©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Gastric Housekeeping Cl Channel Activated via Prostaglandin EP Receptor-mediated Ca/Nitric Oxide/cGMP Pathway (*)

(Received for publication, March 14, 1995; and in revised form, May 18, 1995)

Hideki Sakai (§) Eiichi Kumano Akira Ikari Noriaki Takeguchi

From theDepartment of Pharmaceutical Physiology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Prostaglandin E(2) (PGE(2)) has a cytoprotective role in the gastric parietal cell. PGE(2) opened a housekeeping basolateral Cl channel of rabbit gastric parietal cells, the single channel conductance of which was about 0.3 picosiemens. In the present patch-clamp and Fura 2 fluorescence studies, we found that PGE(2) increased the intracellular free Ca concentration ([Ca]) and that PGE(2)-induced opening of the Cl channel depended on the increase in [Ca]. A novel bifunctional prostaglandin EP(3) agonist/EP(1) antagonist, 5(Z)-7-[(1S, 2S, 3S, 5R)-3-(trans-beta-styren)sulfonamido-6,6-dimethylbicyclo(3.1.1)hept-2-yl]-5-heptenoic acid, also increased both [Ca] and channel opening. The PGE(2)-induced effect was mediated via production of nitric oxide (NO); that is, N^G-monomethyl-L-arginine, an inhibitor of NO production, markedly inhibited the PGE(2)-induced channel opening, and nitroprusside, a NO donor, induced the channel opening in the absence of PGE(2). Both PGE(2) and A23187, a Ca ionophore, elevated the cGMP content of isolated parietal cells. The A23187-induced channel opening was abolished by methylene blue, a guanylate cyclase inhibitor. In conclusion, we found that the PGE(2)-induced opening of the housekeeping Cl channel in the parietal cell involves the EP(3) receptor-mediated increase in [Ca] via a pertussis toxin-sensitive GTP-binding protein, resulting in successive production of NO and cGMP.


INTRODUCTION

Cytoprotection is one of the newly found functions of the NO/cGMP (^1)pathway; however, targets of this pathway are unknown(1, 2) . In stomach, PGE(2) shows morphological and functional cytoprotection against ethanol in deep regions of gastric glands, particularly in parietal cells(3, 4, 5) , but its molecular mechanism and the targets are unknown.

We have recently found a housekeeping Cl channel in the basolateral membrane of rabbit parietal cells(6, 7, 8, 9) . This Cl channel with small single channel conductance of 0.3-0.4 picosiemens is present abundantly in the basolateral membrane (6) and is a major determinant of the cell membrane potential(7) . Although K channels are present, they do not significantly contribute the membrane potential in rabbit parietal cells(7) . The opening of the Cl channel was stimulated by PGE(2)(6) and inhibited by intracellular superoxide production, which was coupled to a PTX-insensitive GTP-binding protein (8, 9) .

In this paper, we studied why PGE(2) can stimulate the opening of the housekeeping Cl channel. We tested whether PGE(2) increased [Ca], because PGE(2)-induced opening of the channel was inhibited by the intracellular presence of a strong Ca chelator, and it was unknown whether PGE(2) increased the [Ca] of gastric parietal cells. We found that PGE(2) mobilized the Ca/NO/cGMP pathway, resulting in the opening of the Cl channel in the basolateral membrane. Therefore, the Cl channel is the target of the cytoprotective NO/cGMP pathway in the parietal cell.


EXPERIMENTAL PROCEDURES

Preparation of Gastric Glands

Gastric glands were prepared by collagenase digestion from male Japanese white rabbits (weighing 1-3 kg)(10) . Isolated glands were further treated with 500 tyrosine units/ml of Pronase (Actinase E; Kaken Pharmaceutical Co., Tokyo, Japan) at 25-27 °C for 5 min(6, 7, 8, 9) . As a result of the additional digestion, parietal cells protruded from the base of glands and had no leaky connection between the intracellular canaliculi and the lumen (6) . The glands were suspended in a respiratory medium containing 132.4 mM NaCl, 5.4 mM KCl, 5 mM Na(2)HPO(4), 1 mM NaH(2)PO(4), 1.2 mM MgSO(4), 1 mM CaCl(2), 2 mg/ml bovine serum albumin, 2 mg/ml glucose, and 10 mM HEPES (pH 7.35).

