Pharmacology and modulation of KATP channels by
protein kinase C and phosphatases in gallbladder smooth
muscle
T. A.
Firth1,
G. M.
Mawe1,2, and
M. T.
Nelson2
Departments of 1 Anatomy and Neurobiology
and 2 Pharmacology, College of Medicine,
University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
ATP-sensitive
K+ (KATP) channels exhibit pharmacological
diversity, which is critical for the development of novel therapeutic agents. We have characterized KATP channels in gallbladder
smooth muscle to determine how their pharmacological properties compare to KATP channels in other types of smooth muscle.
KATP currents were measured in myocytes isolated from
gallbladder and mesenteric artery. The potencies of pinacidil,
diazoxide, and glibenclamide were similar in gallbladder and vascular
smooth muscle, suggesting that the regions of the channel conferring
sensitivity to these agents are conserved among smooth muscle types.
Activators of protein kinase C (PKC), however, were less effective at
inhibiting KATP currents in myocytes from gallbladder than
mesenteric artery. The phosphatase inhibitor okadaic acid increased the
efficacy of PKC activators and revealed ongoing basal activation of
KATP channels by protein kinase A in gallbladder. These
results suggest that phosphatases and basal kinase activity play an
important role in controlling KATP channel activity.
mesenteric artery; okadaic acid; electrophysiology; adenosine
5'-triphosphate-sensitive potassium channel
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INTRODUCTION |
ATP-SENSITIVE POTASSIUM (KATP) channels
exhibit significant functional and pharmacological diversity, which
reflects, in part, the molecular diversity of the channel structure.
Functional KATP channels are heteromultimers composed of
four pore-forming inwardly rectifying K+ channel (Kir)
subunits of the Kir 6.0 subfamily (Kir 6.1 or 6.2) and
four regulatory sulfonylurea receptor subunits (SUR1, 2A, or 2B) (4). Based on pharmacology, tissue distribution, and expression
of recombinant KATP channels, three broad classes of KATP have been identified: pancreatic
-cell (Kir 6.2;
SUR1) (7), cardiac-skeletal muscle (Kir 6.2; SUR 2A) (8), and smooth
muscle KATP channels. The molecular composition of smooth
muscle KATP channels is unclear because Kir 6.1, Kir 6.2, SUR 1, and SUR 2B have all been identified in smooth muscle (5, 9, 21).
The functional properties of KATP channels in smooth muscle
differ substantially from those in other cell types. Notably, they
exhibit a high sensitivity to K+ channel opening drugs such
as pinacidil and levcromakalim and exhibit profound modulation by
protein kinases (1, 2, 6, 11, 17, 24, 25). Smooth muscle relaxants
[e.g., calcitonin gene-related peptide (CGRP) and
adenosine], which act through stimulation of protein kinase A
(PKA), activate KATP channels in vascular smooth muscle
[VSM; mesenteric artery (10, 18), coronary artery (15, 23),
cerebral artery (11), and nonvascular smooth muscle
(gallbladder)] (24, 25). Smooth muscle constrictors (e.g.,
neuropeptide Y, angiotensin II, serotonin, acetylcholine, and
histamine) that act through stimulation of protein kinase C (PKC) have
been shown to inhibit KATP channels in mesenteric and
cerebral arteries and esophageal and urinary bladder smooth muscle
(1-3, 6, 11, 12). Indeed, PKA and PKC modulation of
KATP channels may be a significant mechanism for
physiological and pathophysiological regulation of smooth muscle
function. Despite the apparent similarity of KATP channel
function in various types of smooth muscle, there has been little
information in terms of quantitative comparison of KATP
channels in this tissue. Uncovering diversity of smooth muscle
KATP channel function may point to avenues for the
development of smooth muscle type-selective openers of KATP
channels that could have significant clinical implications.
