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Institute of Cell Signalling (B.L., S.J.H.), University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom; and Centre for Reproduction and Early Life (R.N.K.), Institute for Clinical Research, University of Nottingham, Nottingham NG7 2RD, United Kingdom
Address all correspondence and requests for reprints to: Raheela N. Khan, Academic Division of Obstetrics and Gynaecology, University of Nottingham, The Medical School, Derby City General Hospital, Uttoxeter New Road, Derby DE22 3DT, United Kingdom. E-mail: raheela.khan{at}nottingham.ac.uk.
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Abstract |
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Objective: The objective was to characterize the effect of OT on the T-type calcium channel in human decidual stromal cells before and after the onset of labor.
Design: The nystatin-perforated patch-clamp technique was used to record inward T-type calcium current (ICa(T)) from acutely dispersed decidual stromal cells obtained from women at either elective cesarean section [CS (nonlabor)] or after normal spontaneous vaginal delivery [SVD (labor)].
Setting: These studies took place at the University of Nottingham Medical School.
Results: ICa(T) of both SVD and CS cells were blocked by nickel (IC50 of 5.6 µM) and cobalt chloride (1 mM) but unaffected by nifedipine (10 µM). OT (1 nM to 3.5 µM) inhibited ICa(T) of SVD cells in a concentration-dependent manner, with a maximal inhibition of 79.0% compared with 26.2% in decidual cells of the CS group. OT-evoked reduction of ICa(T) was prevented by preincubation with the OT antagonist L371,257 in the SVD but not CS group. OT, in a concentration-dependent manner, displaced the steady-state inactivation curve for ICa(T) to the left in the SVD group with no significant effect on curves of the CS group.
Conclusion: Inhibition of ICa(T) by OT in decidual cells obtained during labor may signify important functional remodeling of uterine signaling during this period.
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Introduction |
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-smooth muscle-actin staining (1) and display contractile activity that may play a role in expulsion of the trophoblast in spontaneous miscarriage (2). Due to its privileged location at the maternal-fetal interface, the decidua is uniquely sited to receive and transduce a variety of chemical and mechanical stimuli pivotal in the control of pregnancy. Hence, decidual physiology may be intimately linked to the timing and control of parturition. The decidua, as an endocrine organ, is also a source of many hormones, including prolactin, a marker of decidualization (3), and the neurohypophyseal nonapeptide hormone oxytocin (OT) (4). Although indisputably identified as one of the most potent uterotonic agents, scientific and clinical findings no longer support an unequivocal role for OT as a trigger for the initiation of labor. However, it continues to be of clinical significance as a therapeutic for the augmentation and induction of labor, principally through its actions on myometrial contractility. This property has been exploited in the development of OT receptor antagonists (atosiban, barusiban), as tocolytics for the treatment of preterm labor (5, 6), a leading cause of preterm birth. Although the majority of infants born prematurely (>32 wk) have excellent survival rates, of those babies that survive after birth at an extremely preterm gestation (<26 wk), many may suffer considerable associated disability and cognitive impairment (7, 8). Hence, new therapeutic targets for preventing or treating the onset of preterm labor are needed to reduce the incidence of morbidity caused by prematurity. An understanding of decidual function coupled with better predictive methods for detecting those women at risk of preterm labor may permit earlier clinical intervention before myometrial contractility is triggered.
Several distinct genes, CaV1, CaV2, and CaV3, that encode the -subunit of Ca2+ channels have been identified that give rise to a diversity of physiologically and pharmacologically distinct Ca2+ currents (9). The secretion of hormones and neurotransmitters (10), differentiation of myoblasts into skeletal myotubes (11), and burst firing in neurons (12) are attributed to the involvement of T-type Ca2+ channels. These channels are encoded by the CaV3 gene family, expression of which has been detected in human placenta (13, 14), although the exact location of CaV3 expression within the placenta was not defined.
