Muscarinic receptor subtypes on rat pancreatic acini: secretion and binding studies

1986 ◽  
Vol 251 (2) ◽  
pp. G275-G279 ◽  
Author(s):  
D. S. Louie ◽  
C. Owyang
Keyword(s):  
Receptor Antagonist ◽  
Muscarinic Receptor ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Receptor Subtypes ◽  
Muscarinic Receptor Subtypes ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Rightward Shift

Characterization of muscarinic receptor subtypes on rat pancreatic acinar cells was examined by using specific muscarinic receptor antagonists to study amylase secretion and binding of [N-methyl-3H]scopolamine ([3H]NMS). Rat pancreatic acini were dispersed in HEPES-Ringer buffer and incubated with acetylcholine +/- 4-diphenylacetoxy-N-methylpiperadine-methiodide (4-DAMP, a specific M2 muscarinic receptor antagonist) or +/- pirenzepine (a specific M1 muscarinic receptor antagonist). 4-DAMP (10(-9) to 10(-6) M) caused a progressive parallel rightward shift in the acetylcholine dose-response curve without a change in maximal amylase release. Only high concentrations of pirenzepine (10(-6) to 10(-4) M) caused a rightward shift in the dose-response curve to acetylcholine. Schild analysis of the data indicated an inhibitory constant (Ki) of 200 pM for 4-DAMP and 183 nM for pirenzepine. The slope of the Schild regression lines was not different from unity, suggesting competitive inhibition. Binding of 50 pM [3H]NMS was specific, rapid, and saturable. [3H]NMS binding was displaced by increasing concentrations of 4-DAMP or pirenzepine with apparent Ki's of 102 pM and 330 nM, respectively, and similar maximal binding levels of 60 fmol/mg prot. We have demonstrated that 4-DAMP has an approximately 1,000-fold greater potency than pirenzepine to inhibit amylase release and binding, indicating that cholinergic-stimulated amylase release from pancreatic acini is mediated by M2 muscarinic receptors.

Effects of inhibitors of cyclic nucleotide phosphodiesterase on actions of cholecystokinin, bombesin, and carbachol on pancreatic acini

1983 ◽  
Vol 245 (5) ◽  
pp. G676-G680
Author(s):  
J. D. Gardner ◽  
V. E. Sutliff ◽  
M. D. Walker ◽  
R. T. Jensen
Keyword(s):  
Dose Response ◽  
Cyclic Nucleotide ◽  
Response Curve ◽  
Dose Response Curve ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Enzyme Secretion ◽  
Amylase Secretion ◽  
Pancreatic Acini ◽  
Rightward Shift ◽  
Stimulation Of

In dispersed acini from guinea pig pancreas two inhibitors of cyclic nucleotide phosphodiesterase, Ro 20-1724 and 3-isobutyl-1-methylxanthine (IBMX), augmented the increase in amylase secretion caused by supramaximal concentrations of cholecystokinin but did not alter the stimulation of enzyme secretion caused by bombesin. The augmentations of the action of cholecystokinin caused by Ro 20-1724 or IBMX could be reproduced by 8-bromo-cAMP. When tested alone or with theophylline, cholecystokinin did not alter cAMP in pancreatic acini; however, with Ro 20-1724 or IBMX, concentrations of cholecystokinin that were supramaximal for stimulating amylase secretion caused a significant increase in cellular cAMP. These findings indicate that Ro 20-1724 and IBMX augment the action of cholecystokinin on enzyme secretion by inhibiting cyclic nucleotide phosphodiesterase and allowing a significant cholecystokinin-induced increase in cellular cAMP. IBMX but not Ro 20-1724 caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion caused by carbachol. IBMX also caused a parallel rightward shift in the dose-response curve for the stimulation of outflux of 45Ca caused by carbachol. These results indicate that IBMX, but not Ro 20-1724, can function as a muscarinic cholinergic antagonist.

Effect of a Rab3A effector domain-related peptide, CCK, and EGF in permeabilized pancreatic acini

1994 ◽  
Vol 267 (3) ◽  
pp. G350-G356
Author(s):  
S. Zeuzem ◽  
D. Stryjek-Kaminska ◽  
W. F. Caspary ◽  
J. Stein ◽  
A. Piiper
Keyword(s):  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Pancreatic Acinar Cells ◽  
Amylase Secretion ◽  
Small Molecular Weight ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Effector Domain ◽  
Domain Peptide