We are particularly interested in the regulation of KATP
channels in gallbladder smooth muscle (GBSM). Modulation of
KATP channels in GBSM could provide a novel means for
controlling gallbladder motility, which in turn would affect gallstone
formation. KATP channels within GBSM may be an important
physiological target of CGRP, which is contained within sensory fibers
in the gallbladder wall (13, 14). CGRP induces a membrane potential
hyperpolarization and relaxation of intact gallbladder that is blocked
by the KATP channel inhibitor glibenclamide (25). CGRP
causes gallbladder relaxation through activation of adenylyl cyclase,
elevation of cAMP, stimulation of PKA, and activation of
KATP channels (24). In contrast to VSM from mesenteric
artery (18), the actions of CGRP on gallbladder appear to be limited by
high levels of dephosphorylation by a phosphatase, such that
KATP currents immediately deactivate upon removal of CGRP
(24). Although activators of PKC have been shown to cause pronounced
inhibition of KATP channels in smooth muscle from arteries
(2, 11), esophagus (6), and urinary bladder (1), their effects on
gallbladder are unknown.
The goal of this study was to determine the uniqueness of
KATP channels in GBSM, with the ultimate hopes of designing
tissue-selective approaches to modulating this channel. The first
objective was to provide a pharmacological profile for key
K+ channel openers (pinacidil and diazoxide) and
glibenclamide on KATP channel currents in isolated myocytes
from gallbladder, and the second objective was to determine the effects
of activators of PKC on these currents. We found that gallbladder
KATP channels resembled those of VSM with regard to their
pharmacology. In contrast, however, gallbladder KATP
channels were much less sensitive to inhibition by PKC activators than
those in VSM and responded differently to phosphatase inhibition.
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METHODS |
GBSM cell isolation.
Guinea pigs between 2 and 4 wk old (250-350 g) and of either sex
were euthanized with halothane and exsanguinated in a manner approved
by the Institutional Animal Care and Use Committee of the University of
Vermont. Gallbladders were dissected free from the liver and placed
into ice-cold modified Krebs solution (in mM): NaCl 121, KCl 5.9, CaCl2 2.5, MgCl2 1.2, NaHCO3 25, NaH2PO4 1.2, and glucose 8 buffered to pH 7.4 with 95% O2-5% CO2. Gallbladders were
transferred to Ca2+-free cell isolation solution containing
(in mM): NaCl 55, monosodium glutamate 80, MgCl2 2, KCl 6, glucose 10, and HEPES 10 (adjusted to pH 7.3 with NaOH). Gallbladders
were cut open longitudinally and pinned mucosal side up in a
Sylgard-coated dish (Dow Corning, Midland, MI). The mucosa was removed
with blunted forceps under a dissecting microscope. The remaining
tissue was cut into small (1 × 3 mm) strips and placed into cell
isolation solution containing: 1 mg/ml BSA, 1 mg/ml papain (23 U/mg;
Worthington, Lakewood, NJ), and 1 mg/ml dithioerythritol (Sigma, St.
Louis, MO). The mixture was incubated at 37°C for 30-35 min,
and the tissue was subsequently transferred to a solution containing 1 mg/ml BSA, 1 mg/ml collagenase (1.01 U/mg; Fluka, Milwaukee, WI), and
100 µM CaCl2 for a further 8-12 min. The tissue was
rinsed in cold cell isolation solution and triturated with a glass
Pasteur pipette to yield single smooth muscle cells. Cells were stored
in glass vials on ice until required and used within 6 h of isolation.
Mesenteric artery smooth muscle cell isolation.
Female Sprague-Dawley rats (12-14 wk old) were anesthetized with
pentobarbital sodium (25 mg/kg), and the primary branch of the superior
mesenteric artery was removed and dissected free from adipose tissue in
cell isolation solution. The artery was cut open
longitudinally and then enzymatically dissociated in cell isolation
solution containing papain (0.5 mg/ml) and dithioerythritol (1 mg/ml)
at 37°C for 40 min. The tissue was subsequently transferred to
warmed cell isolation solution containing collagenase (0.7 mg/ml;
Fluka) and 100 µM CaCl2 for 10 min. The tissue was rinsed and triturated as before to produce isolated mesenteric arterial myocytes. For guinea pig mesenteric myocytes, cells were obtained as
above, except arteries were initially incubated in 1 mg/ml papain and 1 mg/ml dithioerythritol at 37°C for 30 min, and then incubated in
cell isolation solution containing collagenase (0.5 mg/ml; Sigma
blended collagenase type F), 0.5 mg/ml hyaluronidase (Worthington,
Lakewood, NJ), and 100 µM CaCl2 for 10 min.
