The diacylglycerol kinase inhibitor, R59022, potentiates cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The putative inhibitor of diacylglycerol kinase activity, 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)-ethyl-7-meth yl-5H- thiazolo[3,2-a]pyrimidin-5-one (R59022), markedly potentiated cholecystokinin-C-terminal-octapeptide(CCK-8-)stimulated enzyme secretion from isolated rabbit pancreatic acini. Maximal potentiation occurred when acini were stimulated in the presence of 5-10 microM R59022. Potentiation depended both on the concentration of R59022 and CCK-8. No potentiation was observed when acini were half-maximally stimulated, whereas the secretory response to maximal and supramaximal concentrations of secretagogue was increased by 50-60%. R59022 alone had no effect on basal enzyme secretion and the drug did not potentiate the secretory response to the Ca2+ ionophore A23187 or to the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Moreover, no increase in basal secretion was observed when acini were incubated in the presence of both R59022 and forskolin. These observations strongly suggest that receptor-mediated activation of the inositol phospholipid pathway is required for R59022-induced potentiation. R59022 inhibited the CCK-8-stimulated incorporation of 32Pi into phosphatidic acid dose dependently, without affecting the CCK-8-stimulated hydrolysis of 32P-labelled phosphatidylinositol 4,5-bisphosphate. This is consistent with an inhibitory effect of R59022 on acinar cell diacylglycerol kinase activity. The potentiating effect of R59022 was mimicked by 12-O-tetradecanoylphorbol 13-acetate added simultaneously with CCK-8. Therefore, it is concluded that in the presence of 5-10 microM R59022 the receptor-mediated increase in acinar cell diacylglycerol content is enhanced leading to enhanced activation of protein kinase C and to potentiation of the secretory response. The fact that the secretory response to maximal and supramaximal concentrations of CCK-8 is potentiated by R59022 suggests that at these concentrations of secretagogue the diacylglycerol/protein kinase C branch of the signal-transduction route is rate-limiting.     

Stimulus-dependent inhibition of platelet aggregation by the protein kinase C inhibitors polymyxin B, H-7 and staurosporine

Thrombin, 1-oleoyl-2-acetyl-rac-glycerol (OAG), cis- or trans-octadecadienoic acids (linoleic and linolelaidic acid) and the synergistic combination of octadecadienoic acids plus OAG lead to the activation of gel-filtered human platelets, i.e. aggregation via protein kinase C (PKC). Platelet activation by thrombin was only slightly suppressed by polymyxin B, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or staurosporine, all being potent inhibitors of PKC in vitro. The OAG-induced aggregation, however, was strongly inhibited by H-7 or staurosporine but not by polymyxin B. In contrast, octadecadienoic acid-induced aggregation was substantially inhibited only by polymyxin B. Synergistic activation by OAG plus octadecadienoic acids was strongly suppressed by all three PKC inhibitors. Our results indicate (1) that the ability of various compounds to inhibit PKC in vitro does not correlate with their inhibitory effects in intact cells and (2) that platelet activation induced by various PKC activators exhibits differential PKC-inhibitor sensitivity.     

1,2-Diacylglycerol, protein kinase C, and pancreatic enzyme secretion

To determine the role of 1,2-diacylglycerol (1,2-DAG) and protein kinase C in pancreatic enzyme secretion, we measured the effect of various pancreatic secretagogues on the cellular mass of 1,2-DAG and amylase release in dispersed pancreatic acini from the guinea pig. In addition, we measured the effect of a recently described protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, 5036-5041), on secretagogue-stimulated amylase release from the acini. Cholecystokinin-octapeptide (CCK-OP), cholecystokinintetrapeptide, and carbachol each increased 1,2-DAG 2-3-fold but the increases occurred only with concentrations of these secretagogues that were supramaximal for amylase release and that had an inhibitory effect on stimulated amylase release. Supramaximal concentrations of bombesin stimulated only a small increase in 1,2-DAG and did not cause inhibition of stimulated amylase release. When the action of carbachol was terminated with atropine or CCK-OP with dibutyryl cyclic GMP, stimulated amylase release ceased immediately but cellular 1,2-DAG required at least 15 min to return to the basal level. Increasing cytosolic free Ca2+ with the Ca2+ ionophore, A23187, in Ca2+-containing incubation media augmented amylase release stimulated by 4 beta-phorbol 12-myristate 13-acetate but inhibited amylase release stimulated by CCK-OP, carbachol, and bombesin without decreasing the cellular content of 1,2-DAG. H-7 inhibited protein kinase C activity in a pancreatic homogenate but augmented amylase release from acini stimulated by either CCK-OP, carbachol, or 4 beta-phorbol 12-myristate 13-acetate. These findings indicate that 1,2-DAG and protein kinase C do not have a stimulatory role in pancreatic stimulus-secretion coupling but may have an inhibitory one.     