Preparation of the Parietal Cell-rich Suspension

Isolated rabbit gastric glands prepared as above were treated with 4,000 units/ml Actinase E at 35-37 °C for 50 min. Then parietal cells in the suspension were separated using a continuous Percoll gradient as described elsewhere(11) . The gradient was formed by centrifuging a mixture of Percoll, the respiratory medium (pH 7.0), and 1.5 M NaCl (45:50:5) (v/v/v) at 23,000 g for 50 min. The fraction of parietal cells was further purified with a Beckman J2-21 M elutriator centrifuge(12) . It consisted of 88 ± 1% parietal cells (mean ± S.E., 10 animals).

Patch Clamp Analysis

Whole-cell currents recorded from rabbit parietal cells in the isolated gastric glands were previously established to be due to the current through the basolateral membrane (6, 8) . An EPC-7 patch clamp system (List Electronic, Darmstadt, Germany) was used for whole-cell recordings, and necessary corrections were made as described elsewhere(6, 13) . The following extracellular bathing and intracellular pipette solutions were used(7) . The 133K-142Cl bathing solution contained (in mM): 133 KCl, 7 NaCl, 1 MgCl(2), 1 CaSO(4), 0.1 ouabain, and 10 HEPES. In the Ca-free 133K-142Cl solution, CaSO(4) was omitted, and 0.1 mM EGTA was added. The 140choline-146Cl bathing solution contained (in mM): 140 choline chloride, 2 MgCl(2), 1 CaCl(2), 0.1 ouabain, and 10 HEPES. All bathing solutions were adjusted to pH 7.3 with KOH or Tris. The 133K-13Cl pipette solution contained (in mM): 133 potassium aspartate, 7 NaCl, 3 MgCl(2), 0.062 CaSO(4), 0.1 EGTA, 2 ATP (Oriental Yeast Co., Tokyo, Japan), and 10 HEPES (pCa 7, pH 7.3). The 140choline-146Cl pipette solution contained (in mM): 140 choline chloride, 3 MgCl(2), 0.062 CaSO(4), 0.1 EGTA, 2 ATP, and 10 HEPES (pCa 7, pH 7.3). The 133K-13Cl (pCa 8) pipette solution contained (in mM): 133 potassium aspartate, 7 NaCl, 3 MgCl(2), 0.467 CaSO(4), 5 BAPTA, 2 ATP, and 10 HEPES (pCa 8, pH 7.3). After whole-cell configuration was achieved, the seal between the cell and the patch electrode and the cell activity were checked by measuring the cell resistance and the membrane potential, respectively. This procedures took 10-30 s before the start of the current recording. Current-voltage relations were obtained with the 140choline-146Cl bathing and 140choline-146Cl pipette solutions(6, 7, 8) . Whole-cell Cl currents were recorded continuously with the 133K-142Cl bathing and 133K-13Cl pipette solutions at a holding potential of 0 mV, the zero-current potential for K and non-selective cation channels. Effects of chemicals on the whole-cell Cl current were assessed 6 min after the start of the recording or the addition of chemicals to the bath and shown as a percentage of the Cl current (I) immediately after the recording: 100 (I/I)(8, 9) . Experiments were performed at 35-37 °C.

