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Instituto de Investigaciones Químicas,3 41092 Sevilla, Spain
Department of Physiology,4 The University of Liverpool, Liverpool L69 3BX, United Kingdom
Instituto de Química Orgánica General,5 CSIC, 28006 Madrid, Spain
Laboratorios del Dr. Esteve,6 08041 Barcelona, Spain
ABSTRACT
We investigated the function and expression of voltage-gated Na+ channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca2+] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50–60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca2+]i, which were abolished by treatment with 0.1 µM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.
female reproductive tract, signal transduction, uterus
The generation and propagation of action potentials in a variety of excitable cells depends on the activity of voltage-gated Na+ channels (VGSC) [1]. These complex proteins are composed of one 
subunit and one or more auxiliary ß subunits [2, 3]. The subunits are large proteins with a high degree of amino acid sequence identity; they contain an ion-conducting aqueous pore and can function without the ß subunit as a Na+ channel [1, 2]. Nine different VGSC subunits have been cloned in mammals, each of which is encoded by a different gene (see Table 1 for rat genes). They can be further characterized by their sensitivity to the highly selective blocker tetrodotoxin (TTX). The TTX-sensitive subunits are inhibited by TTX in the nanomolar range and include SCN1A (also known as Nav1.1), SCN2A1 (also known as Nav1.2), SCN3A (also known as Nav1.3), SCN4A (also known as Nav1.4), SCN8A (also known as Nav1.6), and SCN9A (also known as Nav1.7). The TTX-resistant subunits are inhibited by TTX in the micromolar range and include SCN5A (also known as Nav1.5), SCN10A (also known as Nav1.8), and SCN11A (also known as Nav1.9) [2, 4]. A tenth, related, nonvoltage-gated atypical isoform, SCN7A (also known as Nax), has also been cloned and expressed [5, 6]. Four different ß subunits, SCN1B, SCN2B, SCN3B, and SCN4B (also named ß1–4) are currently known [7–9]. The roles of the ß subunits are less well established, although they appear to modulate the cellular localization, functional expression, kinetics, and voltage-dependence of channel gating [3, 7, 10].
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For many years, it was considered that VGSC were mostly expressed in nerves, skeletal muscle, and heart, and many studies have therefore focused on the analysis of channel function in these tissues. These studies have revealed that changes in the expression 0 function of these channel proteins represent an important step in the development of different pathologies, such as familial generalized epilepsy, hyperkalemic periodic paralysis, and Brugada syndrome [11, 12]. In contrast, little is known about the functions of these Na+ channels in other tissues, despite recent evidence suggesting that they may also be present in other types of cells [13, 14].
It is generally accepted that VGSC do not play a major role in the regulation of smooth muscle function. As an exception, several studies have shown the presence of these channels in smooth muscle cells from human and rat myometria [15–18]. However, the precise role of VGSC in regulating uterine contractile activity remains unknown and, to the best of our knowledge, the molecular identity of Na+ channels present in the myometrium has not been established. Therefore, the major aims of the present study were to characterize the effects of modulating VGSC activity on uterine contractility and Ca2+ signals and the expression of voltage-gated Na+ channels in uterine longitudinal smooth muscle from nonpregnant rats.
Animals and Tissue Preparation
The current investigation was approved by the Ethics Committee of Consejo Superior de Investigaciones Científicas (CSIC, Spain) and conforms to the National Institutes of Health guidelines for the care and use of laboratory animals. Three-month-old, virgin, female Wistar rats (Charles River Laboratories) were housed with a 12L:12D cycle and provided with food and water ad libitum. Animals at an undetermined stage of the ovarian cycle or in the estrous stage of the ovarian cycle (as determined by microscopic examination of a vaginal smear), were killed humanely and the uterine horns were rapidly removed and dissected free of adhering fat and mesentery. Each horn was opened longitudinally and whole-tissue strips (approximately 2 mm in width, 2 mm in thickness, and 10 mm in length) were prepared. We also obtained small uterine strips (approximately 1.0 x 0.1 x 5.0 mm), which were excised from the longitudinal smooth muscle layer under binocular observation. Samples of brain cortex, used as a positive control for amplification in the RT-PCR studies, were obtained from the same rats.
