic fibers release several neurotransmitters:
norepinephrine, galanin, and NPY (3, 51, 89, 90, 91). A rich
supply of adrenergic nerves in close proximity of the islet cells has
been observed in several mammalian species (53, 54, 55, 92).
The net physiological effect of splanchnic nerve stimulation is a
lowering of plasma insulin concentration (93, 94, 95, 96). This
effect is attributed to release of norepinephrine from nerve fibers
close to ß-cells and to elevation of catecholamine (epinephrine and
norepinephrine) plasma levels because of the stimulation of the adrenal
medulla. Catecholamines have long been known to inhibit insulin
secretion in vivo (1, 2, 3, 97) and in
vitro (1, 2, 3, 98, 99, 100, 101). Their action is mediated by
2-adrenoceptors (102), probably
of the 2a- and
2c-subtypes (103), which have
been identified in ß-cells by both pharmacological (104)
and molecular approaches (103, 105). Activation of
2-adrenoceptors interferes with the secretory
process through several mechanisms that are all prevented by pertussis
toxin treatment and are, thus, likely mediated by
Gi or Go
(106): an inhibition of adenylate cyclase leading to a
lowering of ß-cell cAMP, an opening of K+
channels of small conductance leading to partial membrane
repolarization and decrease in Ca2+
influx, and a major inhibition of a late step of exocytosis
(106). Similar pathways are implicated in the inhibitory
action of galanin, which may cooperate with catecholamines to inhibit
insulin secretion in response to splanchnic nerve stimulation (3, 106). In contrast, an increase in plasma insulin can be
evoked by selective ß-adrenergic agonists, particularly of the
ß2-subtype (2, 95, 107, 108), that
activate adenylate cyclase and increase cAMP. However, these usually
have little effect on insulin secretion by isolated islets
(109). Moreover, the presence of
ß2-adrenoceptors in ß-cells remains
controversial (105, 110). It is also important to
emphasize that a number of pharmacological studies have been
misinterpreted because antagonists of adrenoceptors can influence
insulin secretion by acting on other targets, e.g., on
ATP-sensitive K+ (K+-ATP)
channels (111). The mechanisms by which NPY inhibits
insulin release are not clearly known and might involve a decrease in
cAMP levels (3).
The sympathetic nervous system exerts profound effects on the secretion
of the other islet hormones. Splanchnic nerve stimulation increases
glucagon secretion (93, 94, 95, 96, 112, 113), and epinephrine
stimulates glucagon secretion in vivo and in
vitro (84, 100, 114, 115). This effect results from
the activation of ß-adrenoceptors (101), probably of the
ß2-subtype (110), although one
report implicates -adrenoceptors (96). It has been
shown that pancreatic -cells express 1-,
2-, and 3-subtypes
(103). Splanchnic nerve stimulation decreases
somatostatin secretion (80, 96, 116), and
norepinephrine inhibits somatostatin release by isolated rat islets
(100). The results are less clear for PP secretion. Thus,
splanchnic nerve stimulation has been reported to increase (3, 113, 117) or inhibit PP secretion (2, 116).
Catecholamines stimulate PP release by isolated islets
(118).
Overall, the sympathetic nervous system serves to maintain or increase
glycemia in various conditions of stress such as neuroglycopenia,
hypovolemia, or physical exercise (3). Its pancreatic
action not only involves inhibition of insulin secretion, but also
stimulation of glucagon secretion (3, 119).
D. Sensory fibers
Calcitonin gene-related peptide (CGRP) and substance P (SP) are
thought to report sensory information in many systems
(120). CGRP (51, 121, 122)- and
SP-immunoreactive (51, 123, 124) nerve fibers have been
observed in both the exocrine and endocrine pancreas. Vanilloid
receptors, activated by heat, low pH, and various vanilloid agents
(such as capsaicin), are localized in sensory fibers and generally
report pain information (120). Neonatal treatment of mice
with capsaicin destroys the majority of capsaicin-sensitive neurons and
has often been used to identify sensory fibers (120). This
treatment was followed by a marked reduction of CGRP-immunoreactive
fibers in both the endocrine and exocrine pancreas (122)
and by a partial reduction in SP-immunoreactive fibers (36, 125).
It is thought that sensory afferents leave the pancreas along the
sympathetic fibers within the splanchnic nerves and that the perikarya
of the sensory fibers are present in dorsal root ganglia, mainly at the
level of the lower thoracic segments of the spinal cord, transmitting
noxious information to the central nervous system by synapsing on
second-order neurons of the dorsal horn of the spinal cord (2, 86, 126, 127). The existence of such an anatomical route is
supported by experiments of retrograde labeling (36, 86, 126, 128). It has been suggested that the pancreas is also innervated
by sensory afferents that run within the vagus nerve, the perikarya of
which are in the nodose ganglion and transmit information to the
nucleus tractus solitarius (30, 35, 36, 129, 130).
There is no doubt that sensory nerve fibers report pain information
associated with diseases of the exocrine tissue, such as pancreatic
cancer and pancreatitis (127, 131), but there are no
reports of sensations of pain associated with a destruction of the
endocrine pancreas. However, it is possible that sensory fibers play a
role in the control of insulin secretion. Thus, neonatal treatment of
mice with capsaicin (to destroy these fibers) results in more
glucose-stimulated insulin secretion than in nontreated mice,
suggesting that sensory fibers exert a direct, tonic inhibition of
insulin secretion (132). CGRP may inhibit insulin
secretion through a direct action on the islets (121, 133), whereas both inhibitory (134) and stimulatory
(124, 135) effects of SP have been reported. Indirect
effects of capsaicin-sensitive fibers are also possible. Indeed, it has
been reported that removal of endogenous sensory neuropeptides by
deafferentation of capsaicin-sensitive sensory nerves improves
glucose tolerance by increasing in vivo insulin sensitivity
(136, 137).
E. Other types of nerves
Immunocytochemistry has revealed the presence of
neurotransmitters other than those described above in pancreatic
nerves: cholecystokinin (138), 5-hydroxytryptamine (5-HT
or serotonin) (139, 140), and methionine-enkephalin
(3, 51). These might also influence insulin secretion:
cholecystokinin stimulates insulin release by activating PLC and
PLA2 (138), but the effects of 5-HT
are controversial, as both inhibition (141, 142) and
stimulation (143) of insulin secretion have been reported.
Enkephalin also exerts variable effects depending on the concentration
used and the species studied (144, 145).
The pancreatic innervation presents other interesting features. The
section of extrinsic pancreatic nerves has revealed that many of the
intrinsic pancreatic neurons are independent of the integrity of the
extrinsic nerves (146), suggesting that the pancreatic
innervation might behave as an independent system. This is supported by
the observation that intrapancreatic ganglia are interconnected
with one another, as are enteric ganglia (37, 140). It has
also been suggested that intrapancreatic ganglia are connected with the
duodenal myenteric plexus by nerve fibers (50), suggesting
the existence of an entero-pancreatic innervation. On the other hand,
ganglia from the myenteric plexus of the stomach and duodenum send
nerve fibers toward the pancreas (50, 139). Many of these
nerves are immunoreactive for 5-HT. Whether these innervations play a
physiological role in the regulation of hormone secretion by the
endocrine pancreas remains to be investigated.
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III. Physiological Role of the Parasympathetic Control of
ß-Cells
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In 1927, Zunz and LaBarre (147), using a
cross-perfused canine model, showed that stimulation of the vagus nerve
in one dog induced hypoglycemia in the other animal. In 1967, three
in vivo studies performed in the dog and the baboon reported
that stimulation of the vagus nerve increased plasma insulin, and that
this effect involved muscarinic receptors because it was inhibited by
atropine (148, 149, 150). At the same time, an in
vitro study showed that cholinergic agonists stimulated insulin
release from pieces of rat pancreas, and this effect was also
antagonized by atropine (99).
The most important characteristic of the influence of ACh on insulin
secretion is a tight dependence on the ambient glucose concentration.
In vivo, electrical stimulation of the vagus nerve has
little effect on the concentration of plasma insulin during
hypoglycemia, but increases it more and more efficiently as the
concentration of plasma glucose augments (41, 151, 152, 153, 154, 155).
Similar observations have been made in vitro when the
perfused pancreas (81, 156, 157, 158, 159, 160) or isolated islets
(161, 162, 163, 164) were used to study insulin secretion directly
(Fig. 1A). This behavior is typical
of a potentiating agent. The mechanisms underlying this potentiation
will be explained in detail in Section IX.
From here, it is important to bear in mind that the majority of
in vitro experiments were conducted with rodent islets, and
that the concentration dependence of glucose-induced insulin secretion
is different in rodent and human islets. Indeed, the threshold glucose
concentration and the half-maximal effective concentration for
insulin secretion are, respectively, around 4 and 9
mM for human islets as compared with 7 and 15
mM for mouse islets (165).
A. Difficulties and pitfalls of in vivo studies
Species differences must be considered when interpreting the
effects of ACh on insulin secretion in vivo. As already
mentioned in Section II, vagal stimulation can release at
least five neurotransmitters (ACh, VIP, PACAP, GRP, and NO), the
relative contribution of which differs between species. In the dog
(49, 60, 76, 149, 166), rat (78), albino
mouse (167), and calf (41), vagal stimulation
of insulin secretion is mediated mainly or exclusively by muscarinic
receptors because it is largely or fully prevented by atropine. This is
not the case in the pig (113, 168) and in the cat
(10, 169), in which neurotransmitters other than ACh are
probably implicated.
A second difficulty is linked to the number of physiological events
that are under parasympathetic control. Cholinergic agonists or
antagonists, stimulation of the vagus nerve, and vagotomy induce
multiple effects that can indirectly interfere with insulin secretion.
Cholinergic agents influence the secretion of the other islet hormones
(see Section II), but it is difficult to establish to what
extent the observed changes in insulin secretion are influenced by
paracrine or endocrine interactions. Such interactions depend on the
organization of the microvascularization and the direction of blood
flow, which are still a matter of debate (170, 171, 172). In
addition, clear evidence for intraislet paracrine influences has not
yet been reported (172). Importantly, the effects of ACh
on insulin secretion are observed at glucose concentrations that are
substimulating or stimulating for insulin release but inhibitory for
glucagon release.
Several intestinal hormones, particularly glucose-dependent
insulin-releasing peptide (GIP) and glucagon-like peptide-1, potently
increase insulin secretion (173, 174, 175). GIP- and
glucagon-like peptide-1-secreting cells possess muscarinic receptors
(176, 177), and both the stimulation of the vagus nerve
and cholinergic agonists stimulate their release
(178).
ACh also stimulates gastric emptying (179), which may
affect the rate of glucose absorption, change in glycemia, and hence,
insulin secretion (180, 181). It has also been reported
that the increase in islet blood flow produced by a rise in blood
glucose is mediated by the central nervous system, which senses the
changes in glycemia and sends signals to islet vessels through the
vagus nerves (182).
B. Physiological situations
1. Role of the vagus nerve on glucose tolerance. In lean
animals or humans, basal insulin secretion is not affected or is only
slightly decreased by vagotomy or atropinization (9, 149, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196), which indicates that there is no significant tonic
stimulation of the ß-cells by the parasympathetic system in the
fasting state. In contrast, it is generally agreed that the vagus
nerves participate in the control of insulinemia during the periods of
feeding. The difficulty is to assess their contribution to the overall
insulin-secretory response. Thus, depending on the study, the tolerance
to a glucose load is unaffected or impaired by atropine or vagotomy,
whereas the associated insulin response is larger, similar, or smaller.
The results become more consistent when the insulinogenic index (
insulinemia/ glycemia) is calculated, and the mode of administration
of glucose (oral or iv) is taken into account (197, 198, 199).
Thus, when glucose is administered iv, the insulinogenic index is not
affected or is hardly modified by atropine or vagotomy (185, 187, 191, 193, 195, 197, 198, 199, 200, 201). In contrast, when glucose is given
orally, the insulinogenic index is significantly decreased by atropine
or vagotomy (184, 197, 199, 200, 202). In addition,
the rise in plasma insulin is delayed, which also contributes to the
glucose intolerance of vagotomized or atropine-treated rats (1, 199, 203). These results suggest that ACh potentiates the
insulin response to glucose after a glucose load, a conclusion that is
supported by experiments using animal models without parasympathetic
innervation of the ß-cells. After destruction of their ß-cells by
alloxan or streptozotocin, rats were transplanted with isolated islets.
The vagus nerves of the receivers were intact, but the transplanted
ß-cells were presumably denervated at the time of test
(203, 204, 205). Meal ingestion induced a glucose increase that
was larger in transplanted than in normal control rats, and that was
associated with a delayed insulin response (40, 206, 207).
This confirms that a direct parasympathetic innervation of ß-cells
improves glucose tolerance.
2. The vagus nerves transmit signals of several origins. When
evaluating the physiological role of the muscarinic control of
ß-cells, it is important to bear in mind that the vagus nerves are
the parasympathetic effectors of signals that are all integrated in the
brain but come from at least four sources: cephalic sensory organs
including those of the oral cavity and the visual and olfactory
systems, the gut, the liver, and the brain itself (208).
The sequential activation of all these inputs will affect insulin
secretion in a time-dependent manner upon meal ingestion.
a. Cephalic sensory organs and the gastrointestinal tract.
The preabsorptive insulin phase corresponds to the earliest plasma insulin
rise during the first minutes of food ingestion. It does not depend on
nutrient assimilation, as it occurs before the glycemia has increased
(1, 200, 206, 207, 209, 210, 211, 212, 213, 214, 215, 216) and is sometimes associated
with a transient hypoglycemia (28, 203, 217). The
amplitude of the preabsorptive insulin phase is highly variable from
one study to another, but it is consistently much smaller than the
postabsorptive insulin phase occurring when glycemia starts increasing.
It corresponds to a rise of approximately 20% (28, 216, 217) or more (1, 206) above basal insulinemia,
which may be an underestimation of the reality, because insulin is
measured in peripheral or heart blood and not directly in the portal
circulation. Insulin is indeed very rapidly degraded by the liver (50%
during the first passage of blood), and the amplitude of the changes in
insulinemia is much smaller in peripheral than in portal blood
(192, 218). The preabsorptive insulin response involves
both cholinergic and noncholinergic mechanisms (3, 216).
It can be subdivided into the cephalic phase and the enteric phase.