Measurement of [Ca] in Single Parietal Cells

Isolated gastric glands were suspended in the dye-loading buffer (40-50 mg wet wt/ml). The buffer contained (in mM) 100 NaCl, 5.4 KCl, 1.2 MgCl(2), 1 CaCl(2), 20 HEPES, 10 pyruvate, 10 glutamate, 10 fumalate, and 10 ascorbate (pH 7.35). Then Fura 2/pentaacetoxymethyl ester (5 µM) with a detergent Pluronic F127 (0.025%, w/v) was added to the suspension and incubated for 40 min at 22 °C. After loading, the glands were washed and resuspended in the ice-cold respiratory medium. Then they were warmed at 35 °C in the bovine serum albumin-free appropriate solution before the measurement. Fura 2-loaded single parietal cells in gastric glands were observed under an inverted microscope (Nikon TMD-EFQ). The total fluorescence intensity from one single parietal cell was monitored at excitation wavelengths of 340 and 380 nm with an emission wavelength of 505 nm (interference filter) using a photon-counting technique (Spex Fluorolog-2 spectrofluorometer, Edison, NJ). After corrections for background fluorescence, the intensity ratio (340/380 nm) and [Ca] were calculated as described previously (14, 15) .

Measurement of Intracellular cGMP Content ([cGMP] ) of Parietal Cells

Isolated cells rich in parietal cells were suspended in respiratory medium or 133K-142Cl bathing solution (1 10^6 cells/ml), and preincubated for 10 min at 35 °C. 1, 3, and 5 min after the addition of PGE(2) or A23187, trichloroacetic acid (6%, w/v) was added to the cell suspension. As a control (0 min), trichloroacetic acid was added to the suspension before the addition of PGE(2) or A23187. The reaction mixtures were kept on ice for 10 min and were centrifuged at 8500 g for 10 min (at 4 °C). Then the supernatant was collected and washed three times with 2 ml of diethyl ether and freeze-dried for 12-15 h. The cGMP content of preparations was determined using a cGMP enzyme immunoassay system (Amersham, Buckinghamshire, UK).

Chemicals

PGE(2) (Toray Industries, Tokyo, Japan) and ONO-NT-012 (ONO Pharmaceutical Co., Osaka, Japan) were generous gifts. They were dissolved in ethanol and were diluted with the appropriate solutions just before use. A23187 (Wako Pure Chemical Industries, Osaka, Japan) and NPPB were dissolved in dimethyl sulfoxide and were diluted to final concentrations just before use. Ethanol and dimethyl sulfoxide concentrations in the final solutions never exceeded 0.5%, the concentration at which the vehicles per se did not affect whole-cell Cl currents, [Ca], and [cGMP] of parietal cells. NPPB was synthesized in this laboratory following a method described elsewhere(16) . PTX (List Biological Laboratories, Campbell, CA), L-NMMA (Sigma), cGMP sodium salt (Sigma), methylene blue (Wako), and sodium nitroprusside dihydrate (Wako) were dissolved in the appropriate solutions just before use. Fura 2/pentaacetoxymethyl ester was obtained from Dojindo Laboratories Co. (Kumamoto, Japan), and Pluronic F127 was from Molecular Probes (Eugene, OR).

Statistics

Statistical significance was evaluated by Student's t test or Cochran-Cox test. A p value below 0.05 was considered to be significant. Data are expressed as the means ± S.E. of a number of observations.


RESULTS

Role of Ca on the PGE-induced Activation of the Cl Channel

The activation of the Cl channel was evidenced by an increase in the whole-cell Cl current. PGE(2) (10 µM) increased the whole-cell Cl current (Fig.1), which arose from the opening of one kind of Cl channel (0.3 picosiemens)(6) . This effect was significant; outward and inward Cl currents at ±100 mV increased during the 6 min period from 565 ± 99 to 1120 ± 114 pA and from -598 ± 102 to -1073 ± 91 pA, respectively (p < 0.05, n = 3). PGE(2) did not change linearity of the current-voltage relationship (Fig.1), indicating that the characteristics of the Cl channel observed before and after stimulation by PGE(2) were the same(6) . PGE(2) at concentrations as low as 1 nM and 0.1 µM increased the Cl current during the 6-min period by 33.8 ± 8.8 and 58.7 ± 11.9% (n = 5), respectively.


Figure 1: PGE(2)-induced increase in whole-cell Cl currents recorded from a gastric parietal cell. Relations between whole-cell Cl currents (I) and membrane potential (Vm) of a parietal cell equilibrated with the 140choline-146Cl pipette solution and the 140choline-146Cl bathing solution are shown. Typical relations from three similar experiments are shown. Vm was changed by a step of ± 20 mV from the holding potential (0 mV). Currents were recorded before the extracellular application of 10 µM PGE(2) (bullet) or 3 () or 6 min (up triangle, filled) after and were measured 350-400 ms after application of voltage steps. Inset, corresponding current traces.