Some experiments were carried out in female rats treated neonatally with capsaicin (50 mg/kg body weight, dissolved in a vehicle of 80% saline, 10% Tween-80, and 10% ethanol). Rat neonates received an s.c. injection, under ice anesthesia, of either capsaicin solution or its vehicle in a final volume of 0.05 ml on Days 1, 2, 3, and 7 of life. This treatment has been shown to produce selective and permanent degeneration of sensory nerves [19, 20]. Six capsaicin-treated and 6 vehicle-treated rats were used. The efficacy of capsaicin treatment in depleting C-fibers was confirmed by the corneal chemosensitivity test, as described previously [20].
Longitudinally arranged whole-tissue uterine strips were prepared and mounted in tissue baths that contained 4 ml of physiological solution (118 mM NaCl, 4.7 mM KCl, 1.9 mM or 1.1 mM CaCl2 [as indicated in the text], 1.1 mM MgSO4, 1.18 mM KH2PO4, 25 mM NaHCO3, 11.6 mM glucose). Some experiments were carried out in a physiological solution with a low (1 mM) concentration of glucose or in a Ca2+-free (1 mM EGTA) solution.
Uterine strips were suspended under an initial tension of 5 mN, gassed with 95% O2/5% CO2, and maintained at 37°C. Mechanical responses were recorded isometrically by means of force-displacement transducers (Grass FT-03). The preparations were allowed to equilibrate until the disappearance of spontaneous phasic contractions (60–90 min) before addition of single or cumulative concentrations of the selective VGSC activators: veratridine (1–100 µmol/L); brevetoxin-B (0.1 nmol/L to 0.1 µmol/L); grayanotoxin (10–60 µmol/L); and aconitine (1–300 µmol/L). Only one compound was tested on each tissue. At the end of the experiment, the preparations were washed repeatedly and induced to contract by the addition of 40 mM KCl and 0.1 mM methacholine at 30-min intervals. We analyzed the effects of different inhibitors on uterine contractions elicited by the following Na+ channel activators: the VGSC blocker TTX; the L-type Ca2+ channel blocker nifedipine; the nicotinic acetylcholine-receptor antagonist mecamylamine; the muscarinic receptor antagonist atropine; the P2-purinoceptor antagonist suramin; the 1-adrenoceptor antagonist prazosin; the 2-adrenoceptor antagonist yohimbine; the ß-adrenoceptor antagonist propanolol; the specific SCN4A blocker µ-conotoxin GIIIB; the class IC antiarrhythmic drug flecainide; and the purported blocker of SCN10A, vinpocetine. Single or cumulative concentrations of an inhibitor or its vehicle (time-matched, paired controls) were added to uterine strips exposed to a VGSC activator for different time periods. The effects of inhibitors or the corresponding solvent were also assessed in uterine strips mounted in parallel and precontracted by treatment with 40 mM KCl.
Some experiments were performed in small strips of uterine longitudinal smooth muscle, and the experimental conditions were similar to those described for whole-tissue strips.
