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MOLECULAR AND GENOMIC |
1 Clinical Sciences Research Institute, Division of Clinical Sciences, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK
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Abstract |
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1G), Cav3.2(1H), and Cav3.3(1I). QRT-PCR, immunohistochemistry, electrophysiology and invitro contractility were performed on human myometrial samples from term, preterm, labour and not in labour. QRT-PCR analysis of Cav3.1, Cav3.2 and Cav3.3 demonstrated expression of Cav3.1 and Cav3.2 with no significant change (P > 0.05) associated with gestation or labour status. Immunohistochemistry localized Cav3.1 to myometrial and vascular smooth muscle cells whilst Cav3.2 localized to vascular endothelial cells and invading leucocytes. Voltage clamp studies demonstrated a T-type current in 55% of cells. Nickel block of T-type current was voltage sensitive (IC50 of 118.57 ± 68.9 µM at –30 mV). Activation and inactivation curves of ICa currents in cells expressing T-type channels overlapped demonstrating steady state window currents at the resting membrane potential of myometrium at term. Current clamp analysis demonstrated that hyperpolarizing pulses to a membrane potential greater than –80 mV elicited rebound calcium spikes that were blocked reversibly by 100 µM nickel. Contractility studies demonstrated a reversible decrease in contraction frequency during application of 100 µM nickel (P < 0.05). We conclude that the primary T-type subunit expressed in some MSMCs is Cav3.1. We found that application of 100 µM nickel to spontaneously contracting human myometrium reversibly slows contraction frequency.
(Received 21 March 2007;
accepted after revision 16 April 2007;
first published online 19 April 2007)
Corresponding author A. M. Blanks: Clinical Science Research Institute, Division of Clinical Sciences, Warwick Medical School, Coventry CV4 7AL, UK. Email: andrew.blanks{at}warwick.ac.uk
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Introduction |
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Although much research has been dedicated to the mechanism by which the sex steroids mediate their influence on myometrium little is known about the physiological mechanism or the molecular identity of currents that manifest spontaneous contractions at term. Some investigations have explored the role of calcium activated K+ channels (Anwer et al. 1993; Khan et al. 1993, 1997; Perez et al. 1993) and voltage activated K+ channels (Knock et al. 1999) in control of resting membrane potential and repolarization. However, the molecular identity or physiology of a pacemaker current remains elusive.
What is clear is that control of resting membrane potential and rises in intracellular calcium are essential for contraction in human myometrial smooth muscle (MSM) (Parkington et al. 1999b). Calcium is not only an important second messenger for the generation of force via myosin light chain kinase, but also depolarizes the plasma membrane allowing for activation of other voltage-dependent ion channels. This latter property is an important function for T-type or low voltage activated (LVA) calcium channels, which are responsible for generating low threshold spikes that in neurons lead to burst firing and oscillatory behaviour (Kim et al. 2001). The subfamily of T-type calcium channels currently comprises of three differing subunits termed Cav3.1 (1G), Cav3.2 (1H), and Cav3.3 (1I) (Cribbs et al. 1998; Perez-Reyes et al. 1998; Lee et al. 1999a; Monteil et al. 2000). Cav3.1 and Cav3.2 demonstrate similar activation and inactivation kinetics but can be differentiated by sensitivity to nickel and recovery from inactivation. Cav3.3 by contrast is easily distinguishable by its slower activation and inactivation kinetics.
In a recent molecular study in the rat it was demonstrated that both Cav3.1 and Cav3.2 were expressed in circular and longitudinal layers of myometrium and that the relative expression profile of these channels differed, dependent on gestational age, layer and subunit (Ohkubo et al. 2005b).
