Insulin secretion by gastrin-releasing peptide in mice: ganglionic versus direct islet effect

Sven Karlsson1, Frank Sundler2, and Bo Ahrén1

1 Department of Medicine, Lund University, Malmö University Hospital, S-205 02 Malmö; and 2 Department of Physiology and Neuroscience, Section of Neuroendocrine Cell Biology, Lund University, S-22185 Lund, Sweden

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Gastrin-releasing peptide (GRP) stimulates insulin secretion by a direct islet effect. In this study, we initially demonstrated, by immunocytochemistry of the mouse pancreas, GRP immunoreactive nerve fibers within exocrine tissue, islets, and intrapancreatic ganglia. A more pronounced GRP innervation was found in ganglia compared with in islets. We therefore studied whether indirect cholinergic mechanisms contribute to the insulinotropic action of GRP. In mice, the insulinotropic response to GRP (4.25 nmol/kg iv) was inhibited by the m3-selective, muscarinic receptor antagonist 4-diphenylacetoxy-N-methyl piperidine methobromide (4-DAMP, 0.21 mol/kg; by 68%, P < 0.05) and by the ganglionic blocker hexamethonium (28 mol/kg; by 98%, P < 0.05). In contrast, in isolated islets, 4-DAMP or hexamethonium (10 or 100 µM) did not inhibit GRP (100 nM)-induced insulin secretion. Furthermore, afferent denervation by neonatal capsaicin did not affect the insulin response to GRP. We conclude that the insulinotropic effect of GRP in the mouse is mediated by both direct islet effects and through activation, at the ganglionic level, of postganglionic cholinergic nerves. In vivo, the indirect cholinergic mechanism predominates.

glucagon secretion; pancreatic nerves; islets of Langerhans

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SEVERAL PREVIOUS STUDIES have suggested that gastrin-releasing peptide (GRP) might participate in the regulation of insulin secretion as a pancreatic neurotransmitter exerting a direct insulinotropic effect on the islet B cells. Thus GRP has been shown to stimulate insulin secretion both under in vivo conditions (4, 20, 22, 23, 30) and in vitro from the perfused pancreas (6, 16) as well as in isolated islets and insulin-producing B cell lines (5, 28, 29). Furthermore, specific GRP-binding sites have been demonstrated in mouse islets (28). In addition, in several species, GRP immunoreactivity has been demonstrated in nerve fibers within the islets (2, 14, 21, 24). GRP has also been demonstrated to be released from the porcine pancreas upon electrical activation of the vagal nerve, and the insulin response to electrical activation of the vagal nerve in pigs has been shown to be inhibited by a GRP receptor antagonist (7, 14). This suggests that GRP contributes to vagally induced insulin secretion.

GRP has previously been shown to stimulate exocrine pancreatic secretion through an indirect mechanism involving activation of cholinergic nerves, since this effect of the peptide was inhibited by cholinergic blockade (3, 15). Whether cholinergic mechanisms are involved also in the effect of GRP on the endocrine pancreas is, however, not established. In one previous study, in the perfused porcine pancreas, GRP-stimulated insulin secretion was unaffected by muscarinic receptor blockade by atropine (16), whereas another study, in the mouse, showed that methylatropine diminished both the insulin and glucagon responses to intravenous GRP (22). In the present study, we investigated in more detail the possible involvement of indirect cholinergic mechanisms in GRP-stimulated insulin secretion in the mouse.