The cephalic phase does not even require ingestion of nutrients, as it
can occur in response to oral saccharin or water intake in animals
(1, 204), but not in humans (219). Its
mechanisms involve stimulation of oropharyngeal receptors (210, 220, 221) and probably also conditioned visual and olfactory
reflexes, because an early peak of insulin secretion can be observed in
animals that simply see or smell food (28). A cephalic
phase exists in humans (Refs. 28 and 222, 223, 224, 225 ,
but see Ref. 226), but is less easily conditioned
than in animals (227, 228). Because no cephalic phase
occurs after vagotomy, nor is this phase observed in diabetic animals
transplanted with denervated islets, it is ascribed to a direct
stimulation of ß-cells by both cholinergic and noncholinergic fibers
of the vagus nerves (3, 203, 204, 205, 206). The sympathetic nervous
system might also contribute to the cephalic phase in the dog by
activating ß2-adrenoceptors
(229).
The enteric phase has been much less extensively studied because of the
difficulty in separating it from the preceding cephalic phase and the
following postabsorptive phase, which rapidly causes an increase in
glycemia (28, 203, 217, 230, 231). This phase has been
observed after direct infusion of a meal into the stomach or the
duodenum (176, 203, 232) and is sometimes reflected by a
single preabsorptive insulinemia peak (203). Abolition of
this phase by vagotomy and atropine implicates the vagus nerve
(176, 217, 232), but it remains unclear whether the
response is mediated indirectly by a vagally induced release of
incretins (203), or more directly by a reflex involving
gut glucoreceptors augmenting efferent activity of the pancreatic
branch of the vagus nerve (233, 234). Glucoresponsive
neurons equipped with K+-ATP channels similar to
those of ß-cells have recently been identified in the myenteric
plexus of the guinea pig ileum (235).
The role of the preabsorptive phases of insulin secretion was initially
addressed by comparing the insulin and glucose responses to oral
vs. iv glucose administrations (203, 236).
However, it was later found that this type of comparison might be
misleading because a lesser glucose tolerance after iv administration
of the sugar could result from the lack of incretin effect rather than
the lack of preabsorptive insulin secretion. The importance of the
cephalic phase for glucose homeostasis was established by experiments
showing that plasma glucose and insulin concentrations increased more
after direct administration into the stomach than after oral intake of
the same amount of nutrients (237, 238). These results
were confirmed in a more recent study that compared the insulin and
glucose responses to gastric glucose administration in humans allowed
or not allowed to taste food (239). Prevention of cephalic
phase during food intake diminished the glucose tolerance without
changing insulin secretion during the 3-h period after the beginning of
food intake. This glucose intolerance was attributed to differences in
the kinetics of the changes in insulinemia, and possibly also to a
larger glucagon secretion over the same period of time
(239).
That the cephalic phase exerts a beneficial, long-lasting effect
has also been elegantly demonstrated after oral glucose administration
to insulin-deficient rats transplanted with denervated islets. The
missing cephalic phase in these rats was mimicked by a small premeal iv
injection of insulin. This restored early insulin peak did not affect
subsequent plasma insulin levels during the period of glucose intake,
but attenuated (without normalizing) the rise in plasma glucose levels
(40).
With the exception of two reports (196, 240), all the
above-described studies suggest that the timing of insulin secretion is
important for optimal glucose homeostasis. By promoting anticipatory
use of glucose by the liver, muscles, and adipose tissue, and by
inhibiting glucose production by the liver, the preabsorptive insulin
phases restrain the changes in glycemia and insulinemia within a narrow
range. This may serve as a protection against overworking of the
ß-cells.
b. Liver.
Like ß-cells, the liver receives efferent vagal
stimuli in response to an oral stimulus (220, 241).
Neurophysiological studies have also revealed that portal glucose
injection increases efferent vagus activity innervating the pancreas,
and it has been suggested that hepatic glucosensitive mechanisms may
affect pancreatic function by involving hepatic vagus afferents and
pancreatic vagus efferents (208). These results are
supported by some physiological experiments demonstrating that a rise
of the glucose concentration in the portal circulation to the liver
induces an increase of insulin secretion that is prevented by vagotomy
of the hepatic branch (242) and is mimicked by hepatic
vagal stimulation (243). However, other studies have
reported that a rise in insulinemia is mimicked by a section of
the hepatic branches of the vagus nerve, whereas a drop in insulinemia
is induced by electrical stimulation of this branch (241, 244). Therefore, it has been suggested that afferent fibers
exert a tonic inhibition in brainstem centers of an efferent vagal
branch innervating the pancreas (241, 244). This implies
that the afferent hepatic nerve activity is inversely related to the
portal glucose level, which is confirmed by neurophysiological data
(245, 246, 247). It is difficult to establish how glucose
homeostasis is influenced by these vagally mediated messages from the
liver to the endocrine pancreas.
c. Brain.
Several studies suggest that an increase in the
glucose concentration in the brain can increase vagal tone. Indeed,
injection of glucose in the carotid artery of rats, in an amount
insufficient to modify systemic plasma glucose concentration, induced a
rapid increase in insulin secretion that was abolished by vagotomy
(182). This effect likely involves glucoresponsive neurons
in the hypothalamus and the nucleus tractus solitarius (220, 248).
3. Is there a long-lasting vagal stimulus during the absorptive
phase?Whereas it is clear that the parasympathetic system
contributes to the preabsorptive insulin response, it is less obvious
whether a parasympathetic stimulus of the endocrine pancreas persists
during the meal. Because ACh is quickly degraded by cholinesterases in
plasma, it is impossible to reliably measure the pancreatic ACh
spillover as an index of parasympathetic neural activity of the
pancreas (229). Measurements of plasma PP provide an
alternative approach to evaluate the parasympathetic activity. Indeed,
PP secretion is predominantly under vagal control because its secretion
in vivo is nearly completely prevented by atropine or
vagotomy (249, 250, 251). Meal ingestion induces a biphasic PP
secretion characterized by a rapid first phase followed by a second
sustained response. Both phases are prevented by atropine
(229). The first phase has sometimes been correlated to
the preabsorptive insulin response (250, 252). The
presence of the second sustained phase supports the existence of a
long-lasting cholinergic stimulus. Because cholinergic nerve fibers
innervating PP and ß-cells likely have a common origin, it is highly
plausible that ß-cells are also under the influence of a long-lasting
vagal stimulus during meals. This suggestion is corroborated by the
observation that atropine markedly suppressed insulin response to a
meal (251). However, recent data obtained with mice
lacking the M3 muscarinic receptor (the main muscarinic
receptor on ß-cells, see Section X) do not clarify the
issue. These mice do not show any signs of impaired glucose intolerance
after oral or ip glucose administration (253), but it is
unclear to which extent other factors that were observed in these
mice, such as increased insulin sensitivity, hypoleptinemia,
and hypophagia, contributed to glucose tolerance.
C. Pathophysiological situations: hyperinsulinemia, obesity, and
insulin resistance
The net effect of the central nervous system on insulin secretion
is the result of a balance between the influence of the inhibitory
sympathetic system and the stimulatory parasympathetic system. The
metabolic consequences of a dysregulation of this subtle balance have
been reviewed recently (254). Only the troubles associated
with an anomaly of the parasympathetic system will be briefly mentioned
here.
Two areas in the brain play a major role in the control of the efferent
autonomic pathways, the ventromedial hypothalamic nuclei (VMH, also
called the satiety center) and the lateral hypothalamic area (LHA, also
called the feeding center) (241, 254). The VMH
increases the activity of the sympathetic nervous system and decreases
that of the parasympathetic nervous system, whereas opposite effects
are produced by the LHA. The VMH and the LHA reciprocally inhibit each
other.
Several animal models of hyperinsulinemia are characterized by a
dysregulation of the sympathetic and parasympathetic pathways. A lesion
of the VMH in the rat causes an exaggerated insulin response to an iv
or intragastric glucose load. This hyperinsulinemia occurs rapidly, 10
min after the lesion (255), and is abolished by atropine
or vagotomy (256, 257, 258, 259). In animal models of
hyperinsulinemia associated with a defect in leptin signaling, such as
the ob/ob mice (abnormal leptin) or fa/fa
(Zucker) rats (abnormal leptin receptors), the earliest detectable
alteration of insulin secretion is a hyperresponsiveness to glucose
that occurs before the animals become hyperphagic (193, 260, 261, 262, 263, 264, 265). It is mediated by the vagus nerve, as it is abolished
by atropine (194, 255, 260, 266) or vagotomy (193, 266). NPY is a potent physiological stimulator of feeding that
is present at abnormally high levels in the hypothalamus of
fa/fa rats and ob/ob mice. Rats that undergo a
chronic intracerebroventricular infusion of NPY display basal and
glucose-induced hyperinsulinemia that is prevented by vagotomy
(267, 268). A common feature of all these animal models is
a hyperinsulinemia that results from an excessive vagal cholinergic
tone and an attenuation of the inhibitory sympathetic tone
(254). The chronic influence of hyperglycemia on the
autonomic system may also aggravate the syndrome
(269).
An increased sensitivity of ß-cells to ACh might also contribute to
hyperinsulinemia. This has been reported in ob/ob mice
(262) and in genetically normal mice subjected to a
high-fat diet (46, 270, 271). The hyperinsulinemia brought
about by an enhanced cholinergic over sympathetic tone and/or an
exaggerated sensitivity of ß-cells to ACh might be a compensatory
mechanism for insulin resistance.
This fairly clear picture obtained in experimental animals cannot
readily be extrapolated to human subjects. Although indirect evidence
suggests that insulin secretion is more sensitive to cholinergic
stimulation in insulin-resistant obese subjects than in lean subjects
(272), it is widely agreed that atropine does not correct
the hyperinsulinemia of obese subjects (195, 196).
Moreover, the preabsorptive phase of insulin secretion was found to be
enhanced (273), normal (224, 274, 275), or
absent in obese subjects (276). In type 2 diabetes, the
initial rise in insulin levels after a meal is often delayed or
deficient (277, 278), but it is unknown whether an
impaired preabsorptive vagal stimulus contributes to this defect.
|
IV. General Characteristics of Acetylcholine (ACh) Effects on
Insulin Secretion in Vitro
|
|---|
The glucose dependence of the effects of ACh on insulin release
has already been emphasized (see Section III and Refs.
162 and 163) and is illustrated in Fig. 1A
.
At the concentration of 1 µM, ACh does not
affect basal insulin secretion (3 mM glucose),
but causes a rapid, marked, and sustained potentiation of insulin
secretion induced by 15 mM glucose (the
half-maximally effective concentration in this model). The effect of
ACh starts to appear between 5 and 7 mM glucose,
i.e., around the threshold concentration of the sugar (not
illustrated), and persists in the presence of a maximally effective
concentration of glucose (30 mM). ACh also
increases insulin secretion in the presence of nutrients other than
glucose, e.g., leucine (279). However,
nutrients can be replaced by tolbutamide to unmask the insulinotropic
effect of ACh. As shown in the lower panel of Fig. 1A, the
addition of 100 µM tolbutamide to a medium
containing 3 mM glucose evokes a small increase
in insulin secretion (mediated by K+-ATP channel
closure and membrane depolarization), and the subsequent addition of 1
µM ACh slightly potentiates insulin secretion.
The larger efficacy of ACh in the presence of high glucose than in the
presence of low glucose plus tolbutamide can largely be ascribed to the
amplification of insulin secretion (increase in
Ca2+ efficiency in exocytosis) that the sugar
produces (280). This glucose dependence persists when ACh
is used at high concentrations (e.g., 100
µM), which, however, also induce a small
sustained elevation of basal insulin secretion (not shown).
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|
Figure 1. General characteristics of ACh effects on insulin
secretion in vitro. Mouse islets were perifused with a
medium containing 2.5 mM CaCl2 (Ca2.5) or no
CaCl2 (Ca0) and 3, 15, or 30 mM glucose (G3,
G15, and G30, respectively). A, Experiments with freshly isolated
islets. In the experiments shown in the lower panel, 100
µM tolbutamide (Tolb) was added to the medium to close
K+-ATP channels and depolarize the ß-cell membrane
despite the low glucose concentration. [The lower panel
was redrawn from M. P. Hermans et al.:
Endocrinology 120:1765–1773, 1987 (279 ).]
B, Experiments with cultured islets. In the experiments shown in the
lower panel, the medium was supplemented with 100
µM diazoxide (Dz) to open K+-ATP channels and
hold the membrane hyperpolarized despite the high glucose
concentration.
|
|
The pattern of ACh-induced insulin secretion critically depends on
whether Ca2+ influx can occur
(281, 282, 283, 284, 285, 286, 287, 288). In the presence of a control medium containing
extracellular Ca2+, the stimulation of secretion
is sustained. When Ca2+ is omitted from the
medium, only high ACh concentrations (
10 µM) trigger a
rapid, transient peak of secretion that also requires the presence of a
high concentration of glucose (or other nutrients) (Refs.
162 and 289, 290, 291 and Fig. 1B
).
Ca2+ influx can also be prevented by opening
K+-ATP channels with diazoxide and holding the
membrane at the resting potential. Under these conditions, the effect
of ACh is similar to that produced in the absence of extracellular
Ca2+ (289) (Fig. 1B, lower
panel). Blockade of voltage-operated Ca2+
channels similarly affects the action of ACh on secretion (not shown)
(287).
The mechanisms underlying these glucose, membrane potential, and
Ca2+ dependencies of ACh-induced insulin
secretion will be explained in the following paragraphs.
|
V. Effects of ACh on ß-Cell Phospholipases
|
|---|
A. Activation of PLC
1. Type of PLC and mechanisms of activation. PLC enzymes
hydrolyze the phosphodiester bond on the third (sn-3) position of
phosphoglyceride molecules to release diacylglycerol (DAG) and a
phosphorylated polar head group (Figs. 2 and 3). There exist three main groups of PLC (PLC-ß, PLC-
,
and PLC-), each containing several
subtypes (292, 293, 294). PLC-ß are activated by
heterotrimeric G proteins, whereas PLC- are activated by tyrosine
kinases. The mechanisms of activation of PLC- are unknown
(294). All three types hydrolyze phosphatidylinositol
(PI), PI 4-phosphate (PIP), and PI 4,5-bisphosphate
(PIP2) in a Ca2+-dependent
manner to produce DAG and inositol 1-phosphate (1 IP),
inositol 1,4-bisphosphate, and IP3, respectively. At low
[Ca2+]c,
PIP2 is the preferred substrate
(293). In some tissues, certain PLC isoforms can hydrolyze
plasmenylcholine, phosphatidylethanolamine, or phosphatidylcholine (PC)
(295, 296, 297, 298).
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Figure 2. Influence of ACh on phosphoinositide metabolism in
pancreatic ß-cells. See text (Section V.A.2) for
explanations.
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Figure 3. Influence of ACh on phospholipid metabolism in
pancreatic ß-cells and its role in the control of insulin secretion.
Arrows with solid lines represent metabolic or
biophysical pathways. Arrows with dashed and dotted
lines illustrate stimulatory and inhibitory influences.
