Glutathione depletion inhibits amylase release in guinea pig pancreatic acini

1983 ◽  
Vol 244 (3) ◽  
pp. G273-G277
Author(s):  
W. F. Stenson ◽  
E. Lobos ◽  
H. J. Wedner
Keyword(s):  
Guinea Pig ◽  
Reduced Glutathione ◽  
Glutathione Depletion ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Stimulus Secretion Coupling ◽  
Dose Dependent ◽  
Higher Doses

Isolated guinea pig pancreatic acini were specifically depleted of glutathione by treatment with 2-cyclohexene-1-one (2-CHX-1). Untreated acini contained 4.3 +/- 0.6 micrograms of glutathione per milligram protein. Incubation with 1 mM 2-CHX-1 for 5 min at 37 degrees C depleted glutathione to 17% of control values; 5 mM 2-CHX-1 depleted glutathione to less than 4% of control values. Incubation with 2-CHX-1 also impaired the ability of the isolated acini to secrete amylase in response to stimulation with carbachol and the ionophore A23187. The depletion of glutathione and the inhibition of amylase secretion by 2-CHX-1 were both dose dependent and time dependent. Incubation of acini with 2 mM 2-CHX-1 for 15 min at 37 degrees C reduced glutathione levels to 6.6% of control and reduced carbachol-stimulated amylase release to 63% of control. Higher doses of 2-CHX-1 or longer incubations resulted in greater depletion of glutathione and greater inhibition of carbachol-induced amylase release. These data indicate that specific depletion of glutathione impairs the ability of isolated acini to secrete amylase in response to physiological and pharmacologic stimuli and suggest that glutathione has a role in stimulus-secretion coupling in the exocrine pancreas.

Potentiation of carbachol-induced amylase release by propionate in guinea pig and vole pancreatic acini

1999 ◽  
Vol 277 (3) ◽  
pp. R767-R775
Author(s):  
Etsumori Harada ◽  
Megumi Mitani ◽  
Takashi Takeuchi
Keyword(s):  
Guinea Pig ◽  
Secretory Pathway ◽  
Direct Action ◽  
Camp Level ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Camp Content ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Intracellular Ca

The action of propionate, one of the major end products of microbial fermentation in herbivores was investigated in isolated, perifused pancreatic acini of guinea pigs, voles, and mice. With the use of guinea pig acini, 100 μM propionate had no effect, whereas 300 and 600 μM increased amylase release by six- and ninefold, respectively. Simultaneous perifusion of carbachol (CCh) 10 μM plus propionate 100 μM in guinea pig acini produced a potentiated secretory response that was 130% higher than the summated value obtained with CCh and propionate alone. The potentiation by propionate (100 μM) of CCh (10 μM)-induced amylase release was also obtained in vole pancreatic acini, but the mouse pancreatic preparation did not exhibit a similar potentiation. In contrast to CCh, propionate (100–600 μM) alone had no significant effect on intracellular Ca2+ concentration ([Ca2+]i) and did not alter [Ca2+]ielicited by CCh. Ca ionophore A23187 (5 μM)-induced amylase release in guinea pig acini was enhanced twofold by the addition of propionate. Cellular cAMP content was increased slightly by propionate, but did not alter dose dependently. The cAMP level with combinations of CCh and propionate was almost same as that with CCh alone and propionate alone. Staurosporine did not modify amylase secretion induced by a combination of CCh and propionate. These results suggest that propionate, in addition to a direct action on amylase release, potentiates CCh-induced amylase release in guinea pig and vole acini via a secretory pathway not associated with an increase in [Ca2+]iand cellular cAMP.