Patch-clamp recordings.
Isolated smooth muscle cells suspended in cell isolation solution were
placed into a recording chamber (1 ml vol) on the stage of an inverted
phase-contrast microscope. Whole cell patch-clamp recordings were
carried out as previously described (24) using an Axopatch 1D amplifier
(Axon Instruments, Foster City, CA). Currents were sampled at 6.6 Hz
with a Digidata A-D board attached to an IBM PC-compatible computer
using Axotape 2 software (Axon Instruments). Electrodes were pulled
from borosilicate glass (Sutter Instruments, Novato, CA), coated with
dental wax, and fire polished to a final resistance of 3-8 M
.
Solutions and drugs.
Electrodes were filled with a solution containing (in mM): KCl 102, KOH
38, NaCl 10, MgCl2 1, CaCl2 1, EGTA 10, Na2ATP 0.1, ADP 0.1, Na2GTP 0.2, glucose 10, and HEPES 10 adjusted to pH 7.2 with KOH. Cells were bathed in external
solution containing (in mM): KCl 5, NaCl 135, MgCl2 1, HEPES 10, glucose 10, and CaCl2 0.1 (pH 7.4). When stable,
the bathing solution was exchanged for a solution with the same
composition as above, except that NaCl was substituted for KCl. All
recordings described were performed at
60 mV in symmetrical 140 mM K+.
Pinacidil (RBI, Natick, MA) and glibenclamide (RBI) were prepared as 10 mM stock solutions in 100% DMSO, and diazoxide was prepared as a 100 mM stock in DMSO. Phorbol 12-myristate 13-acetate (PMA), 5-
-phorbol
12, 13-didecanoate (5
PDD), and 1,2-dioctanoyl-sn-glycerol (DOG) were prepared as 1 mM stock solutions in DMSO. Okadaic acid and
adenosine 3',5'-cyclic monophosphothioate
(Rp-cAMPS; Calbiochem, San Diego, CA) were prepared as 100 µM
and 10 mM stock solutions in DMSO and distilled water, respectively.
Unless stated otherwise, all chemicals were purchased from Sigma.
Data analysis.
All data are expressed as the mean ± SE of n cells.
Glibenclamide-insensitive components of the current were subtracted
before analysis with a custom-written analysis program.
Glibenclamide-sensitive currents were taken as a measure of
KATP currents (24). Concentration-response relationships
were fitted with the equation I = Imax/[1 + (K/D)n]
where I is current, Imax is maximal
current, K is concentration of drug (D) required for half activation,
and n is the Hill coefficient. Unpaired Student's
t-tests were used to perform statistical analysis, and
significance was reported at the 0.05 level.
 |
RESULTS |
Pharmacological modulation of KATP currents in GBSM.
Pinacidil is reasonably selective for smooth muscle, as an activator of
KATP channels (19). Diazoxide, in contrast, is equipotent in activating KATP channels in smooth muscle and pancreatic
-cells, but is rather ineffective on KATP channels in
cardiac and skeletal muscle. The effects of these pharmacological
fingerprints are unknown for KATP channels in GBSM. We
therefore tested the effects of pinacidil and diazoxide, as well as the
classic inhibitor, glibenclamide, on KATP currents in GBSM.
Whole cell KATP currents were measured at a holding
potential of
60 mV in symmetrical 140 mM K+. Cells
were dialyzed with a pipette solution containing 0.1 mM ATP, 0.2 mM
GTP, and 0.1 mM ADP. Under these conditions, pinacidil activated
KATP currents in gallbladder myocytes in a
concentration-dependent manner between 0.1 and 30 µM (Fig.