It has previously been demonstrated in electrophysiological studies that a T-type-like current is present in human myometrium (Young et al. 1993) and that it is larger in magnitude than the more extensively investigated L-type current. In a recent study it was demonstrated that administration of mibefradil, a partially selective T-type inhibitor, reduced the force generated during a contraction whilst decreasing the magnitude of the initiation spike of tissue-level electrical activity (Young & Zhang, 2005). This suggests that the T-type calcium channel may be involved in the initiation of action potentials in uterine smooth muscle. We sought to investigate the role of the T-type calcium channel in human myometrium by characterizing the expression, electrophysiological properties and function of channel subunits Cav3.1, Cav3.2, and Cav3.3.
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Methods |
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All procedures were conducted within the guidelines of The Declaration of Helsinki and were subject to local ethical approval (REC-05/Q2802/107). Prior to surgery, informed written consent for sample collection was obtained. Subjects were recruited into two groups, spontaneous labour (LAB) and elective cesarean section not in labour (NIL) between 32 and 40 weeks gestation. Term was defined as > 37 completed weeks gestation and preterm labour defined as < 37 completed weeks. The LAB group was undergoing caesarean section for reasons of presumed fetal distress. LAB was defined as regular contractions (< 3 min apart), membrane rupture, and cervical dilatation (> 2 cm) with no augmentation.
Sample collection
At caesarean section, samples were collected before syntocin administration by knife biopsy from the lower uterine segment incision. Samples were washed briefly in saline and flash-frozen in liquid nitrogen for mRNA immunohistochemistry. Samples for cell isolation were placed in ice cold modified Krebs–Henseleit solution (see below) and utilized the same day.
Solutions
Fresh samples were stored in ice cold modified Krebs–Henseleit (m-KHS) solution containing (mmol l–1): NaCl, 133; KCl, 4.7; Tes, 10; glucose, 11.1; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; adjusted to pH 7.4 at 25°C with 5 M NaOH. Krebs–Henseleit (KHS) solution contained (mmol l–1): NaCl, 118; NaHCO3, 25; KCl, 4.7; glucose, 11.1; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; pH 7.4 was maintained by constant aeriation with 5% CO2–95% O2 at 37°C. Ca2+-free Tyrode solution contained (mmol l–1): NaCl, 136; KCl, 5.4; MgSO4, 1.0; NaH2PO4, 0.33; glucose, 5; Hepes, 10; adjusted to pH 7.4 at 25°C with 5 M NaOH. Digestion solution (DS) was Ca2+-free Tyrode solution containing: Sigma type IX collagenase (1790 IU mg–1), 1.25 mg ml–1; Sigma type IA collagenase (535 IU mg–1) 1.25 mg ml–1; bovine serum albumin, 1 mg ml–1. Kraftbrühe (KB) solution was as described in Klockner & Isenberg (1985). Ca2+-free physiological salt solution (PSS) contained (mmol l–1): NaCl, 130; KCl, 5; MgCl2, 1.2; Hepes, 10; and glucose, 10; adjusted to pH 7.4 at 25°C with 5 M NaOH. The electrode (interal) solution for voltage clamp contained (mmol l–1): CsCl,135; MgCl2, 2.5; MgATP, 5; Hepes, 10; and EGTA, 10; adjusted to pH 7.2 with CsOH. The bath solution was composed of (mmol l–1): NaCl, 120; CsCl, 1.0; tetraethylammonium chloride (TEA-Cl), 4.0; MgCl2, 1.2; CaCl2, 2.0; Hepes, 10.0; and glucose, 10.0; the pH was adjusted to pH 7.4 with 5 M NaOH. For current clamp experiments bath solution was m-KHS. Electrode solution used in these experiments contained (mmol l–1): NaCl, 12; KCl, 40; potassium glutamate, 90; Tes, 10; sodium pyruvate, 1; MgSO4, 1; EGTA, 0.2; CaCl2, 0.0803; pH 7.2. Nifedipine stock solution was 10 mmol l–1 in DMSO. Nickel stock solution was 100 mmol l–1 in water. Final DMSO concentrations were less than 0.01%. All reagents were obtained from Sigma (Sigma-Aldrich Co., UK) unless otherwise stated.