Question marks denote PKC-stimulating pathways that are
still debated or are not clearly demonstrated in ß-cells. See text
(Section V) for explanations.
|
|
In pancreatic ß-cells, PLC is both cytosolic and membrane associated
(299, 300, 301) and specifically hydrolyzes
phosphoinositides (300, 302). Stimulation of normal
ß-cells (5, 286, 303, 304, 305, 306, 307, 308, 309) and insulin-secreting tumor
cells (310, 311, 312, 313) with cholinergic agonists has long been
shown to cause DAG and IP3 accumulation. By analogy with other tissues
(314, 315), it is assumed that this results from
activation of a PLC-ß isozyme. However, it is not known which of the
three PLC-ß isoforms (ß1, ß2, or ß3) identified in ß-cells
(316, 317, 318, 319, 320) is coupled to the muscarinic receptor.
Activation of PLC by cholinergic agonists involves a G protein
(310, 321, 322). One single study, performed with rat
islets, reported that the G protein activated by carbachol is pertussis
toxin sensitive and suggested that it corresponds to a
Go protein (323). All other
studies, performed with RINm5F cells (324, 325), rat
islets (321, 326, 327), and ß-TC3 cells
(328), found the G protein coupled to the muscarinic
receptor to be pertussis and cholera toxin insensitive. It is thought
to belong to the Gq subfamily (328),
like the G protein that couples muscarinic receptors to PLC-ß in
other tissues. Activation of PLC-ß has been reported to be directly
mediated by the -subunit of the Gq protein
(329). The Gq subfamily contains
several members (Gq,
G11, G14,
G15, and G16)
(330, 331) that seem to specifically interact with the
different PLC-ß subtypes (292). The nature of the
G-subunit involved in the coupling of the
muscarinic receptors to PLC-ß in ß-cells is unknown.
Phospholipid hydrolysis and inositol production is larger in ß-cells
maintained in a Ca2+-containing medium than in a
Ca2+-free medium (5, 162, 290, 303, 332), and are markedly reduced in insulin-secreting cells loaded
with the Ca2+-chelator
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA) (333). Two hypotheses have been put forward
to explain this Ca2+ dependence. Because PLC is
strictly Ca2+ dependent (293), the
enhanced hydrolysis of phosphoinositides observed in cells bathed in a
Ca2+-containing medium has sometimes been
attributed to a direct activation of PLC by Ca2+
(300, 305, 307, 322, 332, 333, 334, 335, 336, 337, 338), independently from
calmodulin (300, 337). This proposal was supported by the
observations that high K+, which induces a large
rise in [Ca2+]c,
increased IP3 or total IPs levels (332, 339, 340) and
accelerated the efflux of radioactivity from rat islets prelabeled with
[3H]inositol (335). However, these
results must be interpreted with caution. First, the effect of high
K+ on IP3 levels is species dependent and larger
in the rat than in the mouse (340), perhaps because of the
expression of different PLC isoforms (317, 320, 341, 342).
Second, even in the rat, phosphoinositide breakdown is much larger in
response to carbachol than to high K+ (316, 332, 335, 339). Therefore, a second hypothesis suggests that the
potentiation by Ca2+ of ACh-induced IP3
production results from a synergistic effect between
Ca2+ and muscarinic activation (309, 316). As G proteins have been reported to enhance the
Ca2+ sensitivity of PLC (343, 344, 345, 346),
the Ca2+ requirement would be reduced to the
resting [Ca2+]c levels.
This would also explain how muscarinic agonists can elicit a
significant phosphoinositide hydrolysis in cells bathed in a
Ca2+-free medium. Finally, three studies have
proposed that PLC activity can also be controlled by the membrane
potential, independently from a change in
[Ca2+]c. Depolarization
in a medium supplemented with methoxyverapamil or in a
Ca2+-free medium was reported to increase PLC
activity in ob/ob islets (347) or in
insulin-secreting ß-TC3 cells loaded with
1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic
acid (BAPTA) (338). The opposite effect, a decrease in PLC
activity, was measured in rat islets (339). Thus, the
question remains unanswered.
2. Phosphoinositol and phosphoinositide metabolism.
Experiments with RINm5F cells or rat islets have shown that IP3 is very
rapidly transformed into inositol 1,3,4,5-tetrakisphosphate (307, 332, 348) by a kinase activated by
Ca2+-calmodulin (305, 349, 350), and
into inositol 1,4-bisphosphate by a phosphatase (305, 348, 351, 352) (Fig. 2). Inositol 1,3,4,5-tetrakisphosphate can then
be degraded into inositol 1,3,4-trisphosphate. All inositol phosphate
isomers can be further metabolized through complex pathways (350, 353). In several studies in which the different isomers of
inositol phosphate were not separated (5, 290, 311, 313),
cholinergic agonists caused a monotonic increase of inositol
trisphosphate levels to a plateau that was reached within approximately
1 min and thereafter maintained with only a minor decline. When the two
major isomers of inositol trisphosphate, i.e., IP3 and
inositol 1,3,4-trisphosphate, were separated, very different time
courses of accumulation emerged (305, 311, 352). Indeed,
IP3 accumulation consisted in a burst, reaching a peak within the first
5 sec of stimulation, followed by a decrease and then a lower sustained
phase. By contrast, inositol 1,3,4-trisphosphate levels increased
slowly, reached a maximum after approximately 30 sec of stimulation,
and plateaued at that level thereafter. The biphasic increase in IP3
probably involves both a negative feedback effect of PKC on PLC
activity (see Section V.A.3) and a rapid degradation of IP3
(352, 354). Indeed, the two-step conversion of IP3 into
inositol 1,3,4-trisphosphate initiated by the
Ca2+-calmodulin-sensitive IP3 kinase was markedly
attenuated if the rise in
[Ca2+]c that IP3 produces
(see Section VIII.A.1) was abolished (305).
This suggests that the rise in
[Ca2+]c contributes to
the rapid degradation of IP3 and, therefore, also contributes to the
transient nature of its accumulation (305, 352).
Whereas it is well established that muscarinic stimulation of insulin
secretion increases with the glucose concentration, it remains unclear
whether glucose, per se, potentiates ACh-induced IP3
accumulation. The larger effect that carbachol produces in the presence
of high glucose (164, 304, 316, 355) might well result
from an additional Ca2+-dependent activation of
PLC due to a greater increase in
[Ca2+]c (see
Section VIII.A.3). This interpretation is supported by the
observation that glucose failed to enhance carbachol-induced
accumulation of inositol trisphosphate in rat islets incubated in a
Ca2+-free medium (339). Glucose
itself and various intermediates of its metabolism were without
effect on PLC activity in a cytosolic fraction of mouse islet
homogenate (300). However, other reports have suggested
that glucose metabolism might interact with phosphoinositol and
phosphoinositide metabolism. Thus, glucose metabolites inhibited
IP3 degradation (356, 357) and increased IP3 production
(358) in rat islet and RINm5F cell homogenates, and
directly stimulated Ca2+ release from
intracellular Ca2+ stores of HIT-T15 cell
homogenates (359). Moreover, glucose has been reported to
stimulate the de novo synthesis of DAG, inositol phosphate,
phosphoinositides, or polyphosphoinositides (4, 302, 305, 311, 358, 360, 361, 362, 363, 364, 365, 366). More experiments must be performed to evaluate
the possible direct effects of glucose on ACh-induced IP3
accumulation.
In many tissues, PLC mainly hydrolyzes PIP2. In
pancreatic ß-cells, cholinergic agonists also stimulate PI hydrolysis
as shown by the rapid accumulation of 1 IP independently from the
other phosphoinositol intermediates in rat islets challenged with
carbachol (348) (Fig. 2). In agreement with this
observation, PI was found to be a better substrate than
PIP2 for PLC from the cytosolic fraction of mouse
islet homogenates (300). Surprisingly,
carbachol-induced accumulation of 1 IP in rat islets is stimulated
by hyperpolarization and is inhibited by depolarization of the plasma
membrane (339). The underlying mechanisms are not known.
The predominance of PI hydrolysis over that of
PIP2 during prolonged stimulation
(348) implies that DAG can be formed independently of IP3
formation. However, the potential importance of this pathway for the
stimulation of insulin release has yet to be established.
At the same time cholinergic agonists hydrolyze phospholipids, they
also accelerate PI turnover. DAG can be resynthesized back to PI by the
following steps (Fig. 2): 1) diacylglycerol kinase converts DAG into
phosphatidic acid (PA) at the expense of an ATP; and 2) PA then reacts
with CTP to form CMP-phosphatidate (CDP-DAG), which in turn reacts with
inositol to form PI (346, 367, 368, 369). The enzymes involved
in this cycle are present in rat islets (370, 371).
Because the cycle requires phosphorylated nucleotides, its turnover was
estimated by labeling islets with
32PO43-.
It was found that carbachol stimulates the labeling of PA and decreases
that of PIP and PIP2, which suggests that
cholinergic agonists accelerate PI turnover after PLC activation in rat
islets (5, 312, 360, 372, 373). This effect was strongly
inhibited in a Ca2+-free medium, which might
result from the Ca2+ dependence of PLC
(334, 372). Concomitantly, there is an enhanced flux from
PI 
PIP PIP2, which is necessary for
resynthesis of PIP2 (322). This
increased flux might result from a direct stimulation of the activity
of PI 4-kinase, the enzyme responsible for the synthesis of PIP
from PI (322). Because PIP kinase is inhibited by its
product, PIP2, hydrolysis of
PIP2 by PLC could also relieve this inhibition,
therefore stimulating the flux for PIP2
synthesis. This latter mechanism has been demonstrated in other cell
types (374), but not in ß-cells. Cholinergic agonists
not only accelerate PI turnover, they also stimulate de novo
synthesis of phospholipids, as deduced from the enhanced incorporation
of [3H]glycerol into DAG (375),
PA, and PI in rat islets (360). Again, this de
novo synthesis pathway is very much Ca2+
dependent (360).
Of all inositol species formed upon ACh stimulation, IP3 is the
physiologically more important isomer. Its effects will be described
later (see Section VI.A.1).
3. Diacylglycerol and PKC. DAG is liposoluble and remains in
the plasma membrane. It causes the translocation of its target, PKC,
from the cytosol to the membrane. This translocation also requires
Ca2+ and an acidic phospholipid, such as
phosphatidylserine (376, 377). Metabolism of DAG, either
by DAG lipase-catalyzed deacylation (which yields arachidonic acid) or
by DAG kinase-catalyzed phosphorylation (which yields PA), terminates
its action on PKC (299, 378) (Fig. 3).
Cholinergic agonists produce two major species of DAG that are enriched
in either arachidonate (a polyunsaturated fatty acid) or palmitate (a
saturated fatty acid), and accumulate with different time courses in
ß-cells (Fig. 3). The concentration of arachidonate-enriched DAG
increases quickly during the first seconds of stimulation, before
declining and remaining at a lower sustained level. This type of DAG
probably originates from PIP2 that mostly
contains arachidonate at the sn-2 position of the phospholipid
(302, 379). By contrast, the palmitate-enriched DAG
accumulates monotonically during the first minutes of stimulation
(302, 375). This species resembles that produced upon
glucose stimulation (365, 375), but its source upon ACh
stimulation is unknown. It might originate from PLD activation (see
Section V.C) and hydrolysis of PC, a phospholipid
enriched in palmitate in islets, from a synergistic effect with glucose
on de novo DAG synthesis, or from other undefined pathways
(302, 380). A biphasic increase in polyunsaturated DAG and
a delayed accumulation of saturated DAG have been documented in many
other cell types (381). The differential time course of
accumulation of the two DAG species might have an impact on PKC
activation. Indeed, polyunsaturated DAG is a much more potent PKC
activator than saturated DAG in HIT cells (365, 382) and
other cell types (379).
The PKC family comprises a number of phosphatidylserine-binding
isoforms that can be classified in four groups. The conventional
isoforms, or cPKC, are activated by Ca2+ and DAG
or phorbol esters; they include , ßI, ßII, and , of which
ßI and ßII refer to the two gene products resulting from
alternative splicing of the same PKCß gene. The novel isoforms, or
nPKC (, 
, 
, and 
), are unresponsive to
Ca2+ but are activated by DAG alone or phorbol
esters. The atypical isoforms, or aPKC (
, and 
/
; PKC is the
mouse homolog of human PKC), are Ca2+
independent and do not bind DAG or phorbol esters. The PKCµ isoform
is also Ca2+ independent and is activated by
phorbol esters, but has a structure different from the other isoforms
(298, 381, 383, 384, 385, 386). The nature of the PKC isoforms
present in insulin-secreting cells remains controversial (reviewed in
Ref. 376). In pancreatic islets, the isoform
predominates, but one or several of the isoforms ß, , , ,
and may also be present (376, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396). This does not
necessarily mean that all these isoforms are expressed in ß-cells
because approximately 20–35% of the islet cells are non-ß-cells.
Insulin-secreting cell lines express one or several of the isoforms
, ß, , , , , /, and µ (382, 387, 392, 394, 397, 398, 399, 400).
Several studies demonstrate that cholinergic agonists induce the
translocation of PKC to membranes (163, 312, 365, 382, 401), but they do not establish which isoforms are translocated.
Because cholinergic agonists induce DAG accumulation (302, 339, 375), it seems reasonable to assume that they stimulate all
DAG-sensitive isoforms present in normal and tumoral insulin-secreting
cells. However, a recent study suggests that this might not be the
case. Carbachol was found to translocate the , ß, and isoforms
without affecting the , , µ, and isoforms in RINm5F cells
(400). These data must be interpreted with caution because
they were obtained with an insulin-secreting cell line whose responses
to cholinergic agonists differ from those of normal ß-cells (see
Section IX.D).
PKC phosphorylate their substrates on serine and/or threonine residues.
It has been suggested that the targeting of PKC isoforms to particular
membranes is mediated by specific anchoring proteins including the
receptors for activated C kinases (384, 402), which might
explain why a PKC isoform is translocated either to the nucleus or the
plasma membrane. It is possible that the multiple phospholipid-derived
second messengers produced upon ACh stimulation activate different PKC
isoforms that, after being translocated to specific targets, activate
different pathways (403). Such a differential activation
of PKC isoforms has been reported upon glucose stimulation, with PKC
and PKC being translocated to the cell periphery and PKC and
PKC being translocated to perinuclear sites (396).
Many proteins are phosphorylated by PKC in islets (for review, see Ref.