Simultaneous Measurement of Contractile Force and [Ca2+]i
Small strips of uterine longitudinal smooth muscle were incubated with the acetoxymethyl ester form of Fura-2 (20 µM Fura-2/AM, Molecular Probes, Eugene, OR) for 2–4 h at room temperature. The noncytotoxic detergent pluronic acid (0.02%; Molecular Probes) was added to increase the solubility of the Ca2+-sensitive fluorophore. Following Fura-2/AM loading, the preparation was rinsed and placed in the quartz cuvette of a spectrofluorometer designed to measure changes in the fluorescence signals concomitantly with force development in biological tissues (SLM Aminco-Bowman, Series 2; Microbeam, Barcelona, Spain). The tissue was incubated at 37°C in oxygenated (100% O2) physiological salt solution of the following composition: 154 mM NaCl, 5.6 mM KCl, 2 mM CaCl2,1.2 mM MgSO4, 11 mM glucose, and 11 mM Hepes (pH 7.4). Force was measured as described for whole-tissue strips, except that the equilibration period after application of the resting load was 15 min. To measure [Ca2+]i, the muscle strip was alternatively illuminated with two excitation wavelengths (340 nm and 380 nm) and the fluorescence ratio (F340:F380) was recorded continuously. The emitted fluorescent light from the two excitation wavelengths was measured by a photomultiplier through a 510-nm filter. After subtracting the autofluorescence signal, obtained by adding 5 mM MnCl2 at the end of the experiment, the F340/F380 ratio was used as an indicator of [Ca2+]i, essentially as described by Taggart et al. [21]. Both contractile force and [Ca2+]i were recorded using the data acquisition system and data recording software provided by the manufacturer.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from uterine whole-tissue strips and from longitudinal smooth muscle strips obtained from the same rats and prepared in parallel to those used in the functional studies. The tissues were immersed in RNAlater (Ambion, Huntingdon, UK) immediately after dissection, i.e., at time 0 (H0), or were incubated in physiological solution that contained 60 µM veratridine or its vehicle for 1 h (H1), 2 h (H2), 6 h (H6), or 12 h (H12), before immersion in RNAlater. Samples of brain cortex from the same animals were used as positive controls for the amplification. All tissues were stored at –80°C until use.
DNase-treated total RNA (2 µg) was reverse-transcribed using a first-strand cDNA synthesis kit (Amersham Biosciences, Essex, UK). The resulting cDNA samples were amplified by PCR with specific oligonucleotide primer pairs designed using the Primer 3 software [22]. Based on previous experiments, Actb (ß-actin) and Polr2a (polymerase [RNA] II [DNA directed] polypeptide A) were chosen as internal standards [23–25]. The sequences of the primers used to amplify the VGSC and ß subunits and the reference genes are shown in Tables 1 and 2. All primers were synthesized and purified by Sigma-Genosys (Cambridge, UK).
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End-point PCR and agarose gel electrophoresis were used to detect the presence and to establish the identity of the different genes following normalization by real-time PCR of the mRNA quantities for the reference genes Actb and Polr2a in the different tissues [25]. The PCR reaction mixture contained 0.2 µmol/L of the primers, 1.5 U of JumpStart Taq DNA polymerase (Sigma Chemical Co., St. Louis, MO), the supplied buffer, 2.5 mM MgCl2, 200 µM dNTP, and cDNA in a total volume of 25 µl. The parameters used for PCR amplification were 10 sec at 94°C, 20 sec at 60°C, and 30 sec at 72°C for 35 (VGSC and ß subunits), 30 (Polr2a), or 24 (Actb) cycles. Amplicon sizes were verified by comparison with a DNA molecular mass ladder, and the identity of each PCR product was established by DNA sequence analysis [26].
Real-time PCR assays were run in the presence of SYBR green I (Molecular Probes, Leiden, The Netherlands) using the iCycler iQ real-time detection apparatus (Bio-Rad Laboratories, Hercules, CA). The fold-change in expression of each target gene relative to Actb or Polr2a was calculated in each sample by the formula: 2–
CT, where CT is the threshold cycle, calculated by the iCycler software, CT = (CTtarget gene – CTreference gene), and CT = (CTtest sample - CTcontrol sample). A pool of all the uterine cDNAs at time 0 was used as a control sample throughout the study, and the target gene mRNA to reference gene (ß-actin or Polr2a) mRNA ratio in this control sample was designated as 1. Each assay was performed in triplicate and three negative controls were run for each assay: no template (sample lacking cDNA), no reverse transcriptase, and no RNA in the reverse transcriptase reaction.
