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Modulation of Potassium Current Characteristics in Human Myometrial Smooth Muscle by 17ß-Estradiol and Progesterone¹

^a The London Myometrium Group, Centre for Cardiovasular Biology and Medicine, New Hunt's House, Guy's Campus, London SE1 1UL, United Kingdom ^b Fetal Health Research Group, Division of Obstetrics and Gynaecology, St. Thomas' Campus, London SE1 7EH, United Kingdom ^c Guy's, King's, and St. Thomas' Schools of Biomedical Sciences and Medicine, London SE1 1UL, United Kingdom

ABSTRACT

The K⁺ channel currents are important modulators of smooth muscle membrane potential and excitability. We assessed whether voltage-gated K⁺ currents from human myometrium are regulated by placental steroid hormones during pregnancy and labor. Pregnant human myometrial cells were isolated from samples obtained at cesarean section. Primary cultured cells were treated with 100 nM 17ß-estradiol, 1 µM progesterone, or both hormones in combination for 24 h. Acute effects of the two hormones were also determined. The K⁺ currents were recorded using the standard whole-cell, patch-clamp technique. Primary cultures possessed both delayed rectifier (I_KV) and A-like (I_KA) voltage-gated K⁺ currents. The 24-h 17ß-estradiol treatment caused a hyperpolarizing shift in the steady-state inactivation of both I_KV and I_KA. Progesterone treatment also shifted the inactivation of I_KA and increased I_KV amplitude by 60%–110%. Conversely, the combined treatment had no effect on these currents. Neither 17ß-estradiol (0.1–1 µM) nor progesterone (1–5 µM) had any effect on the K⁺ current when applied acutely. These results show that 17ß-estradiol should inhibit myometrial K⁺ channel activity, whereas progesterone is likely to have the opposite effect. These results are consistent with the respective procontractile and proquiescence roles for 17ß-estradiol and progesterone in human uterus during pregnancy.

estradiol, hormone action, pregnancy, progesterone, signal transduction, uterus

INTRODUCTION

The suppression of uterine motility during pregnancy and its activation toward term and in labor have been hypothesized to result from "progesterone block" and "estrogen domination" [1, 2], respectively. These hypotheses arose from the fact that during pregnancy, plasma concentrations of these hormones increase steadily until just before parturition, when, at least in most nonprimate species, 17ß-estradiol rises sharply and, conversely, progesterone falls [3, 4]. In human pregnancy, plasma 17ß-estradiol and progesterone levels rise gradually [5, 6], but during labor, instead of a dramatic reduction in plasma progesterone levels [7], a functional withdrawal of progesterone may occur [8, 9].

Functional evidence in animals and humans supports an in vivo association between the 17ß-estradiol:progesterone ratio in pregnancy, with 17ß-estradiol enhancing and progesterone inhibiting uterine electrical and mechanical activity [3, 4, 10, 11]. At the cellular level, resting membrane potential and, hence, excitability of the cell, as well as the frequency and duration of action potentials in myometrial smooth muscle, are differentially influenced by 17ß-estradiol and progesterone [12], again with progesterone generally suppressing activity.

17ß-Estradiol and progesterone are thought to exert their effects on the uterus by regulating the expression of "contraction-associated proteins," which may include ion channels. In excitable cells, enhancement of current through K⁺ channels (I_K) will hyperpolarize the resting membrane potential and generally suppress action potential generation [13]. Thus, K⁺ channel modulation may be expected to have a profound effect on contraction.

At least four distinct types of K⁺ current are found in myometrium. Two of these are delayed rectifier currents (I_KV), which we have termed I_K1 and I_K2 in human myometrium [14]. The I_K2 undergoes steady-state inactivation over a relatively positive range of membrane potentials and is inhibited by 4-aminopyridine (4-AP), a nonselective blocker of most voltage-gated K⁺ (K_V) channels. Conversely, I_K1 inactivates over a relatively negative range of membrane potentials and is enhanced by 4-AP. Two equivalent K_V currents also occur in pregnant rat myometrium [15]. The third current, a transient 4-AP-sensitive K⁺ current (I_KA), is present in nonpregnant rat uterine myocytes but is very rare in pregnant cells [15]. In humans, this current appears to be abundant in leiomyomas from nonpregnant women [16] but less prominent in normal tissue [14, 16]. Finally, the Ca²⁺-activated K⁺ current (I_K(Ca)) has reduced Ca²⁺ sensitivity in pregnant rat myometrium compared to nonpregnant animals [15]. In pregnant human myometrium, an apparent loss of Ca²⁺-sensitivity of the I_K(Ca) is found with the onset of labor [17].