Here, we tested the role of Ca on the increase of the Cl current induced by PGE(2) (10 µM). The PGE(2)-induced effect was almost completely inhibited when intracellular Ca was chelated strongly with BAPTA (pCa 8) (Fig.2, B and D) but was not inhibited when weakly buffered with 0.1 mM EGTA (pCa 7) (Fig. 2, A and D). On the other hand, deletion of Ca from the extracellular solution did not affect the PGE(2)-induced increase of the current (Fig.2, C and D). As reported previously(6) , PGE(2)-induced current was blocked by the Cl channel blocker, NPPB (Fig.2, A and C). Here, 500 µM NPPB was used because a high concentration of NPPB (IC = 300 µM) was required to inhibit the activity of this Cl channel(6) . These results suggest that the elevation of [Ca] is necessary for the PGE(2)-induced effect and that Ca is released from intracellular Ca stores.


Figure 2: Role of Ca on the PGE(2)-elicited Cl current. A-C, representative traces of the whole-cell Cl current. The 133K-13Cl pipette solution contained 10M [Ca] (weakly buffered at pCa 7 with 0.1 mM EGTA) (A and C) or 10M [Ca](buffered with 5 mM BAPTA) (B). The 133K-142Cl bathing solution contained 1 mM Ca (A and B) or 0.1 mM EGTA (C). 10 µM PGE(2) was perfused from the time indicated by the arrows. 500 µM NPPB was added as indicated (A and C). D, the effect was assessed 6 min after the addition of PGE(2). Three experimental protocols for PGE(2) (n = 6), (BAPTA)+PGE(2) (n = 5), and (EGTA)+PGE(2) (n = 3) correspond to panels A, B and C, respectively.**, significantly different from the effect of PGE(2) alone (p < 0.01).



Elevation of [Ca] by PGE in Single Parietal Cells

We measured [Ca] in single Fura 2-loaded parietal cells. Corresponding with the results from the whole-cell recording (Fig.2), PGE(2) (10 µM) increased [Ca] in gastric parietal cells (Fig.3A), and the effect was independent of extracellular Ca (Fig.3B). The increase in [Ca] was induced immediately after the addition of PGE(2), and the transient peak was observed within 30 s (Fig.3). The magnitude of [Ca]increase (Delta[Ca]) with the Ca-free bathing solution (44 ± 8 nM, n = 9) was not significantly different (p > 0.05) from that with the 1 mM Ca bathing solution (47 ± 12 nM, n = 7) (Fig.3). PGE(2) at low concentrations of 10 nM and 0.1 µM PGE(2) increased [Ca] by 17 ± 6 and 21 ± 5 nM (n = 4), respectively. Similar results were obtained using the respiratory medium, where Delta[Ca] was 44 ± 15 nM (n = 3) at 10 µM PGE(2). This is the first report that PGE(2) elevates the [Ca] in parietal cells.


Figure 3: PGE(2)-induced increase in [Ca] in single parietal cells. Representative traces of the change in [Ca] of single parietal cells in gastric glands are shown. The cells were warmed at 35 °C in the 133K-142Cl bathing solutions containing 1 mM Ca (A) or 0.1 mM EGTA (B). 10 µM PGE(2) was perfused from the time indicated by the arrows. The data represent 7-9 similar experiments.



How does the rapid transient increase in [Ca]relate to the very long and sustained increase in the Cl current such as shown in Fig.2? We explain this mechanism hereafter.