Cell isolation
Strips of myometrium (2 x 2 x 4–5 mm) were incubated for 30 min in Ca2+-free Tyrode solution followed by 40–45 min at 37°C in DS. Digestion was terminated by dilution in prewarmed Ca2+-free Tyrode solution followed by centrifugation for 10 min at 250 g. Cells were dispersed by slow tritutration through a wide bore fire polished glass pipette in KB solution. Single myometrial cells were filtered through a 200 µM gauze and stored in KB solution for at least 1 h before experimental procedures. All experiments were performed within 6 h after isolation.
Electrophysiology
Voltage clamp.
A drop of myometrial cell suspension was placed in a cell chamber (1.0 ml) onto a glass coverslip mounted on the stage of an inverted microscope (IX51, Olympus). After settling (approx 20 min) cells were superfused with bath solution for 10 min at a rate of 1–2 ml min–1 at room temperature. Transmembrane currents were recorded with an amplifier (Axopatch 200B; Axon Instruments). Patch pipettes were fabricated (Model P-87; Sutter Instruments, Novato, CA, USA) from 1.5 mm glass capillaries with a resistance of 2.0–4.0 M
when filled with pipette solution. Liquid junction potential was zeroed prior to seal formation.
Following the formation of a gigaseal, the membrane was ruptured by gentle suction obtaining the whole-cell voltage-clamp configuration. Membrane capacitance and series resistance were compensated after membrane rupture. Inward current was elicited by depolarizing voltage steps at a frequency of 0.1 Hz from a holding potential (HP) of either –50 mV or –80 mV to +60 mV in 10 mV increments. Currents were filtered at 1 kHz and sampled at 2 kHz. Voltage protocols were delivered via a Digidata 1320 computer interface using pCLAMP 9.0 software (Molecular Devices, Sunnyvale, CA, USA). Passive leakage currents were subtracted using a positive/negative (P/N) protocol. All experiments were carried out at 22–24°C.
Current clamp.
Cells were treated similarly to voltage clamp experiments. The resting potential and action potentials were recorded using 4–6 M pipettes connected to a headstage of a discontinous voltage/current clamp amplifier (SEC-05, npi electronic GmbH, Tamm, Germany) operated in discontinous current clamp mode. The current and voltage outputs of the amplifier were recorded and the current stimuli delivered via an ITC-18 computer interface (InstruTECH, Port Washington, NY, USA) controlled by PatchMaster software (HEKA Electronik GmbH, Lambrecht, Germany). Liquid junction potential of 15 mV was subtracted from the obtained records. All experiments were carried out at 35°C.
RNA analysis
RNA was isolated and reverse transcribed into cDNA for PCR analysis as previously described (Astle et al. 2005). The expressions of r18S, Cav3.1, Cav3.2 and Cav3.3 were verified by real-time RT-PCR using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. TaqMan gene expression assay primer/probe identification numbers were Cav3.1 (Hs00367969_pm1), Cav3.2 (Hs00184168_m1), and Cav3.3 (Hs00184168_m1) (Applied Biosystems). Expression was normalized using the 
CT method to r18S and non-pregnant myometrium. Amplification efficiency was determined over a linear cDNA titration for each probe set.
Immunohistochemistry
Sample preparation. Frozen sections (8 µm) of myometrium were briefly air dried and fixed for 30 min in ice cold 4% paraformaldeyde in phosphate buffered saline. Isolated myocytes were plated onto coverslips in HBSS and placed overnight in a culture incubator. Plated cells were fixed for 5 min in ice cold 4% paraformaldeyde in phosphate buffered saline.
Antibody incubation. Detection of Cav3.1, Cav3.2 and Cav3.3 was performed according to the manufacturer's instruction using a 1 : 200 dilution (Alomone Labs, Jerusalem, Israel).