Little is known about the long-term effects of 17ß-estrogen and progesterone treatment on K⁺ channel currents. To our knowledge, only a single study that examined the effects of long-term 17ß-estradiol and progesterone treatment on myometrial K⁺ currents in immature rats demonstrated that 17ß-estradiol treatment, administered in vivo, reduced the proportion of myometrial cells expressing I_KA (from 79% to 30%) as well as reduced the overall amplitude and accelerated the current decay rate [18]. In the present study, therefore, we assessed whether chronic, 24-h treatment of human myometrial myocytes in primary culture influenced the functional expression of voltage-gated K⁺ currents in these cells.

MATERIALS AND METHODS

Tissue Collection

Written consent was obtained for the removal of myometrial tissue from women undergoing elective (nonlabor) cesarean section at term (38–41 wk of gestation). None of the pregnant women included in this study had underlying disease, and the indications for cesarean section were breech presentation, placenta previa, or maternal request. Biopsy specimens were taken from the midline of the upper edge of a lower segment incision and placed immediately in cold physiological salt solution (PSS). The study was approved by the Ethics Committee of St. Thomas' Hospital, London, UK (EC94/037).

Isolation and Primary Culture of Human Myometrial Smooth Muscle

Myometrial cells were isolated enzymatically using previously described procedures [14, 19]. For primary cell culture, small segments of muscle were washed in Hanks balanced salt solution and then incubated in Dulbecco modified Eagle medium (DMEM) containing 1 mg/ml of collagenase 1A, 1 mg/ml of collagenase XI, and 0.1% w/v bovine serum albumin, penicillin (25 U/ml), and streptomycin (25 µg/ml) for 30–40 min. The cell suspension was then triturated through a narrow-bore glass pipette, filtered through a 45-µm sterile filter, and washed twice in DMEM containing 10% w/v fetal calf serum (FCS) by centrifugation (450 x g, 5 min). The cell pellet was suspended in DMEM supplemented with 5% FCS, penicillin (25 U/ml), and streptomycin (25 µg/ml). Myocytes were seeded into six-well tissue culture plates and flooded with culture medium. The myocytes were maintained as a primary culture until confluent at 37°C in a humidified atmosphere of 95% v/v air/5% v/v CO₂, with the medium being changed every 2–3 days. Once confluent, cells were serum-deprived (0.5% FCS) for 24 h in phenol red-free DMEM and then treated for a further 24 h (in the same culture medium) with either 100 nM 17ß-estradiol, 1 µM progesterone, or both hormones simultaneously. These concentrations were chosen to resemble those found in maternal serum at the end of pregnancy [5, 6]. Individual cells were then obtained by incubating with trypsin and resuspending in PBS. Immunofluorescent labeling of cells, with -actin and calponin monoclonal antibodies and fluorescein isothyocyanate-labeled secondary antibody, was routinely performed to verify the purity of the myocyte culture.

Electrophysiology

The K⁺ currents were recorded at room temperature using the whole-cell, patch-clamp technique with pCLAMP-6 software and Axopatch 200B amplifier (Axon Instruments, Foster City, CA). The intracellular solution contained KCl (110 mM), MgCl₂ (2.5 mM), MgATP (1.0 mM), Hepes (10 mM), and EGTA (10 mM), and the pH was adjusted to 7.2 with KOH. The bath solution (PSS) contained NaCl (130 mM), KCl (5.0 mM), MgCl₂ (1.2 mM), CaCl₂ (1.5 mM), Hepes (10 mM), and glucose (10 mM), and the pH was adjusted to 7.4 with NaOH.

Drugs and Chemicals

17ß-Estradiol and progesterone (water-soluble, cyclodextrin-encapsulated) were prepared as 1 mM stock solutions in PBS, paxilline was prepared as a 10 µM stock solution in dimethyl sulfoxide, and 4-AP was dissolved directly in PSS, with readjustment of the pH to 7.4 with HCl. Each of these was obtained from Sigma (Poole, UK), as were the cell culture buffers and reagents. All other chemicals were obtained from BDH (Poole, UK).