Involvement of an EP Receptor and a PTX-sensitive GTP-binding Protein in the Cl Channel Activation

There are four known subtypes of prostaglandin E receptor: EP(1), EP(2), EP(3), and EP(4)(17) . Among them, activations of EP(1) and EP(3) receptors are associated with increases in [Ca]. ONO-NT-012, a novel bifunctional EP(3) agonist/EP(1) antagonist(18) , significantly increased the Cl current during the 6-min period by 83 ± 14% (n = 5, p < 0.01) (Fig.4A). Furthermore, ONO-NT-012 transiently increased [Ca] by 52 ± 2 nM (n = 6) (Fig.4B). In Fig. 5, the involvement of a GTP-binding protein in the responses to PGE(2) was investigated with PTX. The PGE(2)-induced Cl current and [Ca] increase were both completely abolished in the cells pretreated with PTX (Fig.5, B and E) but not in control cells (Fig.5, A and D).


Figure 4: Effects of ONO-NT-012 on the Cl current and [Ca]. A, a typical trace of the whole-cell Cl current from five similar experiments. Whole-cell configuration was achieved as described in the legend for Fig.2. The bathing solution that contained 10 µM ONO-NT-012 was perfused from the time indicated by the arrow. 500 µM NPPB was used. B, a trace of [Ca] from a parietal cell in a gastric gland. Typical trace from six similar experiments is shown. 10 µM ONO-NT-012 was perfused as indicated.




Figure 5: Inhibition by PTX of the PGE(2)-induced Cl current and the increase in [Ca]. A and B, typical current traces. Cells were preincubated without (A) or with (B) 500 ng/ml PTX for 160 min at 32 °C. Whole-cell currents were recorded as described in the legend for Fig.2. 10 µM PGE(2) was perfused from the time indicated. C, these effects were assessed 6 min after the addition of PGE(2) (n = 4-6).**, p < 0.01 versus PGE(2) alone. D and E, typical traces of [Ca]. Cells were preincubated without (D) and with (E) 500 ng/ml PTX for 120 min at 32 °C. 10 µM PGE(2) was perfused as indicated by arrows. F, the averaged values of Delta[Ca] from similar experiments shown in D (n = 5) and E (n = 9). **, p < 0.01 versus PGE(2) alone.



Effects of NO- and Guanylate Cyclase-related Compounds on the Cl Channel

When the cells were preincubated with 1 mML-NMMA, an inhibitor of NO production(19) , the PGE(2)-induced increase in the current was completely inhibited (Fig.6). An intracellular application of methylene blue (10 µM), a guanylate cyclase inhibitor(20, 21) , almost completely inhibited the PGE(2)-induced increase in the Cl current (Fig.7, A and C). Methylene blue per se did not increase the Cl current (Fig. 7, B and C). In the absence of PGE(2), an intracellular application of nitroprusside (30 µM), which is a NO donor and a soluble guanylate cyclase activator(20, 21) , significantly increased the Cl current (Fig.8, A and C). Furthermore, cGMP (50 µM) also increased the current (Fig.8, B and C). NPPB, which inhibits the PGE(2)-induced opening of the present Cl channel (Fig.2), inhibited these nitroprusside- and cGMP-induced Cl currents (Fig.8, A and B). These results suggest that the generation of NO and the subsequent production of cGMP by a guanylate cyclase are necessary for PGE(2)-induced activation of the Cl channel.


Figure 6: Inhibition of the PGE(2)-elicited Cl current by L-NMMA. A, a representative trace of the whole-cell Cl current from 5 similar experiments. The parietal cell in the respiratory medium was preincubated with 1 mML-NMMA for 75 min at 32 °C. Then the cell was dialyzed with the 133K-13Cl pipette solution containing 100 µML-NMMA. The 133K-142Cl bathing solution supplemented with 10 µM PGE(2), plus 1 mML-NMMA was perfused from the time indicated by the arrow. B, the effect was assessed 6 min after the addition of PGE(2). The cells were preincubated (70-110 min) in the presence of L-NMMA for the experiment indicated with L-NMMA + PGE(2) and in the absence of L-NMMA for that indicated with PGE(2).**, significantly different from the effect of PGE(2) alone (p < 0.01).