Due to the significance of OT in many facets of pregnancy and lactation as well as its mobilization of Ca2+ from intracellular stores, we hypothesized that this nonapeptide modulates the electrical excitability of decidual stromal cells in a manner that could impact directly on the control of myometrial contractility, thereby determining the timing of labor. Our recent studies have demonstrated differential expression of voltage-dependent K+ currents with the onset of labor in decidual stromal cells (15), suggesting possible functional adaptations to delivery of the fetus. We describe here the modulation of T-type Ca2+ current by OT in freshly dispersed stromal cells obtained before and after the onset of uncomplicated labor. Our findings bring to the fore the complexity of receptor-channel interactions in this stromal cell preparation and their relevance to the timely onset of labor.
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Materials and Methods |
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Fragments of decidua parietalis (
2 g) were gently peeled away from the chorionic surface of fresh placentae, placed in cold physiological salt solution (PSS), and transported to the laboratory. Isolated decidual stromal cells were obtained by finely mincing the decidua into 1–2 mm3 pieces, followed by incubation in 1 mg/ml collagenase in Ca2+/Mg2+-free Hanks’ balanced salt solution at 37 C for 1 h. Thereafter, the cell preparation was layered onto 60% (v/v) Percoll and centrifuged at 800 x g for 20 min, followed by two additional washes in PSS (100 x g at 4 C) to yield a cell pellet. The latter was then incubated with CD45-coated Dynal beads to remove leukocytes as described previously (15). The purified decidual stromal cell preparation was immediately transferred into a second tube for electrophysiological studies after assessment of cell viability by trypan blue exclusion.
Whole-cell perforated-patch-clamp recordings
Freshly dispersed cells were placed directly in a cell chamber mounted on the stage of an inverted microscope (Eclipse TE200; Nikon, Tokyo, Japan), allowed to settle, and then washed with PSS at a constant speed (6 ml/min). This solution was changed to one designed to facilitate recordings of and maximize inward current by inclusion of tetraethylammonium chloride (TEA) and 4-aminopyridine (4-AP) (for composition, see Solutions and drugs below). The nystatin-perforated whole-cell configuration of the patch-clamp technique (16) was used to record macroscopic current through voltage-activated Ca2+ channels in dispersed decidual stromal cells. Pipettes were pulled from borosilicate glass (Heka, Lambrecht, Germany) and had resistances of 2–5 M
when filled with internal solution containing nystatin (150–300 mg/ml). Electrodes were initially placed in nystatin-free electrode solution before backfilling with nystatin-containing electrode solution to ensure that the antibiotic did not interfere with seal formation. Automatic series resistance and capacitance compensation were performed routinely and monitored continuously. Series resistance was typically less than 10 M, and recordings were terminated if this changed by more than 25% during the recording period. All voltage pulses were delivered through an EPC-9 amplifier using Pulse/PulseFit software (version 8.53; Heka).
After formation of a gigaohm seal, cell capacitance was monitored as nystatin perforated the membrane patch circumscribed by the electrode. Access to the cell as seen by an increase in the slow capacitance was obtained within 15 min. Inward current was elicited by depolarizing cells from a holding potential (Vh) of either –80 or –50 mV in 10 mV steps to +60 mV (100-msec duration) at a frequency of 0.1 Hz. Digital subtraction of macroscopic currents generated at the two different Vh values isolates the fast-inactivating inward T-type calcium current (ICa(T)) component. Leak subtraction, using an automated positive/negative protocol, was performed on all currents. Currents were filtered at 1 kHz and sampled at 2 kHz.
For studies of current activation of ICa(T), short 10-msec pulses from Vh values of –80 to +60 mV in 10 mV increments were used to obtain the activation curve. Steady-state inactivation of ICa(T) was performed using a double-pulse protocol in which a 10 sec prepulse of 10 mV incremental depolarizations from a Vh of –100 mV was followed by a test pulse to a fixed membrane voltage of +60 mV. Inactivation and activation curves derived were fitted to the Boltzmann equation as follows: I/Imax = 1/[1 + exp((Vm – V0.5)/k)], where I/Imax represents current during the prepulse normalized with respect to maximal test current, V0.5 is the voltage at which 50% inactivation of current occurs, Vm is the prepulse voltage, and k describes the slope constant of the curves.