Secondary antibody (anti-rabbit Alexa 635) (Molecular Probes, Invitrogen Ltd, Paisley, UK) was used according to manufacturer's instruction using a 1 : 200 dilution. Positive staining was compared with secondary antibody alone. F-actin staining was determined by addition of Phalloidin-488 (Molecular Probes, Invitrogen Ltd, Paisley, UK) according to the manufactures instructions.
Confocal microscopy
After preparation, tissue sections or cells were examined using a Zeiss 510 META confocal microscope with x40 (NA 1.3, Oil DIC) and x63 (NA 1.4, Oil DIC) objectives. The pinhole was set to 1 Airy unit and data were captured in 512 x 512 format.
Myometrial contractility
Contractility experiments were performed as previously described (Woodcock et al. 2006). Myometrial strips were allowed a 2 h equilibration period to establish spontaneous contractions prior to addition of 100 µmol l–1 NiCl2.
Data analysis and statistics
All raw electrophysiological, expression and contractility data were imported directly into Igor Pro v5 (Wavemetrics Inc, Lake Oswego, OR) for graphical and statistical analysis. Statistical significance was determined by one-way ANOVA (Newman–Keuls test) for expression data and one-tailed unpaired Student's t test for contractility and voltage clamp.
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Results |
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subunit mRNAQuantitative RT-PCR analysis of the expression of Cav3.1, Cav3.2 and Cav3.3 in human myometrium demonstrated subunit-dependent differences. Expression of Cav3.1 and Cav3.2 was detected in myometrial cDNA from all gestational ages whilst Cav3.3 remained below the limit of detection in all samples tested. The level of expression of Cav3.1 (Fig. 1A) and Cav3.2 (Fig. 1B) demonstrated no significant change (P > 0.05) between preterm not in labour (n = 9), preterm labour (n = 5), term not in labour (n = 12) and term labour (n = 10).
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After determining that mRNA encoding Cav3.1 and Cav3.2 was expressed throughout gestation in myometrial samples we sought to establish the cell types in which the proteins were expressed. Immunohistochemistry utilizing an antibody raised to a 22-amino-acid epitope of the rat Cav3.1 demonstrated specific binding in myometrial (Fig. 2A) and vascular smooth muscle cells (Fig. 2D). In contrast, antibody raised to a 15-amino-acid epitope of the rat Cav3.2 demonstrated specific binding in vascular endothelial cells (Fig. 2E) and invading leucocytes (Fig. 2H). The precise localization of Cav3.1 was further investigated by high magnification confocal microscopy in dissociated myometrial smooth muscle cells (MSMCs). Detailed scans demonstrated localization of Cav3.1 protein in discreet punctate regions of the plasma membrane and in the peri-nuclear area of the sarcoplasmic reticulum (Fig. 2J–L).
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To determine the LVA ICa in freshly dissociated MSMCs we employed conventional patch clamp techniques combined with pharmacological manoeuvres designed to separate the LVA and HVA ICa. Depolarizing pulses above –40 mV elicited an inward current in all cells tested. In 54 of 98 cells tested, an inward current became apparent at voltages positive to –60 mV indicating the presence of LVA ICa. In a number of control cells (n = 12 for HVA expressing cells and n = 12 for HVA/LVA expressing cells) we utilized two current–voltage (I–V) protocols from a holding potential of –50 mV and –80 mV, respectively, to distinguish the LVA and HVA components. From the resultant I–V plots we determined the approximate contribution of LVA and HVA channels to the overall inward current (Fig. 3A–C). This analysis yielded a population of cells with HVA current (Fig. 3A) and a different population of cells demonstrating both LVA and HVA current (Fig. 3B). After determination of the LVA ICa by I–V plot at HP –50 and HP –80 we added 1 µM nifedipine to remove the dihydropiridine sensitive component of the HVA current (Figs 3D–F and 4Aa and b) and repeated the I–V protocol. This established that the peak LVA response in the presence of nifedipine could be estimated in a more rapid protocol by an assay for current density at –30 mV from a holding potential of –80 mV. Under these conditions LVA current was present in only 55% of cells tested (54/98 cells) which was in contrast to 100% (98/98 cells) for the HVA current. The mean cell capacitance of HVA expressing cells (140 ± 15 pF) was not significantly different from HVA/LVA expressing cells (142 ± 13 pF) (P > 0.05).
