Effects of phorbol ester treatment on dibutyryl cyclic adenosine-5 ' monophosphate- and carbachol-stimulated aminopyrine accumulation in isolated ratparietal cells

Background: The functional role of the intracellular diacylglycerol/protein kinase C second-messenger pathway in the regulation of gastric acid secret ion and the effects on the involved inositotrisphosphate/ Ca2+/calmodulin s ystem are not well understood, and contradictory data have been reported. W e therefore evaluated the effects of phorbol ester treatment (tetradecanoyl phorbol-12,13-acetate (TPA)) on dibutyryl cyclic adenosine-5' monophosphate (dBcARIP)- and carbachol-stimulated aminopyrine (AP) accumulation in compa rison with intracellular alterations of the phospholipase C/inostol phospha te signal transduction pathway in isolated rat gastric parietal cells. Meth ods: [C-14]AP accumulation was determined as an indirect measure of gastric acid secretion. Inositolphosphate second-messenger activation was investig ated with [H-3]inositolmonophosphate release in [H-3]-myoinositol prelabele d rat gastric parietal cells. Results: TPA at a low concentration of 5 nM c aused a small (45%) but significant increase in carbachol (0.1 mM)-stimulat ed AP accumulation, which was dose-dependently inhibited by higher concentr ations of TPA with corresponding shifts in the dose-response curve for carb achol-stimulated AP accumulation. AP uptake stimulated by dBcAMP (0.1 mM) a nd the synergistic stimulatory effect induced by carbachol together with dB cAMP were inhibited by TPA at all concentrations investigated. In the prese nce of increasing concentrations of the calcium ionophore ionomycin (10(-8) -10(-5)M) TPA at 5 nM increased AP accumulation (AP ratio was 4.02 with 5 n M TPA versus 1.23 in the absence of TPA; P < 0.05), indicating that phorbol ester stimulates AP uptake in rat parietal cells. Simultaneous investigati on of [C-14]AP accumulation and [H-3]inositol monophosphate release showed that inhibitory effects of TPA on carbachol- and carbachol plus dBcAMP-stim ulated cells are mediated by an inhibition of the receptor/G-protein/phosph olipase C interaction, leading to a reduction of inositolphosphate release. The costimulation of rat parietal cells with dBcAMP, ionomycin, and TPA (5 nhl) did not reproduce the synergistic effects of carbachol together with dBcAMP on AP accumulation, suggesting that carbachol-stimulated AP uptake s eems to be additionally mediated by a still unknown pathway independent of intracellular calcium release or protein kinase C activation.