Contractile responses were characterized by measuring the frequency (contractions/min, measured during a 10- or 60-min period), the amplitude (mean amplitude of the rhythmic contractions during a 10- or 60-min period, expressed in mN or as a percentage of the control response to KCl), and the area under the force-time curve (measured during 10- or 60-min periods and expressed as a percentage of the area for KCl during the same time period). The effects of inhibitors were measured as the area under the force-time curve during the 10-min period that each concentration of inhibitor remained in contact with the tissue and expressed as a percentage of the contractile area of the response to veratridine or KCl during the 10-min period before the addition of the inhibitor.
All values are expressed as the mean ± SEM and, except where otherwise stated, n represents the number of rats used. Statistical analyses of the data from functional studies were performed by one-way ANOVA, followed by the Dunnett or Tukey multiple comparison test and unpaired Student t-test to compare the means of two groups, using the GraphPad Prism 4.0 software. Statistical analyses of real-time PCR values were performed by two-way ANOVA followed by Bonferroni posttest using the GraphPad Prism 4.0 software. Statistical significance was accepted when P < 0.05.
The drugs used were veratridine, aconitine, grayanotoxin III Hemi (ethyl acetate), nifedipine, mecamylamine hydrochloride, suramin sodium salt, yohimbine hydrochloride, atropine sulphate, prazosin hydrochloride, propranolol hydrochloride, flecainide acetate salt, vinpocetine, and methacholine chloride—all from Sigma; tetrodotoxin was obtained from Laboratorios del Dr. Esteve (Barcelona, Spain), and µ-conotoxin GIIIB was from Alomone Laboratories (Jerusalem, Israel). Brevetoxin-B was a generous gift from Prof. K. Nakanishi (Columbia University).
In a solution that contained 1.9 mM Ca2+, longitudinally arranged whole-tissue uterine strips exhibited rhythmic contractions, which disappeared during the first 90 min of the experiment in 80% of the tissues. In a medium that contained 1.1 mM Ca2+, the spontaneous contractions ceased during the first 90 min of the experiment in 99% of the tissues. Experiments were continued only in those tissues in which spontaneous activity had ceased. We analyzed the mechanical effects produced by cumulative addition of 1–100 µM veratridine, 0.1 nM–0.1 µM brevetoxin-B, 10–60 µM grayanotoxin, or 1–300 µM aconitine. The four VGSC agonists induced rhythmic contractions of irregular amplitude and frequency at concentrations 
10 µmol/L for veratridine (n = 25), 10 nmol/L for brevetoxin-B (n = 6), 100 µmol/L for aconitine (n = 5), and 20 µmol/L for grayanotoxin (n = 4). The time-course and characteristics of the contractile response elicited by each compound were similar in strips isolated from the medial, ovarian, or cervical regions of the uterine horn. The maximal response, measured during a 60-min application, was reached with 60 µM veratridine, 30 nM brevetoxin, 100 µM aconitine, and 30 µM grayanotoxin and was, for each compound, virtually identical in uterine strips incubated in 1.9 mM or 1.1 mM Ca2+-containing solution (Fig. 1A). In all cases, the addition of TTX (0.1 µmol/L, n = 75 uterine strips from 25 different rats) caused the immediate abolition of the rhythmic contractions (see Fig. 1B for a representative tracing). Rhythmic contractions were also inhibited by nifedipine (1 µmol/L, n = 3 for each VGSC agonist), and they did not appear in uterine strips incubated in a Ca2+-free physiological solution (n = 3 for each agonist). Subsequent experiments were carried out with uterine strips isolated from any region of the uterine horn and obtained from rats in estrus. The physiological solution contained 1.1 mM Ca2+. Due to the limited availability of brevetoxin and the lower efficacies of aconitine and grayanotoxin, veratridine was chosen as the VGSC activator.
