cGMP abolishes agonist-induced [Ca2+]i
oscillations in human bladder epithelial cells
H. Y.
Kwan1,
Y.
Huang1,
S. K.
Kong2, and
X.
Yao1
Departments of 1 Physiology and 2 Biochemistry,
Chinese University of Hong Kong, Hong Kong, China
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ABSTRACT |
First published
August 9, 2001; 10.1152/ajprenal.00031.2001.
Cytosolic calcium
oscillations may permit cells to respond to information provided by
increases in intracellular Ca2+ concentration
([Ca2+]i ) while avoiding prolonged exposure
to constantly elevated [Ca2+]i. In this
study, we demonstrated that agonists could induce Ca2+
oscillations in human bladder epithelial cells. Application of 10 µM
acetylcholine or 200 nM bradykinin triggered an initial Ca2+ transient that was followed by periodic
[Ca2+]i oscillations. The oscillations did
not depend on extracellular Ca2+. 8-Bromoguanosine
3',5'-cyclic monophosphate abolished acetylcholine- or
bradykinin-induced oscillations. Elevation of cellular cGMP by
dipyridamole, an inhibitor of cGMP-specific phosphodiesterase, also
terminated the [Ca2+]i oscillations. The
inhibitory effect of cGMP could be reversed by KT-5823, a highly
specific inhibitor of protein kinase G (PKG), suggesting that the
action of cGMP was mediated by PKG. Comparison of the effect of cGMP
with that of xestospongin C, an inhibitor of the inositol
1,4,5-trisphosphate (IP3) receptor, revealed similarities between the action of cGMP and xestospongin C. Therefore, it is likely
that cGMP and PKG may target a signal transduction step(s) linked to
IP3 receptor-mediated Ca2+ release.
protein kinase G; inositol 1,4,5-triphosphate; calcium release; nitric oxide; intracellular calcium concentration; guanosine
3',5'-cyclic monophosphate
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INTRODUCTION |
OSCILLATORY
CHANGES IN INTRACELLULAR Ca2+ concentration
([Ca2+]i), or
[Ca2+]i oscillations, occur in a variety of
nonexcitable cell types (10, 14, 18, 34).
[Ca2+]i oscillations can be triggered by a
great variety of stimuli, including neurotransmitters, hormones, growth
factors, and mechanical stress. Of the natural stimuli, many are
calcium-mobilizing agents that bind to cell surface receptors and then
activate phospholipase C. This leads to the breakdown of
phosphatidylinositol 4,5-biphosphate into two important second
messengers, inositol 1,4,5-triphosphate (IP3) and
diacylglycerol (4). IP3 binds to its receptor,
which acts as a Ca2+ channel in the endoplasmic reticular
membrane and triggers the releases of Ca2+ into cytoplasm
(11).
A number of mechanistic models for [Ca2+]i
oscillations have been proposed (9, 25). The single-pool
model hypothesizes that IP3 activates its receptor and
releases Ca2+ from a single intracellular Ca2+
pool. The resulting elevation in cytosolic Ca2+ then feeds
back to inhibit further release of Ca2+ by the
IP3 receptor (28). Rapid activation of the
IP3 receptor by IP3 and slow inactivation of
the IP3 receptor by Ca2+ as
[Ca2+]i increases to higher values, along
with the functioning of Ca2+-ATPase at the endoplasmic
reticular membrane, lead to cytosolic Ca2+ oscillations
(9, 24, 25). An alternative two-pool model suggests that
two separate intracellular Ca2+ stores may be
involved in the generation of cytosolic Ca2+ oscillations,
one being an IP3-sensitive store and the other an
IP3-insensitive one (3).