The effects of the Ca2+ channel blockers 1 mM Co2+, 10 µM nifedipine, and varying concentrations of Ni2+ (0.1, 10, 50, and 100 µM) on ICa(T) were tested under voltage clamp using the pulse protocol described above. The sensitivity of evoked ICa to OT and the OT receptor antagonist L371,257 (17) was also evaluated. Drugs were applied by gravity perfusion of the drug directly into the bathing chamber containing the cell under investigation. Experiments were performed at room temperature (20–24 C).
Solutions and drugs
With the exception of L371,257, all chemicals and drugs were purchased from Sigma-Aldrich (Poole, UK). The OT antagonist L371,257 was a generous gift from Prof. S. Thornton (Warwick University, Coventry, UK). External PSS consisted of 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.4. For measurement of ICa(T), 0.1 µM tetrodotoxin, 20 mM TEA, 4 mM 4-AP, and 5 mM Ba2+ were added to PSS to block voltage-dependent Na+ and K+ channels. The concentration of extracellular Na+ was adjusted to take account of the addition of 4-AP and TEA. Electrode (internal) solution contained cesium chloride as the main charge carrier in place of KCl and consisted of 140 mM CsCl, 1 mM CaCl2, 1 mM Mg-ATP, 11 mM EGTA, and 10 mM HEPES, pH7.2. Nystatin was prepared as a stock solution in dimethyl sulfoxide, which was kept at –20 C for no more than 1 wk and sonicated for 30 sec before use. L371,257 and nifedipine were prepared in dimethyl sulfoxide, and the latter was protected from light. Tetrodotoxin was prepared as a stock solution in 0.5 M acetic acid. All solutions were freshly made on the day of experiment from stock solutions kept at –20 C.
Data and statistical analysis
Data acquisition and analysis were performed with Pulse/PulseFit software (version 8.53; Heka) and Microsoft Excel (Microsoft, Redmond, WA) on a Pentium III personal computer. Curve fitting of activation and inactivation curves was performed using IgorPro (WaveMetrics, Lake Oswego, OR). Concentration-response curves were analyzed in Prism version 3.0 (GraphPad Software, San Diego, CA), and the concentration required to produce IC50 was obtained. Results are expressed as the mean ± SEM of n observations. Statistically significant differences were evaluated by using paired or unpaired two-tailed Student’s t test as appropriate. A P value of less than 0.05 was taken to be significant.
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Results |
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Figure 1
shows decidual cells prepared before (A) and after (B) incubation with anti-CD45-coated Dynabeads. Before treatment, a mixed cell population of leukocytes and decidual cells distinguishable according to size and morphology were observed. Very few leukocytes are identifiable in this highly pure decidual cell preparation. Over 95% of decidual cells from both the SVD and CS cells were viable after trypan blue exclusion. Moreover, our previous findings showed that this isolation procedure removed 97% of leukocytes as determined by flow cytometry (15). Decidual stromal cells as in Fig. 1B, identified by their large size, stellar appearance, and staining for vimentin (data not shown), were used exclusively for electrophysiology.
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Nystatin-perforated whole-cell recordings of decidual stromal cells proved to be stable for a maximum of 2 h. Representative rapidly inactivating inward currents, ICa(T), from SVD cells were observed after digital subtraction of inward currents evoked from Vh values of –50 and –80 mV (Fig. 1C
). The threshold for current activation of ICa(T) occurred around –60 mV, with currents peaking at approximately –30 mV (Fig. 1D), and had a mean peak amplitude of 82.1 ± 6.9 pA (n = 14) when cells were depolarized from a Vh of –80 to –30 mV. There was no significant difference in mean ICa(T) between SVD and CS cells (P > 0.05).
Stromal ICa(T) from both the SVD and CS groups was unaffected (P > 0.05) by the organic L-type Ca2+ channel blocker nifedipine (10 µM) compared with control (n = 6 per group) (Fig. 2A). Extracellular addition of Co2+ (1 mM) significantly reduced ICa(T) activity in both SVD (n = 8; P < 0.05) and CS (n = 8; P < 0.05) cells (Fig. 2A).