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The uterine contraction to 60 µM veratridine could be clearly divided into two different phases. The initial response was observed during the first 50–60 min of exposure and consisted of transient contractions of irregular amplitude and frequency (Figs. 1B and 2). In the continued presence of veratridine, a second, late response appeared that consisted of rhythmic contractions of very regular frequency (Figs. 1B and 2A). The amplitudes of these late contractions, and thus the area under the force-time curve, which gives a measure of the whole contractile response, slightly increased over approximately 180 min, i.e., during the first 240 min of contact of the preparation with veratridine (Figs. 1B, 2B, and 3). From this point, the contractile response remained very regular and was sustained for at least 12 h after the addition of veratridine (24 uterine strips from 12 different animals; Fig. 3). The response was identical and had a similar duration time in a physiological solution that contained only 1 mM glucose (n = 4; data not shown).
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To determine whether neurotransmitter(s) released from nerve endings contribute to rhythmic contractions elicited by veratridine, we tested the effects of different antagonists of neuronally mediated responses. After 40 min of exposure to veratridine, i.e., during the early phase of the response, the contraction was unaffected by the nicotinic acetylcholine receptor antagonist mecamylamine (1 µmol/L), the P2-purinoceptor antagonist suramin (100 µmol/L) or the 2-adrenoceptor antagonist yohimbine (1 µmol/L), whereas it was partially inhibited by the addition of the muscarinic receptor antagonist atropine (1 µmol/L) or the 1-adrenoceptor antagonist prazosin (1 µmol/L) (Fig. 4). The combined addition of atropine and prazosin caused an additive inhibition of the contraction to veratridine (Fig. 4). Subsequent addition of the ß-adrenoceptor antagonist propanolol (1 µmol/L) caused partial reversion of the inhibitory effect (Fig. 4). Conversely, the late phase of the contractile response to veratridine was unaffected by any of these compounds. Thus, mecamylamine, suramin, yohimbine, atropine, prazosin, and propranolol had no effects when added to the preparation 90 min or 240 min after veratridine exposure (not shown).
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We performed additional experiments to investigate the contribution of neurotransmitters stored in capsaicin-sensitive sensory nerves. The vanilloid receptor antagonist 6-iodo-nordihydrocapsaicin (10 µmol/L) [27] failed to modify the response to veratridine. In uterine strips isolated from rats treated neonatally with capsaicin, the early and late components of the veratridine-induced contraction were similar to those observed in rats treated neonatally with the capsaicin vehicle or in untreated, control rats (data not shown).
In a further set of experiments, we analyzed the effects of the specific SCN4A blocker µ-conotoxin GIIIB [28]; the class IC antiarrhythmic drug flecainide, which blocks SCN5A and SCN4A [29, 30]; and the purported blocker of SCN10A, vinpocetine [31]. In uterine strips exposed for 90 min (late response) to 60 µmol/L veratridine, application of cumulative concentrations of µ-conotoxin GIIIB (0.1 nmol/L–10 µmol/L, n = 3), flecainide (1 nmol/L–30 µmol/L, n = 7) or vinpocetine (1 nmol/L–30 µmol/L, n = 9) did not modify the veratridine-induced rhythmic contractions. Similar results were obtained when these blockers were applied to preparations exposed to veratridine for 40 min (n = 3 for each compound). Cumulative addition of TTX (0.3 nmol/L–1 µmol/L) caused a concentration-dependent inhibition of the early and late contractions to veratridine with –log IC50 values of 8.20 ± 0.19 (n = 15) and 8.02 ± 0.17 (n = 11 uterine strips from seven animals), respectively. Application of cumulative concentrations of TTX (0.3 nmol/L–10 µmol/L, n = 4) had no effect on the KCl-induced uterine contraction (Fig. 5). The L-type Ca2+ channel blocker nifedipine (0.01 nmol/L–10 µmol/L) relaxed in a concentration-dependent manner the contractions induced by both veratridine and KCl (Fig. 5) and was significantly more potent as an inhibitor of the response to KCl (-logIC50 = 8.63 ± 0.07 vs. KCl, n = 4, and 7.76 ± 0.08 vs. veratridine, n = 4, P < 0.001, Student t-test for unpaired data).
