|
Volume 270,
Number 32,
Issue of August 11, pp. 18781-18785, 1995
©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 (PGE) has a
cytoprotective role in the gastric parietal cell. PGE 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 increased the
intracellular free Ca concentration
([Ca] ) and that
PGE-induced opening of the Cl channel
depended on the increase in
[Ca]. A novel
bifunctional prostaglandin EP agonist/EP antagonist, 5(Z)-7-[(1S, 2S,
3S,
5R)-3-(trans- -styren)sulfonamido-6,6-dimethylbicyclo(3.1.1)hept-2-yl]-5-heptenoic
acid, also increased both [Ca] and channel opening. The PGE-induced effect was
mediated via production of nitric oxide (NO); that is, N -monomethyl-L-arginine, an inhibitor of
NO production, markedly inhibited the PGE-induced channel
opening, and nitroprusside, a NO donor, induced the channel opening in
the absence of PGE. Both PGE 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-induced opening of the housekeeping
Cl channel in the parietal cell involves the EP 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 ()pathway; however, targets of this pathway are
unknown(1, 2) . In stomach, PGE 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(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 can stimulate the opening of
the housekeeping Cl channel. We tested whether
PGE increased
[Ca], because
PGE-induced opening of the channel was inhibited by the
intracellular presence of a strong Ca chelator, and
it was unknown whether PGE increased the
[Ca] of gastric
parietal cells. We found that PGE 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 GlandsGastric 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 NaHPO , 1 mM NaHPO, 1.2 mM MgSO, 1
mM CaCl, 2 mg/ml bovine serum albumin, 2 mg/ml
glucose, and 10 mM HEPES (pH 7.35).
Preparation of the Parietal Cell-rich
SuspensionIsolated 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 AnalysisWhole-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, 1
CaSO, 0.1 ouabain, and 10 HEPES. In the
Ca-free 133K-142Cl solution, CaSO was omitted, and 0.1 mM EGTA
was added. The 140choline-146Cl bathing solution contained (in mM): 140 choline
chloride, 2 MgCl, 1 CaCl, 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, 0.062 CaSO, 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,
0.062 CaSO, 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, 0.467 CaSO, 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 CellsIsolated 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, 1 CaCl, 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 CellsIsolated cells
rich in parietal cells were suspended in respiratory medium or
133K-142Cl bathing solution (1
10 cells/ml), and preincubated for 10 min at 35
°C. 1, 3, and 5 min after the addition of PGE 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 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).
ChemicalsPGE (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).
StatisticsStatistical 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 ChannelThe activation of the Cl channel
was evidenced by an increase in the whole-cell Cl current. PGE (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 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 were the
same(6) . PGE 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-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 ( ) or 3 ( ) or 6 min ( ) 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 (10
µM). The PGE-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-induced increase of the
current (Fig.2, C and D). As reported
previously(6) , PGE-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-induced effect and that
Ca is released from intracellular Ca stores.
Figure 2:
Role of Ca on the
PGE-elicited Cl current. A-C,
representative traces of the whole-cell Cl current.
The 133K-13Cl pipette solution
contained 10 M
[Ca] (weakly buffered
at pCa 7 with 0.1 mM EGTA) (A and C) or
10 M [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 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. Three experimental protocols for
PGE (n = 6),
(BAPTA)+PGE (n =
5), and (EGTA) +PGE (n = 3) correspond to panels A, B and C, respectively.**, significantly different from the effect of
PGE alone (p <
0.01).
Elevation of [Ca] by PGE in Single Parietal CellsWe measured
[Ca] in single Fura
2-loaded parietal cells. Corresponding with the results from the
whole-cell recording (Fig.2), PGE (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, and the transient
peak was observed within 30 s (Fig.3). The magnitude of
[Ca]increase
( [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 at low concentrations of 10 nM and 0.1 µM PGE increased
[Ca] by 17 ± 6
and 21 ± 5 nM (n = 4), respectively.
Similar results were obtained using the respiratory medium, where
[Ca] was 44
± 15 nM (n = 3) at 10 µM PGE. This is the first report that PGE elevates the [Ca] in parietal cells.
Figure 3:
PGE-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 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
ActivationThere are four known subtypes of prostaglandin E
receptor: EP, EP, EP, and
EP(17) . Among them, activations of EP and EP receptors are associated with increases in
[Ca]. ONO-NT-012, a
novel bifunctional EP agonist/EP 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 was
investigated with PTX. The PGE-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-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 was perfused from
the time indicated. C, these effects were assessed 6 min after
the addition of PGE (n = 4-6).**, p < 0.01 versus PGE 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 was
perfused as indicated by arrows. F, the averaged
values of [Ca] from similar experiments shown in D (n = 5) and E (n = 9). **, p < 0.01 versus PGE alone.
Effects of NO- and Guanylate Cyclase-related
Compounds on the Cl ChannelWhen the cells
were preincubated with 1 mML-NMMA, an inhibitor of
NO production(19) , the PGE-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-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, 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-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-induced activation
of the Cl channel.
Figure 6:
Inhibition of the PGE-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, 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. The cells were preincubated (70-110
min) in the presence of L-NMMA for the experiment indicated
with L-NMMA + PGE and in the absence
of L-NMMA for that indicated with PGE.**,
significantly different from the effect of PGE alone (p < 0.01).
Figure 7:
Inhibition of the PGE-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 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 alone (p <
0.01).
Figure 8:
Effects of nitroprusside and cGMP on the
Cl current in the absence of PGE. 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 CellFrom experiments shown in
Figs. 7 and 8, PGE was speculated to increase
[cGMP]. PGE (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. This time course was slower
than that of the PGE-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 leads to activation of a guanylate cyclase.
Figure 9:
PGE- 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 (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 BlueIn
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. 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 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 was not
mediated by cAMP(7) . In the present study, we have found that
the PGE-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-induced inhibition of gastric acid
secretion, in which the G /adenylate cyclase/cAMP pathway is
involved(24, 25) . Interestingly, PGE was
reported to have dual effects in bovine adrenal chromaffin cells;
PGE inhibits cAMP accumulation and stimulates
phosphoinositide metabolism (26) . The
PGE-induced increase of whole-cell Cl current of the parietal cell depended on intracellular
Ca and not on extracellular Ca (Fig.2). PGE transiently elevated
[Ca] in the cell, and
this increase was due to mobilization from intracellular Ca stores (Fig.3). Previously, PGE 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, EP, EP, and EP(17) . Among them, activations of the EP and
EP receptors are associated with increases in
[Ca], the EP receptor is associated with an increase in
[cAMP], and the EP receptor
is associated with a decrease in [cAMP].
ONO-NT-012, a novel EP agonist/EP antagonist(18) , induced increases in both Cl current and [Ca] (Fig.4), suggesting the involvement of EP receptor in the response to PGE. This finding is
consistent with a report that shows that the stomach is enriched in
EP receptors(29) . Four isoforms of bovine EP 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-induced Cl current and [Ca] were both completely abolished when pretreated with PTX (Fig.5). We suggest that the elevation of
[Ca] by PGE 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-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.
-
- The abbreviations used
are: NO, nitric oxide; PGE, prostaglandin E; ONO-NT-012,
5(Z)-7-[(1S,2S,3S,5R)-3-(trans--styren)sulfonamido-6,6-dimethylbicyclo(3.1.1)hept-2-yl]-5-heptenoic
acid; PTX, pertussis toxin; L-NMMA, N-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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