Initially, we therefore, by immunocytochemistry, investigated the occurrence of GRP immunoreactivity in sections of mouse pancreata. The possible contribution of cholinergic and ganglionic mechanisms to GRP-stimulated insulin secretion was investigated both under in vivo conditions in the mouse and in isolated mouse pancreatic islets. We thereby used the selective m3-muscarinic receptor antagonist 4-diphenylacetoxy-N-methylpiperadine methobromide (4-DAMP) and the ganglionic nicotinic receptor antagonist hexamethonium. Moreover, to investigate the involvement of afferent nerves, we examined whether the insulinotropic effect of GRP was affected by neonatal capsaicin treatment, which induces a permanent sensory denervation (9, 13).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. Female mice of the Naval Medicine Research Institute (NMRI) strain (Bolmholdtgaard Breeding and Research Center, Ry, Denmark) weighing 25-35 g were used throughout the study. The animals were fed a standard pellet diet and tap water ad libitum. The study was approved by the Ethic Committee for Animal Research of Lund University.

Immunocytochemical studies. Immunocytochemistry for GRP immunoreactivity was undertaken as described previously (21). In short, tissue specimens taken from mouse pancreas were fixed overnight in Stefanini's fixative (15% saturated picric acid and 2% formaldehyde in 0.1 M phosphate-buffered saline). After rinsing, the specimens were frozen on dry ice and were thereafter cut at 10 µm in a cryostat and collected on chrome alum-coated slides. The sections were then processed for immunocytochemistry, using a rabbit anti-GRP antibody (code no. R6902; dilution 1:640). For control sections, the antiserum was inactivated by excess of synthetic GRP. The antiserum does not cross-react with substance P, vasoactive intestinal polypeptide, Leu- and Met-enkephalin, cholecystokinin-8, or neurotensin (21). Ganglionic nerve cell bodies were identified by an antibody raised against the neural cell soma marker protein gene product (PGP; Ultraclone, Cambridge, UK; see Ref. 26) 9.5. Double staining for GRP and PGP was used to discriminate between GRP immunoreactivity located to nerve cell bodies versus to in nerve fibers of the ganglia.

In vivo experiments. Animals were given an intravenous bolus injection of GRP at a dose level that previously was shown to be maximal in regard to stimulation of insulin secretion (4.25 nmol/kg; Peninsula, Belmont, CA; see Ref. 22). Controls were given saline. At 15 min before the intravenous injection, animals were injected intraperitoneally with either the muscarinic m3-selective receptor antagonist 4-DAMP (21 µmol/kg; Research Biochemicals, Natick, MA) or hexamethonium (28 µmol/kg; Sigma Chemical, St. Louis, MO). Controls were injected intraperitoneally with saline. The dose levels of these antagonists were selected from previous studies in our laboratory. Thus, at the dose level of 0.21 µmol/kg, 4-DAMP inhibits carbachol-stimulated insulin and glucagon secretion in the mouse (11), and hexamethonium inhibits neurally induced insulin and glucagon secretion in the mouse at the dose level of 28 µmol/kg (10). At 2 min after the intravenous injection, blood was sampled from the retrobulbar venous plexus. At this time point, the plasma levels of insulin and glucagon reach their peak after an intravenous injection of GRP as previously demonstrated in the mouse (22). Plasma was separated by centrifugation and was stored at -20°C until assayed for its content of insulin, glucagon, and glucose.

Neonatal capsaicin treatment. Under buprenorphine (Themgesic; Reckitt & Colmar, Hull, UK; 0.45 mg/kg) analgesia, capsaicin (8-methyl-N-vanillyl-6-nonenamide; 50 mg/kg) was given as a subcutaneous injection at days 2 and 5 of life as described previously (13). Capsaicin was dissolved in a vehicle consisting of 10% ethanol and 10% Tween 80 in 0.9% NaCl. The volume load was 20 µl/animal. Controls were treated with buprenorphine and were given vehicle alone. This capsaicin treatment of neonatal mice has previously been shown to induce a permanent destruction of sensory nerves of the C-fiber type (8, 9) and to cause a permanent destruction of nerves immunoreactive for calcitonin gene-related peptide and a substantial reduction in the number of substance P immunoreactive pancreatic nerves in the mouse, thus creating a permanent sensory denervation (13). We have previously demonstrated that neonatal capsaicin treatment of mice results in increased glucose tolerance (12). To verify that capsaicin, in the present study, acted as expected from previous studies, an intravenous bolus injection of glucose (2.8 mmol/kg) was given to the animals used for the present experiments. Thereby, basal plasma glucose levels did not differ between vehicle-treated and capsaicin-treated animals, whereas at 10 min after the glucose injection the plasma glucose levels were 13.8 ± 0.9 mmol/l in vehicle-treated animals and 9.5 ± 0.5 mmol/l in capsaicin-treated animals (P < 0.01; n = 6 for each group), verifying the increased glucose tolerance. For the subsequent experiments on islet hormone secretion, female mice were used at the age of 10-12 wk.

