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