Stimulatory Pathways of the Calcium-Sensing Receptor on Acid Secretion in Freshly Isolated Human Gastric Glands
Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca<sup>2+</sup>-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd<sup>3+</sup> leads to activation of the H<sup>+</sup>/K<sup>+</sup>-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H<sup>+</sup>/K<sup>+</sup>-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G<sub>i</sub> and G<sub>o</sub>) with pertussis toxin during stimulation of the CaSR with Gd<sup>3+</sup> only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H<sup>+</sup>/K<sup>+</sup>-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca<sup>2+</sup>, showed that intracellular Ca<sup>2+</sup> plays an important role in the signalling cascade. TMB-8, a ER Ca<sup>2+</sup>store release inhibitor, prevented the stimulation of H<sup>+</sup>/K<sup>+</sup>-ATPase activity. Also verapamil, an inhibitor of L-type Ca<sup>2+</sup>-channels reduced stimulation suggesting that both the release of intracellular Ca<sup>2+</sup> from the ER as well as Ca<sup>2+</sup> influx into the cell are involved in CaSR-mediated H<sup>+</sup>/K<sup>+</sup>-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Gö 6976 which selectively inhibits Ca<sup>2+</sup>-dependent PKC<sub>α</sub> and PKC<sub>βI</sub>-isozymes completely abolished the stimulatory effect of Gd<sup>3+</sup>. In contrast, Ro 31-8220, a selective inhibitor of the Ca<sup>2+</sup>-independent PKCε and PKC-δ isoforms reduced the stimulatory effect of Gd<sup>3+</sup> only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H<sup>+</sup>/K<sup>+</sup>-ATPase activity which was only about 60 % of the effect observed with Gd<sup>3+</sup>. Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd<sup>3+</sup> effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca<sup>2+</sup> dependent and Ca<sup>2+</sup>-independent PKC isoforms are part of the down-stream signalling cascade.