cGMP has distinct effects on intracellular Ca2+ levels in
different cells, decreasing free [Ca2+]i in
smooth muscles (26), cardiac myocytes (28),
platelets (33), and megakaryocytes (39) and
increasing [Ca2+]i in hepatocytes
(34) and sea urchin eggs (13). Multiple targets for cGMP have been identified. cGMP inhibits IP3
formation in smooth muscle cells (26), inhibits
Ca2+ entry into smooth muscle and endothelial cells
(26, 43), and inhibits IP3 receptor-mediated
Ca2+ release from endoplasmic reticulum in smooth muscle
cells and megakaryocytes (26, 39). There are only a few
studies on the effect of cGMP on [Ca2+]i
oscillations. In rat megakaryocytes, application of cGMP inhibits ATP-induced [Ca2+]i oscillations (38,
39). In contrast, cGMP is reported to initiate
[Ca2+]i oscillations via stimulating
IP3 receptor-mediated Ca2+ release in rat
hepatocytes (34).
In the present study, we used laser scanning confocal microscopy to
measure acetylcholine- or bradykinin-induced
[Ca2+]i oscillations in human bladder
epithelial cells. We found that both acetylcholine- and
bradykinin-induced cytoplasmic Ca2+ oscillations in human
bladder epithelial cells could be inhibited by cGMP via a protein
kinase G (PKG)-dependent mechanism.
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MATERIALS AND METHODS |
Materials.
Fluo 3-acetoxymethyl ester (AM) and Pluronic F127 were obtained from
Molecular Probes. Tissue culture media and materials were from GIBCO
BRL. Culture flasks and culture plates were from Becton Dickinson.
Acetylcholine, bradykinin, atropine, carbachol, 8-bromoadenosine
3',5'-cyclic monophosphate (8-BrcAMP), 8-BrcGMP, KT-5823,
xestospongin C (XeC), cyclopiazonic acid (CPA), and dipyridamole were from Calbiochem. HEPES and EDTA were purchased from Sigma. HOE-40
was from RBI.
Cell culture.
ECV304 is a human bladder epithelial cell line identical to T24/83
(6). Cells were cultured in 90% RPMI-1640 and 10% fetal bovine serum (FBS) containing 100 U/ml penicillin and 100 µg/ml streptomycin and incubated in T-75 tissue culture flasks at 37°C in
an atmosphere of 5% CO2-95% air. Confluent cell
monolayers were passaged using 0.25% trypsin containing 2.5 mM EDTA.
[Ca2+]i measurement.
ECV304 cells were grown overnight in 90% RPMI-1640 supplemented with
10% FBS containing 100 U/ml penicillin and 100 µg/ml streptomycin on
circular disks (Fisher 25 CIR-1) at 37°C and 5% CO2-95%
air. Cells were loaded with fluo 3-AM for 1 h in the dark at room
temperature by incubation with 10 µM membrane-permeant fluo 3-AM and
0.02% Pluronic F127 in a normal physiological saline solution (N-PSS)
containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 5 HEPES (pH 7.4). After loading of
fluo 3-AM, cells were transferred to Ca2+-free PSS to
remove excessive external fluo 3-AM. The circular disks containing the
ECV304 epithelial cells were then pinned in a specially designed
chamber. The chamber was placed on the stage of an inverted microscope
(Nikon Diaphot 200), and the fluorescence signal was recorded by the
MRC-1000 laser scanning confocal imaging system with MRC-1000 software
(Bio-Rad). Experiments were performed without flow. Cells were bathed
in Ca2+-free PSS that contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 0.2 EGTA, 10 glucose, and 5 HEPES (pH 7.4). All
agents were applied directly to the bath at the side of the chamber;
solutions were then mixed by gentle pipetting. As a control, pipetting
with bath media in the absence of agonists did not produce any change
in [Ca2+]i. Data analysis was performed with
the Confocal Assistant and Metafluor systems (Bio-Rad). Changes in
[Ca2+]i in response to all agents were
displayed as the ratio of fluorescence relative to the fluorescence
before the application of agents (F0).
 |
RESULTS |
Responses to acetylcholine and bradykinin.