Cyclic GMP does not inhibit protein kinase C-mediated enzyme secretion in rat pancreatic acini

In pancreatic acini, cGMP can be increased by secretagogues such as cholecystokinin (CCK), cholinergic agents, and bombesin, whose actions on enzyme secretion are believed to be mediated by protein kinase C. However, the role of cGMP in acinar cell function has been unclear. A recent paper by Rogers et al. (Rogers, J., Hughes, R.G., and Matthews, E. K. (1988) J. Biol. Chem. 263, 3713-3719) reported that two analogues of cGMP, N2,O2-dibutyl guanosine 3':5'-monophosphate (Bt2cGMP) and 8-bromoguanosine 3':5'-monophosphate (8Br-cGMP), at concentrations in the nanomolar range, inhibited the stimulation of amylase secretion caused by CCK-8, bethanechol, bombesin, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Rogers et al. also reported that sodium nitroprusside inhibited the stimulation of enzyme secretion caused by CCK-8 or TPA. These authors concluded that cGMP inhibits protein kinase C-mediated secretion in pancreatic acini. In the present study we attempted to confirm the findings of Rogers et al., We found, however, that Bt2cGMP inhibited CCK-8-stimulated amylase release only at concentrations of the nucleotide above 10 microM. Moreover, there was a close correlation between the ability of Bt2cGMP to inhibit CCK-8-stimulated amylase release and its ability to inhibit binding of 125I-CCK-8. Bt2cGMP, at concentrations as high as 3 mM, did not alter the stimulation of amylase release caused by carbachol, bombesin, TPA, or A23187. 8Br-cGMP, at concentrations up to 1 mM, did not inhibit the stimulation of amylase release caused by CCK-8 or TPA. At concentrations above 0.1 mM, 8Br-cGMP augmented the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Sodium nitroprusside, at a concentration that causes a 60-fold increase in cGMP, did not inhibit the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Our results do not confirm the findings of Rogers et al. and indicate that cGMP does not inhibit protein kinase C-mediated secretion in pancreatic acini.     

Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in rabbit pancreatic acini.

Treatment of rabbit pancreatic acini with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), resulted in a time- and dose-dependent decrease of soluble protein kinase C activity coinciding with an increase of protein kinase C activity in the particulate fraction. After 5 min, soluble protein kinase C activity had decreased to almost 10% of the corresponding control. Total extractable protein kinase C activity, however, remained unchanged, indicating that the decrease of soluble protein kinase C activity was not due to TPA-induced inactivation of the enzyme. The biologically inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not induce such a translocation of protein kinase C. The half-maximal concentration for TPA-induced translocation of protein kinase C was 40 nM, and was equal to that for TPA-induced amylase secretion from isolated acini. This suggests that translocation of protein kinase C to the particulate fraction is an important step in TPA-induced activation of protein kinase C and enzyme secretion. On the other hand, cholecystokinin, a secretagogue of the calcium-mobilizing type, whose secretory action is thought to be mediated, at least in part, by protein kinase C, did not change the subcellular distribution of protein kinase C. In the presence of R59022 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl ) ethyl-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one, an inhibitor of diacylglycerol kinase activity, cholecystokinin produced a small but significant translocation of protein kinase C, suggesting that the inability of the hormone to induce translocation is not due to a rapid conversion of the diacylglycerol formed into phosphatidic acid.     

Cholecystokinin-induced residual stimulation of enzyme secretion from mouse pancreatic acini