376), but their nature is largely unknown. One identified
target for PKC in ß-cells is the myristoylated alanine-rich C kinase
substrate (MARCKS) (404), a protein that binds actin and
Ca2+-calmodulin and that has been implicated in
cell movement and vesicle transport (405, 406). This
substrate is phosphorylated in response to carbachol (407, 408). Other PKC substrates might be the G proteins that are
associated with the 2-adrenoceptor and
uncouple from the receptor after phosphorylation. This mechanism might
explain how phorbol esters and carbachol reduce the ability of
adrenoceptors to inhibit glucose-induced insulin secretion from rat
islets (409, 410). In view of the importance of the
PKC-dependent pathway in the stimulation of insulin secretion by
ACh (see Section IX.B.1), identification of the targets of
PKC in ß-cells is an important question.
It has been suggested that PKC activation also exerts a negative
feedback control on the signal transduction linked to PLC and activated
by ACh. Indeed, stimulation of PKC inhibits the production of inositol
phosphates induced by cholinergic agonists (290, 312, 322, 352, 393). This effect occurs within 10 min (perhaps within even
less time) of stimulation with phorbol esters (352). It
likely contributes to the biphasic time course of accumulation of IP3
and arachidonate-enriched DAG upon stimulation with ACh. This
PKC-mediated negative feedback might result from the uncoupling of PLC
from the ACh receptor (411), either by a direct
phosphorylation of PLC by PKC (412) and/or
Ca2+-calmodulin kinase (413), by a
modification of the G protein coupling the ACh receptor to PLC
(414, 415), or by phosphorylation of the receptor itself
(416). A muscarinic receptor kinase has recently been
identified that might fulfill this role leading to decreased PLC
activity (315, 417). Alternatively, cholinergic
stimulation could cause a down-regulation of muscarinic receptors via
(312, 418) or independently (419) of PKC
activation.
B. Activation of PLA2
PLA2 enzymes hydrolyze the sn-2 ester
linkages in phosphoglyceride molecules to release a lysophospholipid
and a free acid, such as arachidonate (Fig. 3). They can hydrolyze
various substrates, such as PC, phosphatidylethanolamine,
phosphatidylserine, PI, PA, and plasmalogens (420).
There exist several types of mammalian PLA2
(421). Types I, II, V, and VII are associated with
membranes and, because they are secreted, are referred to as
secretory PLA2
(sPLA2). Types I, II, and V are stimulated by
millimolar Ca2+ concentrations, whereas type VII
is Ca2+ independent (421). Types IV,
VI, and VIII are cytosolic. Type IV is Ca2+
dependent, requiring micromolar Ca2+
concentrations to be translocated to the membrane, whereas types VI and
VIII are Ca2+ independent (421).
Types IV and VI PLA2 display a specificity for
phospholipids with arachidonic acid esterified to the second carbon of
the glycerol backbone (422, 423), whereas types I and II
show little specificity for the hydrolyzed fatty acid chain (293, 423).
The presence of sPLA2 in insulin-secreting cells
is well documented, but controversies persist concerning the type of
sPLA2 that is expressed (424, 425, 426).
sPLA2 might be associated with insulin-secretory
granules (427). Pancreatic islets and insulin-secreting
cell lines also contain type IV PLA2 and type VI
cytosolic, ATP-stimulatable Ca2+-independent
PLA2 (ASCI-PLA2)
(422, 425, 426, 428, 429, 430, 431, 432).
Several studies suggest that ACh activates PLA2
in islets (Fig. 3). Thus, cholinergic agonists stimulate efflux of
radioactivity from rat or mouse islets prelabeled with radioactive
arachidonic acid (5, 433, 434, 435), and arachidonate
represents the major metabolite present in the effluent fractions
(433). This effect is largely Ca2+
dependent, as the stimulated efflux of radioactive arachidonic acid was
markedly reduced by verapamil or removal of external
Ca2+ (433, 434, 436). The
persistence of a small stimulation of the efflux in a
Ca2+-free medium is compatible with the
activation of a Ca2+-independent pathway
(433, 434). Similar results were obtained by studying
carbachol-induced production of PGE2, an
eicosanoid derived from arachidonic acid (327). Muscarinic
activation of PLA2 is also supported by the
demonstration that both lysophosphatidylcholine (434) and
arachidonic acid (375, 434, 437) accumulate in rat islets
upon stimulation with carbachol. However, because the accumulation of
arachidonic acid was larger than that of lysophosphatidylcholine and
was approximately 65% inhibited by RG80267, an inhibitor of DAG
lipase, it is likely that only a fraction of arachidonic acid
accumulation results from PLA2 activation
(434). The other fraction of arachidonic acid could derive
from DAG lipase-catalyzed deacylation of DAG formed after PLC
activation (299, 434). Because PLC can be activated by ACh
in Ca2+-dependent and
Ca2+-independent ways, this pathway could explain
how some arachidonic acid accumulates even in the absence of
Ca2+. Carbachol has also been suggested to
activate ASCI-PLA2, but this proposal was based
on the use of haloenol lactone suicide substrate (HELSS), an
inhibitor of ASCI-PLA2 (438) that
has since been shown to exert nonspecific effects in ß-cells
(435).
The transduction mechanisms leading to activation of
PLA2 in ß-cells are not known. It is likely
that the increase in
[Ca2+]c produced by
cholinergic agonists activates the cytosolic
Ca2+-dependent PLA2
(439). Indeed, high K+ and the
Ca2+ ionophore A23187 also increased arachidonic
acid accumulation within rat islets and also increased
PGE2 release from rat islets (440).
Other mechanisms might also activate PLA2,
including emptying of intracellular Ca2+ stores
(441), G protein regulation (420), PKC
(297, 298), and MAPK (442).
The two primary products formed upon PLA2
activation are arachidonic acid and lysophosphatidylcholine (Fig. 3).
Arachidonic acid has been reported to exert various effects in
ß-cells (380). These include Ca2+
mobilization from the endoplasmic reticulum (Refs.
443, 444, 445, 446, 447, 448 , but see Refs. 324 and
448), facilitation of voltage-dependent
Ca2+ entry (380, 449), increase in
[Ca2+]c through
voltage-independent Ca2+ channels
(449), activation of K+-ATP channels
(450), and stimulation of PKC (446, 451).
Among all these effects, the last one deserves particular attention
because it also exists in other cell types and consists of a direct
activation of PKC or a potentiation of the PKC stimulation by DAG
(297, 298, 381, 403, 452, 453). Arachidonic acid is also
the precursor of cyclooxygenase (PGs, prostacyclins, and
thromboxane) and lipoxygenase products (hydroperoxyeicosatetraenoic
acids, hydroxyeicosatetraenoic acids, and leukotrienes)
(297) that seem to exert various, although not major,
modulatory effects on insulin secretion (380, 454, 455). Lyso-phosphatidylcholine also activates PKC in the
presence of DAG in various cell types (381). In ß-cells
and insulin-secreting cell lines, it stimulates
45Ca2+ efflux and insulin
secretion (456, 457, 458, 459).
C. Activation of PLD
PLD catalyzes the hydrolysis of the terminal diester bond of the
membrane glycerophospholipids, resulting in the formation of PA and a
free polar head group (Fig. 3). Different PLD isoforms hydrolyze
various substrates, such as PC, PI, phosphatidylserine, or
phosphatidylethanolamine (460, 461). PC is the most
abundant phospholipid in pancreatic islets (462, 463, 464), and
its hydrolysis by PLD yields PA and choline. PLD can be activated by
numerous pathways, including tyrosine kinases, PKC, or small G
proteins, and seems to require various cofactors, such as fatty acids,
or Ca2+, for its activation (461, 465, 466).
The studies of PLD activation in islets have yielded conflicting
results. Carbachol has been reported to increase the production of
[3H]choline in mouse islets prelabeled with
[methyl-3H]choline
(467), which suggests that PLD was activated. Because
this effect was mimicked by sodium fluoride, which activates PLC,
and by a phorbol ester, it might result from PKC activation (467, 468). Activation of PLD by PKC has been documented in other cell
types (293, 460, 461, 466). However, carbachol did not
affect [3H]choline production in rat islets
prelabeled with [3H]choline (302).
Therefore, the carbachol-induced accumulation of PA in rat islets
(437) was not ascribed to PLD activation, but to
phosphorylation by DAG kinase of DAG derived from PLC activation
(318).
In various cell types, PLD activation is implicated in the regulation
of vesicular trafficking, and its main product, PA, is involved in
secretion, mitogenesis, and inflammation (461). PA
has been reported to directly activate PKC (469). However,
early suggestions that PA can act as a second messenger to stimulate
insulin release (470, 471) still await confirmation. The
action of PA is terminated by its conversion into lyso-PA by a
PLA2 or DAG by PA-phosphohydrolase
(472). The resulting DAG accumulation might theoretically
activate PKC. However, this mechanism is probably of minor importance
because DAGs derived from PLD contain saturated or mono-unsaturated
fatty acids at the sn-2 position and are poor activators of PKC
(379).
|
VI. Effects of ACh on the Membrane Potential of ß-Cells
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|---|
To understand how ACh influences the membrane potential of
ß-cells, it is important to bear in mind the mechanisms by which
glucose regulates this membrane potential. Glucose enters the ß-cell
by a facilitated transport system belonging to the GLUT family
(473, 474, 475), and its metabolism leads to a rapid increase
in the ATP/ADP ratio (476), which closes
K+-ATP channels in the plasma membrane. In the
absence of glucose or at a nonstimulating glucose concentration, the
ATP/ADP ratio is low. Enough K+-ATP channels are
open to confer a low electrical resistance to the plasma membrane and
to keep it at the resting potential, close to the equilibrium potential
of K+. In the presence of a stimulating glucose
concentration, the ATP/ADP ratio is high, and
K+-ATP channels are largely closed, which
increases the resistance of the membrane. The decrease of the
K+ permeability allows a yet unidentified current
to depolarize the plasma membrane. When the threshold potential for
activation of voltage-dependent Ca2+ channels is
reached, an oscillating electrical activity starts (477, 478). Each oscillation of the membrane potential is
characterized by a sustained depolarizing phase, commonly called slow
wave, on top of which Ca2+ spikes occur. The
effect of glucose on the membrane potential can be mimicked by other
nutrients, e.g., leucine, that are metabolized by the
ß-cell. It can also be reproduced by a pharmacological agent, such as
tolbutamide, that directly closes K+-ATP channels
(479). In contrast, the effect of glucose on the
membrane potential can be antagonized by diazoxide, which directly
opens K+-ATP channels even when the ATP/ADP ratio
has been increased by glucose.
A. Dependence on the electrical resistance of the plasma
membrane
The effects of ACh on the membrane potential depend on the glucose
concentration (Fig. 4, A–C). In
the presence of a low glucose concentration (<5 mM), when
the membrane potential is high (resting potential), cholinergic
agonists (1–100 µM) produce only a small and sustained
depolarization and do not induce electrical activity (279, 480, 481, 482, 483) (Fig. 4A). In contrast, when the membrane has already
been partially depolarized by a stimulatory concentration of glucose,
the depolarizing effect of ACh is larger and is accompanied by an
increase of the electrical activity (Fig. 4B). However, if
glucose-induced depolarization is reversed by diazoxide, the effect of
ACh on the membrane potential is again similar to that produced in low
glucose (Fig. 4C). This difference is not due to the absolute level of
the membrane potential before addition of ACh. Thus, the effect of ACh
remains small when the membrane is depolarized by high
K+ or arginine in the presence of diazoxide, and
is large when the membrane is depolarized by tolbutamide in low glucose
(279). These results indicate that the depolarizing action
of ACh critically depends on the resistance of the plasma membrane.
When K+-ATP channels are open, either because the
glucose concentration is low or because of the presence of diazoxide,
the plasma membrane has a low resistance, and ACh produces only a minor
depolarization. The depolarizing action of ACh is much larger when the
plasma membrane has a high resistance because of the closure of
K+-ATP channels by glucose or tolbutamide. In the
presence of a stimulating glucose concentration, cholinergic agonists
accelerate the slow waves of membrane potential or produce a sustained
depolarization with continuous electrical activity (161, 279, 482, 484, 485, 486, 487) (Fig. 4B). This depolarizing effect is already
manifest at low concentrations of ACh (
0.1 µM) or
cholinergic agonists (161, 279, 482). One report has
described a peculiar inhibitory effect of muscarinic agonists on
glucose-induced electrical activity in ß-cells
(488).
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Figure 4. Effects of ACh on the membrane potential (A–C)
and voltage-dependent Ca2+ current (D) of mouse pancreatic
ß-cells. A–C, The membrane potential of a single cell within an
islet was recorded with a high resistance microelectrode. A, Sodium
dependence of the effect of 20 µM ACh on the membrane
potential of ß-cells perifused with a medium containing 3
mM glucose (G) and 2.5 mM Ca2+. ACh
was added when indicated to a medium containing 135 mM
Na+ (Na 135 mM) or to a medium in which
Na+ has been replaced by
N-methyl-D-glucamine (Na 0). [Redrawn from
J. C. Henquin et al.: Endocrinology
122:2134–2142, 1988 (480 ) © The Endocrine Society.] B,
Effects of two concentrations of ACh (1 and 100 µM) on
the membrane potential of a ß-cell perifused with a medium containing
15 mM glucose throughout. The two recordings are shown
without interruption. [Redrawn from P. Gilon et
al.: Biochem J 311:259–267, 1995
(545 ). © the Biochemical Society.] C, Effect of 1
µM ACh on the membrane potential of a ß-cell perifused with a medium
containing 10 mM glucose throughout. Diazoxide (100
µM) was added when indicated. By reducing the resistance
of the plasma membrane, it decreases the depolarizing action of ACh.
[Redrawn from M. P. Hermans et al.:
Endocrinology 120:1765–1773, 1987 (279 ).
© The Endocrine Society.] D, Inhibition of voltage-dependent
Ca2+ current in an isolated ß-cell. The current, recorded
in the whole-cell mode of the patch-clamp technique, was elicited by a
depolarization from -80 to +10 mV every 10 s. The upper
trace shows control current and current after the application
of ACh. The lower trace represents the time course of
the peak Ca2+ current. An upward deflection
corresponds to a decrease of its amplitude. The inhibitory effect of
ACh depends on the concentration used and is reversible. [Redrawn from
P. Gilon et al.: J Physiol
499:65–76, 1997 (630 ).]
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B. Mechanisms of the depolarization
Several ionic mechanisms may depolarize the plasma membrane: a
decrease of K+ permeability, an increase of
Na+, Ca2+, or
Cl- permeability, or an inhibition of the
electrogenic Na+ pump.