We report here that a synthetic peptide of the effector domain of the small-molecular-weight GTP-binding protein Rab3A (EDRab3AL) is a potent stimulator of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase secretion in digitonin-permeabilized pancreatic acini. Moreover, the Rab3A effector domain peptide caused phosphatidylinositol 4,5-bisphosphate breakdown, indicating that the observed increase in Ins(1,4,5)P3 is due to stimulation of a phosphoinositide-specific phospholipase C (PLC). The dose-response curve for EDRab3AL-induced amylase release was biphasic, showing a maximum at 0.3 nM EDRab3AL and a decline at higher peptide concentrations. By contrast, the dose-response curve for EDRab3AL-induced Ins(1,4,5)P3 production was monophasic, showing stimulation with increasing EDRab3AL concentrations. A peptide of the effector domain of Rab1A, EDRab1AL, had no effect, indicating that the response to EDRab3AL is specific. Cholecystokinin octapeptide (CCK-8) and EDRab3AL had additive effects on the acinar Ins(1,4,5)P3 level. Epidermal growth factor (EGF), which has recently been shown to inhibit CCK-8-induced Ins(1,4,5)P3 production in pancreatic acinar cells, also decreased EDRab3AL-induced Ins(1,4,5)P3 production. These results suggest that EDRab3AL and CCK-8 act on the same EGF-inhibitable PLC by independent mechanisms. CCK-8 increased and EGF decreased amylase release in response to submaximal EDRab3AL concentrations. By contrast, at supramaximal EDRab3AL concentrations EGF increased and CCK-8 decreased EDRab3AL-stimulated amylase release. EDRab3AL had no effect in intact acini, indicating that the site of action of EDRab3AL is intracellular. We conclude that EDRab3AL regulates phosphoinositide-specific PLC activity and thereby amylase secretion in an analogous fashion to CCK-8, but from within the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein synthesis inhibitors enhance secretagogue sensitivity of in vitro rat pancreatic acini

1982 ◽  
Vol 243 (4) ◽  
pp. G285-G290
Author(s):  
M. Otsuki ◽  
J. A. Williams
Keyword(s):  
Protein Synthesis ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Pancreatic Acinar Cells ◽  
Inhibitory Influence ◽  
Ionophore A23187 ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Inhibitor Of Protein Synthesis

Isolated rat pancreatic acini were treated with cycloheximide and amylase release was measured. This agent increased the sensitivity to both synthetic octapeptide of cholecystokinin (CCK8) and carbamylcholine, the major secretagogues known to utilize Ca2+ as a second messenger. The mechanism of the cycloheximide effect was via inhibition of protein synthesis, as indicated by the following: 1) the concentration of cycloheximide used inhibited leucine incorporation by greater than 90%; 2) this effect was not instantaneous but increased up to a 2-h pretreatment; and 3) a similar effect was obtained with puromycin, a chemically different inhibitor of protein synthesis. Cycloheximide acted on the steps by which secretagogues mobilize cellular Ca2+ because the dose-response curve for 45Ca2+ efflux was shifted to the same extent as that for amylase release, whereas the dose-response curve for amylase release induced by the Ca2+ ionophore A23187 was not altered. The results suggest, therefore, that a rapidly turning-over protein present in pancreatic acinar cells exerts an inhibitory influence on Ca2+ mobilization by secretagogues.

scholarly journals Inhibition by somatostatin of amylase secretion induced by calcium and cyclic AMP in rat pancreatic acini

1994 ◽  
Vol 304 (2) ◽  
pp. 531-536 ◽  
Author(s):  
H Ohnishi ◽  
T Mine ◽  
I Kojima
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Pancreatic Acini

It has recently been shown that somatostatin inhibits amylase secretion from isolated pancreatic acini by reducing cyclic AMP (cAMP) production [Matsushita, Okabayashi, Hasegawa, Koide, Kido, Okutani, Sugimoto and Kasuga (1993) Gastroenterology 104, 1146-1152]. To date, however, little is known as to the other mechanism(s) by which somatostatin inhibits amylase secretion in exocrine pancreas. To investigate the action of somatostatin independent of cAMP generation, we examined the effect of somatostatin in isolated rat pancreatic acini stimulated by 1 microM calcium ionophore A23187 and 1 mM 8-bromo-cyclic AMP (8Br-cAMP). Somatostatin inhibited amylase secretion evoked by a combination of A23187 and 8Br-cAMP in a dose-dependent manner. The maximum inhibition was obtained by 10(-7) M somatostatin, and at this concentration somatostatin inhibited the effect of A23187 and 8Br-cAMP by approximately 30%. In electrically permeabilized acini, an elevation of free calcium concentration resulted in an increase in amylase secretion and cAMP enhanced the secretion evoked by calcium. cAMP shifted the dose-response curve for calcium-induced secretion leftwards and elevated the peak value of secretion. Somatostatin inhibited the effect of cAMP on calcium-induced amylase secretion by shifting the dose-response curve to the right. To determine the involvement of a G-protein(s), we examined the effect of somatostatin in acini pretreated with pertussis toxin. Pretreatment of acini with pertussis toxin completely blocked somatostatin-inhibition of amylase-secretion evoked by A23187 and 8Br-cAMP. These results indicate that somatostatin decreases amylase secretion induced by cAMP and calcium by reducing the calcium sensitivity of exocytosis. A pertussis toxin-sensitive G-protein is also involved in this step.

