Different actions of protein kinase C isoforms alpha and epsilon on gastric acid secretion

Abstract

1. The phorbol ester TPA, an activator of protein kinase C (PKC), inhibits cholinergic stimulation of gastric acid secretion but increases basal H(+) secretion. 2. Since these contradictory findings suggest the action of different PKC isozymes we analysed the role of calcium-dependent PKC-alpha, and calcium-independent PKC-epsilon in gastric acid secretion. 3. Inhibition of PKC-alpha by the indolocarbazole Gö 6976 revealed that about 28% of carbachol-induced acid secretion was inhibited by PKC-alpha. In the presence of Gö 6976 approximately 64% of the carbachol-induced signal transduction is mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), and 14% is conveyed by PKC-epsilon as deduced from the inhibition with the bisindolylmaleimide Ro 31-8220. 4. Inhibition of carbachol-induced acid secretion by TPA was accompanied by a decrease in CaMKII activity. 5. The stimulation of basal acid secretion by TPA was biphasic with a peak at a very low concentration (10 pM), resulting in an activation of the calcium-sensor CaMKII. The activation was determined with a phosphospecific polyclonal antibody against active CaMKII. The TPA-induced increase of H(+) secretion was sensitive to the cell-permeable Ca(2+)-chelator BAPTA/AM, Ro 31-8220, and the CaMKII-inhibitor KN-62, but not to Gö 6976. 6. Since TPA induced the translocation of PKC-epsilon but not of PKC-alpha in resting parietal cells, PKC-epsilon seems to be at least responsible for an initial elevation of free intracellular calcium to initiate TPA-induced acid secretion. 7. Our data indicate the different roles of two PKC isoforms: PKC-epsilon activation appears to facilitate cholinergic stimulation of H(+)-secretion likely by increasing intracellular calcium. In contrast, PKC-alpha activation attenuates acid secretion accompanied by a down-regulation of CaMKII activity.

Figure 1

Effects of the PKC inhibitors Gö 6976 and Ro 31-8220 on carbachol-induced acid secretion. Parietal cells were pretreated with Gö 6976 (10 μM) or Ro 31–8220 (10 μM) for 15 min, then with TPA (1 μM) or the CaMKII-inhibitor KN-62 (20 μM, grey bars) or both for 15 min with subsequent stimulation of aminopyrine uptake by carbachol (0.1 mM). Basal aminopyrine-uptake which means in the absence of any secretagogue is represented by the white-filled bar. Results represent means±s.d. from 4–6 experiments. ^*Significantly different from the corresponding control group (+KN-62); ^**significantly different from carbachol alone, P<0.05.

Figure 2

PKC-α and PKC-ε differ in translocation to the parietal cell membrane. Gastric parietal cells were incubated with TPA (1 μM) or carbachol (0.1 mM) or both for 15 min. After subfractionization of parietal cells equal amounts of protein (15 μg) of membrane fraction were subjected to SDS–PAGE and Western-blotting. PKC-α (a) and PKC-ε (b) were immunolabeled with each a specific monoclonal antibody. PKC-ε was translocated to the cell membrane even in the absence of carbachol. PKC-α showed no translocation to the parietal cell membrane in the absence of carbachol. Detection was performed with the ECL system.

Figure 3

Quantitation of relative importance of signal transduction intermediates in acid secretion. Gastric acid secretion via the M₃ muscarinic receptor is illustrated as arrow. The quantity of three different protein kinases (PKC-α, PKC-ε, CaMKII) in carbachol-stimulated H⁺ secretion and in the presence of Gö 6976 (10 μM) is deduced from inhibitor studies presented in Figure 1. About 36% of the signal is transmitted through CaMKII, and about 14% through PKC-ε in carbachol-induced acid secretion. About 22% are mediated by unidentified signalling components (question mark). PKC-α activity suppresses CaMKII activity, and attenuates about 28% of carbachol-stimulated acid secretion (grey section). Thus, if PKC-α activity is inhibited, CaMKII transduces about 64% (36+28%) of total signalling.