When dispersed acini from mouse pancreas are first incubated with cholecystokinin octapeptide, washed and then reincubated with no additions there is significant stimulation of amylase secretion during the second incubation (residual stimulation of enzyme secretion). Cholecystokinin-induced residual stimulation of enzyme secretion is modified, but not abolished, by reducing the temperature of the first incubation from 37°C to 4°C. Measurement of binding of ¹²⁵I-labeled cholecystokinin octapeptide indicated that maximal cholecystokinin induced residual stimulation of enzyme secretion occurs when 12–20% of cholecystokinin receptors are occupied by cholecystokinin octapeptide. Moreover, maximal cholecystokinin-induced residual stimulation of amylase secretion is 25% greater than maximal cholecystokinin-induced direct stimulation of amylase secretion. Cholecystokinin tetrapeptide, which causes the same maximal direct stimulation of amylase secretion as does cholecystokinin octapeptide, causes a maximal residual stimulation of enzyme secretion that is only 30% of that caused by a maximally effective concentration of cholecystokinin octapeptide. Adding dibutyryl cyclic GMP to the second incubation can reverse the residual stimulation caused by adding cholecystokinin to the first incubation. The pattern and extent of the dibutyryl cyclic GMP-induced reversal of residual stimulation varies, depending on the temperature and concentration of cholecystokinin octapeptide in the first incubation. The present results are compatible with the hypothesis that mouse pancreatic acini possess two classes of cholecystokinin receptors. One class has a relatively high affinity for cholecystokinin and produces stimulation of enzyme secretion; the other class has a relatively low affinity for cholecystokinin and produces inhibition of enzyme secretion.     

Cholecystokinin-induced residual stimulation of enzyme secretion from mouse pancreatic acini

When dispersed acini from mouse pancreas are first incubated with cholecystokinin octapeptide, washed and then reincubated with no additions there is significant stimulation of amylase secretion during the second incubation (residual stimulation of enzyme secretion). Cholecystokinin-induced residual stimulation of enzyme secretion is modified, but not abolished, by reducing the temperature of the first incubation from 37 degrees C to 4 degrees C. Measurement of binding of 125I-labeled cholecystokinin octapeptide indicated that maximal cholecystokinin induced residual stimulation of enzyme secretion occurs when 12-20% of cholecystokinin receptors are occupied by cholecystokinin octapeptide. Moreover, maximal cholecystokinin-induced residual stimulation of amylase secretion is 25% greater than maximal cholecystokinin-induced direct stimulation of amylase secretion. Cholecystokinin tetrapeptide, which causes the same maximal direct stimulation of amylase secretion as does cholecystokinin octapeptide, causes a maximal residual stimulation of enzyme secretion that is only 30% of that caused by a maximally effective concentration of cholecystokinin octapeptide. Adding dibutyryl cyclic GMP to the second incubation can reverse the residual stimulation caused by adding cholecystokinin to the first incubation. The pattern and extent of the dibutyryl cyclic GMP-induced reversal of residual stimulation varies, depending on the temperature and concentration of cholecystokinin octapeptide in the first incubation. The present results are compatible with the hypothesis that mouse pancreatic acini possess two classes of cholecystokinin receptors. One class has a relatively high affinity for cholecystokinin and produces stimulation of enzyme secretion; the other class has a relatively low affinity for cholecystokinin and produces inhibition of enzyme secretion.     

The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation

The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) were examined for their ability to inhibit human neutrophil activation. At concentrations up to 100 micromolar, these compounds failed to inhibit either respiratory burst or the secretory response of neutrophils stimulated with particulate (serum-opsonized zymosan) or soluble (A23187, FMLP, PMA) stimuli. In contrast, the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) inhibited both oxygen radical generation and lysosomal enzyme release in response to the same stimuli. These results suggest that calmodulin-dependent enzymes, rather than protein kinase C, may be essential for neutrophil activation.     

Parallel effects of arachidonic acid on insulin secretion, calmodulin-dependent protein kinase activity and protein kinase C activity in pancreatic islets.

A potential role of arachidonic acid in the modulation of insulin secretion was investigated by measuring its effects on calmodulin-dependent protein kinase and protein kinase C in islet subcellular fractions. The results were interpreted in the light of arachidonic acid effects on insulin secretion from intact islets. Arachidonic acid could replace phosphatidylserine in activation of cytosolic protein kinase C (K0.5 of 10 microM) and maximum activation was observed at 50 microM arachidonate. Arachidonic acid did not affect the Ca2+ requirement of the phosphatidylserine-stimulated activity. Arachidonic acid (200 microM) inhibited (greater than 90%) calmodulin-dependent protein kinase activity (K0.5 = 50-100 microM) but modestly increased basal phosphorylation activity (no added calcium or calmodulin). Arachidonic acid inhibited glucose-sensitive insulin secretion from islets (K0.5 = 24 microM) measured in static secretion assays. Maximum inhibition (approximately 70%) was achieved at 50-100 microM arachidonic acid. Basal insulin secretion (3 mM glucose) was modestly stimulated by 100 microM arachidonic acid but in a non-saturable manner. In perifusion secretion studies, arachidonic acid (20 microM) had no effect on the first phase of glucose-induced secretion but nearly completely suppressed second phase secretion. At basal glucose (4 mM), arachidonic acid induced a modest but reproducible biphasic insulin secretion response which mimicked glucose-sensitive secretion. However, phosphorylation of an 80 kD protein substrate of protein kinase C was not increased when intact islets were incubated with arachidonic acid, suggesting that the small increases in insulin secretion seen with arachidonic acid were not mediated by protein kinase C. These data suggest that arachidonic acid generated by exposure of islets to glucose may influence insulin secretion by inhibiting the activity of calmodulin-dependent protein kinase but probably has little effect on protein kinase C activity.     

