1 Abteilung Pharmakologie für Pharmazeuten and 3 Frauenklinik, Universitäts-Krankenhaus Eppendorf, D-20246 Hamburg; and 2 Institut für Pharmakologie und Toxikologie der Technischen Universität München, D-80802 Munich, Germany
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ABSTRACT |
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We investigated the influence of pregnancy on large-conductance calcium-activated potassium channel (BKCa) activity (NPo) and on channel expression in membranes of isolated human myometrial smooth muscle cells. NPo in inside-out patches was higher in pregnant myometria (PM) compared with nonpregnant myometria (NPM), and the half-maximal activation potential was shifted by 39 mV to more negative potentials. This effect was not due to an enhanced BKCa channel expression. In the presence of cAMP kinase (PKA) or cGMP kinase (PKG), NPo increased in patches from PM but decreased in those from NPM. Western blot analysis and use of a specific PKG inhibitor (1 µM KT-5823) verified the existence of a partially active membrane-associated PKG. Inhibition of PKA by 100 nM PKI, the inhibitory peptide of PKA, had no effect on NPo. 8-p-Chlorophenylthio-cGMP (8-pCPT-cGMP) hyperpolarized cells from PM. This effect was abolished by iberiotoxin, a specific blocker of BKCa channels. It is concluded that an endogenous, membrane-bound PKG in myometrial cells specifically enhances BKCa channel activity during pregnancy and thus may contribute to uterine quiescence during pregnancy.
large-conductance calcium-activated potassium channels; cyclic nucleotide-regulated protein kinases; protein kinase inhibitors; human myometrium; pregnancy maintenance
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INTRODUCTION |
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THE NONPREGNANT
MYOMETRIUM (NPM) contracts in response to stretch. During uterine
distension that accompanies fetal growth, however, myometrial smooth
muscle remains remarkably quiescent. Although the mechanism responsible
for this physiological adaptation is not clearly defined, it has been
proposed that hormonally induced changes in cell-to-cell coupling
(29) and in cellular cyclic nucleotide levels may serve to
facilitate uterine quiescence (22, 30, 40). Although the
precise mechanism of cyclic nucleotide-induced relaxation remains
unclear, possible targets of phosphorylation by cAMP kinase (PKA) and
cGMP kinase (PKG) include phospholamban, Ca2+-ATPase, inositol 1,4,5-trisphosphate production and
action, and contractile proteins such as myosin light-chain kinase
(34). Through phosphorylation of these proteins, cAMP and
cGMP are thought to cause a decrease in cytosolic Ca2+
([Ca2+]i) and in the Ca2+
sensitivity of contractile proteins. Another mechanism by which cAMP
and cGMP may promote relaxation of smooth muscle cells is membrane
hyperpolarization as a consequence of K+ channel
activation. Myometrial smooth muscle cells are richly endowed with
large conductance, voltage-dependent and Ca2+-sensitive
K+ (BKCa) channels (31) and their
dependence on [Ca2+]i and depolarization
provokes the suggestion that they may function as negative feedback
regulators of [Ca2+]i by hyperpolarizing the
membrane potential in close proximity to voltage-activated
Ca2+ channels. BKCa channel activity is under a
complex metabolic control that may involve G proteins (17)
and a balance between phosphorylation-dephosphorylation mechanisms
catalyzed by different classes of protein kinases and phosphatases
(20, 46). cAMP has been shown to phosphorylate via cAMP
kinase (PKA) BKCa channels (26) and to
increase their activity specifically in the pregnant myometrium (PM)
(30), and it has been suggested that this mechanism contributes to the tocolytic action of
2-adrenergic agonists in preterm laboring women. Nitric
oxide (NO) produced from nerve plexuses, decidua (27), or
resident cells of the myometrium increases the cellular cGMP level and
is thought to modulate myometrial function. Carbon monoxide, another
cGMP elevating agent, also has been proposed as an endogenous inhibitor
of myometrial contractility during pregnancy, since expression of
carbon monoxide-generating hemeoxygenases in the myometrium is
stimulated by progesterone (1). These findings suggest a
role of cGMP for uterine quiescence during pregnancy. The relief of
cGMP action may lead to retreat from uterine quiescence with the onset
of spontaneous uterine contractions of labor (36). In
fact, cGMP levels are elevated during pregnancy (5, 13,
43), whereas NO and cGMP levels are transiently downregulated at
delivery (25). In turn, the application of the NO donor
glyceryl trinitrate to laboring women induced cessation of uterine
contractions (7, 10, 19). Although it is well established
that the downstream effector of cGMP, the PKG, activates
BKCa channel activity in various smooth muscle cells
(4, 32, 46), its action on BKCa channels in human NPM and PM is presently unknown. The observations made in this
study provide a new insight into the mechanism involved in the
maintenance of uterine quiescence during pregnancy and may have
therapeutic relevance in preventing preterm labor.