1A), with a Hill
coefficient of 1.7 and EC50 of pinacidil at 1.4 µM
(n = 6; Fig. 1B). The KATP channel opener
diazoxide was a less potent activator of KATP channels than
pinacidil (Fig. 2A). The
EC50 concentration for diazoxide calculated from the mean
concentration response data was 97.5 µM, and the Hill coefficient was
1.3 (n = 6 cells; Fig. 2B). Glibenclamide inhibited
KATP currents elicited by 10 µM pinacidil with an
IC50 concentration of 77.6 nM and a Hill coefficient
of 0.94 (n = 6 cells; Fig. 3,
A and B).

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Fig. 1.
Pinacidil activates KATP currents in gallbladder smooth
muscle (GBSM) cells. A: pinacidil activated
glibenclamide-sensitive currents in gallbladder myocytes. Dotted line
represents 0 current level. All currents were recorded at a holding
potential of 60 mV and in symmetrical 140 mM K+.
B: concentration-response curve for activation of
glibenclamide-sensitive currents by pinacidil (mean ± SE, n = 6). EC50 concentration of pinacidil, calculated from mean
concentration response curve, was 1.4 µM, and Hill coefficient
was 1.7.
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Fig. 2.
Diazoxide activates KATP currents in GBSM. A:
diazoxide activated glibenclamide-sensitive currents in gallbladder
myocytes. Dotted line indicates 0 current level. Currents were recorded
at a holding potential of 60 mV and in symmetrical 140 mM
K+. B: concentration-response curve for activation
of glibenclamide-sensitive currents by diazoxide (mean ± SE,
n = 6). EC50 concentration for diazoxide was 97.5 µM, and Hill coefficient was 1.3.
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Fig. 3.
Glibenclamide inhibits KATP currents in GBSM. A:
glibenclamide inhibited KATP currents in gallbladder
myocytes. Currents were recorded at 60 mV in symmetrical 140 mM
K+. Dotted line indicates 0 current level. B:
concentration-dependent inhibition curve illustrating inhibition of
KATP currents by glibenclamide (mean ± SE, n = 6). IC50 concentration for glibenclamide was 77.6 nM, and
Hill coefficient was 0.94.
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Activators of PKC inhibit KATP currents in smooth muscle
cells from gallbladder and mesenteric artery.
In vascular, urinary bladder, and esophageal smooth muscle, activators
of PKC inhibit pinacidil-induced KATP channel currents (1,
2, 6, 16, 20). The consequence of PKC stimulation on GBSM
KATP channels is not known. The activators of PKC, PMA (100 nM), and the membrane-permeable analog of diacylglycerol, DOG (1 µM),
have been shown to inhibit KATP currents in rabbit mesenteric artery by 86% and 87%, respectively (2). Similarly, 100 nM
PMA inhibited KATP currents in urinary bladder smooth
muscle by 87% (1). Figure 4, B and
C, illustrate inhibition of KATP currents in guinea
pig mesenteric artery myocytes. KATP currents were
inhibited by 22.9 ± 6.4% (n = 6) with 100 nM DOG and 62.7 ± 5.9% (n = 6) with 1 µM DOG, respectively.
KATP currents were also inhibited by 100 nM (36.5 ± 3.1%, n = 5) and 1 µM (84.4 ± 2.9%, n = 6) DOG in rat mesenteric arteries. In contrast, 100 nM DOG had no
significant effect on KATP currents in gallbladder myocytes
(44.9 ± 5.3 pA; control, 45.6 ± 6.3 pA; 100 nM DOG, n = 6).
Increasing the DOG concentration to 1 µM, however, did inhibit gallbladder KATP currents by 34.5 ± 10% (n = 6;
Fig. 4, A and C).

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Fig. 4.
The protein kinase C (PKC) activator, 1,2-dioctanoyl-sn-glycerol (DOG),
inhibits KATP channels in gallbladder and mesenteric artery
smooth muscle cells. A: DOG (1 µM) elicited a modest
inhibition of pinacidil (Pin)-evoked KATP currents in
gallbladder myocytes. B: DOG (1 µM) produced a substantial
inhibition of pinacidil-evoked KATP currents in guinea pig
mesenteric arterial myocytes. C: summary showing PKC activator
DOG is less effective at inhibiting KATP currents in
gallbladder myocytes than in mesenteric artery myocytes.