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In small strips of uterine longitudinal smooth muscle, the addition of veratridine caused the rapid appearance of rhythmic contractions (n = 15 strips from three different rats). The veratridine-induced contractile responses were similar to those observed in whole-tissue strips, except that the early component was absent or shorter (about 5–10 min).
Simultaneous Measurement of Contractile Force and [Ca2+]i
In experiments with longitudinal smooth muscle strips loaded with Fura-2, the equilibration period after application of the resting load was 15 min and many preparations exhibited spontaneous contractions. The effects of veratridine were only tested on those preparations that were not contracting (10/21 muscle strips, obtained from 12 different rats). Veratridine again caused rhythmic contractions accompanied by transient increases in [Ca2+]i. The mean amplitude during a 10-min period (8.48 ± 1.20 mN) and the frequency of contractions (0.75 ± 0.09 contractions/min) were similar to the values observed in whole-tissue uterine strips. Both the contractile force and the [Ca2+]i transients were abolished by the addition of 0.1 µM TTX (Fig. 6).
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Expression of Voltage-Gated Na+ Channel and ß Subunit Genes
Using end-point PCR and agarose gel electrophoresis, we detected the presence of single bands of the predicted sizes for the subunits Scn2a1 (431 bp), Scn3a (324 bp), Scn4a (282 bp), Scn5a (265 bp), Scn8a (307 bp), Scn9a (350 bp), and the related isoform Scn7a (487 bp), which appeared in all the cDNAs from whole-tissue uterine strips assayed (n = 5; Fig. 7A). The mRNAs of Scn1b (258 bp), Scn2b (295 bp), Scn3b (233 bp), and Scn4b (215 bp), and of the reference genes Polr2a (226 bp) and Actb (362 bp) were also expressed (Fig. 7B, not shown for the reference genes).
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Because the uterine wall is composed of two distinct smooth muscle cell layers and the endometrium, we performed additional experiments to analyze the mRNA expression in the outer longitudinal smooth muscle. Among the target genes, the mRNAs of Scn2a1, Scn3a, Scn5a, Scn7a, and Scn8a and of the ß subunits were found in cDNAs isolated from the longitudinal muscle strips (Fig. 7).
In an attempt to investigate the molecular identity of the VGSC that mediates contractions in response to veratridine, we used real-time PCR to quantify the mRNA levels of the TTX-sensitive channels Scn2a1, Scn3a, and Scn8a in the cDNAs from uterine longitudinal muscle strips incubated for different time periods with veratridine or its vehicle. The Scn2a1 mRNA levels decreased with the time of incubation in physiological solution (P < 0.001, two-way ANOVA; see Fig. 8 for results using Actb as the reference gene) and had virtually disappeared at H12. Veratridine had no additional effects (P > 0.05) (Fig. 8). Scn3a mRNA expression was slightly but significantly decreased by both the time of incubation (P < 0.05) and the presence of veratridine (P < 0.05) (Fig. 8). The Scn8a mRNA levels increased with the time of incubation in physiological solution (P < 0.05) and were further increased by the presence of veratridine (P < 0.05) (Fig. 8).
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Quantitative real-time RT-PCR showed that the mRNA of Scn2a1 was present at similar levels in uterine whole-tissues and longitudinal muscles from nonpregnant rats at all the time-points assayed. The same pattern was observed for the Scn3a or Scn8a mRNA. Real-time RT-PCR also showed that, relative to Actb, the levels of Polr2a mRNA were not altered by the incubation time and/or veratridine treatment. The mRNA levels for Scn2a1, Scn3a, and Scn8a, respectively, at H0, H1, H2, H6, and H12 were similar to those described above when Polr2a was used instead of Actb as the internal standard (data not shown).
