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Department of Obstetrics and Gynaecology (H.C.D., D.D.H., N.R., J.J.M.), National University of Ireland Galway, Clinical Science Institute, University College Hospital Galway, Galway, Ireland; and National Centre for Biomedical Engineering Science (T.J.S.), National University of Ireland, Galway, Ireland
Address all correspondence and requests for reprints to: Professor John J. Morrison, Department of Obstetrics and Gynaecology, National University of Ireland Galway, Clinical Science Institute, University College Hospital Galway, Galway, Ireland. E-mail: john.morrison{at}nuigalway.ie.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein produced primarily in the placenta (6). Although the importance of hCG in maintenance of early pregnancy has been widely accepted, recent reports have highlighted a potential role of hCG in maintaining uterine quiescence in the third trimester. The hCG exerts a potent concentration dependent inhibitory effect on human myometrial contractions in vitro (7). The hCG receptors in human myometrium are down-regulated following the onset of labor, in both term and preterm deliveries (7, 8). Although possible explanations for the inhibitory effect of hCG in human myometrial smooth muscle have included a direct reduction of intracellular calcium availability (9, 10), down-regulation of myometrial gap junctions (11) and inhibition of uterotonic agents such as eicosanoids (8), little is known of the endogenous mechanisms of hCG regulation of uterine contractility during pregnancy.
The state of contractility of the myometrium is ultimately dependent on both the concentration of and sensitivity to intracellular calcium (2, 12). Cell membrane ion channels play a major role in regulation of smooth muscle activity by controlling cell membrane potential and in the regulation of intracellular calcium concentration. For myometrium, Ca2+, Na+, K+, and Cl- channels all contribute to the regulation of the resting membrane potential and the action potential, and both directly and indirectly contribute to the regulation of intracellular free Ca2+ concentrations (12). The K+ channels are the largest category of ion channels in the cell and hence are of primary importance in the regulation of membrane potential and cell excitability in the myometrium (13, 14, 15, 16). The large conductance calcium-activated potassium channel (BKCa or maxi-K channel) is the predominant K+ channel type encountered in nonpregnant and pregnant human myometrium (14, 15) and has been reported as playing a significant role in modulating myometrial function in both animal models and in humans (12, 16). Although it is established that BKCa channel function in smooth muscle is closely linked to membrane conductance and cellular excitability, little is known about the actual mechanisms or role of BKCa modulation by endogenous substances. This study was undertaken to investigate whether hCG exerted an effect on open-state probability of BKCa in single channel electrophysiological recordings of human uterine myocytes and, second, to evaluate whether the relaxant effect of hCG on myometrial contractions was altered by BKCa blockade in functional studies using iberiotoxin (IbTX).
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Materials and Methods |
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Biopsies of human myometrium were obtained from women undergoing elective cesarean section in the third trimester of pregnancy in the Department of Obstetrics and Gynecology, University College Hospital, Galway, Ireland. The biopsies were excised from the midline portion of the upper lip of the incision in the lower uterine segment. Women who had received exogenous prostaglandins, oxytocin, or corticosteroids were excluded from the study. Recruitment was by written informed consent. Ethics committee approval for the study was obtained from the Research Ethics Committee, University College Hospital, Galway. For electrophysiological studies, tissue samples were immediately placed in sterile Ham’s F-12 medium (Sigma-Aldrich, Dublin, Ireland) supplemented with 100U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich). Tissue samples for isometric recordings were placed in fresh Krebs-Henseleit physiologic saline solution (PSS) of the following composition (mmol/liter): 4.7 KCl, 118 NaCl; 1.2 MgSO4, 1.2 CaCl2, 1.2 KH2PO4, 25 NaHCO3, and 11 glucose (Sigma-Aldrich). Tissue was stored at 4 C and used within 6 h of collection.