Statistics and Data Analysis

Data analysis, statistics, and curve fitting were performed with Microsoft Excel (Microsoft, Redmond, WA) and SigmaPlot 4.01 (Jandel Scientific, San Rafael, CA) software on an Elonex (London, UK) MTX-6233/II computer. Differences were considered to be statistically significant at P < 0.05 using Student unpaired or paired t-test, as appropriate. For multiple point analysis, summary measures analysis was performed before Student t-tests [20].

RESULTS

Initial Current Recordings

The overall K⁺ current was initially inspected by depolarizing cells to +60 mV for 250 msec, from a holding potential of -80 mV, using PSS without the addition of pharmacological agents. It soon became apparent that in all treatment groups, a considerable heterogeneity of current shapes existed. These differed principally in the rate of current decay, possibly because of the existence of both transient, rapidly decaying (i.e., I_KA) and sustained, slowly decaying (i.e., I_KV) currents that differed in their relative proportions between cells (see below). Figure 1, A–C, shows examples from three control cultured cells. A typical outward current recorded in a cell freshly isolated from pregnant human myometrium is also presented for comparison (Fig. 1D).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Appearance of whole-cell K+ current at +60 mV in cultured and freshly isolated myometrial cells. Example traces of three outward currents elicited by 250-msec depolarizations to +60 mV from a holding potential of -80 mV in control-treated culture cells (A–C) and a typical freshly isolated cell (D). The dotted lines represent zero current

Comparison of K⁺ Currents in Freshly Isolated and Primary Cultured Myometrial Myocytes

As shown in Figure 1, the outward current in freshly isolated cells tended to demonstrate an oscillatory or "noisy" appearance, which was less apparent in the cultured cells and is typically associated with the presence of BK_Ca channels [21]. To determine if the amplitude of this current (here termed I_K(Ca)) was, indeed, larger in the freshly isolated cells, we used paxilline, a selective blocker of BK_Ca channels [22]. Current-voltage (I-V) relationships were constructed by stepping to a range of membrane potentials between -80 and +60 mV, from a holding potential of -80 mV, and then repeating the procedure in the presence of 1 µM paxilline. Figure 2 shows the resulting mean (± SEM) I-V relationships for control cultured cells (Fig. 2A) and freshly isolated pregnant cells (Fig. 2B). Although in some cultured cells paxilline appeared to cause slight inhibition, the current was only significantly inhibited in the freshly isolated cells, as also illustrated in the example traces at +60 mV shown in Figure 2C. Figure 2D illustrates that compared to freshly isolated cells, the paxilline-sensitive current (measured at +60 mV) represented only a very small fraction of the overall outward current, not only in the control cultured cells, but also in cultured cells that had been hormone-treated. These data indicate that the outward current in both control and hormone-treated cultured cells was almost entirely caused by voltage-gated K⁺ currents, because it was paxilline-insensitive and depolarization-activated. Figure 2, A and B, also shows that this voltage-gated current (I_KV) was much smaller in the cultured cells than in the freshly isolated cells. For example, I_KV current density at +60 mV was 8.8 ± 0.9 pA/pF in 31 freshly isolated cells but only 1.35 ± 0.16 pA/pF in 25 control cultured cells.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Effect of paxilline on K+ currents in cultured and freshly isolated myometrial cells. A and B) Mean (± SEM) current densities, measured at the end of 250-msec depolarizations to between -80 and +60 mV from a holding potential of -80 mV, in control-treated cultured cells (A, n = 17) and freshly isolated cells from term pregnant women (B, n = 11) in the absence (PSS) and presence of 1 µM paxilline (PAX). An asterisk indicates significant inhibition (P < 0.001 by repeated measures, paired t-test). C) Examples of the effect of 1 µM PAX (arrows) at +60 mV in a control-treated cultured cell (left) and the same freshly isolated cell as shown in Figure 1D (right). The dotted lines represent zero current. D) Mean (± SEM) percentage inhibition at +60 mV in control (n = 17), 17ß-estradiol-treated (estr, n = 15), progesterone-treated (prog, n = 20), combined hormone-treated (estr & prog, n = 13), and freshly isolated cells (n = 31). Block was only significant for freshly isolated cells