M2-Receptor Subtype Does Not Mediate Muscarine-Induced Increases in [Ca2+]i in Nociceptive Neurons of Rat Dorsal Root Ganglia

2000 ◽  
Vol 84 (4) ◽  
pp. 1934-1941 ◽  
Author(s):  
Rainer Haberberger ◽  
Reas Scholz ◽  
Wolfgang Kummer ◽  
Michaela Kress
Keyword(s):  
Receptor Antagonist ◽  
Muscarinic Receptor ◽  
Sensory Neurons ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Muscarinic Receptor Subtypes ◽  
Rt Pcr ◽  
Nociceptive Neurons

Multiple muscarinic receptor subtypes are present on sensory neurons that may be involved in the modulation of nociception. In this study we focused on the presence of the muscarinic receptor subtypes, M2 and M3 (M2R, M3R), in adult rat lumbar dorsal root ganglia (DRG) at the functional ([Ca2+]i measurement), transcriptional (RT-PCR), and translational level (immunohistochemistry). After 1 day in culture exposure of dissociated medium-sized neurons (20–35 μm diam) to muscarine was followed by rises in [Ca2+]i in 76% of the neurons. The [Ca2+]i increase was absent after removal of extracellular calcium and did not desensitize after repetitive application of the agonist. This rise in [Ca2+]i may be explained by the expression of M3R, which can induce release of calcium from internal stores via inositoltrisphospate. Indeed the effect was antagonized by the muscarinic receptor antagonist atropine as well as by the M3R antagonist, 4-diphenylacetoxy-N-(2 chloroethyl)-piperidine hydrochloride (4-DAMP). The pharmacological identification of M3R was corroborated by RT-PCR of total RNA and single-cell RT-PCR, which revealed the presence of mRNA for M3R in lumbar DRG and in single sensory neurons. In addition, RT-PCR also revealed the expression of M2R, which did not seem to contribute to the calcium changes since it was not prevented by the M2 receptor antagonist, gallamine. Immunohistochemistry demonstrated the presence of M2R and M3R in medium-sized lumbar DRG neurons that also coexpressed binding sites for the lectin I-B4, a marker for mainly cutaneous nociceptors. The occurrence of muscarinic receptors in putative nociceptive I-B4-positive neurons suggests the involvement of these acetylcholine receptors in the modulation of processing of nociceptive stimuli.

M1 and M3 muscarinic antagonists inhibit human nasal glandular secretion in vitro

1992 ◽  
Vol 73 (5) ◽  
pp. 2069-2073 ◽  
Author(s):  
J. Mullol ◽  
J. N. Baraniuk ◽  
C. Logun ◽  
M. Merida ◽  
J. Hausfeld ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Muscarinic Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Receptor Subtypes ◽  
Muscarinic Antagonists ◽  
Muscarinic Receptor Subtypes ◽  
Mucus Production ◽  
Glandular Secretion ◽  
M3 Receptor

Mucus glycoproteins (MGP) are high-molecular-weight glycoconjugates that are released from submucosal glands and epithelial goblet cells in the respiratory tract. Muscarinic receptors have an important role in the regulation of human nasal glandular secretion and mucus production, but it is not known which of the five muscarinic receptor subtypes are involved. The effect of nonselective and M1-, M2-, and M3-selective muscarinic antagonists on methacholine (MCh)-induced MGP secretion from human nasal mucosal explants was tested in vitro. MGP was assayed by enzyme-linked immunosorbent assay using a specific anti-MGP monoclonal antibody (7F10). MCh (100 microM) induced MGP secretion up to 127% compared with controls. MCh-induced MGP release was significantly inhibited by atropine (100 microM), the M, receptor antagonist pirenzepine (10–100 microM), and the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 1–100 microM). 4-DAMP significantly inhibited MCh-induced MGP release at a lower concentration (1 microM) than pirenzepine (10 microM). The M2 receptor antagonists AF-DX 116 and gallamine (both at 100 microM) had no effect. No antagonist alone had a significant effect on MGP release. These results indicate that the M1 and M3 muscarinic receptor subtypes regulate MGP secretion from human nasal mucosa and suggest that the M3 receptor has the predominant effect.