Figure 7: Inhibition of the PGE(2)-elicited Cl current by methylene blue. A and B, typical traces of the whole-cell Cl current. Whole-cell configuration with the 133K-13Cl pipette solution in the presence of 10 µM methylene blue was achieved. 10 µM PGE(2) was perfused from the time indicated by the arrow (A). C, these effects were assessed 6 min after the start of the recording (n = 4-7). MB, methylene blue.**, significantly different from the effect of PGE(2) alone (p < 0.01).




Figure 8: Effects of nitroprusside and cGMP on the Cl current in the absence of PGE(2). A and B, typical traces of the whole-cell Cl current. Parietal cells incubated in the 133K-142Cl bathing solution were dialyzed with the 133K-13Cl pipette solution supplemented with 30 µM nitroprusside (A) or 50 µM cGMP (B). 500 µM NPPB was added as indicated. C, the effects were assessed 6 min after the start of the recording (n = 4-5). The control (hatched columns) shows the level before the addition of nitroprusside or cGMP. * and**, significantly different from the control (p < 0.05 and 0.01, respectively).



Increase of [cGMP] by PGE and A23187 in the Parietal Cell

From experiments shown in Figs. 7 and 8, PGE(2) was speculated to increase [cGMP]. PGE(2) (10 µM) really increased [cGMP] in the isolated cell fraction rich in the parietal cells (Fig.9A). The maximal effect was observed 1-3 min after the addition of PGE(2). This time course was slower than that of the PGE(2)-induced elevation of [Ca] (Fig.3). Similar results were also obtained using the respiratory medium (n = 4, data not shown). A23187 (2 µM), a calcium ionophore, also elevated the [cGMP] in the cells (Fig.9B), suggesting that the [Ca] elevation by PGE(2) leads to activation of a guanylate cyclase.


Figure 9: PGE(2)- and A23187-induced increases in [cGMP] in parietal cell-rich suspensions. A and B, isolated cells rich in the parietal cell were suspended in the 133K-142Cl bathing solution. [cGMP] was measured before (0 min) and after (1, 3, and 5 min) the application of 10 µM PGE(2) (A) or 2 µM A23187 (B). The data represent means ± S.E. from 4 rabbits. * and **, significantly different from the value at 0 min (p < 0.05 and 0.01, respectively).



Combined Effects of A23187 and Methylene Blue

In the whole-cell recording, A23187 alone (2 µM) increased the Cl current (Fig.10A and C). This effect was abolished by an intracellular application of 50 µM methylene blue (Fig.10, B and C), confirming that the elevation of [Ca] does not di-rectly activate the Cl channel, but activates a guanylate cyclase, leading to activation of the channel.


Figure 10: Effect of A23187 on the Cl current in the absence of PGE(2). A and B, representative traces of the whole-cell Cl current. Whole-cell configuration with the 133K-13Cl pipette solution (pCa 7) in the absence (A) or presence (B) of methylene blue was achieved. The 133K-142Cl bathing solution supplemented with 2 µM A23187 was perfused from the time indicated by the arrows. 500 µM NPPB was added as indicated (A). C, these effects were assessed 6 min after the addition of 2 µM A23187 (n = 4). *, significantly different from the effect of A23187 alone (p < 0.05).




DISCUSSION

Activation of Cl channels by PGE(2) is known to be mediated by the adenylate cyclase/cAMP pathway in T84 human colonic carcinoma cells (22) and human skin fibroblasts(23) . In contrast, activation of the gastric basolateral Cl channel by PGE(2) was not mediated by cAMP(7) . In the present study, we have found that the PGE(2)-induced activation of the Cl channel is mediated by the elevation of [Ca] ( Fig.2and Fig. 3) and by the subsequent production of NO ( Fig.6and Fig. 8) and cGMP (Fig. 7-9) in gastric parietal cells. This intracellular signaling mechanism differs from that of the PGE(2)-induced inhibition of gastric acid secretion, in which the G(i)/adenylate cyclase/cAMP pathway is involved(24, 25) . Interestingly, PGE(2) was reported to have dual effects in bovine adrenal chromaffin cells; PGE(2) inhibits cAMP accumulation and stimulates phosphoinositide metabolism (26) .