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In agreement with the immunohistochemistry data suggesting that Cav3.1 is the predominant LVA ICa in MSMCs, the majority of cells with LVA ICa (53/54) tested were relatively insensitive to 120 µM nickel. Furthermore, a dose-dependent response to nickel (Fig. 4Ac and B) demonstrated an IC50 of 118.57 ± 68.9 µM (n = 3) at –30 mV, which is similar to that (167 ± 15 µM) in Xenopus oocytes expressing Cav3.1 (Lee et al. 1999b). The inhibition of LVA current by nickel was voltage dependent with IC50 decreasing to 73.741 ± 17.5 at –40 mV. In separate control experiments prior to nifedipine, addition 100 µM nickel did not significantly inhibit HVA ICa (Fig. 4Ab) (mean inhibition versus control at –30 mV from –80 mV holding potential = 0.5 ± 0.01% (n = 3), P > 0.05).
After establishing that the LVA ICa in human MSMCs was most likely to be Cav3.1 we attempted to determine the physiological function of the current. The precise function of a LVA ICa in smooth muscle has remained the subject of much conjecture due to the resting membrane potential lying near steady state inactivation of these channels. This property could theoretically lead to the establishment of a ‘window’ current at the resting membrane potential, which may either contribute to calcium dependent cellular processes or lead to a slow depolarization. We therefore utilized a two-pulse protocol to measure the parameters of steady state activation and inactivation of both LVA and HVA ICa in MSMCs to determine the ‘window’ current. Cells were held at a HP of –80 mV followed by incremental pulses of 10 mV for 500 ms (conditioning pulses) followed by re-polarization to –80 mV and finally a 60 ms pulse to 0 mV (test pulse). To evaluate the steady-state inactivation, peak currents recorded during the test pulse to 0 mV after each conditioning pulse were normalized to a maximum and plotted as a function of the conditioning pulse amplitude. Steady-state activation was determined from the peak amplitude of inward current during conditioning pulses. The means and standard errors of the mean of six cells expressing both HVA and LVA currents are shown in Fig. 4C. A Boltzmann fit revealed half-activation of inward current at –46 ± 0.4 mV with a slope factor of approximately 5 mV. Half-inactivation of inward current was observed at –47 ± 0.5 mV with 9 mV slope factor. A pronounced overlap between steady-state activation and inactivation curves was evident, peaking at approximately –50 mV. At the reported resting membrane potential of approximately –55 mV in human myometrium at term (Parkington et al. 1999a) this overlap would lead to a persistent inward current.
A well established role for Cav3.1 in the central nervous system is to participate in burst firing whereby a combination of Ih and Cav3.1 elicit repetitive depolarization through low threshold calcium spikes (Kim et al. 2001) upon which fast sodium spikes are superimposed. In our hands, under standard current clamp conditions, there was no repetitive firing of action potentials in human isolated myocytes, which is in contrast to both rat and mouse myocytes. A typical current clamp recording of isolated human MSM in response to a 15 s, 10 pA depolarizing pulse is depicted in Fig. 5A. A depolarizing current pulse of 15 s duration resulted in a single action potential with a threshold around –40 mV. However a subsequent 5 s hyper-polarization to a potential greater than –80 mV led to a rebound (anode break) action potential at a threshold of approximately –65 mV. This rebound LVA spike was completely inhibited by 100 µM nickel and recovered after subsequent washout.