Histamine Receptors in Normal Human Bronchi

1980 ◽  
Vol 58 (6) ◽  
pp. 537-544 ◽  
Author(s):  
Noemi M. Eiser ◽  
Jane Mills ◽  
K. D. McRae ◽  
P. D. Snashall ◽  
A. Guz
Keyword(s):  
Receptor Antagonist ◽  
Dose Response ◽  
Response Curve ◽  
Normal Subjects ◽  
Dose Response Curve ◽  
Significant Shift ◽  
H2 Receptor Antagonist ◽  
Anticholinergic Activity ◽  
H2 Receptor ◽  
The Right

1. Nine normal subjects inhaled increasing concentrations of histamine aerosol from an aerosol generator attached to a breath-actuated dosimeter. The responses were monitored by measuring specific airways-conductance in a body plethysmograph, and the results were expressed as cumulative log dose-response curves. On separate days, histamine challenges were repeated after intravenous injections of sodium chloride solution (placebo), or an H1-receptor antagonist chlorpheniramine, or an H2-receptor antagonist cimetidine, or H1- and H2-receptor antagonists together. The anticholinergic activity of chlorpheniramine was estimated by comparing the effect of chlorpheniramine and atropine on methacholine challenge. 2. In all subjects the response to histamine was reproducible. Analysis of the variance showed that placebo did not alter the histamine dose-response curve significantly. In contrast, chlorpheniramine produced a large shift in the histamine dose-response curve to the right and cimetidine produced a significant shift of this curve to the right only at the highest dose of histamine. A combination of cimetidine and chlorpheniramine produced a shift not significantly different from that seen with chlorpheniramine alone. Chlorpheniramine showed no significant anticholinergic activity in this study. 3. In the normal subjects histamine-induced bronchoconstriction appeared to be mediated predominantly by the H1-receptors. The H2-receptor contributed very little to this bronchoconstriction.

Coexisting beta-adrenoceptor subtypes: significance for thermogenic process in brown fat cells

1994 ◽  
Vol 267 (4) ◽  
pp. C969-C979 ◽  
Author(s):  
J. Zhao ◽  
L. Unelius ◽  
T. Bengtsson ◽  
B. Cannon ◽  
J. Nedergaard
Keyword(s):  
Dose Response ◽  
Response Curve ◽  
Brown Fat ◽  
Dose Response Curve ◽  
Receptor Subtypes ◽  
Fat Cells ◽  
Cgp 12177 ◽  
Beta 2 ◽  
Brl 37344 ◽  
Thermogenic Response

The possible significance of the coexisting beta 1-, beta 2-, and beta 3-adrenoceptors in brown adipose tissue for the thermogenic response was investigated. Oxygen consumption of isolated hamster brown fat cells was analyzed as a measure of thermogenesis. Thermogenesis could be evoked not only by the physiological agent norepinephrine but also by BRL-37344 and CGP-12177. No evidence for biphasic inhibition curves was found with either the selective beta 1-antagonist ICI-89406, the beta 2-antagonist ICI-118551, or the beta 1/beta 2-nonselective beta-antagonist propranolol against 1 microM norepinephrine; pI50 (the negative logarithm of the inhibitory constant for an antagonist, as estimated from the dose-response curve for an antagonist vs. a constant agonist concentration) values for ICI-89406 and ICI-118551 were very low (4-5), implying nonselective inhibition; the pI50 for propranolol was approximately 6 (as expected for the beta 3-receptor). Even with suboptimal norepinephrine, no biphasic inhibition was found. CGP-12177 at concentrations where it is primarily an antagonist to the beta 1-receptor did not influence the dose-response curve for either norepinephrine or BRL-37344. BRL-37344- or CGP-12177-induced thermogenesis was inhibited by the beta-antagonists in a manner similar to norepinephrine-induced thermogenesis. Schild plots for propranolol inhibition of norepinephrine-, isoprenaline-, BRL-37344- and CGP-12177-induced thermogenesis yielded similar pA2 (the negative logarithm of the inhibitory constant for an antagonist, as calculated from a series of agonist dose-response curves at different antagonist concentrations) (approximately 5.5), for interaction with either agonist, implying that the same receptor was stimulated by all agonists. Thus, despite the fact that different beta-receptor subtypes coexist in the tissue, we find no evidence for the participation of beta 1- or beta 2-receptors in the thermogenic response. Within the resolution of the experiments, the results therefore imply that it is predominantly or solely the beta 3-receptor that is coupled to thermogenesis, and it is via this beta-adrenergic receptor that not only norepinephrine but also CGP-12177 and BRL-37344 induce thermogenesis.