The present results show for the first time that the VGSC subunits Scn2a1, Scn3a, Scn4a, Scn5a, Scn8a, and Scn9a and the four known VGSC ß subunits Scn1b, Scn2b, Scn3b, and Scn4b are expressed in the uteri of nonpregnant rats. Among the subunits, Scn2a1, Scn3a, Scn5a, and Scn8a were found in the myometrial longitudinal smooth muscle layer. Our data also show that activation of TTX-sensitive Na+ channels induces uterine rhythmic contractions that were maintained over long periods of time.
The mammalian female reproductive tract is innervated by autonomic sympathetic and parasympathetic nerves, as well as by numerous sensory fibers [32]. Many of these nerves are associated with the myometrium, which suggests that neurotransmitters released from their peripheral endings may influence uterine contractility [32, 33]. In the present study, the veratridine-induced contractions were virtually identical in uterine strips obtained from nonpregnant rats treated neonatally with capsaicin or its vehicle and from untreated rats. Therefore, it appears that neurotransmitters stored in capsaicin-sensitive sensory nerves do not play a major role in mediating uterine contractions to veratridine.
The early phase of the contractile response to veratridine was inhibited by atropine and prazosin, which suggests that it was mediated, at least partially, by the release of neurotransmitters from adrenergic and cholinergic nerve terminals. These neurotransmitters could induce inhibitory and excitatory effects, and this could explain the irregular character of the early contractile response to veratridine. Conversely, the late contraction, developed after approximately 60–90 min of contact with the preparation, was unaffected by any of the inhibitors of neuronally mediated responses assayed. The late phase of the contraction appeared to be mediated by the activation of VGSC present in nonneuronal cells within the rat uterus. These nonneuronal rhythmic contractions were very regular and sustained for more than 12 h. Moreover, veratridine caused contractions of similar time-course and duration in uterine strips incubated in a physiological solution that contained only 1 mM glucose, which suggests that the contractile response is able to proceed with a minimum expenditure of energy. Thus, activation of VGSC appears to represent a highly efficient mechanism to generate motility in the rat uterus.
Early electrophysiological studies have demonstrated the presence of voltage-dependent Na+ currents in cultured and freshly isolated smooth muscle cells from human and rat myometrium [16, 17]. In most of these studies, voltage-dependent Na+ currents were detected in the uteri of late pregnant animals [15, 16]. It has been suggested that VGSC are functional in the late stages of pregnancy and play a role in the initiation of labor [16, 34]. Yoshino et al. [18] have described the presence of Na+ currents in about 50% of smooth muscle cells isolated from the myometria of nonpregnant rats. Our data extend these observations and show that veratridine and other VGSC activators cause TTX-sensitive rhythmic contractions in the uteri of nonpregnant rats. These contractions were observed in whole-tissue strips and in thin strips isolated from the longitudinal smooth muscle layer. These small strips consist of 90% smooth muscle and 10% nonsmooth muscle, including interstitial Cajal-like cells, and these interstitial cells are devoid of inward currents [35]. The previous results and those of the present study strongly suggest that veratridine-induced rhythmic contractions are due to the activation of TTX-sensitive Na+ channels present in the longitudinal smooth muscle cells, i.e., they are myogenic.
In the RT-PCR experiments, we detected the presence of Scn2a1, Scn3a, Scn4a, Scn5a, Scn8a, and Scn9a in the cDNAs from whole uteri. The atypical isoform Scn7a was also expressed, in agreement with previous data [5, 6]. The mRNAs of all known ß isoforms were also expressed in the uterus. The widespread expression of the VGSC and ß subunits argue for a role of these channels in the regulation of uterine functions, although further studies are needed to determine their precise physiological significance. Among the TTX-sensitive subunits, Scn2a1, Scn3a, and Scn8a were present in cDNA isolated from uterine longitudinal smooth muscle. Therefore, these subunits are the main candidates for the mediation of uterine rhythmic contractions induced by VGSC activators. The observation that Scn2a1 and Scn3a mRNA levels decreased with time and/or veratridine treatment while Scn8a mRNA increased led us to hypothesize that Scn8a plays a major role in supporting contractions induced by veratridine.