Isolation and incubation of islets. Islets were isolated by the collagenase digestion technique as initially described by Lacy and Kostianovsky (17) with slight modifications. In short, during pentobarbital sodium (120 mg/kg) anesthesia, a catheter was inserted through the common bile duct, and the duodenal papilla was occluded. Thereafter, the pancreas was retrogradely filled with 3 ml of Hanks' balanced salt solution (Sigma Chemical) supplemented with 0.3 mg/ml of collagenase (collagenase P; Boehringer Mannheim). The pancreas was removed and incubated at 37°C for 20-22 min. Islets were thereafter hand picked under a stereomicroscope and cultured overnight in a RPMI 1640 medium (GIBCO-BRL, Paisley, Scotland) supplemented with 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 2.06 mmol/l L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin (Biological Industries), and 2.5 µg/ml amphotericin B (GIBCO). Single islets were incubated in a volume of 100 µl during 60 min at 37°C in an atmosphere of humidified air saturated with 5% CO2. The incubation buffer contained (in mM) 25 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 125 NaCl, 5.9 KCl, 1.28 CaCl2, and 1.1 MgCl2 and was supplemented with 0.1% human serum albumin and glucose (3.3 or 11.1 mM). The pH (7.36) of the HEPES buffer was adjusted by the use of NaOH. During the experiments, GRP (100 nM), carbachol (0.1 mM; Sigma), 4-DAMP (0.01 and 0.1 mM), or hexamethonium (0.01 and 0.1 mM) was added according to the protocols. The concentration of hexamethonium used in the present experiments has previously been demonstrated to induce ganglionic blockade in vitro in the perfused rat pancreas (27). After the incubation period, 25 µl of the medium were removed for analysis of its insulin content by radioimmunoassay.

Determinations of insulin, glucagon, and glucose. Insulin was determined by radioimmunoassay using a guinea pig anti-rat insulin antibody, 125I-labeled human insulin as tracer, and rat insulin as standard (Linco Research, St. Charles, MO). The separation of free and bound radioactivity was performed by the double antibody technique using a goat anti-guinea pig immunoglobulin G antibody (Linco). Plasma glucagon levels were determined by radioimmunoassay using an antibody specific for pancreatic glucagon, 125I-labeled glucagon, and glucagon as standard (Milab, Malmö, Sweden). Plasma glucose levels were determined with the glucose oxidase method.

Statistics. Data are presented as means ± SE. One-way analysis of variance followed by Newman-Keuls test or Student's t-test for unpaired observations were used for statistical evaluation. A P value of <0.05 was considered significant. For the immunohistochemical studies, at least five sections from the pancreata of nine different mice were examined. The relative frequency of GRP immunoreactivity in the exocrine parenchyma, the islets, and the ganglia were estimated semiquantitatively by two independent observers.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

GRP immunoreactivity in mouse pancreas. In sections of the mouse pancreas, GRP immunoreactive varicose nerve fibers were found within the exocrine parenchyma, the islets, and the intrapancreatic ganglia (Fig. 1). GRP immunoreactive nerve fibers were more frequently observed in ganglia compared with in islets (Table 1). Double staining for GRP and the neuronal cell soma marker PGP 9.5 revealed that the majority of the ganglia showed GRP immunoreactivity confined only to nerve fibers. However, rarely also, ganglionic nerve cell bodies showed a slight GRP immunoreactivity (Fig. 1, B and C). Thus GRP immunoreactive nerve fibers occur in both the islets and ganglia of the mouse pancreas.