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METHODS |
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Materials and drug preparation. The catalytic subunit of PKA and the active fragment of PKG were prepared as described (46). cAMP-dependent protein kinase peptide inhibitor was obtained from Promega (Madison, WI), iberiotoxin (IbTX) was from Latoxan (Rosans, France), KT-5823 was from Biomol (Hamburg, Germany), and 4-aminopyridine was from Sigma (Deisenhofen, Germany). 8-p-Chlorophenylthio-cGMP (8-pCPT-cGMP) was purchased from Biolog (Bremen, Germany). NS-1619 was a generous gift from Dr. Olesen, Department of Cellular Biology, NeuroSearch, Glostrup, Denmark. Collagenase (lot 17449) was from Serva (Heidelberg, Germany), and protease (type XIV) was from Sigma (Deisenhofen, Germany). All drugs were dissolved in physiological saline solution (PSS; see Solutions); solutions with IbTX contained 0.1% bovine albumin fraction V (Sigma).
Tissue collection and preparation. Myometrial samples were obtained from nonlaboring women (37-39 wk gestation) undergoing elective cesarean sections. No tocolytic agents had been administered to the mother 24 h prior to cesarean delivery. The reasons for cesarean section included breech presentation, previous cesarean section, and cephalopelvic disproportion. All biopsies were taken from the upper border of the lower isthmic uterine incision. NPM was obtained from uteri of normal cycling premenopausal patients undergoing hysterectomy for benign disease. Single myometrial smooth muscle cells were enzymatically isolated as described previously (47). Experiments were conducted within 6 h after cell isolation. Informed consent was obtained from the patients, and the study had the approval of the Ethics Committee, Hamburg, Germany.
Recording techniques.
Standard patch-clamp recording techniques were used to measure
single-channel or macroscopic currents in the inside-out patch configuration (11). In some experiments membrane
potentials were measured in the whole cell current-clamp mode. Patch
electrodes were fabricated from borosilicate glass capillaries (World
Precision Instruments) and filled with prefiltered solutions of
different compositions (see Solutions). Currents were
recorded at 25°C with a List Electronics model EPC-7 patch-clamp
amplifier, connected via a 16-bit analog-to-digital interface to a
Pentium IBM clone computer. The data were filtered at 1 kHz by a
10-pole Bessel filter and sampled at 3 kHz. Data acquisition and
analysis was performed with an ISO-3 multitasking patch-clamp program
(MFK, Niedernhausen, Germany). The pipette resistance ranged from 2 to
3 M in whole cell and 8 to 9 M
for the excised-patch experiments. The amplitude of single-channel currents was derived from an amplitude distribution histogram. Determination of the average channel open probability (NPo) in patches has been described
elsewhere (43). After the patch had been equilibrated for
at least 5 min, drugs were superfused for another 5 min before the mean
NPo was determined. Depending on the holding
potential, mean NPo was calculated from continuous records of channel activity sampled over a period of 2-5 min. The cell or patch under investigation was studied during continuous superfusion by a gravity-flow microperfusion device consisting of multiple glass tubes assembling into a common tip with a
80-µm orifice and mounted on a hydraulic micromanipulator. Solution
changes were achieved via remote-controlled solenoid valves (N. Graf,
Planegg, Germany). With the exception of current-clamp experiments, all
drugs were applied to the cytoplasmic side of the patch.
Solutions. For inside-out experiments, the bath solution (cytosolic surface of the patch) contained (in mM) 134 KCl, 6 NaCl, 1.2 MgCl2, 5 EGTA, 11 glucose, 3 dipotassium ATP, and 10 HEPES (pH 7.4), and the patch pipette (extracellular patch surface) was filled with (in mM) 140 KCl and 10 HEPES (pH 7.4). Depending on the experiment, the free Ca2+ concentration was changed from 300 nM to 3 µM by changing the Ca2+ concentration in the corresponding solution. The appropriate amounts of CaCl2 were added, and the pH adjusted according to a computer program (24) on the basis of the binding constants of Fabiato (8) and checked by fura 2 fluorescence.