Thapsigargin defines the roles of cellular calcium in secretagogue-stimulated enzyme secretion from pancreatic acini.

In the present study we used thapsigargin (TG), an inhibitor of microsomal calcium ATPase, to evaluate the roles of free cytoplasmic calcium and intracellular stored calcium in secretagogue-stimulated enzyme secretion from rat pancreatic acini. Using microspectrofluorimetry of fura-2-loaded pancreatic acini, we found that TG caused a sustained increase in free cytoplasmic calcium by mobilizing calcium from inositol 1,4,5-trisphosphate-sensitive intracellular stores and by increasing influx of extracellular calcium. TG also caused a small increase in basal amylase secretion, inhibited the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate, and potentiated the stimulation of amylase secretion caused by 12-O-tetradecanoylphorbol-13-acetate or secretagogues that increase cyclic adenosine 3',5'-monophosphate. Bombesin, which like TG increased free cytoplasmic calcium, also potentiated the stimulation of amylase secretion caused by secretagogues that increase cyclic adenosine 3',5'-monophosphate, but did not inhibit the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate. Finally, TG inhibited the sustained phase of cholecystokinin-stimulated amylase secretion and potentiated the time course of vasoactive intestinal peptide-stimulated amylase secretion. The present findings indicate that stimulation of amylase secretion by secretagogues that increase inositol 1,4,5-trisphosphate does not depend on increased free cytoplasmic calcium per se. In contrast, TG-induced potentiation of the stimulation of secretagogues that increase cellular cyclic adenosine 3',5'-monophosphate appears to result from increased free cytoplasmic calcium per se.     

Specific inhibition of phorbol ester and DiC8-induced histamine release from human basophils by the protein kinase C inhibitors, H-7 and H-9

The release of histamine and other inflammatory mediators from human basophils is triggered by numerous stimuli, including chemical, physical and receptor-mediated activators. Several mechanisms of cell activation including protein kinase C activation have been proposed to operate in these cells. We used phorbol ester and DiC8 to induce histamine release from human basophils and the protein kinase C inhibitors H-7 and H-9 to inhibit this release. Both DiC8 and TPA induced histamine release were inhibited by H-7 (ID 50 = 37 mcM) and H-9 (IC 50 = 20 mcM). However, anti-IgE, fmlp and A23187-induced histamine release were unaffected. In contrast, the calmodulin antagonists W-7 and perphenazine effectively inhibited histamine release by all five stimuli. Therefore, different biochemical pathways appear to be critical for basophil activation depending on the nature of the stimulus used.     

Dissimilar effects of transglutaminase inhibitors on peptide hormone-indiced secretion from testis and pituitary

Inhibitors of transglutaminase (monodansylcadaverine and bacitracin) reduced luteinizing hormone stimulated androgen and adenosine - 3′: 5′ - monophosphate production by testicular tissue but had no inhibitory effect on gonadotropin releasing hormone-stimulated luteinizing hormone secretion by the pituitary. These results indicate that there are differences in the mechanisms by which these polypeptide hormones stimulate hormone secretion and suggest a role for protein cross-linking in the mechanism of luteinizing hormone action in the testis.     

Exendin-4, a new peptide from Heloderma suspectum venom, potentiates cholecystokinin-induced amylase release from rat pancreatic acini.

We examined the actions of exendin-4, a new peptide isolated from Heloderma suspectum venom, on dispersed acini from rat pancreas. Exendin-4 caused a 3-fold increase in cAMP but did not alter cellular calcium concentration. Exendin-4-induced increases in cAMP were inhibited by an exendin-receptor antagonist, exendin (9-39)NH2, but not by VIP-receptor antagonists. Whereas up to 1 microM exendin-4 alone did not alter amylase release, potentiation of enzyme release was observed when the peptide (greater than 30 pM) was combined with cholecystokinin. Potentiation of amylase release was also observed when exendin-4 was combined with carbamylcholine, bombesin or a calcium ionophore, A23187. These results indicate that stimulation of exendin receptors on rat pancreatic acini causes an increase in cellular cAMP. Although this increase in cAMP alone does not result in amylase release, combination of exendin-4 with agents that increase cell calcium results in potentiation of amylase release.     