* Statistical significance (P < 0.05) gallbladder vs.
mesenteric artery; Glib, glibenclamide.
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PMA (100 nM) was also less effective in GBSM than VSM, reducing
currents in gallbladder myocytes by only 45.7 ± 4% (n = 5; Fig. 5), compared with almost complete
inhibition of KATP currents in VSM (2, 11). The
biologically inactive phorbol ester 4
PDD had no measurable effect
(n = 3) in GBSM, suggesting that the inhibition was mediated
through PKC activation (Fig. 5). These results suggest that
KATP channels in GBSM are less sensitive to modulation by
activators of PKC than those in VSM.

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Fig. 5.
The PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibits
KATP currents in GBSM cells. A: PMA (100 nM)
inhibited KATP currents in gallbladder myocytes. Inactive
phorbol ester 4- -phorbol 12,13-didencanoate (4 PDD) had no
significant effect. Dotted line is 0 current level, and recordings were
performed at 60 mV.
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Actions of phosphatases on KATP currents in GBSM and
VSM.
Decreased KATP inhibition by PKC activators in GBSM could
be due to a disruption in the PKCsignaling pathway as a consequence of
differences in KATP channel structure or expression of
different PKC isoforms, or, alternatively, could occur if the action of PKC is counteracted by rapid dephosphorylation by phosphatases. Rapid
dephosphorylation by phosphatases is supported by the observation that
the deactivation of GBSM KATP currents after removal of
activators of PKA is rapid (24), compared with VSM (18).
To test the hypothesis that phosphatase activity plays an important
role in the regulation of KATP channels, the ability of DOG
to inhibit KATP currents in the presence of okadaic acid
was examined in gallbladder and mesenteric artery myocytes. DOG-induced inhibition of KATP currents was significantly enhanced in
GBSM by okadaic acid, from 3.4 ± 5.8% (n = 5) to 29.9 ± 9.0% (n = 4) with 100 nM DOG, and from 34.5 ± 10%
(n = 5) to 65.2 ± 7.4% (n = 6) with 1 µM DOG (Fig.
6, A and C). Similarly,
okadaic acid increased inhibition of pinacidil-induced KATP
currents by 100 nM DOG in rat mesenteric artery smooth muscle from 36.5 ± 3.1% to 69.4 ± 2% (n = 5), and from 22.9 ± 6.4%
(n = 6) to 56.6 ± 15% (n = 3) in guinea pig
mesenteric arteries (Fig. 7, A and
B). These results suggest that phosphatases play an important
role in regulating the activity of KATP channels in smooth
muscle.

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Fig. 6.
Phosphatase inhibition in GBSM. A: okadaic acid (OA; 1 µM)
enhanced KATP currents in gallbladder myocytes and
increased inhibition of these currents by PKC activator DOG (1 µM).
Dotted line indicates 0 current level. B: OA (1 µM) activated
a glibenclamide-sensitive current in absence of pinacidil in
gallbladder myocytes. C: summary showing OA (1 µM) increased
inhibition of KATP currents by PKC activator DOG (100 nM
and 1 µM) in GBSM. * Statistical significance (P < 0.05)
inhibition by DOG in presence of OA vs. in absence of OA. D:
OA-induced activation of glibenclamide-sensitive currents was inhibited
by protein kinase A inhibitor adenosine 3',5'-cyclic
monophosphothioate (Rp-cAMPs) in gallbladder myocytes.
* Statistical significance (P < 0.05).
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Fig. 7.
Phosphatase inhibition in mesenteric artery smooth muscle. A:
OA (1 µM) induced a modest inhibition of glibenclamide-sensitive
current in guinea pig mesenteric artery and increased inhibition of
these currents by PKC activator DOG (100 nM). B: summary
showing OA (1 µM) increased inhibition of KATP currents
by PKC activator DOG (100 nM) in guinea pig mesenteric artery myocytes.