Effects of Long-Term Hormone Treatment on K⁺ Current Amplitudes

We next studied the effect of hormone treatment on the amplitude of the transient (I_KA) and sustained (I_KV) currents. The 1 µM paxilline was routinely included in the bath during current recording to suppress any I_K(Ca), and cells were held at -80 mV, at which potential channels were maximally available for opening (see below). To control for the effects of heterogeneity in cell size, current density was calculated by dividing current amplitude (in pA) by cell capacitance (in pF), a measure of membrane area, which was calculated as described in Materials and Methods. It is noteworthy, however, that no significant differences were found in cell capacitance between the groups of cells having undergone each of the four treatments (control; 80.9 ± 9.8 pF, n = 25; 17ß-estradiol, 89.6 ± 13.6 pF, n = 19; progesterone, 77.7 ± 6.7 pF, n =31; 17ß-estradiol and progesterone combined, 78.5 ± 13.3 pF, n =14). The I-V relationships were constructed by plotting the amplitude of the peak current (I_KA) (Fig. 3A), and the current recorded at the end of the depolarizing step (I_KV) (Fig. 3B), against membrane potential. As depicted in Figure 3A, no significant differences were found in I_KA current densities between the four groups of cells. On the other hand, I_KV was significantly larger in the progesterone-treated group at membrane potentials positive of -20 mV (Fig. 3B); this increase amounted to 109% at 0 mV.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Effects of hormone pretreatment on IKA and IKV current-voltage relationships. Mean (± SEM) measurements of currents in cells from control (n = 25), 100 nM 17ß-estradiol (n = 19), and 1 µM progesterone (n = 31) treatments and in cells treated with 100 nM 17ß-estradiol and 1 µM progesterone in combination (n = 14) are shown. Currents were elicited by 400-msec step depolarizations from a holding potential of -80 mV to between -80 and +60 mV (10-mV increments) at peak (IKA, A) and end of pulse (IKV, B). The 1 µM paxilline was present throughout. The asterisk indicates P < 0.05 for progesterone-treated cells compared with controls

Steady-State Inactivation of K⁺ Currents

The steady-state inactivation (i.e., availability) of I_KA and I_KV was determined by applying 10-sec conditioning prepulses to between -90 and -2 mV (8-mV increments), followed by a 250-msec test pulse to +60 mV. Results of previous studies have shown that A-like K⁺ currents decay well within the duration of the test pulses used [16]. Measurements of the current elicited during the +60-mV test pulse could then be made at the peak and at the end of the pulse to estimate I_KA and I_KV availability, respectively. All measurements were normalized to the current amplitudes at +60 mV following the prepulses to -90 mV, plotted against prepulse membrane potential. Points were then fitted by nonlinear regression to the Boltzmann function:

where I is the normalized current at any potential, C is the fraction of the current not inactivated by the prepulse depolarization, V is the prepulse membrane potential, V_0.5 is the half-inactivation potential, and k is the slope factor. To minimize cross-contamination of A-like current measurements with delayed rectifier current measurements, cells in which I_KA was absent were excluded from peak-current measurements, and cells in which I_KV was too small for accurate curve fitting were excluded from end-of-pulse measurements.

The results for I_KA (peak-current measurements) and I_KV (end-of-pulse measurements) are shown in Figure 4, A and B, respectively, and are quantitated in Table 1. For I_KA, treatment with either 17ß-estradiol or progesterone caused similar and significant leftward hyperpolarizing shifts in the steady-state inactivation (assessed as V_0.5). Unexpectedly, combined treatment with both hormones had no effect on I_KA availability. Both hormones also appeared to cause a similar shift in I_KV availability, but this effect was statistically significant only for 17ß-estradiol. Again, combined hormone treatment did not affect current availability. The fraction of I_KV not inactivated was also slightly, but significantly, smaller in cells from the progesterone-treated group compared with controls (Table 1). No other parameters were significantly altered by hormonal treatment.

View larger version (28K): [in this window] [in a new window]   FIG. 4. Steady-state inactivation of IKA and IKV. Shown are mean (± SEM) currents measured at peak (IKA, A) or end of pulse (IKV, B) at a test pulse to +60 mV following 10-sec preconditioning pulses to between -90 and -2 mV (8-mV increments) and normalized to the amplitude of the test-pulse current following the -90-mV preconditioning pulse (I/IMax). Data are fitted to Boltzmann function by nonlinear regression (see text for details and Table 1 for comparisons). The 1 µM paxilline was present throughout. Control: n = 14 for IKA and 8 for IKV; 100 nM 17ß-estradiol: n = 10 for IKA and 6 for IKV; 1 µM progesterone: n = 13 for IKA and 10 for IKV; 17ß-estradiol and progesterone in combination: n = 12 for IKA and 9 for IKV