Autonomic regulation of somatostatin release: studies with primary cultures of canine fundic mucosal cells

1984 ◽  
Vol 247 (5) ◽  
pp. G567-G573 ◽  
Author(s):  
T. Yamada ◽  
A. H. Soll ◽  
J. Park ◽  
J. Elashoff
Keyword(s):  
Dose Response ◽  
Response Curve ◽  
Primary Cultures ◽  
Dose Response Curve ◽  
Dibutyryl Camp ◽  
Rightward Shift ◽  
Fundic Mucosa ◽  
Adrenergic Antagonist ◽  
Mucosal Cells ◽  
Epinephrine Dose

Using a newly developed system for culturing canine fundic mucosal cells, we examined regulation of the secretion of somatostatinlike immunoreactivity (SLI) by cholinergic and adrenergic transmitters. Enzyme-dispersed canine fundic mucosa cells were enriched in SLI content by counterflow elutriation and cultured on collagen for 42 h. Epinephrine alone, and in combination with gastrin, stimulated SLI secretion in a dose-dependent fashion. Propranolol did not alter the response to dibutyryl cAMP or gastrin but produced a parallel, rightward shift of the epinephrine dose-response curve with the dissociation constant (Ki) determined to be 14 nM by nonlinear curve fitting. Phentolamine, an alpha-adrenergic antagonist, enhanced SLI secretion in response to epinephrine, an effect reversed by the alpha 1-agonist methoxamine but not by the alpha 2-agonist clonidine. However, neither methoxamine nor clonidine alone inhibited the response to the beta-selective adrenergic agonist isoproterenol; thus, the existence of an adrenergic alpha 1-inhibitory receptor remained uncertain. Carbachol noncompetitively inhibited SLI secretion stimulated by gastrin, epinephrine, and dibutyryl cAMP. Atropine produced a parallel rightward shift of the carbachol dose-response curve (Ki = 0.4 nM). Pirenzepine also inhibited the effects of carbachol (Ki = 35 nM). Our studies suggest that SLI-containing cells in the canine fundic mucosa possess stimulatory beta-adrenergic receptors and inhibitory muscarinic receptors.

Effects of inhibitors of cyclic nucleotide phosphodiesterase on the actions of vasoactive intestinal peptide and secretin on pancreatic acini

1982 ◽  
Vol 242 (6) ◽  
pp. G547-G551
Author(s):  
J. D. Gardner ◽  
L. Y. Korman ◽  
M. D. Walker ◽  
V. E. Sutliff
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Dose Response ◽  
Cyclic Nucleotide ◽  
Response Curve ◽  
Phosphodiesterase Inhibitor ◽  
Dose Response Curve ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Amylase Secretion ◽  
Pancreatic Acini ◽  
Stimulation Of

Theophylline, 3-isobutyl-1-methylxanthine (IBMX), and Ro 20-1724 each augmented the increase in cAMP and the stimulation of amylase secretion caused by vasoactive intestinal peptide (VIP) or secretin. With IBMX the dose-response curve for the stimulation of amylase secretion caused by VIP or secretin spanned a range of lower concentrations than did that obtained with Ro 20-1724, which in turn spanned a range of lower concentrations than did that obtained with theophylline. The configuration of the dose-response curve for the action of VIP on cAMP differed with each phosphodiesterase inhibitor tested. With Ro 20-1724 the dose-response curve was monophasic, whereas with the two methylxanthines the dose-response curve was biphasic. With theophylline the magnitude of the second component of the dose-response curve was larger than the first; with IBMX the magnitude of the first component was larger than the second. The configuration of the dose-response curve for the action of secretin on cAMP also differed with each phosphodiesterase inhibitor tested. With theophylline the dose-response curve was monophasic, whereas with Ro 20-1724 and IBMX the dose-response curve was biphasic. With Ro-20-1724 the magnitude of the second component of the dose-response curve was larger than the first; with IBMX the magnitude of the first component was larger than the second. These results indicate that cAMP is compartmentalized in pancreatic acinar cells and that the different compartments of cAMP are affected differently by various inhibitors of cyclic nucleotide phosphodiesterase. These findings also suggest that the different compartments of cAMP are acted on by phosphodiesterases with different sensitivities to various inhibitors.

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