For whole cell (current-clamp) experiments, the intracellular (pipette) solution contained (in mM) 134 KCl, 6 NaCl, 1.2 MgCl2, 5 EGTA, 11 glucose, 3 dipotassium ATP, and 10 HEPES (pH 7.4). The free Ca2+ concentration was 300 nM. The bath was superfused with PSS containing (in mM) 127 NaCl, 5.9 KCl, 2.4 CaCl2, 1.2 MgCl2, 11 glucose, and 10 HEPES adjusted to pH 7.4 with NaOH.Preparation of plasma membranes. Myometrial plasma membranes were isolated as described (37). All solutions and procedures used in the preparation of membranes were at 4°C. In brief, pieces of 400-500 mg and 200-350 mg (in total 2.7 g each) of myometrial muscle were separately pooled from 6 nonpregnant and 10 pregnant women (37-39 wk gestation), respectively, washed twice in buffer A [5 mM MOPS-Tris buffer, pH 7.4, containing 155 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 0.1% glucose, 1 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.2 µg/ml leupeptin] and twice in buffer B (20 mM MOPS-Tris buffer, pH 7.4, containing 8% sucrose, 1 mM benzamidine, 0.2 mM PMSF, and 0.2 µg/ml leupeptin). The two pools of myometrial muscle were homogenized in buffer B using an Ultra Turrax. Homogenates were filtered through two layers of cheesecloth and centrifuged for 20 min at 7,000 g. Supernatants were removed, filtered as aforementioned, and centrifuged for 1 h at 130,000 g. The resulting microsomal membranes were suspended in buffer B and loaded onto a discontinuous sucrose gradient [30% and 40% (wt/vol) sucrose in 20 mM MOPS-Tris buffer, pH 7.4, containing 1 mM benzamidine, 0.2 mM PMSF, and 0.2 µg/ml leupeptin]. The gradient was centrifuged at 160,000 g for 1 h in a Beckman model SW56 rotor. Membranes sedimenting at the interface between the 8% and 30% sucrose layer were collected, diluted in buffer C (20 mM MOPS-Tris buffer, pH 7.4, containing 160 mM NaCl, 1 mM benzamidine, 0.2 mM PMSF, and 0.2 µg/ml leupeptin), and centrifuged at 160,000 g for 1 h. The resulting pellet was resuspended in buffer C, aliquoted, and rapidly frozen. Membrane protein concentration was determined according to Lowry.
Preparation of total cell lysate. Frozen myometrial tissue (120 mg) from individual nonpregnant and pregnant women was powdered in a precooled mortar in the presence of liquid nitrogen. Tissue powder was dissolved in 1× Laemmli buffer at 95°C (18), boiled for 3 min, and centrifuged at 12,000 g to remove insoluble material. Protein concentration in the supernatant was determined according to Lowry.
Immunoblots.
Proteins in plasma membranes and in total muscle homogenates were
separated by SDS-PAGE (7.5%). Proteins were transferred to Immobilon P
and decorated with affinity-purified antibodies directed against a
peptide from -bSlo (amino acids 913-926) (15),
-bSlo (amino acids 118-133) (16), or the common
region of PKG type I
and I
(33). Immunoblots were
developed using a peroxidase-coupled goat-anti-rabbit IgG antibody
(Dianova, Hamburg, Germany) and the ECL reagent from Amersham
(Göttingen, Germany). Relative amounts of BKCa
channel subunits and PKG were quantified using the TINA software from
Raytest. Quantification was obtained from blots in which densitometric
units were linear with respect to amounts of purified PKG standards.
Statistics. Origin for Windows (version 6; Microcal Software, Northampton, MA) was used for statistical analyses. Significance was determined by either paired t-test or one-way ANOVA for repeated measurements on the same patch. When a significant effect was detected with ANOVA, Student's t-test was used for pair-wise comparisons. P < 0.05 was considered statistically significant. Data are expressed as means ± SE.