View larger version (63K):
[in this window]
[in a new window]
 
Fig. 1.   Sections of mouse pancreas demonstrating fine varicose gastrin-releasing peptide (GRP) immunoreactive nerve fibers in the center and in the periphery of a pancreatic islet (A). B: ganglion with GRP immunoreactivity confined to fine varicose nerve fibers (arrow) within the ganglion, and also moderately stained GRP immunoreactive nerve cell bodies are demonstrated. C: same ganglion as in B is shown double stained for the neuronal cell soma marker protein gene product (PGP) 9.5 to identify the ganglionic nerve cell bodies; it is seen that two nerve cell bodies are distinctly double stained for both GRP and PGP. Also in C, PGP 9.5 immunoreactive nerve fibers are seen scattered within the exocrine parenchyma. GRP immunoreactivity was visualized by fluorescein isothiocyanate, and PGP immunoreactivity was visualized by tetramethylrhodamine isothiocyanate. Magnification: A, ×150; B and C, ×200.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Distribution of GRP immunoreactive nerve fibers in the mouse pancreas

Muscarinic and ganglionic antagonism in vivo. Intravenous injection of GRP (4.25 nmol/kg) elevated plasma insulin levels to 246 ± 60 pmol/l compared with 72 ± 18 pmol/l in controls (P < 0.05). The muscarinic receptor antagonist 4-DAMP (0.21 µmol/kg) inhibited this response by ~68% (P < 0.05), whereas hexamethonium (28 µmol/kg) inhibited the insulinotropic effect of GRP by 92% (P < 0.05). Also, plasma glucagon levels were increased by GRP (to 805 ± 70 pg/ml compared with 309 ± 13 pg/ml in controls; P < 0.001). This response was not significantly affected by 4-DAMP but was partially (by 48%) inhibited by hexamethonium (P < 0.05). The plasma glucose levels were not significantly affected by GRP or 4-DAMP, whereas pretreatment with hexamethonium resulted in a slight lowering of the plasma glucose levels in both controls (from 8.8 ± 0.4 to 7.3 ± 0.3 mmol/l; P < 0.01) and GRP-injected animals (from 8.9 ± 0.3 to 7.2 ± 0.3 mmol/l; P < 0.01; Fig. 2).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2.   Plasma levels of insulin (A) and glucagon (B) after intravenous injection of GRP (4.25 nmol/kg;) or NaCl (Control). Mice were pretreated at 15 min before intravenous injection with an intraperitoneal injection of either 4-diphenylacetoxy-N-methyl piperidine methobromide (4-DAMP, 0.21 µmol/kg), hexamethonium (28 µmol/kg), or NaCl. Means ± SE are shown. There were 22-24 animals in each group. * Probability of random difference of <0.05 in plasma insulin levels between GRP-injected control animals and GRP-injected animals pretreated with either 4-DAMP or hexamethonium. NS, not significant.

These results show that GRP-stimulated insulin secretion under in vivo conditions is critically dependent on muscarinic m3-receptors and ganglionic mechanisms, whereas GRP-stimulated glucagon secretion is partially dependent on ganglionic but not on muscarinic (m3) mechanisms.