The Ca2+ responses to acetylcholine and bradykinin in
cultured human bladder epithelial cells loaded with the
Ca2+ indicator fluo 3 were monitored by laser scanning
confocal microscopy. The application of 10 µM acetylcholine greatly
changed the intracellular Ca2+ levels. Three basic
responses to acetylcholine were observed. These consisted of
1) a single Ca2+ transient after exposure to ATP
(Fig. 1D); 2)
initiation of [Ca2+]i oscillations (Fig. 1,
A and B); and 3) a prolonged elevation of baseline [Ca2+]i (Fig. 1C).
Acetylcholine (10 µM) induced an initial Ca2+ transient
in ~85% of cells. After the initial Ca2+ increase
induced by acetylcholine, 35-65% of cells, depending on the
preparations, displayed periodic [Ca2+]i
oscillations. The frequency of oscillations varied from 1 to 2 Hz,
whereas the amplitude of oscillations varied greatly and, in general,
decreased with time. The mean peak amplitude
(F1/F0) of the first
[Ca2+]i oscillation was calculated to be
3.8 ± 0.2 (n = 10). For comparison, the peak
amplitude of the fifth oscillation was ~30% lower at 2.7 ± 0.2 (n = 10). The oscillations did not depend on
Ca2+ influx because the oscillatory activity could be
recorded in cells bathed in Ca2+-free PSS. Acetylcholine
washout immediately halted the oscillatory responses. The
acetylcholine-induced [Ca2+]i oscillations
were not related to the hydrolysis of acetylcholine because 50 µM
carbachol was also able to induce the oscillations. Incubation of cells
for 5 min in atropine (100 nM), a muscarinic receptor antagonist,
completely abolished the initial Ca2+ transient as well as
the subsequent Ca2+ oscillations (Fig. 1E,
n = 3).

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Fig. 1.
ACh-induced intracellular Ca2+ concentration
([Ca2+]i) oscillations in bladder epithelial
cells. A-D: variation in
[Ca2+]i responses to ACh among different
cells. Each trace represents a separate cell. Cells were grown thinly
and placed in a Ca2+-free physiological saline solution
(0Ca-PSS). ACh (10 µM) was added. E: effect of atropine
(100 nM) on ACh-induced [Ca2+]i oscillations.
F1/F0, relative fluorescence. Curves are
typical of the data obtained from 4-5 experiments, comprising a
total of 60-80 cells.
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Similarly, bradykinin was also able to induce
[Ca2+]i oscillations. Application of 200 nM
bradykinin triggered an initial Ca2+ transient in >95% of
cells, and this was followed by periodic [Ca2+]i oscillations in ~80% of cells
(Fig. 2, A-D).
The oscillatory activity elicited by bradykinin ceased immediately on
bradykinin washout. Incubation of cells for 5 min in HOE-140 (1 µM),
a selective B2 bradykinin receptor antagonist, completely
abolished bradykinin-induced initial Ca2+ transient as well
as the subsequent [Ca2+]i oscillations (Fig.
2E, n = 6).

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Fig. 2.
Bradykinin (BK)-induced [Ca2+]i
oscillations in bladder epithelial cells.
A-D: variation in
[Ca2+]i responses to BK among different
cells. Each trace represents a separate cell. Cells were grown thinly
and placed in 0Ca-PSS. BK (200 nM) was added. E: effect of
HOE-140 (1 µM) on BK-induced [Ca2+]i
oscillations. Curves are typical of the data obtained from 6-10
experiments, comprising a total of 90-150 cells.
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In agreement with what has been observed in rabbit airway epithelial
cells (10) and rat pituitary gonadotrophs
(25), the oscillatory responses of
[Ca2+]i in human bladder epithelial cells
were also greatly affected by the agonist concentration applied. As the
concentration of applied ATP increased from low to intermediate to
high, the response of cells shifted from a single Ca2+
transient to [Ca2+]i oscillations and then to
prolonged [Ca2+]i elevation (Table
1). In addition, the peak amplitudes of
Ca2+ responses also increased with increasing ATP
concentrations (Table 1). Similar dose-dependent differential
[Ca2+]i responses could also be observed for
bradykinin at concentrations between 10 and 200 nM (data not shown). We
also attempted to test whether individual cells within a population
exhibited differing sensitivities to agonists by applying increasing
concentrations of agonists in a stepwise manner to a single dish of
cells. However, we found that treatment of cells with ATP or
bradykinin, even at low concentrations, reduced and sometimes abolished
the Ca2+ responses to subsequent agonist challenge. In
other words, the Ca2+ responses of individual cells
desensitized after their preexposure to agonists. For this reason, our
protocol failed to resolve whether individual cells within an exhibited
population had differing sensitivities to agonists.