Redistribution of protein kinase C isoforms in rat pancreatic acini during lactation and weaning

Freshly enzymatically isolated pancreatic acini from lactating and weaning Wistar rats were used to investigate the role of protein kinase C (PKC) isoforms during these physiologically relevant pancreatic secretory and growth processes. The combination of immunoblot and immunohistochemical analysis shows that the PKC isoforms alpha, delta, and epsilon are present in pancreatic acini from control, lactating and weaning rats. A vesicular distribution of PKC-alpha, -delta, and -epsilon was detected by immunohistochemical analysis in the pancreatic acini from all the experimental groups. PKC-delta showed the strongest PKC immunoreactivity (PKC-IR). In this vesicular distribution, PKC-IR was located at the apical region of the acinar cells. No differences were observed between control, lactating and weaning rats. However, the immunoblot analysis of pancreatic PKC isoforms during lactation and weaning showed a significant translocation of PKC-delta from the cytosol to the membrane fraction when compared with control animals. Translocation of PKC isoforms (alpha, delta and epsilon) in response to 12-O-tetradecanoyl phorbol 13-acetate (TPA) 1 microM (15 min, 37 degrees C) was comparable in pancreatic acini from control, lactating and weaning rats. In the control group, a significant translocation of all the isoforms (alpha, delta and epsilon) from the cytosol to the membrane was observed. The PKC isoform most translocated by TPA was PKC-delta. In contrast, no statistically significant increase in PKC-delta translocation was detected in pancreatic acini isolated from lactating or weaning rats. These results suggest that the PKC isoforms are already translocated to the surface of the acinar cells from lactating or weaning rats. In addition, they suggest that isoform specific spatial PKC distribution and translocation occur in association with the growth response previously described in the rat exocrine pancreas during lactation and weaning.     

Comparative effects of a highly specific protein kinase C inhibitor, calphostin C and calmodulin inhibitors on angiotensin-stimulated aldosterone secretion

We have examined the relative roles of the calcium-calmodulin system and protein kinase C in angiotensin-mediated aldosterone secretion. We used a highly specific protein kinase C inhibitor, calphostin C and two well-accepted calmodulin inhibitors, W-7 and calmidazolium. Although both types of inhibitors affected angiotensin-induced aldosterone secretion, as judged by the inhibitory doses of these compounds, angiotensin-evoked aldosterone secretion was more sensitive to calmodulin inhibition than protein kinase C inhibition. Manipulation of intracellular calcium by dantrolene and thapsigargin also modified aldosterone secretion significantly.     

A protein kinase C inhibitor, staurosporine, activates phospholipase D via a pertussis toxin-sensitive GTP-binding protein in rabbit peritoneal neutrophils.

In rabbit peritoneal neutrophils prelabeled with [3H] lyso platelet-activating factor, a protein kinase C inhibitor, staurosporine (> 1 microM), increased [3H]phosphatidylethanol ([3H]PEt) level in the presence of ethanol in a concentration- and time-dependent manner, providing evidence for staurosporine activation of phospholipase D (PLD). The staurosporine activation of the enzyme absolutely required both extracellular calcium and cytochalasin B, and was almost completely inhibited by pretreatment of the cells with pertussis toxin (IAP). In a reconstituted system where the purified Gi1 had been incorporated into phospholipid vesicles, staurosporine activated GTPase activity of Gi1 in a concentration-dependent fashion, with a maximal 4-5-fold effect. ADP-ribosylation by IAP of Gi1 in vesicles significantly suppressed the staurosporine activation. As with the GTPase activity of Gi1, GTPase activities of other purified IAP-sensitive G proteins, such as Gi2 and G(o), were significantly stimulated by staurosporine, but the cholera toxin substrate Gs was appreciably less sensitive to the staurosporine stimulation. The staurosporine activation of GTPase was also observed in rabbit neutrophil membranes from control cells, but not in membranes from IAP-treated neutrophils. From these results, we conclude that the staurosporine activation of PLD in rabbit neutrophils is attributed to the direct activation of an IAP-sensitive G protein in a similar manner to receptors occupied by agonists. By contrast, staurosporine failed to activate phosphoinositide-specific phospholipase C (PI-PLC) under the conditions in which it activated PLD, indicating that there exists a PLD activation pathway independent of PI-PLC. Furthermore, it was found that N-acetyl-beta-glucosaminidase release from the granules of intact neutrophils was evoked by staurosporine to almost the same extent as by fMLP (100 nM), but O2- generation was not affected. These results suggest a possibility that PLD pathway plays an important role in enzyme release, but is not sufficient for O2- generation, in rabbit peritoneal neutrophils.     