Effects of muscarinic (m3) and ganglionic blockade on insulin release from isolated pancreatic islets. To investigate whether the inhibition of GRP-stimulated insulin secretion by 4-DAMP or hexamethonium observed under the in vivo conditions was due to interaction of the receptor antagonists with islet receptors, experiments were performed on isolated mouse islets. Thereby, GRP (100 nM) was found to stimulate insulin secretion in the presence of 11.1 mM glucose (P < 0.05). 4-DAMP at the concentrations of 10 or 100 µM did not affect glucose (11.1 mM) or GRP-stimulated insulin secretion (Table 2), whereas carbachol (0.1 mM)-stimulated insulin secretion was totally abolished by the antagonist. Thus carbachol increased insulin release from 0.16 ± 0.04 to 0.75 ± 0.07 pmol/islet (P < 0.01), and 4-DAMP (100 µM) decreased this response to 0.12 ± 0.04 pmol/islet (P < 0.001). Also, hexamethonium at 10 and 100 µM did not affect the insulin response to glucose (11.1 mM) or to GRP (100 nM; Table 2). Furthermore, at 11.1 mM glucose, hexamethonium did not affect the insulinotropic effect of carbachol (0.67 ± 0.11 pmol/islet after incubation with carbachol vs. 0.67 ± 0.1 pmol/islet after incubation with carbachol + 100 µM of hexamethonium; not significant). Thus the inhibition of the insulinotropic response to GRP by 4-DAMP or hexamethonium observed under in vivo conditions was not due to a nonspecific action of these antagonists on islet GRP receptors. Furthermore, the direct islet insulinotropic effect of GRP does not require intact muscarinic (m3) or nicotinic receptors.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of 4-DAMP or hexamethonium on GRP-stimulated insulin release in vitro

Afferent denervation by neonatal capsaicin treatment. Capsaicin-sensitive afferent nerves have previously been demonstrated to be involved in the regulation of glucose homeostasis and of insulin and glucagon secretion (12, 13). In the present study, we found, however, that GRP-stimulated insulin and glucagon secretion was as efficient in capsaicin-treated animals as in their vehicle-treated controls and that plasma glucose levels did not differ between the groups (Table 3). These results therefore show that the effect of GRP on insulin and glucagon secretion is not dependent on afferent neural mechanisms.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Plasma levels of insulin, glucagon, and glucose after intravenous injection of GRP or NaCl in mice subjected to neonatal capsaicin or vehicle treatment

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study demonstrates that GRP, in addition to exerting a direct effect on the islet B cells, also stimulates insulin secretion by an indirect mechanism that is critically dependent on muscarinic and ganglionic nicotinic mechanisms but not on capsaicin-sensitive afferent nerves. We also revealed that GRP immunoreactive nerve fibers, in addition to innervating the islets also, and in fact more markedly, innervate intrapancreatic ganglia.

The involvement of muscarinic receptors in the insulinotropic effect of GRP in the mouse under in vivo conditions has previously been implicated (22). The present study confirms these previous findings and further demonstrates that the muscarinic receptor subtype involved is of the m3-subtype, which is the muscarinic receptor subtype present on the pancreatic B cells (1, 11). The fact that the insulinotropic response to GRP was found to be inhibited by ganglionic blockade by hexamethonium suggests that GRP acts at the ganglionic level to activate postganglionic cholinergic nerves innervating the islets, since hexamethonium did not affect GRP-induced insulin secretion in vitro in isolated islets.

The present study also confirmed previous studies demonstrating that GRP acts directly on the islets to stimulate insulin secretion (5, 28, 29) and further demonstrated that this effect is unaffected by muscarinic receptor blockade with 4-DAMP or by the nicotinic ganglionic receptor antagonist hexamethonium used at a concentration that has previously been shown to induce ganglionic blockade in the endocrine pancreas (27). This shows that the effects of these receptor antagonists on GRP-stimulated insulin secretion observed in vivo are not due to nonspecific actions on islet GRP receptors and that the action of GRP does not require intact muscarinic receptors at the level of the B cell plasma membrane. The failure of GRP to exert any insulinotropic effect under the in vivo conditions in the presence of hexamethonium, in spite of stimulated insulin secretion in vitro, is probably due to the islets being affected by a high concentration of GRP for a longer period of time in vitro than in vivo. Hence, the major part of the insulinotropic response to GRP under these in vivo conditions of the present study is explained by a ganglionic activation. Such a hypothesis is further supported by our immunohistochemical findings that intrapancreatic ganglia contained GRP immunoreactive nerve terminals, suggesting that the ganglia are innervated by GRP-containing nerves. Therefore, in the mouse, GRP seems to participate in the regulation of insulin release by acting as an intrapancreatic neurotransmitter both at the islet and at the ganglionic level. Previously, the pancreatic ganglia have been demonstrated to participate in the regulation of insulin release (25), and our present results show that GRP is a neurotransmitter involved in this neural regulation of insulin release.