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Table 1.
Effect of acetylcholine concentration on
[Ca2+]i responsiveness in
cultured human bladder epithelial cells
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One previous report suggested that extracellular EGTA might interfere
with histamine-induced Ca2+ release from intercellular
Ca2+ stores in airway epithelial cells (15).
In our experiments, however, no apparent differences in oscillatory
responses could be found between the cells bathed in
Ca2+-free PSS that contained 0.2 mM EGTA and a nominally
Ca2+-free solution containing no EGTA, suggesting that EGTA
at the concentration used did not influence the oscillatory activity elicited by agonists. Changes in cell culture media from RPMI-1640 to
DMEM or
-MEM did not affect the agonist-induced oscillatory responses either.
Besides calcium-mobilizing agonists, flow shear stress is another
factor that may initiate Ca2+ oscillations, at least in
cultured vascular endothelial cells (16, 36). We therefore
tested the effect of mechanical stimulation generated by flow on
Ca2+ signaling in cultured bladder epithelial cells. In
this series of experiments, flow was initiated by pumping N-PSS or
Ca2+-free PSS to a specially designed flow chamber so that
appropriate shear force could be generated. Shear force at the
range of 0.1-10 dyn/cm2 elicited a transient
Ca2+ elevation, but without Ca2+ oscillations,
in cells perfused by Ca2+-containing N-PSS. The
flow-induced Ca2+ transient was dependent on extracellular
Ca2+, because perfusion by Ca2+-free PSS could
not elicit the Ca2+ transient. Therefore, it appears that
flow and agonists may stimulate cytosolic Ca2+ changes
through different mechanisms in bladder epithelial cells.
Effect of cGMP and KT-5823 on
[Ca2+]i oscillations.
cGMP is an intracellular second messenger that activates PKG. We used
8-BrcGMP, a membrane-permeant cGMP analog, to activate PKG and a highly
specific inhibitor, KT-5823, to inhibit PKG. Addition of 2 mM 8-BrcGMP
immediately stopped ongoing acetylcholine- or bradykinin-induced
[Ca2+]i oscillations (Fig.
3, A and B).
Application of 1 µM KT-5823 reversed the effect of cGMP and
reinitiated the transient rise of intracellular Ca2+. In
~50% of cells, this initial Ca2+ transient was followed
by [Ca2+]i oscillations (Fig. 3). As a
control, KT-5823 alone was not able to initiate
[Ca2+]i oscillations (n = 3).
In separate experiments, cells were preincubated in 2 mM 8-BrcGMP for 5 min; the preincubation completely abolished the acetylcholine- or
bradykinin-induced initial Ca2+ transient as well as the
subsequent [Ca2+]i oscillations
(n = 5). If cGMP preincubation was carried out in the
presence of 1 µM KT-5823, cGMP was not able to inhibit acetylcholine-
or bradykinin-induced [Ca2+]i oscillations
(Fig. 4, A and B).

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Fig. 3.
Effect of 8-bromoguanosine 3',5'-cyclic monophosphate
(8-BrcGMP) and KT-5823 on ACh- or BK-induced
[Ca2+]i oscillations. 8-BrcGMP inhibited ACh
(A)- or BK-induced (B)
[Ca2+]i oscillations. KT-5823 reversed the
inhibition. Cells were grown thinly and placed in 0Ca-PSS. Chemicals
were added sequentially as follows: 10 µM ACh; 200 nM BK; 2 mM
8-BrcGMP; and 1 µM KT-5823. Each curve is typical of the data
obtained from 3-9 experiments, comprising a total of 50-150
cells.