In a small number of cells (one control, one 17ß-estradiol-treated, four progesterone-treated, and two combined-treatment cells), the inactivation profile for the end-of-pulse current (I_KV) was clearly biphasic and best fitted by a biphasic Boltzmann function:

(1 + exp[V - V_0.5B]/k_B) + C where I is the normalized current amplitude; V is the prepulse membrane potential; A is the fraction of the first inactivating current component; B is the fraction of the second inactivating current component; C is the fraction of the current not inactivated; and V_0.5A and V_0.5B are the half-inactivation potentials of fractions A and B, respectively; and k_A and k_B are the slope factors for fractions A and B, respectively. For the four progesterone-treated cells, we found that A was 35.5% ± 5.8%, B was 29.3% ± 6.0%, C was 36.5% ± 4.4%, V_0.5A was -61.9 ± 4.4 mV, V_0.5B was -25.2 ± 2.5 mV, k_A was 5.4 ± 1.8 mV, and k_B was 2.9 ± 1.2 mV. Similar values of these parameters were obtained for the one control cell, for one 17ß-estradiol-treated cell, and for two combined-treatment cells (data not shown).

4-AP Sensitivity of K⁺ Currents

We have previously reported that I_KV in freshly isolated myometrial cells comprised two separate voltage-gated currents, termed I_K1 and I_K2. These could be readily distinguished, because I_K1 was unaffected or slightly enhanced by 4-AP whereas I_K2 was blocked. Moreover, I_K1 inactivated over a much more negative potential range than I_K2. In addition, cells tended to express either one or the other current, such that V_0.5 in individual cells was close to either -65 or -30 mV [14]. Although V_0.5 for I_KV in the cultured cells did not demonstrate this obvious bimodal distribution, the presence of two components of I_KV inactivation in some cells suggested that both components might coexist in the cultured cells, and this suggests the possibility that hormone treatment might differentially affect their expression. Therefore, we examined the effect of 4-AP on I_KV in each of the four treatment groups using a concentration of drug (5 mM) that blocked I_K2 by 80% but that had no effect on I_K1 [14].

The mean percentage inhibitory effect of 5 mM 4-AP on peak (I_KA) and end-of-pulse (I_KV) values at +60 mV in the four treatment groups is presented in Figure 5A. In all groups of cells, I_KA was inhibited to a significantly greater degree than I_KV (P < 0.01), and no significant differences were found in block of either current when the four groups were compared. The effect of 4-AP on I_KV demonstrated considerable variability, and when the cells from all four treatment groups were combined, the percentage inhibition appeared to be normally distributed (Fig. 5B). This contrasts with the freshly isolated cells, in which 4-AP had clearly different effects on two subpopulations of cells, causing inhibition in one and activation in the other (Fig. 5C) [14]. Typical examples for the effect of 4-AP on I_KA and I_KV K⁺ currents are shown in Figure 6. Currents are shown before (top traces) and after (middle traces) the addition of 5 mM 4-AP and with the 4-AP-sensitive difference currents (bottom traces). In Figure 6A, the fast I_KA current was nearly abolished by 4-AP, leaving only a very small sustained current. However, as shown in Figure 6B, the slow I_KV current was only partially inhibited, revealing a substantial 4-AP-insensitive I_KV component.

View larger version (29K): [in this window] [in a new window]   FIG. 5. Inhibition of IKA and IKV by 4-AP. A) Mean (± SEM) percentage inhibition of currents at peak (IKA) and end of pulse (IKV) by 5 mM 4-AP is shown in control cells (n = 25) and cells treated with 100 nM 17ß-estradiol (estr, n = 19), 1 µM progesterone (prog, n = 31), or the two drugs in combination (estr & prog, n = 14). Significantly more block of IKA than of IKV currents was observed in all four treatment groups. The asterisk indicates P < 0.05. B) Distribution of percentage inhibition by 5 mM 4-AP of IKV at +60 mV in all cultured cells combined. C) Distribution of percentage inhibition by 5 mM 4-AP of IKV at +60 mV for 14 freshly isolated cells

View larger version (22K): [in this window] [in a new window]   FIG. 6. Examples of the effect of 4-AP on IKA and IKV. Current-voltage relationships in a control-treated cell with a large IKA (A) and a progesterone-treated cell with a relatively large IKV (B). Traces show currents elicited by 400-msec depolarizations to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -80 mV. The top two sets of traces show currents in the absence of 4-AP, and the middle sets show the effect of 5 mM 4-AP. The bottom two sets show the 4-AP-sensitive difference currents, as derived by subtracting currents recorded in the presence of 4-AP from those recorded before 4-AP was applied. The 1 µM paxilline was present throughout

