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.

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.

Downregulation of protein kinase C in guinea pig pancreatic acini: effects on secretion

1988 ◽  
Vol 254 (2) ◽  
pp. G242-G248 ◽  
Author(s):  
C. K. Sung ◽  
S. R. Hootman ◽  
E. L. Stuenkel ◽  
C. Kuroiwa ◽  
J. A. Williams
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Guinea Pig ◽  
Ionophore A23187 ◽  
Response Curves ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Almost All

Pretreatment of guinea pig pancreatic acini with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and concentration-dependent down-regulation of protein kinase C. In control acini almost all of the protein kinase C activity was present in a cytosolic fraction. Incubation with TPA initially shifted protein kinase C activity to a particular fraction which then disappeared over the following 24-h incubation with TPA. To study the role of protein kinase C in stimulus-secretion coupling, acini were pretreated with TPA and then amylase release was studied in response to various secretagogues. Preincubation of acini with TPA led to a time- and concentration-dependent decrease in TPA-stimulated amylase release that correlated with protein kinase C downregulation. Preincubation of acini with 1 microM TPA for 24 h, resulting in complete loss of protein kinase C activity, abolished the secretory effect of subsequently added TPA. By contrast, the secretory effects of cholecystokinin octapeptide (CCK-8) and carbamylcholine chloride (CCh) were only inhibited by 44 and 34%, respectively, and amylase release stimulated by the Ca2+ ionophore A23187 and an adenosine 3',5'-cyclic monophosphate-mediated agonist, vasoactive intestinal peptide, was unaffected. Dose-response curves for CCK-8- or CCh-stimulated amylase release in TPA-pretreated acini revealed attenuation of both maximal efficacy and sensitivity. However, the CCh-stimulated intracellular Ca2+ increase as determined by use of the fluorescent probe fura-2 was not affected by the long-term TPA pretreatment of acini. This study strongly suggests that both protein kinase C and intracellular Ca2+ play a significant role in CCK-8- and CCh-stimulated amylase release.

Somatostatin inhibits VIP-stimulated amylase release from perifused guinea pig pancreatic acini

1988 ◽  
Vol 254 (2) ◽  
pp. G217-G223 ◽  
Author(s):  
P. Singh ◽  
I. Asada ◽  
A. Owlia ◽  
T. J. Collins ◽  
J. C. Thompson
Keyword(s):  
Guinea Pig ◽  
Fold Increase ◽  
Optimal Conditions ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
In Vitro System ◽  
Basal Release ◽  
Perifusion System ◽  
Chamber Size

We have examined the direct effect of somatostatin (SRIF) on basal and stimulated amylase release from guinea pig pancreatic acini using the in vitro method of continuous perifusion. The optimal conditions of flow rate, chamber size, acinar cell volume per chamber, and period of secretagogue infusion were defined for the perifusion system. The kinetic profile of amylase release in response to cholecystokinin-octapeptide (CCK-8), vasoactive intestinal peptide (VIP), and SRIF was studied. Under optimal conditions, the acini were found to remain equally responsive to an ED50 dose of CCK-8 (0.5-0.8 nM) for 12 h of perifusion. The duration of amylase response to any given dose of CCK-8, given for the optimal period of 5 min, was 80-100 min. The total amylase released minus the basal release divided by 90 min (delta response) in response to the maximum effective (Maxeff) dose of CCK-8 (100 nM) was 14,667 +/- 1,433 U/l (amounting to a 10-fold increase compared with basal values). When compared with the amount of total delta amylase released in response to the Maxeff dose of CCK, the total amylase released in response to the Maxeff doses of SRIF (1 microM) and VIP (10 nM) was 10-21% and 51-59%, respectively. SRIF (100 nM) significantly decreased VIP- (0.1-1.0 nM) stimulated amylase release by 45-70% in the perifusion method of study but had no significant effect on the CCK-stimulated amylase release. This suggests that the perifusion method can be used for investigating the mechanism of SRIF-mediated inhibition of VIP effects on amylase release in an in vitro system.

Discovery of a cholecystokinin analogue with partial agonist activity

1984 ◽  
Vol 247 (3) ◽  
pp. G261-G264 ◽  
Author(s):  
J. M. Howard ◽  
M. Knight ◽  
R. T. Jensen ◽  
J. D. Gardner
Keyword(s):  
Guinea Pig ◽  
Partial Agonist ◽  
Effective Concentration ◽  
Amylase Secretion ◽  
Agonist Activity ◽  
Pancreatic Acini ◽  
Rat Pancreas ◽  
Partial Agonist Activity ◽  
Stimulation Of

In dispersed acini from guinea pig pancreas, cholecystokinin-(27-32)-amide [CCK-(27-32)-NH2] did not stimulate amylase secretion and inhibited the stimulation caused by CCK-(26-33). In dispersed acini from mouse or rat pancreas, CCK-(27-32)-NH2 stimulated amylase secretion. The stimulation of amylase secretion caused by a maximally effective concentration of CCK-(27-32)-NH2 was less than that caused by a maximally effective concentration of cholecystokinin-(26-33), and in contrast to the action of cholecystokinin-(26-33) supramaximal concentrations of CCK-(27-32)-NH2 did not cause submaximal stimulation of amylase secretion. Dibutyryl cGMP and proglumide each inhibited the stimulation of amylase secretion caused by CCK-(27-32)-NH2. CCK-(27-32)-NH2 inhibited binding of 125I-labeled CCK to mouse and rat pancreatic acini. These results indicate that, in mouse and rat pancreatic acini, CCK-(27-32)-NH2 is a partial agonist and that this partial agonist activity is produced by occupation of the CCK receptor by CCK-(27-32)-NH2.