Regulation of muscarinic acetylcholine receptors in cultured guinea pig pancreatic acini

1986 ◽  
Vol 251 (1) ◽  
pp. G75-G83 ◽  
Author(s):  
S. R. Hootman ◽  
M. E. Brown ◽  
J. A. Williams ◽  
C. D. Logsdon
Keyword(s):  
Guinea Pig ◽  
Acetylcholine Receptors ◽  
Muscarinic Acetylcholine Receptors ◽  
Amylase Secretion ◽  
Cholinergic Agonists ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Antagonist Binding ◽  
Dose Dependent ◽  
Binding Level

Regulation of muscarinic receptors in cultured guinea pig pancreatic acini was investigated by assessing the effects of cholinergic agonists on binding of [N-methyl-3H]scopolamine [( 3H]NMS) and on amylase release. Freshly dispersed acini bound [3H]NMS with a Kd of 74 pM and a maximal binding level (Bmax) of 908 fmol/mg DNA. Carbachol (CCh) stimulated amylase secretion and inhibited [3H]NMS binding. Incubation of acini for 30 min with 0.1 mM CCh decreased the subsequent efficacy of CCh in stimulating amylase release by threefold but had no effect on its potency. In contrast, amylase release in response to cholecystokinin octapeptide (CCK-8) was not altered by CCh preincubation. [3H]NMS binding to acini was decreased only 15–20% after 30-min incubation with CCh. However, culture of acini with 0.1 mM CCh decreased [3H]NMS binding by 50% at 3–4 h and by 85–90% at 24 h. This decrease was attributable primarily to a reduction in Bmax. [3H]NMS binding also was decreased to a similar extent by the cholinergic agonists bethanechol and methacholine but not by other secretagogues. The decrease in antagonist binding induced by CCh was dose dependent, with the IC50, 5.8 microM, approximating the EC50 for amylase release, 4.3 microM. Culture of acini for 24 h with CCh abolished subsequent amylase release in response to CCh but not to CCK-8. When CCh was removed from the culture medium after 24 h and acini recultured in its absence, [3H]NMS binding increased with a half-time for recovery of 20–24 h; this recovery was blocked by cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of natural glucagon on pancreatic acini: due to contamination by previously undescribed secretagogues

1983 ◽  
Vol 245 (5) ◽  
pp. G703-G710 ◽  
Author(s):  
S. J. Pandol ◽  
V. E. Sutliff ◽  
S. W. Jones ◽  
C. G. Charlton ◽  
T. L. O'Donohue ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Vasoactive Intestinal Peptide ◽  
Biologically Active ◽  
Enzyme Secretion ◽  
Amylase Secretion ◽  
Trypsin Treatment ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Maximal Stimulation ◽  
Threefold Increase

In dispersed acini from guinea pig pancreas, natural glucagon caused a two- to threefold increase in amylase secretion, and this natural glucagon-induced increase was augmented by theophylline. Natural glucagon also caused a sixfold increase in cellular cAMP but did not alter cellular cGMP or outflux of 45Ca. Natural glucagon caused detectable changes in cAMP and amylase secretion at a concentration of 1 microM, half-maximal stimulation at 10 microM, and maximal stimulation at 100 microM. Natural glucagon potentiated the increase in enzyme secretion caused by secretagogues that act by causing mobilization of cellular calcium but did not alter the increase in enzyme secretion caused by secretagogues that increase or mimic the action of cellular cAMP. At concentrations up to 100 microM, natural glucagon did not inhibit binding of 125I-vasoactive intestinal peptide. The potency with which glucagon stimulated amylase release and augmented the increase in amylase release caused by cholecystokinin or carbachol was the same with acini from rat or mouse pancreas as it was with acini from guinea pig pancreas. Biologically active synthetic glucagon did not increase enzyme secretion. On reverse-phase, high-pressure liquid chromatography of natural glucagon, the ability to stimulate enzyme secretion migrated differently from the glucagon. Natural glucagon, at concentrations that stimulated cAMP accumulation, did not react with vasoactive intestinal peptide or secretin radioimmunoassays. Neither insulin nor pancreatic polypeptide, which are known to contaminate natural glucagon preparations, increased enzyme secretion from pancreatic acini. Trypsin treatment abolished the ability of natural glucagon to increase enzyme secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of okadaic acid indicate a role for dephosphorylation in pancreatic stimulus-secretion coupling