Tissue dispersion/primary human myometrial cells
The preparation of single myometrial cells for electrophysiological recordings was performed using methodology previously described (17, 18). Freshly isolated myometrial cells were obtained by enzymatic digestion of finely minced myometrium with 2 mg/ml collagenase (type IA, 300–400 U/mg) (Sigma-Aldrich) in Hanks’ buffered salt solution. The incubation with enzyme was performed at 37 C for 2 h followed by centrifugation (1000 revolutions per minute) in 50% Percoll (Sigma-Aldrich) for 10 min. The cell layer was removed, washed, and spun in physiological solution to remove excess red blood cells. The cell suspension was then triturated and filtered through an 80-µm nylon mesh filter. Single cells were placed in a recording chamber (Warner Instrument Corp., Hamden, CT) and electrophysiological experiments begun immediately. Morphologically, freshly dissociated uterine myocytes were characterized by a long, slender fusiform shape. All myocytes used for this work were relaxed and adhered to the bottom of the recording chamber with no additional substrate.
Electrophysiological recordings of the BKCa channel
Single-channel recordings using the cell-attached configuration of the patch clamp technique were performed. Several drops of cell suspension were placed in the recording chamber containing a solution of the following composition (mmol/liter): 140 KCl, 10 MgCl2, 0.1 CaCl2, 10 HEPES, and 30 glucose (pH 7.4, 22–25 C). Single potassium channel currents were measured in cell-attached patches by filling the patch pipette (2–5 M
) with Ringer solution composed of (mmol/liter): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, and 10 HEPES, and making a gigaohm seal on a single myocyte. Voltage across the patch was controlled by setting the cellular membrane potential to 0 mV using a high-[K+] extracellular solution. Average channel activity in patches was measured as mean open probability (NPo). The effects of hCG (1, 10, and 100 IU/ml) on channel activity were measured by recording about 10 sec of continuous recording at +40 mV before and 15 min after drug treatment. Currents were filtered at 1 kHz and digitized at 10 kHz. In all experiments, voltage-clamp and voltage-pulse generation were controlled with an Axopatch 200-A patch-clamp amplifier (Axon Instruments, Inc., Union City, CA), and data were acquired and analyzed with pClamp 8. For statistical purposes and to ensure accuracy, NPo was calculated for single BKCa channels at a deploarized potential in which channel openings can be clearly differentiated from other channel species. BKCa channels are easily visualized at +40 mV because there is virtually no contamination with other channel species. In addition, at such deploarized voltages, there is no other ion channel expressed in these cells with an amplitude of 7–9 pA, making statistical analysis of BKCa channel activity more accurate (19). Moreover, recording single-channel activity at more deploarized potentials helps compensate the artificial depression of activity otherwise encountered from recording channel activity at room temperature (22–25 C) (19).
Isometric tension recordings
Longitudinal myometrial strips (measuring approximately 2 x 2 x 10 mm) were dissected free from uterine decidua and serosa and mounted isometrically in an organ tissue bath under 2 g tension for recordings, as previously described (20, 21). The tissue baths contained 20 ml of Krebs-Henseleit PSS maintained at 37 C, pH 7.4, and were gassed continuously with a mixture of 95% oxygen/5% carbon dioxide. During a period of equilibration of 1 h, the Krebs-Henseleit PSS in the tissue baths was changed every 15 min. The experiments evaluating effects on myometrial contractility were then designed in four groups as follows: group 1: oxytocin alone; group 2: oxytocin + hCG; group 3: oxytocin + IbTX; group 4: oxytocin + IbTX + hCG. Following equilibration, all myometrial strips were incubated with 0.5 nmol/liter oxytocin to elicit regular rhythmic contractions. After 30 min, strips from groups 3 and 4 were exposed to IbTX at a bath concentration of 100 nmol/liter for a further 30 min, and group 1 strips were exposed to Krebs-Henseleit PSS only. Contractile activity was measured for a 20-min period at which time bath addition of hCG to groups 2 and 4 strips took place. Highly purified hCG was added to the tissue bath in a cumulative manner at bath concentrations 0.1, 0.5, 1, 5, and 10 IU/ml at 20-min intervals, and the resultant contractile activity was measured for each period and expressed as a percentage of the integral obtained in the 20-min period before any hCG addition (i.e. percentage of contractility). Measurement of contractile activity was performed by calculation of the integral of the selected area with the PowerLab (AD Instruments, Hastings, UK) hardware unit and Chart version 3.6 software (AD Instruments).