The outward current in the freshly isolated cells did not demonstrate an obvious I_KA. However, the presence of this current may have been obscured by the initial activation of the large I_KV currents (either I_K1 or I_K2) in these cells. In this case, the high sensitivity of I_KA to 5 mM 4-AP (Figs. 5 and 6A) suggests that this current could be unmasked as an initial 4-AP-sensitive component of current in freshly isolated cells in which the delayed rectifier was 4-AP-insensitive (i.e., cells with I_K1 but not I_K2) [14]. Figure 7 shows that application of 4-AP to one such cell produced, as predicted, a transient initial current component closely resembling I_KA. The 4-AP-sensitive transient components in a group of similar freshly isolated cells lacking I_K2 were, therefore, compared with the initial transient 4-AP-sensitive current in the control cultured cells using a test potential of +60 mV. No significant difference was found in current density between the two groups of cells (control cultured cells, 2.5 ± 0.6 pA/pF, n = 25; freshly isolated cells, 3.9 ± 0.8 pA/pF, n = 16).

View larger version (12K): [in this window] [in a new window]   FIG. 7. Use of 4-AP to unmask IKA in a freshly isolated myometrial cell. A) Currents elicited by 250-msec step depolarizations to +60 mV in 1 µM paxilline (PAX) and then in both PAX and 5 mM 4-AP (arrows). B) The 4-AP-sensitive difference current derived by subtracting the current in the presence of 4-AP from that in the absence of 4-AP. The dotted lines represent zero current

Acute Effects of 17ß-Estradiol and Progesterone on K⁺ Currents

The acute effects of 17ß-estradiol and progesterone on K⁺ currents were determined using both cultured (i.e., serum deprived in phenol red-free media for 48 h before harvesting) and freshly isolated myometrial cells. The 250-msec steps to +60 mV, from a holding potential of -80 mV, were applied every 10 sec, and after an initial control period, 17ß-estradiol (100 nM followed by 1 µM) or progesterone (1 µM followed by 4 or 5 µM) was applied to the bath for 5 min. In the cultured cells, in the presence of 1 µM paxilline, 17ß-estradiol had no significant effect at either concentration (peak: 1.2% ± 3.8% increase at 100 nM, ;n = 8; 1.7% ± 4.9% block at 1 µM, n = 8; not significant [NS]; end of pulse: 0.4% ± 4.9% block at 100 nM, n = 8; 7.8% ± 5.0% increase at 1 µM, n = 8; NS). Progesterone was equally without significant effect in these cells (peak: 8.9% ± 2.2% increase at 1 µM, n = 8; 1.1% ± 4.4% increase at 5 µM, n = 8; NS; end of pulse: 11.5% ± 1.6% increase at 1 µM, n = 8; 0.8% ± 6.4% block at 5 µM, n = 8; NS). In the freshly isolated cells, in the absence of paxilline, 17ß-estradiol had no significant effect at either concentration (end of pulse: 1.8% ± 6.4% increase at 100 nM, n = 4, NS; 4.5% ± 8.5% increase at 1 µM, n = 4, NS). Similarly, progesterone did not significantly alter current amplitude at 1 µM (end of pulse: 4.9% ± 11.8% increase at 1 µM, n = 5, NS), or at 4 µM (end of pulse, 8.5% ± 3.9% block, n = 5, NS). Although these experiments were not designed to evaluate selective effects of these hormones on the different components of current (i.e., I_KV, I_KA, I_K(Ca)), the complete lack of effect of either hormone on the amplitude or shape of the overall outward current suggests strongly that it was unlikely any of the components was markedly affected by these treatments.

DISCUSSION

These experiments demonstrate, to our knowledge for the first time, that long-term treatment of human myometrial smooth muscle cells with 17ß-estradiol and progesterone causes alterations in the functional expression of voltage-gated K⁺ channels in these cells. Given the likely inhibitory function of these channels in regulating smooth muscle excitation-contraction coupling, the nature of these changes are in line with the putative role of both hormones in controlling myometrial excitability.