Recently, GRP-stimulated amylase release from isolated rat pancreatic lobules was demonstrated to partially involve a cholinergic mechanism inhibited by hexamethonium, atropine, as well as tetrodotoxin (3). GRP was in that study also shown to induce the release of [3H]acetylcholine from the lobules, although at concentrations above those required for amylase release. A similar mechanism might underlie the cholinergic dependence of GRP-stimulated insulin release, i.e., GRP might facilitate the release of acetylcholine at the presynaptic ganglionic level or alternatively increase the postsynaptic sensitivity to acetylcholine, as was recently demonstrated for cholecystokinin-8 in intracellular recordings from cat pancreatic ganglion cells (19). However, the exact mechanism by which GRP affects the pancreatic ganglia has to await further studies.

Afferent neural mechanisms have previously been demonstrated to be involved in the regulation of glucose homeostasis, glucose-stimulated insulin secretion, and neuroglycopenia-induced glucagon secretion (12, 13). Furthermore, activation of capsaicin-sensitive afferent nerves has previously been suggested to be a mechanism involved in the satiety effect induced by the GRP-like peptide bombesin (18). However, the insulin or glucagon responses to GRP were in the present study demonstrated to be unaffected by capsaicin treatment. Therefore, our results demonstrate that the mechanism of GRP-stimulated insulin release does not involve capsaicin-sensitive afferent nerves.

GRP is known also to stimulate glucagon release under in vivo conditions in several species, including the mouse (22). In the present study, GRP-stimulated glucagon secretion was not affected by muscarinic antagonism by the m3-selective antagonist 4-DAMP but was partially inhibited by hexamethonium. This is in contrast to a previous study in the mouse in which methylatropine was shown to slightly diminish GRP-stimulated glucagon secretion (22). The lack of effect of 4-DAMP in the present study is unlikely to be due to the use of too low a dose level since, in the mouse, 4-DAMP inhibits carbachol-stimulated glucagon secretion at lower dose levels than insulin secretion (11). Instead, the results might imply that the cholinergic receptor involved in GRP-stimulated glucagon secretion is not of the m3 subtype. Furthermore, the fact that GRP-stimulated glucagon release is not affected by m3-blockade, whereas GRP-stimulated insulin release is, shows that the insulinotropic effect of GRP is not secondary to increased glucagon secretion. The partial inhibition by hexamethonium and the lack of effect of 4-DAMP on the glucagon response to GRP imply, besides an indirect ganglionic mechanism, a major contribution of a direct islet effect of GRP in glucagon secretion in the mouse. Indeed, GRP has previously been demonstrated to potently stimulate glucagon secretion from isolated mouse pancreatic islets (29).

We conclude that, in the mouse, GRP is confined to intrapancreatic nerves of the islets and the ganglia and that the peptide acts as an intrapancreatic neurotransmitter, stimulating insulin release both directly through an action on the islet B cell and indirectly through activation of postganglionic cholinergic nerves, presumably by an action at the ganglionic level. The findings imply that activation of the cholinergic mechanisms is a major determinant for the effect of GRP on insulin secretion under in vivo conditions.

    ACKNOWLEDGEMENTS

We are grateful to L. Bengtsson, L. Kvist, and I. Reimertz for technical assistance.