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RESULTS |
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In inside-out patches of freshly isolated smooth muscle cells from
human NPM and PM, BKCa channels were identified by their high conductance, their voltage dependence, and their sensitivity to
Ca2+ and IbTX (data not shown). Although basic channel
properties such as single-channel conductance did not differ between PM
and NPM cells, the open probability (NPo) of
BKCa channels was always higher in patches from PM cells
(see Fig. 2 and following). To determine whether an increase in
available BKCa channel number was responsible for the
enhanced NPo, macroscopic currents were examined
in inside-out patches from NPM and PM cells exposed to 3 µM
[Ca2+]i. The contribution of delayed
rectifier potassium channels to the macroscopic current was minimized
by holding the patches at 0 mV and by including 5 mM 4-aminopyridine in
the patch pipette. Figure 1A
shows the respective conductance-voltage relationships calculated from
the macroscopic currents obtained at voltages between 160 and +120
mV. With 3 µM Ca2+ on the cytosolic side, the
half-maximal activating voltage (V1/2) in six
NPM patches was
20.0 ± 3.2 mV, and channel opening increased e-fold per 14.9 mV to a maximum of 7.2 ± 0.9 nS. In 10 PM patches, there was a shift in V1/2 to
59.3 ± 6.3 mV, whereas the slope factor (16.3 mV) and the
maximum conductance (7.3 ± 0.8 nS) were unchanged. Thus during
pregnancy the V1/2 of BKCa channels
is shifted by 39 mV to the left, whereas the available channel number and the voltage-dependent activation of these channels remained unchanged. The conclusion of an unchanged channel number was supported by Western blot analysis of membranes derived from NPM and PM specimens, using antibodies specific for the 125-kDa pore-forming
-subunit and the 31-kDa regulatory
-subunit of the
BKCa channel. As shown in Fig. 1B, the
respective
- and
-subunits could be clearly identified in the
membranes of both tissues. Densitometric evaluation of the
immunosignals revealed a nearly unchanged expression of the
pore-forming
-subunit in pregnancy (95% in PM compared with NPM),
whereas the expression of the
-subunit in PM membranes was only 66%
of that measured in NPM membranes. Note that the immunosignals of Fig.
1B represent the mean expression of BKCa channel
subunits from 10 PM and 6 NPM, respectively (for further details see
METHODS).
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BKCa channels are regulated by cyclic nucleotide-dependent
protein kinases. Superfusion of the cytosolic side of inside-out patches with the active fragment of PKG (300 nM) or the catalytic subunit of PKA (300 nM) decreased BKCa channel activity in
NPM cells but increased channel activity in PM cells. This
activation/inhibition pattern by protein kinases is shown in Fig.
2 at potentials varying between 60 and
+80 mV. As can be further seen from Fig. 2, the protein kinases were
not equally effective; PKA decreased NPo in
patches from NPM cells more effectively than PKG (Fig. 2, A and B), whereas PKG enhanced NPo in
patches from PM cells much stronger than PKA (Fig. 2, C and
D). At a holding potential of +40 mV, PKA and PKG decreased
NPo by 67 ± 4 and 49 ± 5% in seven NPM cells, respectively, but increased NPo by
96 ± 13 and 183 ± 19% in seven PM cells, respectively. All
experiments shown in Fig. 2 were carried out at a
[Ca2+]i of 0.3 µM. As has been described
before (30, 47), 15% of NPM and 18% of PM patches
exhibited responses to the kinases opposite to those shown in Fig. 2.
These patches were not further considered in this study. To test
whether endogenous protein kinases were contributing to
BKCa channel activity in excised membranes, 1 µM KT-5823,
a specific inhibitor of PKG at this concentration, and the inhibitory
peptide of PKA, 100 nM PKI, were applied to the cytosolic surface of
inside-out patches. The original traces in Fig.
3 show that at a holding potential of +40
mV, 1 µM KT-5823 increased NPo in the NPM cell
from 0.08 (control) to 0.13 (Fig. 3A) and decreased
NPo in the PM cell from 0.4 (control) to 0.14 (Fig. 3C). A summary of the results with KT-5823 is
presented in Fig. 3, B and D; KT-5823 increased
NPo in NPM cells by 64% (before, 0.14 ± 0.01; after KT-5823, 0.23 ± 0.01; n = 11) and decreased NPo in PM cells by 55% (before
0.31 ± 0.03; after KT-5823, 0.17 ± 0.02; n = 11). To further validate the role of a membrane-associated PKG
intrinsically modulating BKCa channel activity, inside-out patches were superfused at their cytosolic surface with 500 µM 8-pCPT-cGMP, a specific and potent activator of PKG. As shown in Fig.