1992 ◽  
Vol 263 (6) ◽  
pp. C1172-C1180 ◽  
Author(s):  
A. C. Wagner ◽  
M. J. Wishart ◽  
D. I. Yule ◽  
J. A. Williams
Keyword(s):  
Okadaic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Receptor Activation ◽  
Amylase Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Permeabilized Cells ◽  
Late Step ◽  
Stimulus Secretion Coupling ◽  
The Right

Okadaic acid completely inhibits phosphatase 2A at nanomolar concentrations, while complete inhibition of type 1 phosphatases occurs at 1 microM. Phosphatase 2B is significantly inhibited only at concentrations > 1 microM. In rat pancreatic acini, 1 microM okadaic acid shifted the cholecystokinin (CCK) dose-response curve for stimulating amylase release to the right without reducing maximal secretion. At 3 microM, okadaic acid inhibited maximal CCK-induced amylase release to 78 +/- 7% of control, whereas the inactive analogue 1-Nor-okadaone had no effect. Three lines of evidence indicate that this inhibition by okadaic acid occurs at a late step in stimulus-secretion coupling: 1) intracellular Ca2+ signaling in response to agonist stimulation was not appreciably altered by okadaic acid; 2) stimulation with phorbol ester plus thapsigargin (thus by-passing receptor activation), which gave 85 +/- 4% of maximal CCK-induced amylase release, was inhibited 66 +/- 4% by 3 microM okadaic acid; and 3) Ca(2+)-induced amylase secretion in streptolysin O-permeabilized cells was also reduced by 85 +/- 7%. Two-dimensional polyacrylamide gel electrophoresis of 32P-labeled acini and autoradiography demonstrated that okadaic acid dose dependently increased overall protein phosphorylation. Correspondingly, okadaic acid also led to an inhibition of CCK-induced dephosphorylation. These results show that okadaic acid inhibits pancreatic acinar secretion at a step after generation of intracellular messengers and indicate a role for protein dephosphorylation in stimulus-secretion coupling.

Effects of staurosporine on protein kinase C and amylase secretion from pancreatic acini

1989 ◽  
Vol 257 (4) ◽  
pp. G548-G553 ◽  
Author(s):  
T. B. Verme ◽  
R. T. Velarde ◽  
R. M. Cunningham ◽  
S. R. Hootman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Digestive Enzyme ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Functional Protein ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Protein Kinase C Activity

The effects of staurosporine, a recently isolated microbial alkaloid, on amylase secretion and protein kinase C activity of guinea pig pancreatic acini were investigated. Staurosporine at a concentration of 1 microM completely inhibited both acinar protein kinase C activity (IC50 = 5.5 +/- 1.4 nM) and amylase secretion induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (IC50 = 4.1 +/- 0.4 nM). At this concentration, staurosporine reduced amylase secretion elicited by maximally effective concentrations of carbachol and cholecystokinin by approximately 50% but did not appreciably alter the potencies of the two secretagogues. In the presence of staurosporine, amylase secretion induced by carbachol was linear for at least 60 min. Staurosporine had no effect on amylase release elicited by the Ca2+ ionophore A23187. It did, however, inhibit secretion induced by vasoactive intestinal peptide, although with a reduced potency relative to its effects on amylase release stimulated by TPA, carbachol, and cholecystokinin (IC50 = 34 +/- 17 nM). These results indicate that staurosporine is a potent inhibitor of protein kinase C activity in pancreatic acini and that protein kinase C has an important role as an intracellular mediator of digestive enzyme secretion induced by cholecystokinin and carbachol in the acinar cell. In addition, a separate staurosporine-insensitive coupling pathway, most likely involving Ca2+, appears to be equally important and can maintain long-term secretion in the absence of functional protein kinase C activity.

Actions of peptides isolated from amphibian skin on amylase release from dispersed pancreatic acini.