Drugs and solutions
Highly purified hCG (CR-127 14,900 IU/mg) was obtained as a gift from Dr. A. G. Parlow, National Hormone and Pituitary Program, Torrance, CA. A stock solution (10,000 IU/ml) of hCG was prepared in sterile saline. A stock solution of oxytocin (1 mmol/liter; Sigma-Aldrich) was made in ethanol. Series of dilutions were made in deionized water on the day of experimentation and maintained on ice for the duration of the experiment. A stock solution of IbTX (Sigma-Aldrich) (1 x 10-5 mol/liter) was made in saline. Fresh Krebs-Henseleit PSS was prepared daily.
Statistical analysis
For single-channel activity data (NPo values) are expressed as mean ± SE of the mean. Comparisons between groups were made by one-way ANOVA, with a post hoc Tukey test to determine significant differences among data groups. For isometric recordings, multiple comparisons of measured integrals of contractility were performed using two-way ANOVA, followed by post hoc testing using Newman-Keuls test. The statistical packages (SPSS, version 11.0, SPSS Inc., Chicago, IL) and GB Stat (version 6.5; GB Stat, Silver Spring, MD) were used for statistical calculations. A P value <0.05 was accepted as statistically significant.
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Results |
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Myometrial biopsies were obtained from 15 women. The reasons for cesarean section included breech presentation (n = 3), previous cesarean section (n = 9), maternal spine abnormality (n = 1), and cephalopelvic disproportion (n = 2). All cesarean sections were performed before the onset of labor under regional anesthesia. The maternal demographic details were as follows: mean age 31.3 yr (range 23–42); median period of gestation 38 wk (range 37–39); and median parity 1 (range 0–2).
BKCa channel activity
Single-channel recordings from freshly dispersed myocytes, using the cell attached configuration of the patch clamp technique, revealed ion channel activity with an amplitude average of 7–10 pA at +40 mV identifying the BKCa channel (19). NS1619, a specific BKCa channel opener, elicited potent channel activation at applied concentration of 20 µmol/liter (Fig. 1
). Furthermore, application of 10 mmol/liter tetraethylammonium (TEA), which exhibits selectivity for BKCa channels, reversed the NS1619 activation of this channel. These experiments identified the BKCa channel as the predominant K+ channel expressed in human myometrial myocytes, consistent with previous studies on these cells (13, 14, 16).
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. Cumulative additions of hCG resulted in a potent activation of BKCa channel activity in a concentration-dependent fashion, as illustrated. BKCa channel activity for this recording was increased following the addition of 1 (NPo = 0.088), 10 (NPo = 0.121), and 100 (NPo = 0.107) IU/ml hCG, respectively. The average NPo (Fig. 3B) before and after addition of hCG was as follows: control 0.036 ± 0.01 (n = 5); 1 IU/ml hCG 0.065 ± 0.014 (n = 5; P = 0.262); 10 IU/ml hCG 0.111 ± 0.009 (n = 5; P = 0.001); and 100 IU/ml hCG 0.098 ± 0.004 (n = 4; P = 0.007). The hCG stimulated the BKCa channel with a maximal activation effect observed at a concentration of 10 IU/ml, and the effect appeared to plateau at higher concentrations. Open-state channel activity was stimulated 2- and 3-fold by 1 and 10 IU/ml hCG, respectively; however, subsequent addition of 100 IU/ml hCG did not elicit significant further effect. In general, there was a 5- to 10-min latency period before observation of hCG-stimulated channel activity, and this effect appeared to be maximal within 15–20 min. In all patches this activity persisted until either seal integrity was lost, or the experiment was terminated.
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The hCG exerted a cumulative inhibitory effect on contractility in isolated pregnant human myometrium (n = 6). Figure 4A demonstrates a representative recording of oxytocin (0.5 nmol/liter) elicited myometrial contractility, and in Fig. 4B the effects of cumulative additions of hCG (0.1, 0.5, 1, 5, and 10 IU/ml) to the tissue bath are shown. ANOVA revealed a significant effect on contractility of both addition of hCG (group 1 vs. group 2; P < 0.05) and increasing hCG concentrations. Post hoc analysis showed a statistically significant relaxant effect of hCG at bath concentrations of 1 (n = 6; P < 0.01), 5 (n = 6; P < 0.01), and 10 (n = 6; P < 0.01) IU/ml when compared with respective control values measured from strips exposed to oxytocin alone.
