BKCa channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy

Xiao-Bo Zhou1, Ge-Xin Wang1, Peter Ruth2, Bernd Hüneke3, and Michael Korth1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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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 MOmega in whole cell and 8 to 9 MOmega 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 alpha -bSlo (amino acids 913-926) (15), beta -bSlo (amino acids 118-133) (16), or the common region of PKG type Ialpha and Ibeta (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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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 alpha -subunit and the 31-kDa regulatory beta -subunit of the BKCa channel. As shown in Fig. 1B, the respective alpha - and beta -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 alpha -subunit in pregnancy (95% in PM compared with NPM), whereas the expression of the beta -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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Fig. 1.   Basal conductance and expression of large-conductance calcium-activated potassium channels (BKCa) in myometrial smooth muscle cells from pregnant and nonpregnant women. A: conductance-voltage relations in inside-out patches obtained from pregnant (PM, solid circles; n = 8) and nonpregnant (NPM, open circles; n = 6) myometrial cells. Data were fitted by a Boltzmann equation. Patches were held at 0 mV and stepped from -160 to +120 mV in 20-mV increments for 400 ms. [K+] on both sides of the patch was 140 mM; cytosolic Ca2+ ([Ca2+]i) was 3 µM. B: levels of BKCa channel alpha - and beta -subunits in PM and NPM. Purified plasma membranes (10 µg membrane protein) from PM and NPM (for detail see METHODS) were separated by SDS-PAGE and blotted. Immunoblots were stained with antibodies specific for the alpha - and beta -subunits, respectively, and with a peroxidase-conjugated secondary antibody for visualization. Note that the immunosignals represent the mean expression of BKCa channel subunits from 10 PM and 6 NPM.

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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Fig. 2.   Differential regulation of BKCa channel activity by cGMP- and cAMP-dependent protein kinases in inside-out patches from NPM and PM myometrial cells. NPo-voltage relations (-60 to +80 mV) are shown in the absence and presence of 300 nM cGMP kinase (PKG) and 300 nM cAMP kinase (PKA) in patches from NPM (A and B) and PM (C and D) cells. Note the different scaling for NPM and PM patches. Values in A-D are means ± SE of 7 patches, respectively. Insets: representative single-channel current recordings before (control) and after the addition of either PKG or PKA. Channel openings are upward deflections; the membrane potential was +40 mV. PKG and PKA were applied to the cytosolic surface of the patch. [Ca2+]i was 0.3 µM.



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Fig. 3.   The PKG inhibitor KT-5823 has opposite effects on the basal BKCa channel activity in NPM (A and B) and PM (C and D) cells. Single-channel recordings in inside-out patches from an NPM (A) and a PM (C) cell. KT-5823 (1 µM) was applied to the cytosolic surface of the patch. Single-channel openings are upward deflections. Membrane potential was +40 mV, and [Ca2+]i was 0.3 µM. Values are means ± SE of NPo in the absence and in the presence of 1 µM KT-5823 in 11 patches from NPM (B) and PM cells (D), respectively. **P < 0.01. ***P < 0.001.



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Fig. 4.   8-p-Chlorophenylthio-cGMP (8-pCPT-cGMP) regulates BKCa channel activity via an endogenous membrane-associated cGMP kinase in NPM (A and B) and PM (C and D) cells. Single-channel recordings in inside-out patches from an NPM (A) and a PM (C) cell. 8-pCPT-cGMP, 500 µM, alone and in combination with 1 µM KT-5823 was sequentially applied to the cytosolic surface of the patch. Single-channel openings are upward deflections. Membrane potential was +40 mV, and [Ca2+]i was 0.3 µM. B and D: combined results from experiments with 8-pCPT-cGMP alone or in combination with KT-5823. Values are means ± SE of 7 NPM (B) and 8 PM cells (D). *P < 0.05 vs. control. **P < 0.01 vs. control.



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Fig. 5.   Expression of PKG type I in membranes (A) and in total cell lysate (B) of myometria from PM and NPM. A: purified plasma membranes (10 µg of membrane protein) from PM and NPM (for details see METHODS) were separated by SDS-PAGE and blotted. Immunoblots were stained with an antibody specific for PKG I and with a peroxidase-conjugated secondary antibody for visualization. Note that the immunosignals represent the mean expression of PKG I from 10 PM and 6 NPM specimens. B: total cell lysate (150 µg of protein) of three individual PM and NPM specimens, respectively (for details see METHODS), were separated by SDS-PAGE and blotted. Immunoblots were stained with an antibody specific for PKG I and with a peroxidase-conjugated secondary antibody for visualization. The statistical summary of the densitometric analysis is shown on right (means ± SE).

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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Fig. 6.   The inhibitory peptide of PKA (PKI) has no influence on the basal and 8-pCPT-cGMP-modulated BKCa channel activity. Single-channel recordings in inside-out patches from an NPM (A) and a PM (C) cell are shown. The cytosolic surface of the patch was sequentially superfused with 100 nM PKI alone and in combination with 500 µM 8-pCPT-cGMP. The holding potential was +40 mV, and the [Ca2+]i was 0.3 µM. Single-channel openings are upward deflections. B and D: combined results from experiments with PKI alone and in combination with 8-pCPT-cGMP. Values are means ± SE of 7 NPM (B) and PM cells (D). *P < 0.05 vs. control.

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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Fig. 7.   Activators of BKCa channels hyperpolarize the membrane of myometrial smooth muscle cells from pregnant women. Membrane potentials were recorded in the whole cell current-clamp mode. 8-pCPT-cGMP (300 µM) or NS-1619 (30 µM) was applied via a superfusion pipette to the surface of the cell, first in the absence and then in the presence of the BKCa channel blocker iberiotoxin (IbTX, 100 nM) (B and C). Note that the hyperpolarization induced by 300 µM 8-pCPT-cGMP is completely reversed by the PKG inhibitor KT-5823 (1 µM) (A). Control resting membrane potentials were -46.3 mV (A), -43.8 mV (B), and -45.5 mV (C).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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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 alpha -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 beta -subunits, which enhance the apparent Ca2+ sensitivity of the alpha -subunit (23), was decreased during pregnancy (by 34% when compared with NPM membranes). This clearly excludes the possibility that beta -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 alpha -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 alpha -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.


    ACKNOWLEDGEMENTS

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 alpha -bSlo and to Dr. M. Garcia, Dept. of Membrane Biochemistry and Biophysics, Merck Research Laboratories, Rahway, NJ, for the antibody directed against beta -bSlo.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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