Before K+-ATP channels were identified in
ß-cells and were shown to be the target of glucose metabolism,
measurements of 86Rb+
efflux (a tracer of K+ efflux) from mouse islets
indicated that ACh depolarizes the ß-cell membrane by a mechanism
other than a decrease in K+ conductance
(285). Thus, under no experimental condition did ACh
decrease 86Rb+ efflux as do
glucose and tolbutamide. Moreover, the effects of ACh on the electrical
activity were very different from those induced by glucose through
closure of K+-ATP channels. Indeed, a rise in the
glucose concentration increased the duration of the plateau phase
without affecting the frequency of slow waves, whereas low
concentrations of ACh increased the frequency of slow waves of the
membrane potential without affecting the duration of the plateau phase
(279, 484). All available data, except those of one study
(486), speak against an effect of ACh on
K+-ATP channels. However, no direct test with the
patch-clamp technique has been reported. In view of the recent
suggestion that PIP2 might negatively modulate
K+-ATP channels, and that its
hydrolysis by PLC-linked agonists might decrease
K+-ATP channel activity (489, 490),
it would be interesting to evaluate whether ACh indirectly influences
K+-ATP channels in ß-cells.
It has been suggested that ACh inhibits Cl-
channels in outside-out patches of ß-cell membrane
(491). However, ACh was found not to affect
36Cl- efflux from normal
mouse islets (492) and
36Cl- retention by
ob/ob mouse islets (493). Moreover, the
depolarization produced by ACh was unaffected in a
Cl--free medium (492). Overall,
these observations indicate that Cl- plays no
major role in the effect of ACh on the ß-cell membrane potential.
The currently accepted hypothesis is that ACh depolarizes the ß-cell
membrane by increasing its permeability to Na+
(480, 493). The cornerstones of this proposal are the
abolition of the depolarization by omission of extracellular
Na+ (Fig. 4A) (480) and the
activation of a small Na+-dependent inward
current by ACh (494). The hypothesis is also supported by
the observations that ACh increases total Na+
content (495),
22Na+ uptake (480, 493), and free cytosolic Na+ concentration
([Na+]c)
(496) in islet cells. The mechanisms by which ACh
activates a Na+ current are not known. Activation
of voltage-dependent Na+ channels has been ruled
out for two reasons: 1) these channels are already completely
inactivated at the resting potential in mouse ß-cells
(497) or at the plateau potential in the rat
(498); and 2) tetrodotoxin, a blocker of voltage-dependent
Na+ channels, does not prevent the
depolarization, the 22Na+
uptake, the [Na+]c
increase, or the inward current produced by ACh (480, 494, 496). Nicotinic receptors are nonselective cation channels
(416, 499). However, these channels are not present in
pancreatic ß-cells. All effects of ACh on membrane potential,
Na+ current, and
[Na+]c measurements are
completely prevented by atropine, whereas they are unaffected by
tubocurarine or hexamethonium, two nicotinic antagonists, and are not
mimicked by nicotine (480, 484, 486, 493, 494, 496). In
cardiac Purkinje cells, ACh was found to increase
[Na+]c by blockade of the
Na+ pump (500). This is not the case
in ß-cells, because ACh- and ouabain-induced
[Na+]c increases were
additive (495, 496).
In various cell types, emptying of intracellular
Ca2+ pools activates different conductances
(Ca2+, Na+, or
K+) (501, 502, 503, 504, 505) carried by a family
of channels called store-operated channels (SOCs)
(503, 504, 505). The tumoral insulin-secreting MIN6 cells
express the transient receptor potential 1 gene (506),
whose human homolog encodes a nonselective channel permeable to
Na+ and Ca2+ and is
activated by Ca2+ store depletion
(507). In platelets and lymphocytes (508, 509), intracellular Ca2+ store depletion
by thapsigargin and cyclopiazonic acid, two inhibitors of the
sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)
pump, activates Na+ influx. It has been
hypothesized that ACh, which also depletes intracellular
Ca2+ stores (see Section VIII.A.1),
activates Na+ influx by a similar mechanism
(487). However, this pathway accounts for only a small
fraction of the influx of Na+ elicited by ACh in
ß-cells, because thapsigargin or cyclopiazonic acid, which empty
intracellular Ca2+ pools much more
efficiently than does ACh, did not mimic or abolish the rise in
[Na+]c produced by ACh
(510). Likewise, thapsigargin did not prevent ACh from
activating an inward Na+ current
(494).
It is clear that K+ is the major counterion for
the increased Na+ influx in ß-cells. Indeed,
ACh induces a sustained stimulation of
86Rb+ efflux from mouse
islets, which is abolished in a Na+-free medium
(162, 285, 480). This acceleration of
K+ efflux is a very sensitive response to ACh,
similar to that of the membrane potential, as it is almost maximally
stimulated by 1 µM ACh. Its resistance to omission of
extracellular Cl- and to furosemide rules out
the intervention of the
Na+K+2Cl-
cotransport system (492). It remains unclear whether the
channel activated by ACh is highly selective for
Na+ or is nonselective, carrying both
K+ efflux and Na+
influx.
Activation of a Na+ conductance by muscarinic
receptors is not classical, but it has also been reported in other
systems. M2 receptors induce a tetrodotoxin- and
pertussis toxin-resistant Na+ current in
ventricular myocytes (511, 512, 513). Muscarinic stimulation
activates a nonselective cationic conductance in guinea pig gastric and
ileal smooth muscle cells (514, 515, 516, 517), rabbit jejunal
longitudinal cells (518), canine pyloric circular muscle
cells (519), and chromaffin cells (520).
Recently, an inward monovalent cation current activated by carbachol
has been reported in Chinese hamster ovary (CHO) cells expressing the
M3 receptor (521).
It is important to emphasize here that although the SOC current is not
responsible for the influx of Na+ triggered by
ACh, it can depolarize the plasma membrane by stimulating
Ca2+ influx (capacitative
Ca2+ entry; see Section VIII.A.2).
Indeed, thapsigargin has been reported to stimulate
Ca2+ influx and to depolarize ß-cells
(522, 523). This mechanism is activated by high
concentrations of ACh (100 µM), but contributes
much less to the depolarization of the plasma membrane than does the
stimulation of Na+ influx that is already
operative at low concentrations of the neurotransmitter (1
µM).
C. Paradoxical hyperpolarization by ACh
Several (482, 483, 486, 487, 524, 525), but not all
(480, 484), studies have reported that high concentrations
of cholinergic agonists (10 µM) produce an early
transient hyperpolarization of ß-cells when islets are perifused with
a stimulating concentration of glucose. Because this hyperpolarization
is blocked by charybdotoxin, it might result from the transient opening
of large conductance maxi K(Ca) channels
activated during the large
[Ca2+]c increase
resulting from Ca2+ mobilization
(482). Activation of a K+ current
synchronized with Ca2+ release from intracellular
Ca2+ stores is well documented in pancreatic
acinar cells (526) and ß-cells
(527, 528, 529, 530).
The rise in [Na+]c
brought about by ACh activates the sodium pump, which is electrogenic
and produces a repolarizing current. However, the impact of this
current only becomes evident when the depolarizing current produced by
ACh stops. It is responsible for the marked and transient
repolarization of the ß-cell membrane upon washing of ACh (161, 480, 486, 531). It is also possible that this pump current is
involved in the acceleration of the slow waves by ACh (480, 484).
|
VII. Other Effects of ACh in Islet Cells
|
|---|
Many other effects of ACh in ß-cells have been reported, but
they have remained controversial. Only those that were believed to be
important for the control of insulin secretion will be mentioned
briefly.
A. Effects on glucose metabolism
ACh has been reported to slightly increase glucose utilization
(532) and nicotinamide adenine dinucleotide (reduced form)
(NADH) content in rat islets (533, 534). This effect might
result from the [Ca2+]c
increase produced by ACh. However, other studies found glucose
oxidation by mouse islets (493) and reduced
nicotinamide-adenine dinucleotide (phosphate) [NAD(P)H]
fluorescence (535) and glucose utilization
(341) in rat islets to be unaffected by ACh. We have
already emphasized that the effects of ACh on ionic fluxes and ß-cell
membrane potential differ from those induced by an increase in nutrient
concentration.
B. Effects on cyclic nucleotides
ACh induced a small, rapid (284), and transient
(534) increase in cAMP levels in rat islets incubated in
low glucose, probably via the activation of
Ca2+-calmodulin-sensitive adenylate cyclase
(536, 537). However, in the presence of stimulating
glucose concentrations, ACh did not affect islet cAMP levels
(161, 284, 286). In contrast to the situation in the
exocrine pancreas (538) and various other cell types
(539), cholinergic agonists do not increase cyclic GMP
(161) and NO production (540) in islets.
C. Effects on cytoplasmic pH
It has been suggested that alkalinization of ß-cells increases
insulin release under certain conditions (541). ACh
slightly increases intracellular pH in mouse ß-cells and probably
does so through the activation, by PKC, of the
Na+/H+ exchanger, because
the effect was observed in a HEPES-buffered, bicarbonate-free medium
(542, 543).
|
VIII. ACh Controls Free Cytosolic Ca2+ Concentration
([Ca2+]c) in ß-Cells
|
|---|
The rise of [Ca2+]c
in ß-cells serves as a triggering signal for exocytosis of insulin
granules. The complex effects of ACh on this triggering signal were
first deciphered by 45Ca2+
efflux measurements. The conclusions of these experiments were later
confirmed by more direct approaches using fluorescent probes to measure
[Ca2+]c directly inside
the cells (Fig. 5). ACh has only small
effects on ß-cell
[Ca2+]c in the presence
of low, nonstimulatory glucose concentrations (Fig. 5A), but causes a
sustained [Ca2+]c rise in
the presence of high glucose (Fig. 5, B and C) (544, 545, 546).
This sustained response, however, requires the presence of
extracellular Ca2+ and the possibility for
Ca2+ to enter ß-cells through voltage-operated
Ca2+ channels (Fig. 5A). At high concentrations,
ACh also unexpectedly lowers
[Ca2+]c in ß-cells
(Fig. 5C) (545). The following paragraphs describe the
mechanisms by which ACh produces these changes.
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Figure 5. General characteristics of ACh effects on mouse
islet cell [Ca2+]c. Cultured islets were
perifused with a medium without Ca2+ (Ca0) or with 2.5
mM Ca2+ (Ca2.5), and containing 3 or 15
mM glucose (G3 and G15, respectively). A, Left
panel, The biphasic increase in
[Ca2+]c produced by 100 µM ACh
is considerably reduced in a medium containing only 3 mM
glucose. Middle and right panels, The second sustained
[Ca2+]c phase produced by 100
µM is also strongly reduced when Ca2+ influx
is prevented by perifusing the islet with a Ca2+-free
medium (middle panel) or with a medium containing
Ca2+ and the K+-ATP channel opener, diazoxide
(Dz 250 µM), that keeps the plasma membrane at resting
potential. The large initial increase observed under these conditions
results from mobilization of Ca2+ from intracellular stores
as demonstrated by its persistence in a Ca2+-free medium. B
and C, In a medium containing Ca2+ and 15 mM
glucose, ACh induced a biphasic increase in
[Ca2+]c, the characteristics of which
depended on the concentration of ACh. A low concentration of ACh
(1 µM) accelerated the frequency of
[Ca2+]c oscillations induced by 15
mM glucose, whereas a high concentration of ACh (100
µM) transformed [Ca2+]c
oscillations into a sustained phase. Note that 100 µM ACh
induced the largest initial increase but the lowest sustained phase.
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A. Mechanisms by which ACh increases
[Ca2+]c
1. Mobilization of Ca2+ from intracellular
Ca2+ stores (Figs. 5A and 6,
A and B). Mobilization of Ca2+ from intracellular
Ca2+ stores can be studied by monitoring
45Ca2+ efflux from or
[Ca2+]c in islets perifused with a
Ca2+-free medium, i.e., when no
Ca2+ influx can occur. Under these conditions, cholinergic
agonists increase the 45Ca2+ efflux rate
(162, 279, 285, 286, 287, 291, 304, 483, 547, 548) and
[Ca2+]c (545, 546, 549, 550).
The mechanisms underlying this [Ca2+]c rise
have been extensively studied with subcellular fractions or
permeabilized insulin-secreting cells (324, 351, 354, 357, 443, 547, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561). They involve rapid production of IP3, catalyzed
by PLC (see Section V.A.1), and its binding to specific
IP3 receptors located on intracellular Ca2+ stores. The
concentration of IP3 accumulated in response to maximal concentrations
of carbachol has been estimated in experiments performed with RINm5F
cells in which phosphoinositides were labeled to isotopic equilibrium
with [3H]inositol (311). An increase of IP3
of 1.5 µM was calculated, which is close to the reported
half-maximal concentration (0.5–3 µM) that releases
Ca2+ from the endoplasmic reticulum in permeabilized
insulin-secreting cells (351, 552, 562). Accumulation of
IP3 was very fast, in keeping with the rapidity of Ca2+
mobilization by ACh (311, 333). This Ca2+
mobilization is not produced by physiological phosphoinositols other
than IP3 (367, 447, 552, 554, 563) and is prevented by
injecting ß-cells with heparin, an antagonist of IP3 receptors
(523, 557, 564, 565).
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Figure 6. Mechanisms of the effects of ACh on
[Ca2+]c in mouse pancreatic ß-cells. All
experiments were performed in the presence of 15 mM
glucose. A, Mobilization of intracellular Ca2+ in an islet
perifused with a Ca2+-free medium. Atropine (Atr)
suppressed the ACh-induced small sustained elevation of
[Ca2+]c due to mobilization. B,
[Ca2+]c oscillations due to mobilization of
intracellular Ca2+ in a single cell perifused with a
Ca2+-free medium. Thapsigargin (Thapsi 1 µM),
a specific inhibitor of the SERCA pump, abolished the oscillations by
preventing uptake of Ca2+ into the endoplasmic reticulum
and thereby emptying it of Ca2+. [Redrawn from Y. Miura
et al.: Biochem Biophys Res Commun
224:67–73, 1996 (510 ).] C, Mobilization of intracellular
Ca2+ followed by capacitative Ca2+ entry in
clusters of cells whose plasma membrane was hyperpolarized with
diazoxide (Dz 250 µM). Ca2+ mobilization was
observed in a Ca2+-free medium, and capacitative
Ca2+ entry occurred upon Ca2+ readmission to
the medium. A blocker of voltage-dependent Ca2+ channels,
D-600 (100 µM), was added to the medium to ensure that
the sustained [Ca2+]c increase that was
observed upon Ca2+ readmission resulted exclusively from
influx through voltage-independent Ca2+ channels. D,
Sustained [Ca2+]c elevation in an islet
perifused with a medium containing 2.5 mM Ca2+.