Figure 4

The abundance of CaMKII in SA vesicles doubled after carbachol-stimulation whereas the amount of autoactivated CaMKII was strongly increased. (a) The specific CaMKII transphosphorylating activity of SA vesicles was 2 fold increased after carbachol-stimulation compared to the apical membrane of the resting state. Apical membranes of resting or carbachol-stimulated rabbit gastric mucosal cells were incubated under CaMKII phosphorylation conditions to phosphorylate the CaMKII-specific substrate autocamtide-II. (b) A strong increase of autoactivated CaMKII (CaMKII-P) was detected for the postnuclear supernatant as well as for the SA vesicles after carbachol-stimulation compared to each corresponding membrane fraction of the resting state. Autoactivated CaMKII was probed with a phospho-specific, anti-autoactivated CaMKII. The post nuclear supernatant (S₀) represents almost the total of cellular CaMKII activity; (R_S0: postnuclear supernatant of the resting state, TV: tubulovesicles, R_A: apical membrane of the resting state, S_S0: postnuclear supernatant of the carbachol-stimulated state, S_A: SA vesicles).

Figure 5

Effects of Gö 6976 and Ro 31-8220 on the activity of CaMKII in carbachol-stimulated parietal cells. Active CaMKII (CaMKII-P) was detected in membrane fraction with a phospho-specific antibody by immunoblot analysis (each upper panel), and quantified by densitometry (each lower panel). Parietal cells were pretreated with either Gö 6976 (10 μM) (a), or Ro 31-8220 (10 μM) (b), for 15 min. Cells were then incubated with TPA (1 μM) or KN-62 (20 μM) or both for 15 min, and optionally stimulated with carbachol (0.1 mM). Equal amounts of cell membrane proteins (15 μg) per lane were subjected to SDS-gel electrophoresis with subsequent Western blotting. Results represent means±s.d. from three experiments. ^*Significantly different from carbachol alone; ^**significantly different from TPA alone, P<0.05.

Figure 6

Effects of protein kinase inhibitors on TPA-induced acid secretion. Parietal cells were dose-dependently treated with TPA (0–0.1 μM) resulting in a biphasical course of [¹⁴C]-aminopyrine uptake (white-filled points). (a) TPA-stimulated acid secretion was abolished by Ro 31-8220 (10 μM; black triangles) but not by Gö 6976 (10 μM; black points). (b) TPA-induced acid secretion (white-filled points) was abolished by KN-62, a potent, cell-permeable inhibitor of the calcium-sensitive CaMKII at 20 μM (gray-filled points), and the cell-permeable calcium-chelator BAPTA/AM (0.1 mM; black points), respectively. Results are expressed means±s.d. from four experiments.

Figure 7

CaMKII is activated when resting parietal cells are treated with TPA. After treatment of parietal cells with TPA (0–1 μM), activated CaMKII (CaMKII-P) was detected in the membrane by immunoblot analysis (each upper panel). Bands of activated CaMKII were quantified by densitometry (each lower panel). Results are expressed as means±s.d. from 3–5 experiments. Absence of any chemical agent corresponds with basal acid secretion. (a) Resting parietal cells were treated with various concentrations of TPA (0–1 μM), and analysed for active CaMKII (black points). Controls were stimulated with carbachol (0.1 mM; black rectangle). ^*Significantly different from the corresponding control group (absence of any secretagogue or TPA), P<0.05. (b) Parietal cells were incubated with different protein kinase modulators (Gö 6976 (10 μM), KN-62 (20 μM), Ro 31-8220 (10 μM)) for 15 min with subsequent treatment by TPA (1 μM) for 30 min. ^*Significantly different from control (absence of TPA); ^**significantly different from TPA alone; ^***significantly different from TPA+Gö 6976+KN-62, P<0.05. (c) Cells were pretreated with BAPTA/AM (0.1 mM) or KN-62 (20 μM) for 15 min, then incubated with carbachol (0.1 mM) or TPA (1 μM) or both for 30 min. ^*Significantly different from carbachol alone; ^**significantly different from TPA alone, P<0.01.