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Fig. 4.
Effect of preincubation in 8-BrcGMP and KT-5823 on
[Ca2+]i oscillations. Preincubation of cells
in 8-BrcGMP abolished ACh (A)- or BK-induced (B)
[Ca2+]i oscillations. KT-5823 reversed the
inhibition. Cells were grown thinly and placed in 0Ca-PSS. Chemicals
were added as shown: 10 µM ACh; 200 nM BK; 2 mM 8-BrcGMP; and 1 µM
KT-5823. Each curve is typical of the data obtained from 3-7
experiments, comprising a total of 50-110 cells.
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It appeared that KT-5823 treatment also increased the percentage of
cells responding to acetylcholine. After KT-5823 treatment, the
percentage of cells demonstrating the acetylcholine-induced [Ca2+]i oscillations was 98 ± 4%
(n = 5). Without the treatment, only 30 ± 3%
(n = 5) cells displayed the acetylcholine-induced
[Ca2+]i oscillations. On the other hand, the
percentage of cells demonstrating bradykinin-induced
[Ca2+]i oscillations did not change
significantly after KT-5823 treatment (before treatment: 82 ± 7%, n = 10; after treatment: 90 ± 6%, n = 4).
Taken together, our results suggest that acetylcholine- or
bradykinin-induced [Ca2+]i oscillations in
human bladder epithelial cells are regulated by a PKG-dependent
mechanism. Activation of PKG by 8-BrcGMP abolishes [Ca2+]i oscillations, whereas the inhibition
of PKG by KT-5823 reinstates them.
It has been reported that PKG and PKA have similarities in structure
and substrate specificity (2). We therefore tested the effect of 8-BrcAMP on acetylcholine- or bradykinin-induced Ca2+ oscillations. Unlike with cGMP, preincubation of
cells with 2 mM 8-BrcAMP had no effect on the acetylcholine- or
bradykinin-induced [Ca2+]i oscillations,
suggesting that PKA was not involved (n = 3).
Effect of dipyridamole on
[Ca2+]i oscillations.
Dipyridamole is an inhibitor of cGMP-specific phosphodiesterase V. It
raises intracellular cGMP levels by inhibiting cGMP degradation via
phosphodiesterase (41). Incubation of cells for 5 min in
10 µM dipyridamole before the application of 10 µM acetylcholine
(Fig. 5A) or 200 nM bradykinin
(Fig. 5B) completely abolished the agonist-induced initial
Ca2+ transient as well as the subsequent
[Ca2+]i oscillations. These data are
consistent with the inhibitory role of cGMP in agonist-induced
[Ca2+]i oscillations.

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Fig. 5.
Effect of dipyridamole on ACh- or BK-induced
[Ca2+]i oscillations. Incubation of cells in
dipyridamole abolished ACh (A)- or BK-induced (B)
[Ca2+]i oscillations. Cells were grown thinly
and placed in 0Ca-PSS. Chemicals were added as shown: 10 µM ACh, 200 nM BK, and 10 µM dipyridamole. Each curve is typical of the data
obtained from 3 experiments, comprising a total of ~50 cells.
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Effect of CPA and XeC on
[Ca2+]i oscillations.
To define the importance of the IP3 receptor in
acetylcholine-induced [Ca2+]i oscillations,
we used a membrane-permeable blocker, XeC, to selectively inhibit the
receptor (12). Application of 5 µM XeC immediately
ceased ongoing acetylcholine- or bradykinin-induced [Ca2+]i oscillations (Fig.
6A). In separate experiments,
when cells were pretreated with 5 µM XeC, acetylcholine or bradykinin
failed to induce the initial Ca2+ transient as well as the
subsequent [Ca2+]i oscillations (Fig.
6B). These data suggest that IP3
receptor-mediated Ca2+ release is required for
[Ca2+]i oscillations.

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Fig. 6.