4, 8-pCPT-cGMP decreased
NPo from 0.08 (control) to 0.05 in the NPM cell
(Fig. 4A) and increased NPo from 0.36 (control) to 0.67 in the PM cell (Fig. 4C). Additional
superfusion of the patch with 1 µM KT-5823 not only reversed the
effects of the cGMP derivative but significantly (P < 0.05) increased or decreased NPo beyond the
respective control values in NPM and PM cells. A summary of the results
obtained with 8-pCPT-cGMP is shown in Fig. 4, B and
D; 8-pCPT-cGMP decreased NPo in NPM
cells by 44% (before, 0.16 ± 0.03; after 8-pCPT-cGMP, 0.09 ± 0.01; n = 7) and increased
NPo in PM cells by 82% (before, 0.38 ± 0.05; after 8-pCPT-cGMP, 0.69 ± 0.07; n = 8).
Additional superfusion of patches with 1 µM KT-5823 resulted,
relative to the control values, in an increase of
NPo by 31% (0.21 ± 0.03; Fig.
4B) in NPM cells and in a decrease of
NPo by 42% (0.22 ± 0.02; Fig.
4D) in PM cells. Activation and inhibition of
BKCa channels by 8-pCPT-cGMP was preserved at various membrane potentials ranging from
20 to +80 mV (data not shown). The
electrophysiological data obtained with the cGMP derivative and KT-5823
are compatible with the view that an endogenous PKG exhibiting basal
activity is present in excised patches and, being closely associated
with the BKCa channel protein, regulates its activity in
opposite directions in NPM and PM cells. Because of the physiological
relevance of these results, expression of PKG in membranes and in total
cell lysates of NPM and PM was examined by Western blot analysis.
Utilizing an antibody against the common region of PKG type I, a single
immunoreactive species of 78 kDa was detected in all tissues. As shown
in Fig. 5A, immunosignals from
membranes pooled from 10 PM and 6 NPM, respectively, disclosed a lower
density of PKG in PM membranes. Densitometric evaluation showed that
the relative density of PKG in PM membranes was 69% of that in NPM
membranes. When the levels of PKG were evaluated in total cell lysates
from individual NPM and PM specimens, respectively, the mean density in
PM cell lysates was 46 ± 5.1% (n = 3) of that in
NPM cell lysates (Fig. 5B). These findings indicate that the overall expression of PKG declines during pregnancy. This decrease, however, is smaller in the membrane than in the total cell lysate.
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To investigate the role of endogenous PKA in BKCa channel
regulation, inside-out patches were superfused with the PKI. The original recordings in Fig. 6 demonstrate
that in patches from an NPM (Fig. 6A) or a PM cell (Fig.
6B), PKI (100 nM) had a marginal inhibitory effect on
BKCa channel activity. This effect was statistically not
significant when the results from six additional patches were combined
(Figs. 6, B and D). As further shown in Fig. 6,
B and D, simultaneous application of 100 nM PKI
plus 500 µM 8-pCPT-cGMP decreased NPo from
0.15 ± 0.02 (control) to 0.09 ± 0.01 in NPM cells
(n = 7) and increased NPo from
0.34 ± 0.04 (control) to 0.64 ± 0.07 in the patches from PM
cells (n = 7).
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BKCa channels have been implicated in the regulation of
membrane resting potential. When smooth muscle cells from human PM were
superfused with 300 µM 8-pCPT-cGMP, hyperpolarization of the membrane
resting potential by 12 (Fig.
7A) and 10 mV (Fig. 7B) was observed. Because hyperpolarization was completely
abolished by the additional application of 100 nM IbTX,
BKCa channels must have been involved in this membrane
effect (Fig. 7B). The abolition of the hyperpolarization by
1 µM KT-5823 (Fig. 7A) indicates that the effect of the
cGMP derivative was exclusively due to activation of PKG. For
comparison, Fig. 7C shows the marked IbTX-sensitive hyperpolarization of 32 mV, which became apparent when a PM cell was
superfused with 30 µM NS-1619, a benzimidazolone derivative which
activates BKCa channels directly (28).
