Abstract
Dibutyryl cyclic GMP (Bu2cGMP) inhibited agonist-induced secretion of amylase from isolated rat pancreatic acini. In contrast to previous studies, this inhibitory action was not confined to butyryl derivatives of cyclic GMP, since the membrane-permeant cyclic GMP analogues Bu2cGMP and cyclic 8-bromo-GMP (8-Br-cGMP) were equipotent (IC50 2 nM) in their inhibition of amylase secretion stimulated by cholecystokinin-(26-33)-octapeptide (CCK8): at extracellular concentrations up to 1 mM, cyclic GMP itself was devoid of inhibitory activity. Both Bu2cGMP and 8-Br-cGMP also potently inhibited secretion stimulated by 4 beta-phorbol 12-myristate 13-acetate (PMA) (IC50 6 nM), but only partially inhibited responses elicited by bethanechol or bombesin and were without effect on A23187-evoked secretion. Furthermore, agents that are known to raise intracellular cyclic GMP levels (MB22948 (2-o-propoxyphenyl-8-azapurin-6-one) or nitroprusside) or antagonize the actions of protein kinase C (4 alpha-PMA or staurosporine), also inhibited CCK8- or PMA-stimulated secretion but not secretion elicited by bombesin, bethanechol, or A23187. It is concluded from these and other observations reported here that protein kinase C is the major intracellular mediator of amylase secretion stimulated by CCK8 and that this pathway may be regulated by cyclic GMP at a step that follows protein kinase C activation.
Highlights
Dibutyryl cyclic GMP (Bu,cGMP) inhibited agonist- is generally accepted
Previous studies on pancreatic acini have indicated that neither cyclic GMP, nor its membrane-permeant analogues, when added exogenously at concentrations which fall within the intracellular cyclic GMP range (60-80 nM in unstimulated areknown to raise intracellular cyclic GMP levels (MB22948 (2-o-propoxyphenyl-8-azapurin-6-oneo)r cells, 1.2-1.6 PM upon stimulation), affect basal or agoniststimulated secretion (2,14-16)
The inhibition was ascribed to antagonism concluded from these and other observations reportedat theCCK receptor since BuzcGMPinhibited T - C C K bindhere that protein kinase C is the major intracellular ing, membrane depolarization, and Ca2+influx due to CCK, mediator of amylase secretion stimulatbeyd CCKs and these effects being reversed by washing the cells to remove that this pathway may be regulated bycyclic GMP at BuzcGMP.In these earlier studies other membrane-permeant a step thatfollows protein kinase C activation
Summary
Tate 13-acetate; BSA, bovine serum albumin; HEPES, N-P-hydroxyethylpiperazine-N’-2-ethanesulfonicacid; Me2S0, dimethyl sulfox-. Materials ide; MB22948, 2-O-propoxyphenyl-8-azapurin-6-onper;otein kinase Cholecystokinin-(26-33)-octapeptideamide (sulfated) was pur-. Cyclic GMP Inhibition of Protein KinaseC Actions bridgeshire, England; A23187 was purchased from Behring Diagnostics; Carbamoyl-j3-methylcholinechloride (bethanechol)was obtained from Koch Light; PMA, bombesin, 8-Br-cGMP, BuZcGMP, cyclic. GMP, a-amylase, lactate dehydrogenase,bovine serum albumin (fraction V), collagenase (Type XI), basal Eagle'smedium amino acid supplement, and Me2S0 were purchased from Sigma. 4a-PMA was purchased from Scientific Marketing Associates, London, England; staurosporine was obtained from Dr T. MB22948 was a generous gift from May & Baker Ltd., Dagenham, Essex, and quin 2 AMwas a generous gift from Dr G.
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