Gastric cytoprotective Cl − channel activated by prostaglandin E 2 via Ca 2+-dependent cyclic GMP production

Abstract

Prostaglandin E 2 (PGE2) is known to have a cytoproteetive effect against ethanol in gastric parietal ceils (Robert et al., Gut 33: 444-451, 1992), although its mechanism of action is so far unknown. We have recently found that an extracellular application of PGE2 opened a eytoprotective sub-pS C1channel (0.3 pS) in the basolateral membrane of rabbit gastric parietal cells (Sakai et al., J. Physiol. 448: 293-306, 1992; Sakai & Takeguchi, J. Physiol. 461: 201-212, 1993). Here, we investigated the cellular signaling mechanism of the cytoprotective CIchannel activated by PGE2. In the whole-cell patch-clamp recording, the PGE2 (10 lxM)-indueed increase in the C1current was completely abolished when intracellular Ca 2+ was strongly chelated with 5 mM 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA; pCa 8). In contrast, removal of extracellular Ca 2+ did not affect the PGE2-induced opening of the channel. These results suggest that the elevation of [Ca2+]i is necessary for the PGE2-induced effect, and that Ca 2+ is released from intracellular Ca 2+ stores. Indeed, the fluorescent measurement of the intracellular Ca 2+ concentration ([Ca2+]i) of single fura-2 loaded parietal cells showed that PGE2 transiently increased [Ca2+]i, which was firstly found in this study. The peak was observed within 30 sec, and this increase was dependent on intracellular Ca 2+ stores. Low concentration of PGE2 at even 0.01 p.M had a significant effect. On the other hand, an intracellular application of Methylene Blue (10 IxM), a gnanylate cyclase inhibitor, markedly suppressed the PGE2-induced increase in the C1current. Methylene Blue per se did not increase the CIcurrent. In the absence of PGE2, an intracellular application of cyclic GMP (50 lxM) or nitroprusside (30 p.M) which is a soluble guanylate cyclase activator, significantly increased the C1current. The measurement of intracellular cyclic GMP content of isolated cells rich in the parietal cell showed that PGE2 really elevated its level. The maximal effect was observed 1-3 rain after the application of PGE2. A23187 (2 p.M), a Ca 2+ ionophore, also elevated the cyclic GMP level in the cells. In the whole-cell recording, A23187 alone activated the CIcurrent, and this effect was abolished by Methylene Blue, indicating that the elevation of [Ca2+]i does not directly activate the C1channel, and that the elevation of [Ca2+]i followed by the activation of a guanylate cyclase leads to activation of the channel. In the present study of gastric parietal ceils, we have found that PGE2-induced activation of the cytoprotective CIchannel is mediated by Ca2+-cyelic GMP pathway, in contrast to the PGE2-induced inhibition of gastric acid secretion is mediated by cyclic AMP pathway (Soil, J. Clin. Invest. 65: 1222-1229, 1980; Nylander et al., Am. J. Physiol. 250: G607-G616, 1986). PGE2 plays dual important physiological roles in parietal cells with different intracellular signaling mechanisms.