This sustained rise resulted essentially from the plasma membrane
depolarization that ACh produced. It was lower at a high (100
µM) ACh concentration than at a low (1 µM)
ACh concentration because the high concentration of the
neurotransmitter activates mechanisms of
[Ca2+]c decrease that oppose to the
mechanisms of [Ca2+]c increase. [Redrawn
from P. Gilon et al.: Biochem J
311:259–267, 1995 (545 ).] E, Sustained decrease of
[Ca2+]c in islets whose
[Ca2+]c was raised by depolarizing the plasma
membrane with 45 mM K+. Diazoxide (Dz 250
µM) was added to the medium to decrease the plasma
membrane resistance and prevent ACh from affecting the membrane
potential. The initial [Ca2+]c peak upon ACh
addition reflects Ca2+ mobilization from the endoplasmic
reticulum, whereas the transient drop induced by atropine (Atrop 10
µM) reflects Ca2+ sequestration into the
endoplasmic reticulum. [A, D, and E redrawn from P. Gilon
et al.: Biochem J 311:259–267, 1995
(545 ). © the Biochemical Society.]
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The response to ACh is different in whole islets and in single cells.
In whole islets, ACh induces a concentration-dependent transient peak
of [Ca2+]c followed by a
small sustained elevation (545) (Figs. 5A and 6A). A
similar biphasic pattern was reported for
45Ca2+ efflux (162, 279, 285, 287, 547, 548). This contrasts with the two types of
responses occurring in single cells: a rapid single
[Ca2+]c transient
(510, 546, 550, 566) or a series of
[Ca2+]c oscillations
(510, 566, 567) (Fig. 6B). Similar oscillations can be
produced by infusing ß-cells with guanosine
5'-[-thio]triphosphate (527, 529, 568, 569). The
reason why islets do not display
[Ca2+]c oscillations in
response to ACh in a Ca2+-free medium is
attributed to the fact that the recorded Ca2+
signal is the average of the
[Ca2+]c responses of all
ß-cells within the islet. Contrary to glucose-induced
[Ca2+]c oscillations that
result from periodic depolarizations of the plasma membrane and are
coupled between all ß-cells of the islet through gap junctions
(289, 570, 571), IP3-induced
[Ca2+]c oscillations are
not synchronized between electrically coupled ß-cells
(572).
The amplitude of the transient peak of
[Ca2+]c or
45Ca2+ efflux triggered by
ACh largely depends on the glucose concentration present before and
during ACh stimulation (162, 547, 548, 556, 573, 574, 575). It
is much smaller at a low glucose concentration than at a high glucose
concentration (Fig. 5A). This difference is attributed to the filling
of intracellular Ca2+ stores by glucose
(562, 565, 574). Other mechanisms, such as an enhanced
production or a decreased degradation of IP3 in the presence of glucose
(see Section V.A.1.b), might also be involved.
Three isoforms of the IP3 receptor have been described (I, II, and
III) (576) that form both homo- and heterotetramers
(577). Rat islets express more type III isoforms than
types I and II (578, 579, 580), and mouse islets express more
type I isoforms than types II and III (581, 582). Type II
isoform was, however, undetectable in ß-cells by immunocytochemistry
(582). Because of the use of different techniques, it is
unclear whether this difference between the rat and the mouse is real
or only apparent. The contribution of non-ß-cells in this expression
is also unknown.
Studies in various tissues have shown that the three isoforms are
differently regulated by cAMP, IP3, ATP, Ca2+,
and other factors (583, 584). Type II isoform has a higher
affinity for IP3 than types I and III (584, 585, 586, 587). Type I
isoform contains a regulatory domain for PKA (588, 589),
which might explain the observation that cAMP-producing agents enhance
the carbachol-induced mobilization of Ca2+ in
ob/ob mouse ß-cells (347). All isoforms are
regulated by Ca2+, possibly through a
Ca2+/calmodulin complex (590). Type
I and II isoforms are allosterically modulated by
Ca2+ so that the
Ca2+-mobilizing action of IP3 is markedly
amplified when [Ca2+]c
increases from basal (100 nM) to intermediate
levels (typically 
300 nM), whereas it is
inhibited when [Ca2+]c
reaches higher concentrations (584, 591, 592, 593). These
positive and negative feedback mechanisms of Ca2+
are considered important for generation of Ca2+
oscillations from IP3-sensitive Ca2+ stores. In
contrast, type III isoform is only positively modulated by
Ca2+, and this isoform would not be suitable
for [Ca2+]c
oscillations (584, 594).
The subcellular localization of IP3 receptors has not been firmly
established in ß-cells, although subcellular fractionation
experiments show that they are located on Ca2+
stores distinct from the mitochondria. An immunocytochemical study
using an antibody against IP3 receptors of type III suggested that
their preferential localization is on insulin-containing granules
(595, 596). However, this conclusion was subsequently
shown to be incorrect (597), which is consistent with the
observations that IP3 does not release Ca2+ from
subcellular fractions enriched in secretory granules (552, 598), and that granules do not regulate the ambient free
Ca2+ concentration (551, 599, 600, 601, 602)
even though they contain high levels of Ca2+
(536, 600, 603). The endoplasmic reticulum appears
to be the major source of Ca2+ released by IP3.
This is consistent with the following two observations: First, ACh- or
carbachol-induced mobilization of Ca2+ is
completely suppressed by thapsigargin and cyclopiazonic acid, two SERCA
pump inhibitors (510, 545, 546, 549, 600, 604). Second, a
drop in the free Ca2+ concentration in the
endoplasmic reticulum has recently been visualized upon carbachol
stimulation of INS-1 cells expressing the
Ca2+-sensitive photoprotein, aequorin, in the
endoplasmic reticulum. It is likely that the Golgi apparatus can also
release Ca2+ upon ACh stimulation
(605).
Experiments using INS-1 cells expressing aequorin in the endoplasmic
reticulum also revealed that high carbachol concentrations (100
µM) decreased free Ca2+
concentration in the endoplasmic reticulum by only 20–25%, in
contrast to SERCA pump inhibitors that completely emptied the
endoplasmic reticulum (606). This is in agreement with the
observation that thapsigargin can still release
Ca2+ from the endoplasmic reticulum in the
presence of ACh (510, 607). It is unclear why ACh is
unable to empty the endoplasmic reticulum to the same extent as IP3
itself (50% or more in permeabilized cells) (565, 606, 608). Because desensitization of IP3 receptors does not seem to
occur (369, 561, 562, 606, 608), the transient time course
of IP3 elevation may be involved.
In agreement with the widespread localization of the endoplasmic
reticulum within the cell, mobilization of Ca2+
by carbachol produces a rather uniform increase in
[Ca2+]c, contrary to
agents that stimulate Ca2+ influx through
voltage-dependent Ca2+ channels and raise
[Ca2+]c preferentially in
the periphery of the cell (566, 570, 609, 610). This
spatial difference has sometimes been taken as an argument to explain
the poor insulinotropic effect of ACh in a
Ca2+-free medium. Probably because of close
contacts between the endoplasmic reticulum and mitochondria (611, 612), high concentrations of carbachol can also increase the
mitochondrial free Ca2+ concentration in clonal
ß-cells (613).
It is important to emphasize that the process of
Ca2+ mobilization by ACh requires relatively high
concentrations (1 µM) of the neurotransmitter
(162, 545). Even in the presence of optimal glucose
concentrations, the half-maximal effective concentration of
ACh-induced Ca2+ mobilization is approximately 10
µM (545). Stimulation of
Ca2+ influx is much more sensitive to ACh (see
Section VIII.A.3).
2. Capacitative Ca2+ entry (Fig. 6C). In nonexcitable cells, PLC-linked agonists induce a biphasic
rise in [Ca2+]c. The
first phase corresponds to mobilization of Ca2+
from intracellular stores, whereas the second phase corresponds to
Ca2+ influx through voltage-independent
Ca2+ channels belonging to the family of SOCs.
The process by which emptying of intracellular
Ca2+ pools activates Ca2+
influx has been called capacitative Ca2+ entry
(614), but the mechanisms linking
Ca2+ pool depletion to Ca2+
influx are still disputed (615, 616).
A capacitative Ca2+ entry has been documented in
pancreatic ß-cells (522, 550, 617). Indeed, cholinergic
agonists and thapsigargin activate a Ca2+ entry
sensitive to La3+ but resistant to the blockade
of voltage-dependent Ca2+ channels by D-600
(methoxyverapamil) (Fig. 6C) or membrane hyperpolarization with
diazoxide. However, the rise in
[Ca2+]c that this entry
produces is small, approximately 8-fold less than that after the
opening of voltage-dependent Ca2+ channels by
high K+. Moreover, it decreases when the membrane
depolarizes, probably because the driving force for
Ca2+ diminishes as the membrane potential
approaches the equilibrium potential for Ca2+
(522). Contrary to other systems (615, 618),
the capacitative Ca2+ entry in ß-cells is not
affected by the energy state of the cell, PKC activation, or
serine/threonine phosphatase or tyrosine kinase inhibition
(550). The situation is different in RINm5F cells in which
capacitative Ca2+ entry requires activation of
PKC (619).
The concentration dependence of the capacitative
Ca2+ entry elicited by ACh has not been precisely
studied, but high concentrations of agonists (100
µM) seem to be necessary (550).
3. Ca2+ influx through voltage-dependent
Ca2+ channels (Figs. 5 and 6D). Under control
conditions, when extracellular Ca2+ is present,
the effects of cholinergic agonists on
[Ca2+]c and
45Ca2+ efflux largely
depend on the glucose concentration or, more exactly, on the ß-cell
membrane potential set by the glucose concentration. In the presence of
a nonstimulatory glucose concentration, when ß-cells are
hyperpolarized, ACh induces a biphasic change in
[Ca2+]c (Fig. 5A) and
45Ca2+ efflux (162, 285, 480) characterized by an initial slight peak followed by a
small sustained elevation. When ß-cells are depolarized by a
stimulatory or near-stimulatory glucose concentration, cholinergic
agonists also induce a biphasic change in
[Ca2+]c and
45Ca2+ efflux, but both
phases are now much larger than at low glucose (162, 279, 285, 287, 480, 483, 545, 617, 620, 621) (Figs. 5B–C). When
Ca2+ influx is inhibited by keeping the membrane
hyperpolarized with diazoxide or by blocking the voltage-dependent
Ca2+ channels, the initial peak is only partially
reduced, whereas the sustained phase is largely suppressed (Fig. 5A).
This indicates that the contribution of Ca2+
influx through voltage-dependent Ca2+ channels is
much more important to the sustained phase than the early phase.
When Ca2+ influx through voltage-dependent
Ca2+ channels is prevented, the residual initial
peak results from Ca2+ mobilization from the
endoplasmic reticulum, and the small residual sustained phase is caused
by continuous mobilization and capacitative Ca2+
entry.
ACh stimulation of Ca2+ influx through
voltage-dependent Ca2+ channels is explained by
the effects of the neurotransmitter on the membrane potential
(described above). In low glucose or in high glucose plus diazoxide,
the depolarization by ACh is too small to activate voltage-dependent
Ca2+ channels. In contrast, in the presence of
high glucose and other depolarizing secretagogues, ACh further
activates voltage-dependent Ca2+ channels
(279, 480, 545, 546, 617, 622). This constitutes the major
mechanism by which ACh, already at low concentrations (0.01
µM) (545, 546), induces a sustained
[Ca2+]c increase
(545).
4. Relative importance and physiological relevance of these three
mechanisms. Because the rise in
[Ca2+]c resulting from
the capacitative Ca2+ entry is very small,
requires high concentrations of ACh, and decreases when the membrane
depolarizes, its contribution to the overall rise in
[Ca2+]c produced by ACh
is minimal and will not be discussed further.
Although Ca2+ mobilization is by far the most
widely studied effect of ACh on
[Ca2+]c, its importance
in the electrically excitable ß-cell must be qualified. In the
absence of glucose or in the presence of low concentrations of the
sugar (<3 mM), ACh has almost no effect on
[Ca2+]c because little
Ca2+ can be mobilized from nearly empty
intracellular Ca2+ pools, and because the
membrane depolarization is insufficient to open
Ca2+ channels. At glucose concentrations (3–6
mM) that allow refilling of intracellular stores with
Ca2+ (565, 623) but remain below the
threshold for generation of electrical activity (7 mM),
mobilization of Ca2+ is the major mechanism by
which ACh increases
[Ca2+]c. At
near-stimulating glucose concentrations (6–7 mM), the
depolarization that ACh produces also triggers
Ca2+ influx through voltage-dependent
Ca2+ channels. At stimulating glucose
concentrations, this mechanism contributes even more than the
mobilization of Ca2+ to the overall increase in
[Ca2+]c brought about by
ACh. Moreover, the different concentration dependencies of
Ca2+ mobilization and Ca2+
influx for ACh at stimulating glucose concentrations reinforce the role
of the depolarization in the
[Ca2+]c rise. Indeed,
Ca2+ influx is already stimulated by low
concentrations of the neurotransmitter (0.1 µM),
whereas Ca2+ mobilization requires higher ACh
concentrations (1 µM).
The relative contribution of each mechanism to the action of ACh
in vivo is difficult to evaluate, but two reasons reinforce
the view that the changes in membrane potential play a predominant
role. First, without knowing what concentration of ACh can be reached
in the vicinity of ß-cells upon cholinergic nerve stimulation, it is
reasonable to assume that the effect observed with the lower
concentrations is likely to be physiological. Second, because of the
influence of nonglucose stimuli (which are not present in the
experimental buffers), the threshold glucose concentration that
triggers depolarization of ß-cells is lower in vivo than
in vitro (624).
B. Mechanisms by which ACh decreases
[Ca2+]c
When the effects of various concentrations of ACh on
[Ca2+]c were compared in
glucose-stimulated islets, it was unexpectedly found that the
steady-state [Ca2+]c was
higher in the presence of low concentrations (0.1–1 µM)
of ACh than in high (10 µM) concentrations (Figs. 5, B
and C, and 6D). This suggests that ACh might also decrease
[Ca2+]c, an effect that
is clearly demonstrated in islets steadily depolarized with high
K+ (545) (Fig. 6E). The
45Ca2+ efflux measurements
indicate that a slight acceleration of Ca2+
efflux contributes to this effect. This acceleration may be ascribed to
PKC stimulation because phorbol esters also promote
Ca2+ efflux (407, 625, 626) by
activating the plasma membrane Ca2+-ATPase
(627) or the
Na+/Ca2+ exchanger
(628). PA and DAG, which increase in the presence of
muscarinic agonists, stimulated Ca2+-ATPase
activity in an islet cell plasma membrane-enriched fraction
(629).