The PGE(2)-induced increase of whole-cell Cl current of the parietal cell depended on intracellular Ca and not on extracellular Ca (Fig.2). PGE(2) transiently elevated [Ca] in the cell, and this increase was due to mobilization from intracellular Ca stores (Fig.3). Previously, PGE(2) was reported to induce Ca release from intracellular stores in Madin-Darby canine kidney cells (27) and rat osteosarcoma cells (28) .

There are four known subtypes of prostaglandin E receptor, EP(1), EP(2), EP(3), and EP(4)(17) . Among them, activations of the EP(1) and EP(3) receptors are associated with increases in [Ca], the EP(2) receptor is associated with an increase in [cAMP], and the EP(3) receptor is associated with a decrease in [cAMP]. ONO-NT-012, a novel EP(3) agonist/EP(1) antagonist(18) , induced increases in both Cl current and [Ca] (Fig.4), suggesting the involvement of EP(3) receptor in the response to PGE(2). This finding is consistent with a report that shows that the stomach is enriched in EP(3) receptors(29) . Four isoforms of bovine EP(3) receptor have been cloned, two of which (EP and EP) couple to PTX-sensitive and -insensitive GTP-binding proteins, respectively, both resulting in an increase in [Ca](30) . The present increases in the PGE(2)-induced Cl current and [Ca] were both completely abolished when pretreated with PTX (Fig.5).

We suggest that the elevation of [Ca] by PGE(2) does not directly activate the Cl channel but leads to the activation of a guanylate cyclase, because 1) the peak of elevation of [Ca] (within 30 s, Fig.3) was attained faster than that of [cGMP] (1-3 min, Fig.9A), 2) a calcium ionophore, A23187, elevated [cGMP] of parietal cells (Fig.9B), and 3) the A23187-induced increase in [Ca] did not accompany the increase of the Cl current in the presence of a guanylate cyclase inhibitor, methylene blue (Fig.10). The intracellular application of cGMP activated the Cl channel with a slow time course, whereas NPPB, a Cl channel blocker, immediately blocked the channel (Fig.8B). These results suggest that cGMP also does not directly activate the Cl channel, in contrast to cGMP-gated cation channels in retinal rods(31, 32) .

Coupling of the elevation of [Ca] with activation of a guanylate cyclase has been reported in mouse neuroblastoma rat glioma hybrid cells (33, 34) and porcine kidney epithelial cells(35) . These reports demonstrated that the rise of [Ca] by serotonin (33) and endothelin-1 (34, 35) stimulated the NO-forming enzyme and that NO activated a soluble guanylate cyclase. Our present study showed that nitroprusside, which releases NO, activated the Cl channel in the gastric parietal cell (Fig.8A). Furthermore, the PGE(2)-induced opening of the channel was inhibited when the cells were preincubated with L-NMMA, which inhibits NO production (Fig.6).

The present Cl channel is closed by superoxide (O) production mediated by a PTX-insensitive GTP-binding protein(8, 9) . The regulatory system of this Cl channel provides an example of two compounds belonging to the same category exerting opposite effects; the Cl channel is regulated positively by NO and a PTX-sensitive GTP-binding protein and negatively by O and a PTX-insensitive GTP-binding protein.


FOOTNOTES

*
This study was supported in part by grants-in-aid for encouragement of young scientists (to H. S.), for scientific research (B) (to N. T.), and for scientific research on priority areas (to N. T.) from the Ministry of Education, Science, and Culture of Japan and by the grants from ONO Medical Research Foundation, Salt Science Research Foundation, and Ciba-Geigy Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 81-764-34-4656.

^1
The abbreviations used are: NO, nitric oxide; PGE, prostaglandin E; ONO-NT-012, 5(Z)-7-[(1S,2S,3S,5R)-3-(trans-beta-styren)sulfonamido-6,6-dimethylbicyclo(3.1.1)hept-2-yl]-5-heptenoic acid; PTX, pertussis toxin; L-NMMA, N^G-monomethyl-L-arginine; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; BAPTA, O,O`-bis(2-aminophenyl)ethyleneglycol-N,N,N`,N`-tetraacetic acid.


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