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DISCUSSION |
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The main finding of this investigation is that the basal open probability of BKCa channels is enhanced in the human PM. This effect is due to a membrane-bound and channel-associated PKG that activates BKCa channel activity during pregnancy but inhibits it during the nonpregnant state. This mechanism may contribute to uterine quiescence, which is important to maintain pregnancy despite progressive stretch of the myometrium by the rapidly growing fetus.
Myometrial smooth muscle cells are richly endowed with BKCa
channels (3, 31, 47). Because BKCa channels
are sensitive to both Ca2+ and voltage, it has been
postulated that these channels might be involved in the membrane
repolarization that follows depolarization and the accompanying
increase in cytosolic free Ca2+ during an action potential
(14, 29). By utilizing several experimental approaches
such as measurement of [Ca2+]i, membrane
potential, and macroscopic and single-channel currents, evidence has
been presented in a human myometrial cell line that BKCa
channels play an important role in the regulation of membrane potential
and [Ca2+]i (3). The present
study supports this finding. NS-1619, a direct activator of
BKCa channel activity (28), produced a marked hyperpolarization in a smooth muscle cell from the human PM (see Fig.
7C). The membrane electrical and contractile properties of the myometrium vary markedly during the normal ovulatory cycle and
pregnancy, presumably as a consequence of hormonal influences (29). Distension of the NPM causes reflex contraction, and
specific mechanisms must therefore exist in the PM to maintain
electrical and mechanical quiescence despite the increasing stretch
produced by the developing fetus. If BKCa channels play a
role as negative feedback regulators of membrane electrical activity,
then their regulatory function should be adapted to meet the higher
demands of the PM. Indeed, comparison of basal channel activities
disclosed a significant difference; there was a nearly 39-mV left-shift of the conductance-voltage relationship in smooth muscle cells from PM
compared with NPM. Because maximum channel conductance and the
expression density of the pore-forming -subunit were unchanged in
myometrial membranes, increased basal channel activity was not due to
an increase in BKCa channel expression in the PM. Unexpectedly, the expression level of
-subunits, which enhance the
apparent Ca2+ sensitivity of the
-subunit
(23), was decreased during pregnancy (by 34% when
compared with NPM membranes). This clearly excludes the possibility
that
-subunits by their effects on Ca2+ sensitivity
contributed to the higher BKCa channel activity during pregnancy.
Protein phosphorylation is an ubiquitous mechanism of regulating the
activity of many biological processes including membrane excitability.
BKCa channels are important targets for regulation by PKA
and PKG (2, 20, 22, 26). Recently, it has been reported that BKCa channels are differentially regulated in
PM and NPM. In NPM, BKCa channels were mainly inhibited,
whereas in PM BKCa channels were primarily activated in
response to phosphorylation by PKA (30). The present study
confirms this differential regulation by PKA in human myometrium but
shows beyond it that PKG takes part in the differential regulation of
BKCa channels. More importantly, PKG proved to be more
effective than PKA to increase channel open probability in the PM,
whereas PKA was more efficient to inhibit channel openings in the NPM
cell. The mechanism by which BKCa channels are
differentially regulated in the PM and NPM by PKA and PKG is presently
unknown but may involve expression of isoforms of BKCa
channels, different heteropolymeric assembly of channel subunits,
and/or expression of associated proteins with modulatory function. With
respect to channel isoforms, it has been demonstrated that the
proportion of the BKCa channel transcripts containing the
STREX-1 exon at splice site 2 of the pore-forming -subunit is
hormonally regulated in chromaffin cells (42).
BKCa channels of neuroendocrine cells containing the
STREX-1 exon have been shown to be inhibited by PKA, whereas
expression of the
-subunit homolog of the ZERO variant in
Xenopus oocytes resulted in channel activation by PKA
(35, 26). Whether uterine smooth muscle cells express
STREX exons and whether this expression would be affected during
pregnancy is unknown. Recently, hormone-regulated protein phosphatase
2A activity closely associated with corticotroph BKCa
channels was shown to block PKA-mediated inhibition of the channel
(39). Although this observation cannot completely explain the switch from channel inhibition to activation, it points to the
modulatory function of hormonally regulated channel-associated proteins
in the phosphorylation process.