    FOOTNOTES

The work was financially supported by grants from The Swedish Medical Research Council (Grants 14X-6834 and 14X-4499), The Swedish Diabetes Association, Ernhold Lundström, Novo Nordisk, The Emma Ekstrand, Hildur and Jan Tegger, Albert Påhlssson and Crafoord Foundations, Malmö University Hospital, and by the Faculty of Medicine, Lund University, Sweden.

Address for reprint requests: S. Karlsson, The Wallenberg Laboratory, Dept. of Medicine, Malmö Univ. Hospital, S-205 02 Malmö, Sweden.

Received 18 November 1996; accepted in final form 18 September 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Boschero, A. C., M. Szpak-Glasman, E. M. Carneiro, S. Bordin, I. Paul, E. Rojas, and I. Atwater. Oxotremorine-m potentiation of glucose-induced insulin release from rat islets involves M3 muscarinic receptors. Am. J. Physiol. 268 (Endocrinol. Metab. 31): E336-E342, 1995[Abstract/Free Full Text].

2.   De Giorgio, R., C. Sternini, K. Andersson, N. C. Brecha, and V. L. W. Go. Tissue distribution and innervation pattern of peptide immunoreactivities in the rat pancreas. Peptides 13: 91-98, 1992[Medline].

3.   Flowe, K. M., T. H. Welling, and M. W. Mulholland. Gastrin-releasing peptide stimulation of amylase release from rat pancreatic lobules involves intrapancreatic neurons. Pancreas 9: 513-517, 1994[Medline].

4.   Greeley, G. H., Jr., and J. C. Thompson. Insulinotropic and gastrin-releasing action of gastrin-releasing peptide (GRP). Regul. Pept. 8: 97-103, 1984[Medline].

5.   Gregersen, S., and B. Ahrén. Studies on the mechanisms by which gastrin releasing peptide potentiates glucose-induced insulin secretion from mouse islets. Pancreas 12: 48-57, 1996[Medline].

6.   Hermansen, K., and B. Ahrén. Gastrin releasing peptide stimulates the secretion of insulin, but not that of glucagon or somatostatin, from the isolated perfused dog pancreas. Acta Physiol. Scand. 138: 175-179, 1990[Medline].

7.   Holst, J. J. Peptidergic mechanisms in the pancreas. Arch. Int. Pharmacodyn. Ther. 303: 252-269, 1990[Medline].

8.   Holzer, P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 24: 739-768, 1988[Medline].

9.   Holzer, P. Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol. Rev. 43: 143-201, 1991[Medline].

10.   Karlsson, S., and B. Ahrén. Peptide receptor antagonists in the study of insulin and glucagon secretion in mice. Eur. J. Pharmacol. 191: 457-464, 1990[Medline].

11.   Karlsson, S., and B. Ahrén. Muscarinic receptor subtypes in carbachol-stimulated insulin and glucagon secretion in the mouse. J. Auton. Pharmacol. 13: 439-446, 1993.

12.   Karlsson, S., A. W. J. Scheurink, A. B. Steffens, and B. Ahrén. Involvement of capsaicin-sensitive nerves in the regulation of insulin secretion and glucose tolerance in conscious mice. Am. J. Physiol. 267 (Regulatory Integrative Comp. Physiol. 36): R1071-R1077, 1994[Abstract/Free Full Text].

13.   Karlsson, S., F. Sundler, and B. Ahrén. Neonatal capsaicin-treatment in mice: effects on pancreatic peptidergic nerves and 2-deoxy-D-glucose-induced insulin and glucagon secretion. J. Auton. Nerv. Syst. 39: 51-60, 1992.

14.   Knuhtsen, S., J. J. Holst, F. G. A. Baldissera, T. Skak-Nielsen, S. S. Poulsen, S. L. Jensen, and O. V. Nielsen. Gastrin releasing peptide in the porcine pancreas. Gastroenterology 92: 1153-1158, 1987[Medline].