1979 ◽  
Vol 236 (5) ◽  
pp. E571
Author(s):  
E R Uhlemann ◽  
A J Rottman ◽  
J D Gardner
Keyword(s):  
Salivary Gland ◽  
Guinea Pig ◽  
Vasoactive Intestinal Peptide ◽  
Amylase Secretion ◽  
Amphibian Skin ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Stimulation Of

In dispersed acini prepared from guinea pig pancreas, peptides isolated from amphibian skin (caerulein, bombesin, litorin, and physalaemin) as well as eledoisin, a peptide isolated from the posterior salivary gland of a Mediterranean octopod, caused a significant increase in amylase release. Each amphibian peptide potentiated the stimulation of amylase release caused by vasoactive intestinal peptide or secretin in that the increase in amylase release caused by an amphibian peptide plus vasoactive intestinal peptide or secretin was significantly greater than the sum of the increase caused by each secretagogue acting alone. Potentiation of amylase secretion did not occur with an amphibian peptide plus cholecystokinin or carbachol.

Visualization of amylase secretion from individual pancreatic acini

1988 ◽  
Vol 254 (5) ◽  
pp. G664-G670 ◽  
Author(s):  
D. Bosco ◽  
M. Chanson ◽  
R. Bruzzone ◽  
P. Meda
Keyword(s):  
Protein A ◽  
Plaque Assay ◽  
Wide Distribution ◽  
Amylase Secretion ◽  
Functional Heterogeneity ◽  
Active Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Dose Dependent ◽  
Stimulation Of

To assess the secretion of individual rat pancreatic acini, we developed a reverse hemolytic plaque assay that allows for a direct visualization of amylase release. This release was detected around secreting cells by the presence of hemolytic plaques that resulted from the complement-mediated lysis of red blood cells bearing amylase-antiamylase complexes bound to protein A. Controls showed that these plaques reflected specifically the active secretion of amylase. Quantitation of hemolytic plaques showed that after a 30-min incubation approximately 50% of the acini secreted under basal conditions. Stimulation of amylase release by increasing concentrations of carbamylcholine resulted in a dose-dependent recruitment of secreting acini as well as in a time-dependent enhancement in the response of individual acini. Under all conditions, the wide distribution of hemolytic plaque sizes indicated large differences in the secretory output of individual acini. Thus, using a new method to directly visualize and quantitate amylase secretion, we have provided evidence for a functional heterogeneity of pancreatic acini.

Properties of receptors for gastrin and CCK on gastric smooth muscle cells

1989 ◽  
Vol 257 (1) ◽  
pp. G73-G79 ◽  
Author(s):  
D. Menozzi ◽  
J. D. Gardner ◽  
R. T. Jensen ◽  
P. N. Maton
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Amylase Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Cck Receptors ◽  
Gastric Smooth Muscle ◽  
Relative Potencies

Previous studies have demonstrated that cholecystokinin (CCK), gastrin, and structurally related peptides can interact with various types of receptors that can be distinguished by their relative affinities for agonists and antagonists. In the present study we examined the effect of gastrin, the COOH-terminal octapeptide of CCK (CCK-8), and the tetrapeptide of CCK (CG-4) on contraction of dispersed gastric smooth muscle cells from guinea pig and tested the ability of various CCK receptor antagonists to affect agonist-induced muscle cell contraction. For purposes of comparison we tested the effect of each antagonist on CCK-stimulated amylase secretion by pancreatic acini from guinea pig. On gastric smooth muscle cells, CCK-8, gastrin, and CG-4 were all full agonists. CCK-8 and gastrin were equipotent and CG-4 was 6,000-fold less potent. Each antagonist caused inhibition of CCK-stimulated contraction with relative potencies (IC50): L364,718 (4 microM) = CBZ-CCK-(27-32)-NH2 (3 microM) greater than proglumide analogue 10 (90 microM). Inhibition by each of the antagonists was competitive in nature, specific for CCK peptides, and each had the same IC50 whether contraction was stimulated by CCK-8, gastrin, or CG-4. Relative potencies (IC50) of the three antagonists for inhibiting CCK-stimulated amylase release from pancreatic acini were L364,718 (3 nM) greater than proglumide analogue 10 (200 nM) greater than CBZ-CCK-(27-32)-NH2 (3 microM). These results demonstrate that gastric smooth muscle cells possess receptors that differ from CCK receptors on pancreatic acini in terms of affinities for both agonists and certain antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Regulation of protein phosphorylation in pancreatic acini. Distinct effects of Ca2+ ionophore A23187 and 12-O-tetradecanoylphorbol 13-acetate