* Statistical significance (P < 0.05) inhibition by DOG in
presence of OA vs. in absence of OA.
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In addition to enhancing inhibition by PKC activators, application of
okadaic acid alone increased KATP currents in gallbladder myocytes. In the presence of pinacidil, okadaic acid caused a 51.6 ± 10.5% (n = 10) increase in the glibenclamide-sensitive inward
current in GBSM (Fig. 6A). In the absence of pinacidil, KATP currents were increased from 10.0 ± 4.6 pA in
control to 37.55 ± 13.1 pA (n = 4) with 1 µM okadaic acid
(Fig. 6B).
We investigated the possibility that this effect was a consequence of
phosphatase inhibition. KATP channels are under the dual
regulation of PKA and PKC (2, 10, 11, 16), being inhibited by PKC
activators and stimulated by PKA activators. Activators of
cGMP-dependent protein kinase (PKG) have no effect on
glibenclamide-sensitive currents in VSM (18, 23). Blocking phosphatases
should increase the sensitivity of KATP channels to both
PKC and PKA activators. To investigate the possibility that the
increase in the glibenclamide-sensitive current resulted from enhanced
PKA activity, the effect of the PKA inhibitor Rp-cAMPs (100 µM) on KATP currents was examined. Rp-cAMPs had
no observable effect on the amplitude of pinacidil-evoked
KATP currents but dramatically reduced the okadaic
acid-evoked increase in the glibenclamide-sensitive current (Fig.
6D). These results suggest that the okadaic acid-induced increase in current was the result of enhanced PKA activation. In
contrast to GBSM, okadaic acid induced a modest reduction [26 ± 7.2% (n = 3)] in the glibenclamide-sensitive current in
guinea pig mesenteric arteries but had no significant effect in rat
mesenteric arteries [control 34.8 ± 3.8 pA, okadaic acid 30.4 ± 5.3 pA (n = 5)].
 |
DISCUSSION |
Pharmacology of GBSM KATP channels.
GBSM KATP channels resemble those of VSM with regard to
their sensitivity to pinacidil, diazoxide, and glibenclamide. Pinacidil and diazoxide activated KATP currents in gallbladder
myocytes with EC50 values of 1 µM and 97 µM, and Hill
coefficients of 1.7 and 1.3, respectively. These values are similar to
those reported in rabbit mesenteric artery smooth muscle cells where
pinacidil activated KATP currents with an EC50
of 1 µM and a Hill coefficient of 1.5 (19), and rat colonic smooth
muscle where pinacidil activated KATP currents with an
EC50 of 1.3 µM (17). Diazoxide has been shown to open
KATP channels in rabbit mesenteric artery smooth muscle
with an EC50 of 37 µM and a Hill coefficient of 1.0 (19) and in myocytes isolated from rat colon with an EC50 of
34.2 µM (17). In this study, glibenclamide inhibited gallbladder
KATP currents with an IC50 of 77.6 nM and a
Hill coefficient of 0.9, which is also comparable to the
IC50 of 100 nM and the Hill coefficient of 0.8 reported for
glibenclamide in VSM (19). The K+ channel openers pinacidil
and diazoxide, and glibenclamide, are thought to bind to the SUR
subunit of the KATP channel to modulate channel activity.
This is supported by observations showing that recombinant
KATP channels expressing different SUR subunits differ with
regard to their sensitivity to pinacidil, diazoxide, and glibenclamide
(7, 9). Mutations that disrupt the integrity of two nucleotide binding
folds (NBF1 and NBF2) of the SUR subunit abolish sensitivity of
recombinant KATP channels to K+ channel-opening
drugs (21). Kir 6.2 channels without SUR do not exhibit
sensitivity to K+ channel-opening drugs (22), consistent
with SUR being the target of these drugs. We have demonstrated here
that KATP channels in GBSM have similar sensitivities to
pinacidil, diazoxide, and glibenclamide as those in VSM and colon,
suggesting that SUR is likely to be conserved among smooth muscle
types. The molecular identity of SUR in smooth muscle, however, is
unclear, because mRNA for both SUR1 and SUR 2B are found in VSM (3). In
terms of their sensitivity to K+ channel openers and to
glibenclamide, however, smooth muscle KATP channels
resemble recombinant Kir 6.2/SUR 2B channels (9).