Effects of Cell Culture on Functional K⁺ Current Expression

The K⁺ currents in the primary cultures of pregnant human myometrium used in the present study differed in a number of ways from those we have previously characterized in freshly isolated, term pregnant myometrial cells. The freshly isolated cells possessed a prominent Ca²⁺-activated K⁺ current, I_K(Ca), and two distinct types of I_KV, I_K1 and I_K2. The I_K1 was 4-AP insensitive and inactivated over a relatively negative range of membrane potentials (V_0.5 = -61 mV), whereas the I_K2 was 4-AP sensitive and inactivated over a much more positive range of membrane potentials (V_0.5 = -30 mV) [14]. These cells usually possessed either I_K1 or I_K2 as the predominant I_KV, only occasionally having both. In the cultured cells, by contrast, the I_KV component, as measured at the end of the pulse, was much smaller and showed a broad range of 4-AP sensitivities rather than a bimodal sensitivity (Fig. 6B). Current availability was well described by a simple Boltzmann function, with a mean V_0.5 of -53 mV in approximately 90% of the cells, although a small proportion of cells exhibited clearly biphasic availabilities, where V_0.5A was -62 mV and V_0.5B was -25 mV. This change in the properties of I_KV might have resulted if the cultured cells were expressing a random mix of both I_K1 and I_K2. Alternatively, a down-regulation of I_K1 and I_K2 may occur in culture, such that other I_KV currents, which normally make a minor contribution to the overall I_KV, become predominant. It is interesting that the only current appearing not to be down-regulated in the cultured cells is I_KA, defined as the 4-AP-sensitive initial transient component of the current. Although I_KA is much more obvious in the cultured cells, the results shown in Figure 7 suggest that this difference is more apparent than real, and that it results from a reduction in size of the sustained currents that tend to mask it in the freshly isolated cells. Despite cultured cells representing an imperfect representation of the in vivo milieu, the presence of well-defined, voltage-gated K⁺ currents in these cells encouraged us to evaluate the long-term effects of hormone treatment, especially taking into account that it is not possible to mimic the rat experiments of long-term 17ß-estradiol treatment in vivo in humans.

Effects of Long-Term Hormone Treatments on K⁺ Currents

The pregnancy-associated fall in expression of I_KA observed in the rat by Wang et al. [15] is consistent with the finding of Erulkar et al [18] that in vivo treatment of prepubertal rats with 17ß-estradiol reduced the amplitude and occurrence of myometrial A-current. The implication, although not proven, is that increases in circulating 17ß-estradiol during pregnancy may be responsible for down-regulating A-current channel expression. They did not report any changes in delayed rectifier currents.

In contrast to those studies, we did not find that chronic treatment with a physiological concentration of 17ß-estradiol reduced the proportion of cells in which I_KA was functionally expressed. Moreover, it seems that I_KA exists in most (or all) term pregnant human myometrial cultured cells (Fig. 7), although its presence is masked by the activation of the other K⁺ currents in these cells. On the other hand, our results do agree with the general sense of those studies, in that I_KA is likely to be reduced in amplitude by 17ß-estradiol treatment. Although 17ß-estradiol treatment did not inhibit I_KA when the holding potential was set to a level (–80 mV) at which the current was fully available (Fig. 3A), it did cause a significant hyperpolarizing shift in the steady-state inactivation of I_KA (Fig. 4A). Considering that the resting membrane potential is approximately -50 mV in pregnant women at term [23], this shift would result in a decreased proportion of I_KA channels being available for opening on stimulation, from approximately 40% to approximately 15%. We also found that 17ß-estradiol treatment had a similar effect on I_KV (Fig. 3B), such that the proportion of I_KV channels available at -50 mV would fall from approximately 75% to approximately 30%. The overall effect of 17ß-estradiol treatment would, thus, be to depress K⁺ channel function and, therefore, increase cellular excitability.

Similar to treatment with 17ß-estradiol, progesterone treatment reduced I_KA availability without affecting the current density at -80 mV. This contrasts with the result of Erulkar et al. [18], who showed that progesterone treatment had little effect on I_KA inactivation in prepubertal rat myometrium. In contrast to the effect of 17ß-estradiol, however, treatment with progesterone caused a marked increase in the amplitude of I_KV when cells were held at -80 mV. Because I_KV availability was not significantly affected (Fig. 4B), the net effect of progesterone treatment would be an enhancement of I_KV at the resting potential. Because I_KV is both more available at the resting potential and more sustained because of its slower rate of inactivation compared to I_KA, it likely is more important for regulating resting membrane potential and modulating action potential generation and repolarization. The overall effect of progesterone treatment on voltage-gated K⁺ channels should, therefore, lead to a reduction in cell excitability.