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a representative recording demonstrating the effects of preincubation with the BKCa channel antagonist IbTX, on the uterorelaxant effects of hCG, is shown. Comparison of the inhibitory effects of hCG on the oxytocin-induced contractility of myometrial strips, in the presence or absence of IbTX, revealed a significant difference across the groups (group 2 vs. group 4; n = 6; P < 0.05). Post hoc analysis revealed that preincubation with IbTX (100 nmol/liter) significantly attenuated the uterorelaxant effect of hCG at all test concentrations, resulting in no significant difference when compared with control values [group 3 (n = 5) vs. group 4 (n = 6); P > 0.05] at all hCG bath concentrations. In Fig. 5 the effects of cumulative additions of hCG (0.1, 0.5, 1, 5, and 10 IU/ml) on contractions of myometrial strips, in the absence and presence of IbTX, are shown in graphical form alongside the integrals observed in control strips.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our experiments, single-channel patch clamp studies clearly identified BKCa channels in the isolated myocytes and demonstrated that hCG significantly increases their open state probability. These studies are consistent with previous reports indicating that the BKCa channel is the predominant potassium channel in primary myocytes from human pregnant myometrium (13, 14, 15). Because of its large conductance, and high density of expression, this channel plays an important role in the regulation of membrane potential and cellular excitability of the myometrium and hence has been reported as playing a significant role in modulating myometrial function in both animal models and humans. Initial links between the myometrial BKCa channel and parturition were evident with the report outlining that it apparently loses its Ca2+ and voltage sensitivity in human myometrium with the onset of labor (13). In addition, NS1619, the specific BKCa channel activator, exerts a potent relaxant effect on pregnant human myometrium, in in vitro experiments (16). Using both inside-out and outside-out configurations of the patch clamp technique, NS1619 appears to act directly on myometrial BKCa channels and stimulates channel activity by increasing their time spent in the open state (16). The findings from this study indicate that hCG exerts its uterorelaxant effect in a manner similar to that of NS1619.
The results from single BKCa channel recordings concur with the data obtained from isometric tension recordings with human myometrial strips. After preincubation with IbTX, the contractility measured following hCG exposure, was not significantly different to that of control strips (i.e. strips exposed to oxytocin alone or oxytocin and IbTX). This confirms the fact that BKCa channel function is intrinsic to myometrial contractions and also providing evidence that this channel plays a central role in hCG-mediated relaxation. In our study, after the equilibration period, the strips were exposed to oxytocin to achieve regular rhythmic contractions, which are reproducible over a period of hours and hence serve as a reliable control, unlike spontaneous contractions in vitro (23). In our experiments, there was no significant difference observed in integrals of contractility in strips exposed to oxytocin alone vs. those exposed to oxytocin and IbTX, before bath addition of hCG.
Although the physiological and clinical importance of endogenous factors regulating myometrial quiescence during pregnancy are obvious, the potential use of hCG, administered exogenously, as a therapeutic agent for preterm labor, has been recently debated (7, 24, 25). Whole hCG exhibited potent inhibition of PGF2
-induced preterm delivery in mice, and the effect was dose dependent (25). There are no reliable data in relation to clinical studies, to our knowledge. Our findings support the hypothesis that clinical tocolysis using hCG remains a possibility, with a predominant mechanism of action via BKCa channel opening in myometrial cells.
We did not address the possibility that the effects outlined in our results may be altered by labor onset. This remains an exciting study for further evaluation of both hCG effector mechanisms, and BKCa channel activity, in myometrial tissue obtained during spontaneous labor, with similar clinical criteria as outlined for this study.
In conclusion, we report the first direct evidence that hCG exerts a potent uterorelaxant effect in human pregnancy via BKCa channel activation. These novel data raise further questions about a central role of hCG, and BKCa channel function, in association with uterine quiescence and parturition. These findings support the growing hypothesis that hCG may confer therapeutic benefit in preterm labor management.
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Acknowledgments |
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Footnotes |
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Abbreviations: BKCa, Calcium-activated K+ channel; hCG, human chorionic gonadotropin; IbTX, iberiotoxin; NPo, mean open probability; PSS, physiologic saline solution; TEA, tetraethylammonium.
Received February 10, 2003.
Accepted May 27, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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