Effect of xestospongin C (XeC) on ACh-induced
[Ca2+]i oscillations. XeC abolished
ACh-induced [Ca2+]i oscillations. Cells were
grown thinly and placed in 0Ca-PSS. Chemicals were added as shown: 10 µM ACh and 5 µM XeC. Each curve is typical of the data obtained
from 3-4 experiments, comprising a total of 40-60 cells.
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The role of sarcoplasmic or endoplasmic reticular
Ca2+-ATPase (SERCA) in [Ca2+]i
oscillations was examined with the use of CPA, a selective inhibitor of
SERCA (30). Preincubation of cells for 1 min in 10 µM
CPA had no effect on the rising phase of the initial Ca2+
signal elicited by acetylcholine. In the presence of CPA, however, [Ca2+]i remained at an elevated level over
the time course of experiments after it had reached its peak (Fig.
7A). These results suggest that the falling phase of the initial Ca2+ transient is
mostly due to the activity of SERCA.

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Fig. 7.
Effect of cyclopiazonic acid (CPA) on ACh-induced
Ca2+ transient. A: CPA diminished the falling
phase of the ACh-induced Ca2+ transient. B:
8-BrcGMP abolished the ACh-induced Ca2+ transient, and
KT-5823 reversed the inhibition. Cells were grown thinly and placed in
0Ca-PSS. Chemicals were added as shown: 10 µM ACh; 10 µM CPA; 2 mM
8-BrcGMP; and 1 µM KT-5823. Each curve is typical of the data
obtained from 3-4 experiments, comprising a total of 40-60
cells.
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The acetylcholine-induced [Ca2+]i rise in the
presence of CPA was also subjected to the regulation by cGMP and PKG.
Incubation of cells for 5 min in 2 mM 8-BrcGMP abolished the
acetylcholine-induced [Ca2+]i rise. In the
presence of 1 µM KT-5823, cGMP had no effect (Fig. 7B).
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DISCUSSION |
Calcium signaling in nonexcitable cells regulates such diverse
processes as gene regulation, secretion, apoptosis, and cell proliferation. In bladder epithelial cells, intracellular calcium is
known to regulate Na+ reabsorption and proton secretion
(23, 35), alter antidiuretic hormone-mediated osmotic
water flow (7), participate in cell volume regulation
(42), control granule exocytosis (29), and regulate the insertion of H+- ATPase into the apical
membrane (40). Ca2+-sensitive cell functions
are often mediated by oscillatory rather than prolonged sustained
increases in [Ca2+]i (32). The
advantages of these oscillatory signals include 1) favorable
signal-to-noise ratios (32) and 2) avoidance of the adverse effects of sustained elevation in
[Ca2+]i (27). Oscillatory
[Ca2+]i signals can be decoded into changes
in Ca2+/calmodulin-dependent protein kinase II activity
(22) and nuclear factor-kB transcriptional activity
(17). Until now, however, there was still a lack of
evidence for [Ca2+]i oscillations in bladder
epithelial cells. In the present study, we demonstrated the existence
of agonist-induced [Ca2+]i oscillations in
human bladder epithelial cells. The oscillations elicited by
acetylcholine were inhibited by atropine, suggesting that the action of
acetylcholine was mediated by muscarinic receptors. The oscillations
elicited by bradykinin could be abolished by HOE-140, implicating the
involvement of the B2 bradykinin receptor. As in many other
cell types (10, 34), agonist-induced Ca2+
oscillations in bladder epithelial cells did require the presence of
extracellular Ca2+.
The effect of cGMP on [Ca2+]i oscillations
appears to depend on the cell type. cGMP stimulates
[Ca2+]i oscillations in rat hepatocytes,
whereas it inhibits [Ca2+]i oscillations in
rat megakaryocytes (34, 38, 39). The effect of cGMP could
be caused by the direct action of cGMP (19), be mediated
by a G kinase (26), result from the activation of PKA
(2), or be due to increases in cAMP that result from an inhibition of cAMP phosphodiesterase activity (1). In our
experiments, application of cGMP abolished the acetylcholine- or
bradykinin-induced [Ca2+]i oscillations in
cultured human bladder epithelial cells (Figs. 3 and 4). The inhibitory
effect was reversed by a highly specific PKG inhibitor, KT-5823 (Figs.