1986 ◽  
Vol 235 (1) ◽  
pp. 125-131 ◽  
Author(s):  
D B Burnham ◽  
P Munowitz ◽  
S R Hootman ◽  
J A Williams
Keyword(s):  
Guinea Pig ◽  
Protein Phosphorylation ◽  
Phorbol Ester ◽  
Effective Concentration ◽  
Secretory Response ◽  
Maximal Response ◽  
Ionophore A23187 ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Intracellular Messengers

Regulation of protein phosphorylation in isolated pancreatic acini by the intracellular messengers Ca2+ and diacylglycerol was studied by using the Ca2+ ionophore A23187 and the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate. As assessed by two-dimensional polyacrylamide-gel electrophoresis, the phorbol ester (1 microM) and Ca2+ ionophore (2 microM) altered the phosphorylation of distinct sets of proteins between Mr 83,000 and 23,000 in mouse and guinea-pig acini. The phorbol ester increased the phosphorylation of four proteins, whereas the ionophore increased the phosphorylation of two proteins and, in mouse acini, decreased the phosphorylation of one other protein. In addition, the phorbol ester and ionophore each caused the dephosphorylation of two proteins, of Mr 20,000 and 20,500. Administered together, these agents reproduced the changes in phosphorylation induced by the cholinergic agonist carbamoylcholine. The effects of the phorbol ester and ionophore on acinar amylase release were also studied. In mouse pancreatic acini, a maximally effective concentration of phorbol ester (1 microM) produced a secretory response that was only 28% of that produced by a maximally effective concentration of carbamoylcholine, whereas the ionophore (0.3 microM) stimulated amylase release to two-thirds of the maximal response to carbamoylcholine. In contrast, in guinea-pig acini, the phorbol ester and carbamoylcholine evoked similar maximal secretory responses, whereas the maximal secretory response to the ionophore was only 35% of that to carbamoylcholine. Combination of phorbol ester and ionophore resulted in a modest synergistic effect on amylase release in both species. It is concluded that cholinergic agonists act via both diacylglycerol and Ca2+ to regulate pancreatic protein phosphorylation, but that synergism between these intracellular messengers is of limited importance in stimulating enzyme secretion.

Metabolism of arachidonic acid by pancreatic acini: relation to amylase secretion

1982 ◽  
Vol 242 (5) ◽  
pp. G493-G497
Author(s):  
W. F. Stenson ◽  
E. Lobos
Keyword(s):  
Arachidonic Acid ◽  
Guinea Pig ◽  
Thin Layer ◽  
Prostaglandin E2 ◽  
Amylase Secretion ◽  
Pancreatic Acini ◽  
Arachidonate Metabolites ◽  
Stimulus Secretion Coupling ◽  
Layer Chromatography

Isolated guinea pig pancreatic acini were incubated with exogenous [14C]arachidonic acid (10 microM) at 37 degrees C for 3 min. The lipids were extracted and separated by thin-layer chromatography. Radiolabeled metabolites were identified by comigration with standards: 0.024% of the recovered radioactivity comigrated with prostaglandin E2 (PGE2), 0.016% comigrated with PGF2 alpha, 4.9% was incorporated into triglycerides, 1.8% was incorporated into phospholipids, and 93.2% remained as arachidonic acid. The synthesis of PGE2 and PGF2 alpha was inhibited by indomethacin (ID50, 30 nM). Simultaneous addition of carbachol or caerulein with the [14C]arachidonic acid did not alter the metabolism of the arachidonate. Further studies were done on the role of arachidonate metabolites in the secretion of amylase. Exogenously added PGE2 and PGF2 alpha (0.3-100 nM) did not induce amylase secretion from isolated acini. Incubation of isolated acini with indomethacin (0.1-28 microM) did not inhibit the release of amylase induced by carbachol or caerulein. From these data, we conclude that isolated guinea pig pancreatic acini are capable of converting a small percentage of exogenous arachidonate to PGE2 and PGF2 alpha. However, there is no evidence for a role of these compounds in stimulus-secretion coupling.

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