However, membrane potential measurements also revealed that whereas low
ACh concentrations increased the electrical activity elicited by
glucose, high concentrations of the neurotransmitter decreased the
amplitude of the spikes (Fig. 4B). Because spikes reflect
Ca2+ influx through voltage-dependent
Ca2+ channels, this observation suggested that
high concentrations of ACh might inhibit these channels
(545). This was confirmed by patch-clamp experiments
(630) (Fig. 4D). ACh dose dependently inhibited the
whole-cell voltage-dependent Ca2+ current of the
L-type. Maximum inhibition was produced by approximately 100
µM ACh and reached about 35%, whereas the 50%
inhibitory concentration was observed at 5 µM ACh.
This effect was mediated by a pertussis- and cholera toxin-insensitive
G protein. It is unlikely to involve DAG-sensitive PKCs, because
phorbol esters increase voltage-dependent Ca2+
currents in insulin-secreting cells (393, 631, 632, 633, 634). The
inhibitory effect of ACh on the Ca2+ current is
compatible with the inhibition of the L-type current by photorelease of
guanosine 5'-[-thio]triphosphate in ß-cells
(635). Inhibition of L-type current by muscarinic
receptors has also been observed in smooth muscle (636)
and neuronal cells (637, 638, 639, 640, 641). In contrast to the
situation found in normal ß-cells, the muscarinic agonist bethanechol
increased the L-type Ca2+ current by activating
PKC in HIT-T15 cells (634). This discrepancy might be
related to the very different responses to muscarinic agents between
normal and insulin-secreting cell lines (see Section
IX.D).
The decrease in
[Ca2+]c occurring in the
presence of high concentrations of ACh might constitute a protective
mechanism against deleterious Ca2+ overload. As
will be discussed below, it is not accompanied by an equivalent
decrease in insulin secretion.
|
IX. Mechanisms of the Stimulation of Insulin Secretion by ACh
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ACh brings into operation at least two types of
Ca2+-dependent mechanisms: the first one involves
a rise in [Ca2+]c, and
the second one increases the efficacy of Ca2+ on
exocytosis.
A. The rise in [Ca2+]c by ACh triggers
exocytosis
When the stimulation by ACh is applied in the presence of
diazoxide or a voltage-dependent Ca2+ channel
blocker, or in a Ca2+-free medium, there exists a
tight temporal parallelism between the rise in
[Ca2+]c and insulin
secretion. Indeed, insulin release is stimulated only during the
transient elevation of
[Ca2+]c (compare trace
with open circles of Fig. 1B with middle panel of
Fig. 5A). This indicates that ACh triggers exocytosis by increasing
[Ca2+]c. Two effects of
Ca2+ may be involved: a direct action of
Ca2+ on the exocytotic machinery close to the
zone of fusion of secretory granules with the plasma membrane
(642, 643, 644, 645), and a Ca2+-mediated
acceleration of granule movements to sites of release
(646, 647, 648). This second effect, which may serve to amplify
exocytosis upon subsequent stimulation, could be independent from PKC
activation but might involve a
Ca2+-calmodulin-dependent protein kinase
(376), either myosin light chain kinase (647)
or Ca2+/calmodulin-dependent kinase II
(648).
The amplitude of the transient secretory peak in a
Ca2+-free medium depends on the glucose
concentration (162). Two reasons may explain this
glucose-dependence: mobilization of Ca2+ is
greater in the presence of glucose (see Section VIII.A.1),
and Ca2+-induced insulin secretion is increased
by glucose through its amplifying pathway (for a given
[Ca2+]c, more insulin is
secreted at high glucose than at low glucose) (280, 649).
B. ACh increases the efficacy of Ca2+ on exocytosis
In a Ca2+-containing medium, the effects of
ACh on insulin secretion result from a balance between multiple
mechanisms that increase or decrease
[Ca2+]c and amplify the
efficacy of Ca2+ on exocytosis. Indeed, there is
no good temporal or quantitative relationship between the sustained
changes in [Ca2+]c and
insulin secretion induced by ACh in the presence of 15 mM
glucose. During the first minutes of stimulation, both
[Ca2+]c and insulin
responses (initial peaks) increase with the concentration of the
neurotransmitter (Fig. 7). However,
during steady-state stimulation, concentrations of ACh that barely
increase [Ca2+]c strongly
potentiate glucose-induced insulin secretion. This indicates that one
or several mechanisms other than the rise in
[Ca2+]c become operative
and increase the efficacy of Ca2+ on the
secretory machinery. In patch-clamp experiments using membrane
capacitance measurements in which the intracellular
Ca2+ concentration is artificially clamped
(650), it has been clearly demonstrated that ACh
sensitizes the secretory machinery to Ca2+. This
sensitization is also evident in islets depolarized with high
K+ and diazoxide. Under these conditions, ACh
exerts no or minor effects on the membrane potential of ß-cells
(279, 545), lowers
[Ca2+]c
(545), but potentiates insulin secretion
(651).
View larger version (21K):
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Figure 7. Comparison of the effects of various
concentrations of ACh on [Ca2+]c and insulin
secretion measured during the first minutes of stimulation (integrated
over 2 min for [Ca2+]c and 4 min for insulin
secretion) and the steady-state stimulation (integrated over 3 min for
[Ca2+]c and 6 min for insulin secretion) with
ACh. The results are presented as percentages of control values, which
were computed by integrating [Ca2+]c and
insulin secretion during the last 3 and 6 min, respectively, before
addition of ACh. The glucose concentration of the medium was 15
mM throughout. All data were obtained with cultured mouse
islets. (Derived from Ref. 545 for
[Ca2+]c experiments.)
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1. The PKC pathway plays a major role. Whereas accumulation of
phosphoinositols per se is devoid of any stimulatory effect
on insulin secretion (311, 319, 341), activation of PKC
sensitizes the secretory machinery to Ca2+
(326, 625, 632, 652, 653, 654, 655).
Involvement of PKC in the stimulation of insulin secretion by ACh is
suggested by experiments using various PKC inhibitors, including
bisindolylmaleimide, H-7, and staurosporine (400, 408, 656, 657, 658). However, these experiments are not conclusive because
the inhibitors are nonselective kinase inhibitors or exert nonspecific
effects (376, 659). Synthetic pseudosubstrate peptide
inhibitors permit more specific inhibition of certain PKC isoforms. The
insulin response of rat islets to carbachol was completely prevented by
an inhibitory peptide corresponding to the consensus sequence of the
pseudosubstrate regions of the PKC isoforms and ß
(660). Down-regulation of PKC by prolonged exposure (>20
h) of ß-cells to phorbol esters strongly inhibited the insulin
response to a subsequent cholinergic stimulation (163, 290, 399, 573, 661, 662). Because the treatment with the phorbol ester
down-regulated DAG-sensitive PKC isoforms, with the surprising
exception of PKCßII isoform in MIN6 cells (399), it was
suggested that one or several of the three PKC isoforms, , ,
and/or play a major role in the stimulatory effect of ACh on
insulin release. Because PKC isoform is the only isoform that has
been implicated in experiments with both PKC pseudosubstrates and PKC
down-regulation, and it is the major isoform expressed in normal
ß-cells, it is likely that most of the PKC-dependent effects of ACh
on insulin secretion are mediated by this isoform.
A small residual stimulation of insulin secretion by muscarinic
agonists was observed in islets with down-regulated PKC (163, 290, 662, 663). It probably results from the increase in
[Ca2+]c that cholinergic
agents still produce in such islets (290) and from the
activation of PKC-independent pathways. It is important to stress here
that translocation of PKC to the plasma membrane by carbachol does not
stimulate insulin secretion when
[Ca2+]c is low
(163, 657). The PKC-dependent stimulation of insulin
secretion only occurs when
[Ca2+]c is elevated
(Figs. 1, A and B). It is therefore teleologically understandable that
ACh brings into operation separate mechanisms that simultaneously
increase [Ca2+]c
(depolarization) and stimulate PKC.
As described above, PKC activation exerts a negative feedback control
on the signal transduction linked to PLC, which might explain the
biphasic time course of accumulation of arachidonate-enriched DAG upon
stimulation by cholinergic agents. However, this feedback control does
not determine the time course of insulin secretion; cholinergic
agonists can induce a sustained insulin secretion for relatively long
periods (30–60 min) without any sign of desensitization
(161, 162, 263, 286, 288, 664, 665). This suggests that
the decrease in PLC-derived DAG levels is probably not accompanied by a
parallel decrease of PKC activation. Low levels of PLC-derived DAG
levels might be sufficient to maintain a sustained PKC activation.
Other phospholipid-derived products formed during stimulation by ACh
(arachidonic acid, lysophosphatidate, phosphatidate, various DAGs, and
probably several other metabolites) may, alone or in synergy, stimulate
PKC (297, 453) and support the sustained secretion of
insulin. Such a time-dependent, multifactorial activation of PKC has
been reported in various systems (381).
2. The role of the PLA2 pathway is
uncertain. Whereas the role of the PLC-PKC pathway in the
insulinotropic effect of ACh is firmly established, it remains unclear
whether the PLA2 pathway is also involved, and if
so, whether its effects are also mediated by PKC. The reported effects
of arachidonic acid on insulin secretion are extremely controversial
(666, 667). Exogenous arachidonic acid inhibited, had no
effect, or stimulated insulin secretion by mouse or rat islets
depending on the glucose concentration used (435, 440, 446, 451, 668, 669). It also induced insulin release from permeabilized
islets (670, 671). Arachidonic acid-stimulated insulin
secretion has been reported to involve PKC (446), but it
has also been reported not to involve PKC (668, 671, 672).
At the concentrations that induce insulin secretion, arachidonic acid
may also exert toxic effects in islets and inactivate PKC
(672). Its insulinotropic effect is not blocked by
norepinephrine (670), which, in contrast, prevents
ACh-induced insulin secretion (673). Because of all
these controversies, the contribution of the PLA2
pathway to the insulinotropic effect of cholinergic agonists is still
unsettled.
C. Delayed effects of ACh on insulin secretion
It has been suggested that cholinergic agonists also exert
long-lasting effects on insulin secretion. The phenomenon, referred to
as time-dependent potentiation or priming, consists in the enhancement
of the ß-cell secretion response to various stimuli, including
glucose, GIP, cholecystokinin, and tolbutamide, by prior transient
stimulation with cholinergic agonists (164, 342, 366, 674, 675). This effect has been observed in the rat and the mouse
(658) and might play a role during the preabsorptive phase
(see Section III.B.2.a). Because it was mimicked by phorbol
12-myristate 13-acetate (PMA) (658, 676, 677), it has been
ascribed to a persistent activation of PKC, which can then be readily
activated by the rise in
[Ca2+]c that glucose
produces. However, in the perfused rat pancreas, the phenomenon could
be induced by PMA (678), but not by carbachol
(679). Comparison of the effects of both agents is not
easy because PMA, unlike carbachol, exerts irreversible activation of
PKC even after short application.
Whereas brief stimulation with ACh amplifies insulin secretion,
prolonged stimulation might exert adverse affects. Exposure of rat
islets to 10 µM carbachol for 3.5 h has been
reported to desensitize ß-cells to subsequent stimulation by glucose
and cholinergic agonists (263, 665, 680). This
desensitization might result from an impaired phosphoinositide pathway
(342). Ubiquitination is a process whereby ubiquitin, a
76-residue protein, is associated with certain proteins to make them
recognizable by the proteasome pathway that degrades them
(681). Prolonged exposure (6 h) to carbachol has recently
been shown to down-regulate IP3 receptors in mouse islet by the
ubiquitin/proteasome pathway (582).
D. Muscarinic responses are often abnormal in insulin-secreting
cell lines
Insulin-secreting cell lines have been used extensively to study
stimulus-secretion coupling. They can be useful when responses
occurring in non-ß-cells of the islets complicate interpretation of
the results, when large amounts of cells are needed for biochemical
determinations, and for transfection experiments. Their use has yielded
interesting data that can sometimes be extrapolated to normal
ß-cells. However, it is important to bear in mind that they are, in
many respects, different from normal ß-cells. A major difference
between normal ß-cells and some cell lines that were established long
ago is a markedly different glucose dependence (682).
Described below are some important differences regarding
cholinergic effects.
RINm5F cells (a clonal rat ß-cell line) are not depolarized by
cholinergic agonists (311, 607), but are depolarized by
phorbol esters that activate PKC (607, 654, 683, 684).
This is exactly opposite to the situation in normal ß-cells, in which
ACh depolarizes the plasma membrane, whereas PMA lacks this effect
(407, 625). In RINm5F cells, carbachol induces a transient
increase in [Ca2+]c by
mobilizing intracellular Ca2+, but causes a
sustained secretion of insulin that is independent from a rise in
[Ca2+]c and persists
after depleting the Ca2+ content of the
endoplasmic reticulum with thapsigargin (311, 607). The
effect of ACh on insulin secretion is poorly glucose dependent
(685), and PKC down-regulation or inhibition does not
affect (400, 607) or paradoxically enhances
(397) insulin secretion in response to carbachol in RINm5F
cells. Some of these peculiar effects might be explained by the fact
that carbachol also translocates the phorbol ester-insensitive
-isoform of PKC (400).
In MIN6 (a mouse ß-cell line) and HIT cells (a clonal hamster
ß-cell line), ACh and carbachol induce a transient rise in
[Ca2+]c, which mainly
results from Ca2+ influx. Surprisingly, they
stimulate insulin secretion even when
[Ca2+]c has returned to
basal levels (573, 686). The latter effect is markedly
reduced by PKC down-regulation (163, 399, 573). In MIN6
(686) and HIT cells (685), the insulinotropic
effect of ACh is poorly dependent on the glucose concentration.
|
X. Nature of the Muscarinic Receptor Activated by ACh
|
|---|
With the exception of two reports from the same group (687, 688), there is general agreement that all direct effects of ACh
on insulin-secreting cells are exclusively mediated by muscarinic
receptors (288, 480, 486, 621, 689, 690).
Muscarinic receptors belong to the family of receptors with seven
transmembrane domains connected by three cytoplasmic loops and three
extracellular loops (691, 692, 693, 694). Five muscarinic receptor
subtypes, which elicit classical responses, have been cloned so far:
the M1, M3, and
M5 subtypes are linked to G proteins of the
Gq class and activate PLC, and the
M2 and M4 are linked to
pertussis toxin-sensitive G proteins of the Gi or
Go class and initiate several processes such as
inhibition of adenylate cyclase or of voltage-dependent
Ca2+ channels and activation of the atrial
cardiac K+ channel by M2
(314, 416, 513, 637, 638, 640, 693, 694, 695, 696). However,
classification of muscarinic receptors on the basis of the signal
transduction is unreliable because of the overlap between the
transduction pathways activated by the different subtypes (692, 697, 698).
Three strategies have been used to identify the muscarinic receptor
subtypes present in ß-cells: pharmacological blockade of
physiological responses by selective antagonists, binding studies of
selective ligands, and molecular biology studies.