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) and CS groups that was near maximal at 100 µM and characterized by an IC50 of 5.6 µM (data not shown). Compared with control, peak ICa(T) in the SVD group at –30 mV was decreased by 14.5% (P > 0.05; n = 4), 50.4% (P < 0.05; n = 3), 60.6% (P < 0.05; n = 4), and 79.4% (P < 0.05; n = 3) after the cells were treated with 0.1, 10, 50, and 100 µM Ni2+, respectively.
Additional experiments were designed to assess whether ICa(T) activation and inactivation profiles are similar in the two groups using two distinct protocols (Fig. 3, A and B). Control data demonstrated no significant difference between the SVD and CS groups for the activation curves (Fig. 3C). However, the inactivation curve for the CS group was significantly shifted to the left compared with that of the SVD group. V0.5 and k values for the CS group were –60.2 ± 0.6 mV and 8.1 ± 0.5 (n = 8), respectively, compared with –66.2 ± 0.6 mV and 11.0 ± 0.8 for the SVD group (n = 8).
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The effects of increasing concentrations of the hormone OT (0.001–3.5 µM) were tested on ICa(T) of decidual cells from both the SVD and CS groups. We found that OT had a marked inhibitory effect on ICa(T) in the SVD group (Fig. 4A) that was much reduced when tested on ICa(T) in cells of the CS group (Fig. 4B). In the former, OT reversibly reduced peak ICa(T) (at –30 mV) by 33.7% (P > 0.05), 53.3% (P < 0.05), 73.1% (P < 0.05), and 79.0% (P < 0.01) at respective concentrations of 0.1, 0.35, 1, and 3.5 µM OT compared with control peak current (n = 10–12) (Fig. 5). A summary of the degree of inhibition for both the SVD and CS groups is shown in Fig. 5. OT blocked ICa(T) with an IC50 of 0.21 µM in the SVD group and 0.35 µM for the CS group (P > 0.05).
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). In contrast, L371,257 had no significant effect on OT-inhibited ICa(T) in the CS group (P > 0.05; n = 10), nor did L371,257 have any detectable effect on inward currents when applied without OT (n = 3; data not shown).
Effects of varying concentrations of OT on current activation and inactivation were also analyzed separately in the SVD and CS groups. OT shifted the steady-state inactivation curves of the SVD group to the left (Fig. 6). In keeping with the lack of effect of OT on ICa(T) of CS cells, the hormone produced little significant shift in V0.5 and k for activation or inactivation in the CS group (n = 6). Results from both groups are summarized in Tables 1 and 2.
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Discussion |
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OT has varied functions in the uterus. In addition to its role as a powerful contractile agent, OT indirectly augments myometrial contractility by stimulating prostaglandin biosynthesis in the decidua (22, 23). Furthermore, OT in human myometrium is intricately linked to Ca2+ signaling in which its application induces activation of both nonselective cation and chloride conductances in pregnant rat myometrium (24) and mobilization of intracellular Ca2+ after phosphoinositide hydrolysis via phospholipase C and G proteins of the G(q/11) subclass (25). Our novel observation that decidual ICa(T) observed after the onset of labor is much more sensitive to OT than that of nonlabor decidual cells is intriguing and suggests a possible role for this channel in the temporal control of labor. We propose that the inhibitory action of OT on ICa(T) is likely to be mediated by direct coupling of the OT receptor to T-type Ca2+ channels because this current in cells of the SVD group was significantly attenuated in the presence of the selective OT receptor antagonist L371,257. It is interesting to note that the maximal inhibition of approximately 30% of ICa(T) by OT in CS cells mirrors that of the L371,257-resistant component in SVD cells. The similar magnitude of these two responses implies that part of the OT response is unlikely to be mediated directly via the OT receptor and may reflect a lack of functional coupling between decidual T-type Ca2+ channels and the OT receptor before labor is initiated, possibly related to altered expression of Ca2+ channel subunits. OT could also be acting indirectly to modulate ICa(T) because the perforated-patch method used would largely be expected to preserve the integrity of signaling pathways by preventing "washout" of key cytosolic messengers.