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)

Interaction of peptides related to VIP and secretin with guinea pig pancreatic acini

1989 ◽  
Vol 256 (2) ◽  
pp. G283-G290 ◽  
Author(s):  
Z. C. Zhou ◽  
J. D. Gardner ◽  
R. T. Jensen
Keyword(s):  
Guinea Pig ◽  
High Selectivity ◽  
Fold Increase ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Gila Monster ◽  
Secretin Receptor ◽  
Rat Growth ◽  
Releasing Factors ◽  
Relative Potencies

The abilities of human and rat growth hormone-releasing factors (hGHRF, rGHRF), peptide histidine isoleucine or methionine (PHI, PHM) and the Gila monster venom peptides (helospectin I, helospectin II, and helodermin) to interact with guinea pig pancreatic acini were characterized and compared with vasoactive intestinal peptide (VIP) and secretin. Each peptide caused a sevenfold stimulation of amylase release, and the relative potencies were: VIP greater than helospectin I = helospectin II = helodermin = rGHRF greater than PHI = PHM greater than hGHRF greater than secretin. Each peptide inhibited 125I-labeled VIP binding, and the relative potencies agreed closely with those for stimulating enzyme secretion. Each peptide inhibited 125I-labeled secretin binding with the potencies: secretin greater than helospectin I = helospectin II = helodermin greater than rGHRF = PHI = VIP greater than PHM greater than hGHRF. Each peptide caused a 78-fold increase in adenosine 3',5'-cyclic monophosphate cAMP. VIP or rGHRF and PHI or PHM demonstrated high and low selectivity, respectively, for VIP receptors, secretin high selectivity for the secretin receptor, and helospectin I or II and helodermin a relatively high affinity for both VIP and secretin receptors. Correlation of the ability of each peptide to increase cAMP or amylase release and inhibit binding of 125I-VIP or 125I-secretin suggested all the actions of these peptides could be explained by the occupation of VIP or secretin receptors. To investigate this further, 125I-labeled helodermin was prepared, and binding was saturable, specific, and could be accounted for by the binding to VIP or secretin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term cholinergic desensitization of rat pancreatic secretory response

1987 ◽  
Vol 252 (3) ◽  
pp. G392-G397
Author(s):  
J. Asselin ◽  
L. Larose ◽  
J. Morisset
Keyword(s):  
Dose Response ◽  
Recovery Period ◽  
Ionophore A23187 ◽  
Muscarinic Agonists ◽  
Short Term ◽  
Response Curves ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Cholinergic Agent ◽  
The Right

Dispersed pancreatic acini were first exposed to carbamylcholine (10(-7)-10(-4) M) for 60 min, washed, and reexposed to this same agonist (10(-8)-10(-3) M) for 15 min. During this second incubation, the functional secretory capacity of these acini was evaluated by measuring amylase release. Acini preexposed to concentrations of carbamylcholine of 10(-6) M or greater showed shifts to the right in the subsequent carbamylcholine dose-response curves of amylase release. A 3-h recovery period (without carbamylcholine) did not restore the altered carbamylcholine dose-response curve. Ca2+ concentrations of 10(-7) M or 2.5 X 10(-3) M instead of 0.5 X 10(-3) M during the 60-min preincubation did not affect the desensitization process. With use of N-[3H]methylscopolamine to evaluate muscarinic receptors, the only changes observed after desensitization were a significant decrease in the high-affinity and an equivalent increase in that of the low-affinity receptors. After cholinergic exposure amylase release stimulated by caerulein was only slightly modified, whereas amylase release in response to a phorbol ester 12-O-tetradecanoylphorbol-13-acetate and to the ionophore A23187 was not altered. These data indicate that short-term desensitization with a cholinergic agent is relatively specific to muscarinic agonists, causes changes in the muscarinic receptor high- and low-affinity concentration but does not alter intracellular steps after calcium mobilization or protein kinase C activation known to be involved in the secretion process.

Effect of extracellular calcium on amylase release from dispersed pancreatic acini.

1979 ◽  
Vol 236 (6) ◽  
pp. E754
Author(s):  
J D Gardner ◽  
C L Costenbader ◽  
E R Uhlemann
Keyword(s):  
Guinea Pig ◽  
Vasoactive Intestinal Peptide ◽  
Cyclic Nucleotides ◽  
Extracellular Calcium ◽  
Enzyme Release ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Basal Rate ◽  
Derivatives Of ◽  
Stimulation Of

In dispersed acini prepared from guinea pig pancreas, removing extracellular calcium did not alter the basal rate of amylase release but reduced the stimulation of enzyme release caused by cholecystokinin, carbachol, secretin, and vasoactive intestinal peptide as well as that caused by derivatives of cyclic nucleotides. In acini incubated in a calcium-free, EGTA-containing medium the increase in amylase release caused by each secretagogue tested did not change during the initial 10 min of incubation, decreased by approximately 65% during the subsequent 40 min, and remained constant thereafter. Removing extracellular calcium did not alter the maximally effective concentrations of cholecystokinin or vasoactive intestinal peptide but abolished the decrease in stimulated enzyme secretion seen with supramaximal concentrations of cholecystokinin. Incubating pancreatic acini with cholecystokinin or carbachol plus secretin or vasoactive intestinal peptide caused potentiation of amylase release, and removing extracellular calcium reduced the stimulation of enzyme release caused by the two secretagogues in combination but did not alter their potentiating interactions.

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