Inhibitors such as staurosporine,H-7 or polymyxin B cannot be used in skeletal muscle to prove the role of protein kinase C on insulin action

The precise role of protein kinase C in insulin action in skeletal muscle is not well defined. Based on the fact that inhibitors of protein kinase C block some insulin effects, it has been concluded that some of the biological actions of insulin are mediated via protein kinase C. In this study, we present evidence that inhibitors of protein kinase C such as staurosporine, H-7 or polymyxin B cannot be used to ascertain the role of protein kinase C in skeletal muscle. This is based on the following experimental evidences: a) staurosporine, H-7 and polymyxin B markedly block in muscle the effect of insulin on System A transport activity; however, this effect of insulin is not mimicked in muscle by TPA-induced stimulation of protein kinase C, b) H-7 and polymyxin B block insulin action on System A transport activity in an additive manner to the inhibitory effect of phorbol esters, c) staurosporine, H-7 and polymyxin B block the effect of insulin on lactate production, a process that is activated by insulin and TPA in an additive fashion, and d) staurosporine completely blocks the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle.Abbreviations MeAIB a-(methyl)aminoisobutyric acid - TPA 12-O-tetradecanoylphorbol-13-acetate - H-7 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine     

Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.     

The diacylglycerol kinase inhibitor R59022 potentiates superoxide production but not secretion induced by fMet-Leu-Phe: effects of leupeptin and the protein kinase C inhibitor H-7

The addition of low concentrations of the chemotactic factor fMet-Leu-Phe to rabbit neutrophils in the absence of cytochalasin B produces very little superoxide. This level of superoxide can be greatly increased in neutrophils pretreated for 30 min with 10 microM of the diacyl-glycerol kinase inhibitor R59022. This potentiation occurs also in the presence of cytochalasin B. In addition, while the small level of superoxide generated by fMet-Leu-Phe is not inhibited by the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7), the increase by R59022 is completely abolished by this compound. In addition, this increase can be potentiated further by leupeptin. Unlike superoxide generation, the release of lysozyme or N-acetyl-beta-glucosaminidase produced by fMet-Leu-Phe is not stimulated by R59022. The results presented here suggest that stimulation of the oxidative burst requires the generation and the maintenance of a sufficient amount of diacylglycerol and/or the rearrangement of the cytoskeleton such as the inhibition of actin polymerization. Furthermore, the membrane-associated form of protein kinase C is the one responsible for the activation of the oxidative burst. The relationship between protein kinase C activation and the stimulated oxidative burst and the physiological role of chemotactic factors in the functions of the neutrophils are discussed.     

Cholecystokinin receptor occupation and cholecystokinin-induced calcium mobilization in the early phase in rat pancreatic acini.

We examined receptor occupation, calcium mobilization and amylase release for cholecystokinin octapeptide (CCK-8) within a 3-min incubation period at 37 degrees C using dispersed acini from rat pancreas. Analysis of competitive binding inhibition data obtained after a 3-min incubation revealed the presence of only a single class of CCK receptors, while two classes of CCK receptor, i.e., high-affinity and low-affinity CCK receptors, were detected when binding reached a steady-state after a 60-min incubation. The IC50 of CCK receptors calculated from the 3-min binding data was 19.0 +/- 0.5 nM (mean +/- S.D.), close to the Kd of the low-affinity CCK receptors determined by equilibrium binding studies. Exposure of fura-2-loaded acini to 10-1000 pM CCK-8 caused an immediate and dose-dependent increase in [Ca2+]i followed by a gradual decrease in [Ca2+]i. The CCK-stimulated amylase release after 3 min of incubation was biphasic; amylase release increased over the dose range of 3-300 pM CCK-8, peaked at 300 pM CCK-8 and decreased with supramaximal concentrations of CCK-8. Our data suggest that occupation of the low-affinity, but not the high-affinity, CCK receptors is more directly associated with calcium mobilization and subsequent stimulation of amylase release in rat pancreatic acini.