The observed concentration-dependent, hyperpolarizing shift of the steady-state inactivation curves of the SVD group with OT suggests a reduced T-type channel availability. Similar effects for ICa(T) with atrial natriuretic peptide (10) and somatostatin (26), which, like OT, caused displacement of the steady-state inactivation curve to more negative potentials, have been observed. Human decidual cells have a resting membrane potential of approximately –63 mV (15). The mean control V0.5 value (–60.1 mV) obtained for inactivation for the SVD group is close to the reported resting membrane potential and indicates approximately 50% T-type channel availability at rest, which is reduced to 20% at the highest OT concentration tested. This would have the effect of reducing Ca2+ influx, thereby potentially altering downstream Ca2+-dependent processes.
Since the discovery of extrahypothalamic production of OT, debate over its role in the initiation of human labor continues. Chibbar et al. (4) have argued that the presence of mRNA for OT peptide supports intrauterine production of OT for stimulation of myometrial contractions and the timing of birth. This would also explain the lack of raised serum OT levels before the onset of labor (27). However, studies in the rat have questioned whether lower decidual OT peptide levels (in stark contrast to the high levels of decidual OT mRNA) are sufficient or indeed required to sustain myometrial contractility (28). In favor of this, evidence from the human laboring uterus supports high mRNA but low peptide levels of decidual OT (29). Decidual OT levels are also lower than those encountered in fundal and lower segment myometrial biopsies, as shown by positive detection of the functional -amidated form of OT at term (29). It is not clear why this is so, but local spatial gradients in OT levels may facilitate in compartmentalizing tissue-specific functions of OT. Decidual OT may also be acting locally to regulate prostaglandin formation for subsequent actions on the myometrium or it may control hormone secretion, possibly via modulation of ICa(T) rather than any direct actions on myometrial contractility. Indeed, it has been demonstrated in vitro that incubation of rat myometrial strips with or without decidua modifies the pattern of contractility such that, in the presence of decidua, OT and prostaglandin-induced contractions had a much reduced frequency (30), suggesting paracrine regulation via other decidua-derived mediators.
Although speculative, we also considered the notion that the observed inhibitory effect of OT on ICa(T) may represent an autocrine mechanism whereby OT inhibits its own secretion from decidual cells by negative feedback mechanisms through ICa(T) as the need for it diminishes as delivery nears completion. For the present studies, decidual tissue from SVD was obtained after delivery of the fetus and placenta after completion of the third and end stage of labor. During this period, the requirement for decidual OT is likely to be very low. It is plausible that, because of this, the reported inhibitory effects of OT on ICa(T) are simply to prepare the uterus for the puerperium: the period during which the uterus undergoes extensive remodelling for its return to a nonpregnant state and hence reversal of many pregnancy-specific physiological pathways. For this reason, comparison with decidual cells of placentae obtained at emergency CS may help to resolve the issue of whether ICa(T) inhibition by OT also occurs at earlier stages of labor.
In conclusion, the appreciable degree of inhibition of ICa(T) by OT observed in laboring decidua may be a mechanism whereby regulation of stromal cell [Ca2+]i levels is temporally regulated in parallel with the phasic shift from uterine quiescence to myometrial activation and finally termination of labor. We suggest that the differential action of OT implies that key signaling and feedback mechanisms are activated or inactivated preferentially with the onset of labor in human decidua and that this may be governed by ICa(T) regulation.
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Acknowledgments |
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Footnotes |
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First Published Online April 26, 2005
Abbreviations: 4-AP, 4-Aminopyridine; CS, cesarean section; ICa(T), inward T-type calcium current; OT, oxytocin; PSS, physiological salt solution; SVD, spontaneous vaginal delivery; TEA, tetraethylammonium chloride; Vh, holding potential.
Received March 3, 2005.
Accepted April 19, 2005.
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References |
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1H Ca2+ channels are involved in Ca2+ signalling during terminal differentiation (fusion) of human myoblasts. Proc Natl Acad Sci USA 97:7627–76321H from human heart, a member of the T-type Ca2+ channel gene family. Circ Res 83:103–109
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