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1C (with calcium as the permeant ion) we utilized 100 µM nickel to inhibit the LVA ICa in spontaneously contracting myometrial strips. In these experiments addition of 100 µM nickel increased mean contraction interval by 3.5-fold over a 30–50 min period of application (P < 0.05; n = 4) (Fig. 6A and C), an effect that was reversible on washout (Fig. 6B). In addition to an effect on contractile frequency, a small but significant (P < 0.001) increase was observed in activity integral, but there was no significant effect on maximum contractile force (Fig. 6C).
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Discussion |
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subunits, Cav3.1, Cav3.2 and Cav3.3 in human myometrium. Initially, we established that the Cav3.1 and Cav3.2 isoforms are expressed in human myometrial samples. This was in agreement with a previous study utilizing RT-PCR that demonstrated that both Cav3.1 and Cav3.2 are differentially expressed throughout gestation in the different layers of rat myometrium (Ohkubo et al. 2005b). In our study we used a panel of cDNA derived from patients at different times of gestation and labour status to establish if there were gestation related changes in Cav3 isoforms. In contrast with the rat study we found no significant difference in expression in the Cav3 isoforms with either gestation or labour. Following RT-PCR analysis we sought to establish whether there were different types of cells expressing Cav3.1 and Cav3.2. Utilizing immunohistochemistry we found that Cav3.1 was expressed in some MSMCs and in vascular smooth muscle cells. In contrast, Cav3.2 demonstrated positive staining in vascular endothelial cells and leucocytes. This is the first description of the cellular distribution of Cav3 isoforms in the human uterus and demonstrates that the expression of Cav3.2 splice variants as described by Ohkubo et al. (2005a) is unlikely to be physiologically relevant to smooth muscle cells but is more likely to be relevant to invading leucocytes. Interestingly, block of the T-type current in leucocytes has been demonstrated to inhibit adhesion (and subsequent invasion) to vascular endothelial cells by inhibiting the calcium dependent expression of 
2-integrins and L-selectin (Nebe et al. 2002). Given the importance of invading leucocytes in the process of parturition this may warrant further investigation. We sought to characterize the function of the T-type current and establish the density of Cav3.1 current in myometrial cells by electrophysiology. Utilizing a combination of biophysical parameters, nifedipine and nickel, we were able to establish that there was LVA current in 55% of myometrial cells, which was in contrast to 100% of cells demonstrating a HVA current. Our voltage-clamp data were in good agreement with that of Young et al. (1993) and Knock & Aaronson (1999), who demonstrated that both LVA and HVA Ca2+ currents were present in freshly dissociated MSMCs.
The LVA current was blocked, in a voltage-dependent manner, by nickel with an IC50 at –30 mV that is consistent with recordings made from oocytes expressing Cav3.1 (Lee et al. 1999b). This further supported the immunohistochemistry data suggesting that Cav3.1 is the predominant isoform in MSM.
Our data suggest that LVA ICa is present in approximately 50% of cells, which raises the question of whether there is a gestation dependent shift in the proportion of cells expressing LVA ICa. Unfortunately we were unable to address this question by assessing current density per patient because it is not technically possible to assay a sufficient population of cells from any given patient. However, if the overall ratio of cells expressing LVA ICa was changing with gestation and assuming that mRNA reflects protein levels and that a single biopsy is a sample of millions of cells one would expect to see an overall change in expression of Cav3.1 mRNA. Since this was not the case in our study, and in the absence of good population current density data, we assume there to be no overall change in ratio.