Uterine rhythmic contractions depend on extracellular Ca2+ and involve Ca2+ influx through L-type Ca2+ channels, which are the predominant voltage-dependent Ca2+ channels in the uterus [36–38]. Regulated uterine contractions are essential in a diverse range of reproductive functions, including sperm transport, embryo positioning during implantation, and expulsion of the fetus at parturition. However, the cellular mechanisms responsible for uterine spontaneous pacemaker activity are poorly understood. We found that veratridine and other VGSC activators induced contractions that were inhibited by TTX, as well as by nifedipine or Ca2+-free medium. In longitudinal muscle strips, veratridine induced rhythmic contractions concomitant with an increase in [Ca2+]i, and TTX inhibited both contractility and [Ca2+]i rises. These results provide evidence that activation of Na+ channels causes the subsequent opening of L-type Ca2+ channels, leading to Ca2+ influx and the initiation of contraction. Veratridine induced very regular rhythmic contractions that were well maintained for more than 12 h. These data strongly suggest that TTX-sensitive VGSC present in smooth muscle cells within the myometrium play a role in the generation and regulation of rhythmic activity. It has recently been shown that SCN8A can modulate the contractility of the mouse portal vein [14]. This channel also plays an important role in mediating Na+ currents in several classes of neurons with pacemaker roles [39]. It may thus be argued that SCN8A is involved in modulating uterine pacemaker activity, although a role for SCN2A1 and/or SCN3A cannot be excluded.
Different studies have demonstrated the association of certain pathologies with precise mutations in particular VGSC or ß subunits [11, 12]. Therefore, it is essential, from a therapeutic point of view, to find compounds that are able to act selectively on certain VGSC subtypes. In this context, a reliable biological model that allows a broad study of the activities of different compounds on these channels would be extremely useful. The present data show that a) the VGSC activators assayed—grayanotoxin, aconitine, veratridine, and brevetoxin-B [1, 40]—are all able to induce uterine rhythmic contractions; b) these contractions are abolished in the presence of 0.1 µM TTX; c) the contractile response induced by veratridine is unaffected by µ-conotoxin GIIIB, flecainide, or vinpocetine, which block SCN4A, SCN5A, and SCN10A [28–31]; and d) TTX inhibits in a concentration-dependent manner the veratridine-induced contraction, and the –logIC50 value is close to that found for this inhibitor in isolated myometrial cells from pregnant rats [34]. Moreover, TTX had no effect on KCl-induced uterine contraction, whereas nifedipine blocked contractions in response to both veratridine and KCl, being more potent as a KCl inhibitor. Therefore, compounds that act on VGSC can easily be differentiated from compounds that act primarily on L-type Ca2+ channels. Thus, the uterus isolated from a nonpregnant rat may provide a useful model to analyze the effects of drugs on TTX-sensitive Na+ channels, particularly on SCN8A, SCN2A1, and/or SCN3A.
In conclusion, the present findings show that the uterus of the nonpregnant rat expresses different voltage-gated Na+ channel and ß subunits. TTX-sensitive Na+ channels, particularly SCN8A, SCN2A1, and/or SCN3A, appear to play an important role in the regulation of myometrial rhythmic contractions.
FOOTNOTES
1Supported by a grant from Laboratorios del Dr. Esteve (Barcelona, Spain), and fellowships from Laboratorios del Dr. Esteve (to M.S.) and the MRC (to S.W). 
Correspondence: 2Correspondence: Luz Candenas, Instituto de Investigaciones Químicas, Avda Americo Vespucio 49, 41092 Sevilla, Spain. FAX: 34 95 446 0565; e-mail: luzcandenas{at}iiq.csic.es
Received: 22 May 2007.
First decision: 10 June 2007.
Accepted: 30 July 2007.
REFERENCES
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