Recently, it has been proposed that NO may be an endogenous uterine relaxant that contributes to uterine quiescence during pregnancy (13, 27, 44). NO is thought to be the most important endogenous activator of guanylyl cyclase, and cGMP levels have been shown to increase in the rat and human PM (5, 43). A dramatic increase in cGMP levels during pregnancy has also been observed in pregnant guinea pigs, but this rise was independent of NO (40). The downstream target for cGMP is believed to be the PKG, which may finally phosphorylate target proteins that are involved in myometrial relaxation. Whereas increases in cGMP correlate with relaxation in a time- and concentration-dependent manner in vascular smooth muscle (21, 41), the role of cGMP as a uterine relaxant is not well established. For example, no correlation between increases in cGMP and relaxation was found in myometrial smooth muscle (6), and increases in cGMP that produce almost complete relaxation of the precontracted aorta had relatively little effect in uterine smooth muscle (41). Even more surprising, Word and Cornwell (41) conclusively showed that in rat PM the insensitivity to cGMP was accompanied by progesterone-mediated decreases in the level of PKG expression. The authors speculated that the substantial uterine smooth muscle cell hypertrophy that accompanies pregnancy may involve downregulation of cGMP action. For example, antiproliferative properties have been assigned to cGMP based on studies in vascular smooth muscle cells (9, 45). In agreement with Word and Cornwell (41) is the finding of this study that the levels of PKG measured in membranes and in total cell lysates of human myometria were substantially lower during pregnancy, although membranes were apparently less affected by this decline.
The experiments with KT-5823, a selective inhibitor of PKG (12), disclosed that the endogenous PKG was partially active in inside-out patches and contributed to the basal channel activity by enhancing NPo in PM and inhibiting it in NPM cells. This finding is not surprising, because PKG shows basal activity even in the absence of activators (38, 46). Further evidence for a membrane-associated endogenous PKG comes from the finding that 8-pCPT-cGMP, a potent stimulator of PKG, mimicked the effects of exogenously applied PKG in inside-out patches. Contrary to PKG, no regulation of BKCa channel activity by a membrane-associated PKA could be detected in membrane patches from PM and NPM by means of the inhibitory peptide of PKA (PKI). Cross-activation of PKA by 8-pCPT-cGMP was excluded by the observation that the cGMP derivative activated BKCa channels invariably in the absence and presence of PKI.
Although PKG is downregulated in plasma membranes of human PM, its basal activity apparently is sufficient to induce enhancement of BKCa channel activity. This membrane-delimited action of PKG during pregnancy may specifically reinforce the negative feedback regulation of membrane potential by BKCa channels, whereas myometrial relaxation due to other pathways of cGMP signaling may be of minor importance. The efficacy of the cGMP signaling on membrane potential was clearly demonstrated by the ability of 8-pCPT-cGMP to hyperpolarize the membrane of myometrial cells from pregnant women. Because IbTX as well as KT-5823 antagonized this effect, BKCa channel regulation by PKG is an important mechanism that contributes to the determination of the membrane potential.
In summary, the higher basal activity of BKCa channels in the PM is due to a membrane-bound PKG which activates BKCa channels during pregnancy but inhibits the channel activity in the NPM. This mechanism may be physiologically important in protecting the myometrium from stretch-induced electrical activity during pregnancy.
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ACKNOWLEDGEMENTS |
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We thank Simone Kamm for excellent technical assistance. We are
grateful to Dr. H.-G. Knaus, Institut für Biochemische
Pharmakologie der Universität Innsbruck, for the antibody
directed against the -bSlo and to Dr. M. Garcia, Dept. of Membrane
Biochemistry and Biophysics, Merck Research Laboratories, Rahway, NJ,
for the antibody directed against
-bSlo.
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FOOTNOTES |
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This study was supported by grants from the Deutsche Forschungsgemeinschaft, Bundesministerium für Forschung und Technologie, and Fond der Chemischen Industrie.
Address for reprint requests and other correspondence: M. Korth, Abteilung Pharmakologie für Pharmazeuten, Universitäts-Krankenhaus Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany (E-mail: korth{at}uke.uni-hamburg.de).
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.
Received 2 November 1999; accepted in final form 4 August 2000.
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