The precise physiological role of the T-type calcium channel in smooth muscle remains the subject of debate (Perez-Reyes, 2004; Fry et al. 2006). This is largely because, unlike some neurons where resting membrane potentials of –70 to –80 mV and/or contributions of hyperpolarizing currents lead to LVA spikes from the T-type, smooth muscle cells have resting membrane potentials within the range for steady-state inactivation of the T-type channel. However, due to the particular biophysical properties of the channel there remains a possibility that a small population of channels may contribute to a window current (Perez-Reyes, 2003) at the resting membrane potential of the smooth muscle cell and thereby contribute to either a slow wave depolarization or other calcium-dependent intracellular processes. To elucidate the potential for both a contribution to a LVA spike or window current, we undertook a combination of voltage clamp and current clamp experiments. Voltage clamp experiments to determine the steady state activation and inactivation kinetics of the combined LVA and HVA inward currents demonstrated a window current between –60 mV and 0 mV. This is particularly interesting since the resting membrane potential of the myometrium becomes steadily depolarized throughout gestation from –80 mV at mid-gestation to –55 mV at labour and delivery (Parkington et al. 1999a). At term, therefore, due to the resting membrane potential being within the window current, a slow depolarization or calcium ‘leak’ may occur within smooth muscle cells expressing the T-type channel.
To explore the possibility of the T-type channel mediating a LVA spike in myometrial cells we tested cells under current clamp conditions by an initial depolarization to ensure the initiation of an action potential followed by a hyper-polarization to negative potentials sufficient to de-inactivate the T-type channel. Consistent with the classical role of the T-type channel, hyperpolarization (to greater than –80 mV) was followed by a rebound LVA spike that was abolished by nickel and recovered upon wash-out. This suggests that Cav3.1 in MSMCs can elicit LVA spikes although it is unclear as to what the identity of the endogenous hyperpolarizing current might be that could drive de-inactivation. The physiological role of this process requires further investigation.
If there is a slow depolarization via the T-type current in MSMCs, or if a steady calcium leak contributed to the activation of a calcium-dependent inward conductance, one would hypothesize that an inhibition of this current would slow the frequency of contractions since any slow depolarization/activation of an inward calcium conductance would increase the chances of a threshold event. Utilizing a concentration of nickel consistent with specific block of the T-type and not L-Type current in this study and well below the Ki (800 µM) for recombinant L-type subunit 1C (Zamponi et al. 1996) we determined that the frequency of spontaneous contractions of myometrial strips was significantly reduced. Furthermore, the effect was reversible on wash out and caused no significant decrease in maximal contractile force. We observed a small but significant increase in activity integral that was due to an increase in duration of contraction. Whether this was a direct result of T-type blockade or an effect due to reduced contraction frequency remains to be determined. It should be noted that there are a number of plausible hypotheses other than slow depolarization that may lead to the T-type channel affecting contraction frequency. For example, a steady calcium leak may contribute to a slow filling of the sarcoplasmic reticulum, the filling status of which will determine spontaneous release and subsequent activation of the calcium sensitive ICl(Ca) current, previously demonstrated to affect contraction frequency (Jones et al. 2004). The effect of nickel on other conductances that may contribute to contraction frequency cannot be ruled out. However, the concentrations of nickel used in this study are well below the millimolar concentrations required for block of Na+/Ca2+ exchange or Na+,K+-ATP-ase and addition of ouabain actually increases contraction frequency (Parkington et al. 1999b) suggesting a non-specific effect on a sodium pump is unlikely.
We conclude that the primary T-type subunit expressed in some MSMCs is Cav3.1. We show that the LVA ICa conductance is heterogeneously expressed in human myometrial smooth muscle cells and that cells expressing both LVA ICa and HVA ICa demonstrate an extended window current. We found that 100 µM nickel reduces the contraction frequency of spontaneously contracting human myometrium.
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Footnotes |
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Acknowledgements |
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Author's present address
Z-H. Zhao: Department of Pharmacology, Medical School, Xian Jiatong University, China.
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), preterm in labour (PTL; 
), term not in labour (NIL, 
) and term labour (TL, 
). Each point represents individual patients normalized by 2–
) and –80 mV (
). B, HVA/LVA expressing cells (mean ±
S.E.M., n
= 12 per point) current density versus step voltage from a holding potential of –50 mV (