3 and 4). Membrane-permeant 8-BrcAMP had no effect on
[Ca2+]i oscillations. These data suggest that
the effect of cGMP is mediated by PKG and argue against either a direct
effect of cGMP or an indirect effect due to an increase in cAMP or
activation of PKA.
The [Ca2+]i oscillations in human bladder
epithelial cells require the functioning of both the IP3
receptor and SERCA. The rising phase of
[Ca2+]i oscillations may result from
Ca2+ release through the IP3 receptor, whereas
the falling phase of the oscillations may be attributable to
Ca2+ sequestration into intracellular stores as well as
Ca2+ extrusion into the extracellular medium
(24). In our experiments, a membrane-permeant
IP3 receptor inhibitor, XeC, abolished the acetylcholine-
or bradykinin-induced [Ca2+]i oscillations
(Fig. 6). Application of CPA, which blocks SERCA and results in the
inability of the sarcoplasmic reticulum to sequester Ca2+,
diminished the falling phase of Ca2+ signals (Fig.
7A). These data suggest that the Ca2+
sequestration into intracellular Ca2+ stores is the main
mechanism responsible for the falling phase of agonist-induced
[Ca2+]i transients and/or oscillations,
whereas Ca2+ extrusion to the extracellular medium may only
play a minor role.
In our experiments, action of cGMP on intracellular Ca2+
was similar to that of XeC but was apparently different from that of CPA. Preincubation of cells in cGMP or XeC completely abolished the
agonist-induced initial Ca2+ transient as well as the
subsequent [Ca2+]i oscillations. In contrast,
CPA did not influence the rising phase of the initial Ca2+
transient. Furthermore, in the presence of CPA, cGMP could still abolish the acetylcholine-induced Ca2+ transient (Fig.
7B). These results suggest that cGMP and PKG may act like
XeC and target some signaling step(s) linked to the IP3
receptor-mediated Ca2+ release. The present data cannot
distinguish the precise step in which PKG may act. cGMP may inhibit
IP3 formation, as in smooth muscle cells, or it may
directly inhibit the IP3 receptor, as in smooth muscle
cells and megakaryocytes (26, 39).
Nitric oxide (NO) is produced in urinary bladder epithelial cells
(5, 8) and in the nerves supplying the bladder
(20). It can cause smooth muscle relaxation in the lower
urinary tract (20). Changes in the NO level in bladder
epithelium have been implicated in the pathogenesis of bladder tumors
(21). As in many other cell types (26), the
action of NO in uroepithelial cells is likely to be mediated by cGMP
(37). NO may activate guanylate cyclase, leading to the
elevation of cGMP levels in bladder epithelial cells (37,
44). Our present data provide a possible target for this NO-cGMP
signaling pathway in bladder epithelium. It is possible that NO-cGMP
may target the [Ca2+]i oscillations in
bladder epithelium. The change in Ca2+ oscillations may
then regulate other processes, such as gene transcription and cell proliferation.
In conclusion, we find that cultured human bladder epithelial cells
display acetylcholine- and bradykinin-induced
[Ca2+]i oscillations. The oscillatory
activity requires the functioning of the IP3 receptor as
well as SERCA. cGMP, via its action on PKG, may affect the signaling
pathway, leading to IP3 receptor-mediated Ca2+
release, thus regulating [Ca2+]i oscillations.
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ACKNOWLEDGEMENTS |
This study was supported by the Hong Kong Research Grant Council
(CUHK4079/00M) and the Chinese University Research Committee.
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FOOTNOTES |
Address for reprint requests and other correspondence: X. Yao, Dept. of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China (E-mail:
yao2068{at}cuhk.edu.hk).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published August 9, 2001;10.1152/ajprenal.00031.2001
Received 5 February 2001; accepted in final form 23 July 2001.
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