Combined treatment of cells with both progesterone and 17ß-estradiol together produced no measurable changes in any of the K⁺ current parameters investigated. When considering this unexpected result, the influence of these two hormones on expression of their own receptors may be relevant. In several mammalian species, including humans and rats, 17ß-estradiol treatment up-regulates both estrogen receptor (ER) and progesterone receptor (PR) expression [24, 25]. A study in the mouse, however, showed that progesterone treatment does the reverse, inhibiting expression of both ER and PR [26]. This latter effect seems to occur in humans as well, because in term pregnant myometrium, PR levels are higher than in nonpregnant myometrium [27], and during the estrous cycle, both PR and ER are high when 17ß-estradiol levels are high but low when progesterone levels are high [28]. Perhaps these complex interactions in some way account for the lack of response of K⁺ currents to combined 17ß-estradiol and progesterone treatment in our experiments.

To our knowledge, the long-term effects of hormonal treatment on voltage-gated K⁺ channel expression have not previously been investigated. However, in vivo treatment of nonpregnant, ovariectomized rats with 17ß-estradiol results in increased Ca²⁺-channel density [29], whereas in pregnant rats, progesterone and antiprogesterone treatment prevents and enhances, respectively, the normal increase in Ca²⁺-channel expression observed during pregnancy [30]. The only contraction-associated protein that has been studied in great detail at the molecular level is the gap-junction protein connexin 43 (Cx43). The expression of Cx43 is increased toward term, and more so during labor, and the level of expression is correlated with the 17ß-estradiol:progesterone ratio in rats [31] and humans [7]. At least in rats, gap junction expression is clearly stimulated by 17ß-estradiol and inhibited by progesterone [31–33]. The net effect of 17ß-estradiol on Cx43 expression, however, depends on which of the two ER subtypes (ER and ERß) is predominantly expressed in the myometrium, with ER stimulating and ERß inhibiting Cx43 expression [25]. According to Wu et al. [25], primary cultures of nonpregnant human myometrium express comparable levels of both ER and ERß.

Effects of Acute Hormone Treatment on K⁺ Currents

In addition to genomic effects, 17ß-estradiol and progesterone may influence K⁺ currents rapidly, via either a plasma membrane receptor or direct binding to the channel. 17ß-Estradiol activates the large-conductance Ca²⁺-activated K⁺ channel in oocytes transfected with hSlo and ß subunits by binding directly to the ß subunit [34]. This effect may partly explain the cardioprotective properties of acute 17ß-estradiol treatment [35]; however, this effect occurred at concentrations 5- to 10-fold those known to occur physiologically. At even higher concentrations, 17ß-estradiol inhibits I_K(Ca), as well as I_KV and I_Ca, in rat myometrium [36] but is much less effective at concentrations closer to those used by Valverde et al. [34]. In our study, by contrast, acute external application of 17ß-estradiol to either primary cultured or freshly isolated myometrial smooth muscle cells at concentrations much nearer to the physiological level had no measurable effect on K⁺ current. Similar results were obtained with progesterone.

In summary, our results are consistent with those of previous studies showing a relationship between exposure to 17ß-estradiol or progesterone and the respective stimulatory and inhibitory effects of the two hormones on in vitro and in vivo uterine electrical activity and contractions [4, 11, 12]. Furthermore, our work suggests that a reduction in K⁺ current activity may be one mechanism by which functional progesterone withdrawal, occurring in the human uterus with the onset of labor [8, 9], may contribute to the enhanced myometrial excitability and contractility required for parturition.

ACKNOWLEDGMENTS

The authors would like to thank Ruth Brucker, Vasia Dekou, Judy Crowe, and the labor ward staff at St. Thomas' and Guy's Hospitals, London, for their help with the collection of myometrial samples for this study.

FOOTNOTES

First decision: 17 October 2000.

¹ Supported by the Tommy's Campaign, registered charity 1060508.

² Correspondence: Greg A. Knock, Centre for Cardiovasular Biology and Medicine, Room 2.30, New Hunt's House, Guy's, King's, and St. Thomas' Schools of Biomedical Sciences and Medicine, Guy's Campus, London SE1 1UL, UK. FAX: 44 207 848 6371; greg.knock{at}kcl.ac.uk

Accepted: January 8, 2001.

Received: August 28, 2000.

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