15.   Knuhtsen, S., J. J. Holst, S. L. Jensen, U. Knigge, and O. V. Nielsen. Gastrin-releasing peptide: effect on exocrine secretion and release from isolated perfused porcine pancreas. Am. J Physiol. 248 (Gastrointest. Liver Physiol. 17): G282-G286, 1985.

16.   Knuhtsen, S., J. J. Holst, T. W. Schwartz, S. L. Jensen, and O. V. Nielsen. The effect of gastrin-releasing peptide on the endocrine pancreas. Regul. Pept. 17: 269-276, 1987[Medline].

17.   Lacy, P. E., and M. Kostianovsky. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16: 35-39, 1967[Medline].

18.   Ladenheim, E. E., and R. C. Ritter. Capsaicin attenuates bombesin-induced suppression of food intake. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R263-R266, 1991[Abstract/Free Full Text].

19.   Ma, R. C., and J. H. Szurszewski. Facilitating effect of CCK on nicotinic neurotransmission in cat pancreatic ganglion. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G526-G534, 1996[Abstract/Free Full Text].

20.   McDonald, T. J., P. Houghton, J. R. Challis, and I. M. Hramiak. The effect of gastrin-releasing peptide on the endocrine pancreas. Ann. NY Acad. Sci. 547: 242-254, 1988[Abstract].

21.   Moghimzadeh, E., R. Ekman, R. Håkansson, N. Yanaihara, and F. Sundler. Neuronal gastrin-releasing peptide in the mammalian gut and pancreas. Neuroscience 10: 553-563, 1983[Medline].

22.   Pettersson, M., and B. Ahrén. Gastrin releasing peptide (GRP): effects on basal and stimulated insulin and glucagon secretion in the mouse. Peptides 8: 55-60, 1987[Medline].

23.   Pettersson, M., and B. Ahrén. Insulin and glucagon secretion in the rat: effects of gastrin releasing peptide. Neuropeptides 12: 159-163, 1988[Medline].

24.   Shimosegawa, T., T. Asakura, J. Kashimura, K. Yoshida, T. Meguro, M. Koizumi, T. Mochizuki, N. Yanaihara, and T. Toyota. Neurons containing gastrin releasing peptide-like immunoreactivity in the human pancreas. Pancreas 8: 403-412, 1993[Medline].

25.   Stagner, J. I., and E. Samols. Modulation of insulin secretion by pancreatic ganglionic nicotinic receptors. Diabetes 35: 849-854, 1986[Abstract].

26.   Thompson, R. J., J. F. Doran, P. Jackson, A. P. Dihlon, and J. Rode. PGP 9.5---a new marker for vertebrate neurons and neuroendocrine cells. Brain Res. 278: 224-228, 1983[Medline].

27.   Verche, C. B., Y. N. Kwok, and J. C. Brown. Modulation of acetylcholine-stimulated insulin release by glucose and gastric inhibitory polypeptide. Pharmacology 42: 273-282, 1991[Medline].

28.   Wahl, M. A., E. A. Landsbeck, H. P. T. Ammon, and E. J. Verspohl. Gastrin-releasing peptide: binding and functional studies in mouse pancreatic islets. Pancreas 7: 345-351, 1992[Medline].

29.   Wilkes, L. C., C. J. Bailey, M. G. Thompson, J. M. Conlon, and K. D. Buchanan. Effects of gastrin-releasing peptide on the secretion of mouse islet hormones in vitro. J. Endocrinol. 127: 335-340, 1990[Abstract].

30.   Wood, S. M., R. T. Jung, J. D. Webster, M. A. Ghatei, T. E. Adrian, N. Yanaihara, C. Yanaihara, and S. R. Bloom. The effect of the mammalian neuropeptide, gastrin releasing peptide (GRP), on gastrointestinal and pancreatic hormone secretion in man. Clin. Sci. (Lond.) 65: 365-371, 1983[Medline].


AJP Endocrinol Metab 274(1):E124-E129
0193-1849/98 $5.00 Copyright © 1998 the American Physiological Society