Inhibition of smooth muscle KATP channels by PKC: role
of phosphatases.
KATP channels in VSM are regulated by PKA and PKC.
Vasoconstrictors such as histamine, serotonin, angiotensin II,
acetylcholine, and neuropeptide Y that stimulate PKC cause a reduction
in KATP channel activity (2, 11, 12), whereas vasodilators
that stimulate PKA, such as CGRP and adenosine, activate
KATP channels (10, 15, 18). Nitrovasodilators that activate
PKG such as nitric oxide and sodium nitroprusside have not been
demonstrated to activate KATP currents in VSM (18, 23). In
non-VSM of the urinary bladder and esophagus, KATP channels
are inhibited by activators of PKC (1, 6). The effects of PKC
activators on KATP channels in GBSM were previously not known.
We have demonstrated here that GBSM KATP channels are less
sensitive to inhibition by PKC activators than those in either guinea
pig or rat mesenteric artery, suggesting there may be differences in
KATP channel modulation in GBSM and VSM. The phosphatase
inhibitor, okadaic acid, increased the effectiveness of PKC activators
to inhibit KATP currents in GBSM and VSM, suggesting that
phosphatases play an important role in regulating the activity of
KATP channels. However, even in the presence of okadaic
acid, PKC activators were more effective at inhibiting KATP
channels in myocytes from mesenteric arteries than from gallbladder.
This observation suggests that other factors in addition to
phosphatases are responsible for the apparent difference in PKC
activator efficacy.
Phosphatase inhibition activated a glibenclamide-sensitive current in
GBSM that was blocked by the PKA inhibitor Rp-cAMPs (Fig. 6,
A and D). In contrast, phosphatase inhibition by
okadaic acid did not increase KATP currents in myocytes
from mesenteric arteries, but, in fact, slightly decreased the
KATP current (Fig. 7A). One explanation of these
results is that PKA activation of KATP currents is ongoing
in GBSM, but not in VSM, and phosphatase inhibition enhances this
activation by decreasing dephosphorylation of the PKA site. Our results
suggest that, under physiological conditions, KATP channel
activity is dependent on a balance between phosphorylation by PKA and
PKC, and dephosphorylation of their respective phosphorylation sites by phosphatases.
It is unknown what sites on KATP channels or other unknown
regulatory proteins are phosphorylated by PKA and PKC to cause physiological effects. There are several putative PKC phosphorylation sites on both SUR and Kir. Interestingly, there are three putative PKC
phosphorylation sites at the COOH-terminal region of Kir 6.1 that are
absent in Kir 6.2, making the Kir subunit a possible phosphorylation
target of PKC, and may explain why there are tissue-specific differences in regulation of KATP channels by PKC
activators. Similarly, there are four putative PKA phosphorylation
sites on the KATP channel, two on SUR, and two on Kir.
In conclusion, we have demonstrated that gallbladder
KATP channels have a similar pharmacological profile
as VSM KATP channels with respect to their
sensitivity to pinacidil, diazoxide, and glibenclamide. Furthermore,
our results support a key role of phosphatases in the regulation of
KATP channel activity in GBSM and VSM.
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ACKNOWLEDGEMENTS |
We thank Gerald Herrera and George Wellman for critical appraisal
of the manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of Neurological Disorders
and Stroke Grant NS-26995, National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-45410, and National Heart, Lung, and Blood
Institute Grant HL-44455.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. M. Mawe, Dept. of Anatomy and Neurobiology, College of Medicine, Univ. of
Vermont, Burlington, VT 05405 (E-mail:
gmawe{at}zoo.uvm.edu).
Received 2 August 1999; accepted in final form 22 December 1999.
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