A. Pharmacological studies
More than a decade ago, only three muscarinic receptor subtypes
were identified and classified as neuronal M1
(high affinity for pirenzepine), cardiac M2
(M2, low affinity for pirenzepine/high
affinity for AF-DX 116), and glandular M2
(M2ß, low affinity for both pirenzepine and
AF-DX 116). A first study comparing the effects of atropine and
pirenzepine on insulin secretion from the perfused rat pancreas
suggested that the receptor present in ß-cells was different from the
M1 receptor (664). Subsequent
experiments testing atropine, pirenzepine, and AF-DX 116 on insulin
release, 86Rb+ efflux, and
Ca2+ efflux ruled out the presence of
M1 and cardiac M2 receptors
in mouse islets and suggested that the receptor present in ß-cells
was a glandular M2 subtype (699).
This was confirmed by studying the effect of other agonists or
antagonists on the electrical activity (486) and insulin
release (700). Later, when gene receptor analysis revealed
the existence of 5 muscarinic subtypes (691, 692, 701), it
clearly appeared that the glandular M2 receptor
corresponded to a new subtype, the M3 receptor
(702, 703). The observations that the insulin response to
cholinergic agonists is mediated by a M3 subtype
(699) were confirmed in vitro in RINm5F cells
(704) and rat islets (288) and in
vivo in the mouse (705) with more specific
antagonists.
B. Binding studies
The presence of muscarinic receptors in the endocrine pancreas was
clearly demonstrated by measuring the specific binding of the
muscarinic antagonists [3H]-methylscopolamine
or [3H]quinucliolinyl benzilate (QNB) to rat
(321, 700, 706, 707, 708, 709), mouse (46, 485), and
guinea pig islets (710), or to insulin-secreting tumoral
RINr cells (312) and INS-1 cells (711).
Scatchard plot analysis revealed a single population of high affinity
binding sites without any obvious low affinity binding sites
(312, 485, 706, 709). Displacement of the binding of
[3H]-methylscopolamine by various antagonists
indicated the presence of M3 receptors in rat
islets (700).
C. Molecular identification of the receptor subtypes
Using RT-PCR or ribonuclease protection assays, RNA encoding
M3 and M1 receptor subtypes
was detected in rat islets (288, 711). These two receptor
subtypes were much more expressed than the M5
receptor subtype (711). Although similar results were
obtained in INS-1 cells (711), this type of determination
does not prove that the three types of receptors are expressed in
ß-cells because isolated islets contain at least four endocrine cell
types (ß-cells, -cells, -cells, and PP-cells) as well as
vascular muscle and endothelial cells. Immunocytochemical experiments
using a specific antibody against the M3 receptor
subtype indeed indicated that both central (mainly ß-cells) and
peripheral cells (mainly non-ß-cells) express the
M3 receptor (711). On the other
hand, M3 (704, 711) and
M4 receptor subtypes (704, 711), but
not M1 (711), were detected in
RINm5F cells. Interestingly, although several studies reported the
presence of M3 and non-M3
receptors, two of these (288, 704) suggested that only
M3 receptors are involved in the secretion of
insulin in response to cholinergic agonists.
D. One or several receptor subtypes for several transduction
pathways?
On the basis of pharmacological, binding, and RT-PCR studies, it
is clear that the M3 receptor plays a central
role in ß-cells. The idea that this sole subtype activates several
transduction pathways is supported by the observation that three
different parameters of the ß-cell function (insulin secretion,
86Rb+ efflux, and
Ca2+ efflux) displayed a similar antagonistic
profile (699).
Activation of multiple transduction pathways by a single class of
muscarinic receptors is not a unique feature of the pancreatic
ß-cell. Another example of complexity is found in ventricular
myocytes in which cholinergic agonists, likely acting solely on the
M2 subtype, inhibit L-type
Ca2+ current through an inhibition of adenylate
cyclase activity and activate a Na+ current
(512, 513). Activation of two different transduction
pathways by two different parts of the M3
receptor has also been documented in A9 fibroblast cells
(712). The diversity of the effects mediated by ACh not
only depends on the nature of the muscarinic receptor subtype involved,
but also on posttranslational modifications (glycosylation,
phosphorylation, etc.), which might be different from one cell
type to another (314) or on the nature of the effector
system present in the cells (315, 512, 695, 697). Indeed,
when heart M2 muscarinic receptors, which
classically inhibit adenylate cyclase, are expressed in CHO cells,
their activation also produces nonclassical effects such as
phosphoinositide breakdown (713). Similar results were
found for the M1 receptor (697).
Activation of all five muscarinic receptor subtypes expressed in NIH
3T3 cells has recently been shown to inhibit L-type current of this
cell type (714). This suggests that each receptor subtype
elicits preferential rather than specific effects, depending on the
cell type in which it is expressed.
In the same line of ideas, the different concentration dependencies of
the multiple effects of ACh in ß-cells do not necessarily imply that
several muscarinic receptors are involved. They might result from
different sensitivities of the effector systems to G protein activation
or from other unidentified mechanisms. Activation of transduction
pathways with different concentration dependencies has recently been
reported for the M3 receptor expressed in CHO
cells. Moderate concentrations of carbachol (1–10 µM)
elicited maximal capacitative Ca2+ influx,
whereas higher concentrations were necessary to activate an inward
monovalent cation current that depolarizes the plasma membrane
(521).
|
XI. Summary and Conclusions
|
|---|
A. The physiological role of ACh
ACh is released by intrapancreatic nerve endings under the control
of the vagus nerves during the preabsorptive, cephalic, and enteric
phases of feeding and, very likely, also during the absorptive phase.
Vagal stimulation occurs after activation of cephalic sensory organs
including those of the oral cavity and the visual and olfactory
systems, and after activation of glucoreceptors in the gut, brain, and
liver. ACh stimulates insulin secretion in a glucose-dependent manner,
becoming more and more effective as the plasma glucose concentration
increases. This stimulation appears to be important to ensure optimal
glucose tolerance during the periods of feeding.
Several animal models of type 2 diabetes are characterized by an
alteration of the autonomic nervous system with an increased ratio of
the parasympathetic over sympathetic activities leading to
hyperinsulinemia. Hyperinsulinemia is a characteristic of obesity, and
the kinetics of insulin secretion is often altered in type 2 diabetes,
but it is unclear to which extent these abnormalities result from an
impaired activity of the autonomic nervous system. Because their
effects on insulin secretion are glucose dependent, cholinergic
agonists might theoretically be helpful to improve insulin secretion
and glucose homeostasis in certain type 2 diabetic patients (46, 715). Although supported by some animal studies
(46), this idea has not been largely tested because of
insufficient selectivity of the available muscarinic agents for
ß-cells.
B. The mechanisms of action of ACh in ß-cells
At the ß-cell level, ACh binds to M3
receptors and activates several transduction pathways (Fig. 3); one of
the major pathways is PLC, which mainly generates IP3 and
diacylglycerol, a potent PKC activator. ACh also stimulates
PLA2, probably secondary to the
[Ca2+]c rise. This leads
to accumulation of arachidonic acid and lysophosphatidylcholine. ACh
might also activate PLD by a mechanism that possibly depends on PKC
activation. Many of the phospholipid-derived messengers are also, alone
or in synergy with other lipid messengers such as diacylglycerol,
activators of PKC (Fig. 3). Besides these complex effects on lipid
metabolism, ACh also depolarizes the plasma membrane of ß-cells by a
Na+- or nonspecific cationic-dependent mechanism
(Fig. 8A, pathway 3), and possibly also
by a mechanism involving SOCs activated by intracellular
Ca2+ pool emptying (Fig. 8A, pathway 4). This
depolarization is small and reaches the threshold for the
activation of voltage-dependent Ca2+ channels
only if the plasma membrane is already depolarized by secretagogues
such as glucose. The glucose dependence of this depolarization
largely contributes to the glucose-dependence of ACh effects on
insulin secretion.
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|
Figure 8. Mechanisms by which ACh increases (A) or
decreases (B) [Ca2+]c in ß-cells. A, ACh
induces a transient increase in [Ca2+]c by
mobilizing Ca2+ from IP3-sensitive stores (mainly the
endoplasmic reticulum; pathway 1). The resulting Ca2+
depletion of these stores activates a small sustained Ca2+
influx through voltage-independent Ca2+ channels
(capacitative Ca2+ entry; pathway 2). ACh also depolarizes
the plasma membrane of ß-cells by increasing a specific
Na+- or a nonspecific cationic conductance (pathway 3), and
probably also by activating SOCs carrying Ca2+
(capacitative Ca2+ entry) or other ions (pathway 4). This
depolarization is small and reaches the threshold for the activation of
voltage-dependent Ca2+ channels (VDCC) only if the plasma
membrane is already depolarized by secretagogues, such as
glucose, that close K+-ATP channels. Stimulation of
Ca2+ influx through voltage-dependent
Ca2+ channels is the main mechanism by which ACh
induces a sustained increase in [Ca2+]c
at stimulatory glucose concentrations. B, When
[Ca2+]c is already elevated, ACh also
decreases [Ca2+]c by inhibition of
voltage-dependent Ca2+ channels (pathway 5) and stimulation
of Ca2+ efflux, probably via PKC activation (pathway 6).
|
|
All these transduction pathways modulate
[Ca2+]c in ß-cells
(Fig. 8). ACh transiently increases
[Ca2+]c by mobilizing
Ca2+ from IP3-sensitive stores mainly in the
endoplasmic reticulum (Fig. 8A, pathway 1). ACh induces a sustained
increase in [Ca2+]c by
stimulating Ca2+ influx by two pathways: through
voltage-independent Ca2+ channels that open upon
intracellular Ca2+ pool emptying
(capacitative Ca2+ entry; Fig. 8A, pathway 2)
and through voltage-dependent Ca2+ channels that
are activated by depolarization (Fig. 8A, pathways 3 and 4). ACh
decreases [Ca2+]c under
certain circumstances (Fig. 8B). This effect, which is detectable only
after the initial phase of intracellular Ca2+
mobilization and only when
[Ca2+]c is sustained,
requires higher ACh concentrations than those depolarizing the plasma
membrane. It results from a stimulation of Ca2+
efflux that likely involves PKC (Fig. 8B, pathway 6) and a G
protein-mediated inhibition of Ca2+ influx
through voltage-dependent Ca2+ channels (Fig. 8B, pathway 5). It might protect ß-cells against deleterious
Ca2+ overload.
The insulinotropic effect of ACh largely depends on the glucose
concentration and Ca2+ influx. When no
Ca2+ influx can occur, ACh induces a transient,
small, monophasic stimulation of insulin secretion, provided a high
concentration of glucose is present. The tight temporal parallelism
between the rises in
[Ca2+]c and insulin
secretion that occur under these conditions indicates that ACh triggers
exocytosis by increasing
[Ca2+]c. When
Ca2+ influx can occur, ACh induces a biphasic
stimulation of insulin secretion, the amplitude of which, again,
largely depends on the glucose concentration. However, there is no good
temporal and quantitative relationship between changes in
[Ca2+]c and insulin
secretion because, in the steady state, a large stimulation of insulin
secretion occurs with only a moderate increase in
[Ca2+]c (Fig. 7). This
suggests that an additional mechanism becomes operative and increases
the efficacy of Ca2+ on the secretory machinery.
The most important amplifying mechanism involves PKC. This
PKC-dependent mechanism increases insulin secretion only when
[Ca2+]c is sufficiently
elevated above basal levels. Thus, the insulinotropic effect of ACh
results from two Ca2+-dependent mechanisms, one
that involves a rise in
[Ca2+]c and another that
increases the efficacy of Ca2+ on exocytosis
(Fig. 3).
Although the mechanisms of action of ACh have been extensively
studied, many remain incompletely understood. How PKC increases insulin
secretion is unclear. Because of the transient accumulation of the
PLC-derived arachidonate and the multiple interactions between PKC and
various phospholipid-derived products, it is not known which routes
require PKC or lead to PKC activation. Interactions between
PLA2-, PLC-, and PLD-derived products and PKC are
not well defined. Moreover, it is unclear which PKC isoform is
activated by ACh, whether the neurotransmitter translocates specific
isoforms to different targets, and which proteins are phosphorylated by
PKC. Phorbol esters not only stimulate insulin secretion, they also
activate early genes and stimulate cell proliferation
(403). It is unknown whether ACh could exert such effects
physiologically. Many other questions await clear answers: What are the
precise roles of PLA2 and PLD in the action of
ACh? What is the identity of the channel involved in the depolarization
produced by ACh? Is the ACh-induced inhibition of the
Ca2+ current mediated by a cytosolic
diffusible messenger or by a direct interaction with
Ca2+ channels? How can a single subtype of
receptor activate so many different transduction pathways with
different concentration dependencies for ACh? Answers to this last
question might be provided by the use of the recently developed model
of muscarinic receptor-knockout mice (253, 641, 716) and
of systems expressing truncated muscarinic receptors subtypes.
|
Acknowledgments
|
|---|
This work was supported by grants from the Fonds de la Recherche
Scientifique Médicale and the Fonds National de la Recherche
Scientifique (Brussels), of which P. G. is a Senior Research
Associate; the General Direction of Scientific Research of the French
Community of Belgium; and the Federal Office for Scientific, Technical
and Cultural Affairs of Belgium.
|
Footnotes
|
|---|
Abbreviations: -cell, Glucagon-secreting cell; ACh,
acetylcholine; ASCI-PLA2, ATP-stimulatable,
Ca2+-independent PLA2; ß-cell,
insulin-secreting cell; [Ca2+]c, free
cytosolic Ca2+ concentration; CGRP, calcitonin gene-related
peptide; CHO, Chinese hamster ovary; DAG, diacylglycerol; -cell,
somatostatin-secreting cell; GIP, glucose-dependent
insulin-releasing peptide or gastric inhibitory polypeptide; GRP,
gastrin-releasing peptide; 5-HT, 5-hydroxytryptamine (or
serotonin); 1 IP, inositol 1-phosphate; K+-ATP
channel, ATP-sensitive K+ channel; LHA, lateral
hypothalamic area; [Na+]c, free cytosolic
Na+ concentration; NO, nitric oxide; PA, phosphatidic acid;
PACAP, pituitary-adenylate cyclase activating polypeptide;
PC, phosphatidylcholine; PI, phosphatidylinositol; PIP,
phosphatidylinositol 4-phosphate; PIP2,
phosphatidylinositol 4,5-bisphosphate; PMA, phorbol 12-myristate
13-acetate; PP-cell, pancreatic polypeptide-secreting cell; SP,
substance P; SERCA pump, sarco-endoplasmic reticulum
Ca2+-ATPase; SOC, store-operated channel; VMH, ventromedial
hypothalamic nuclei.
|
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Abstract
I. Introduction
