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Biology of Reproduction 63, 1262-1270 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Gestational Changes in Uterine L-Type Calcium Channel Function and Expression in Guinea Pig1

Patricia L. Collins2,,a,d,e,f,g, John J. Moorea,b, David W. Lundgrenb,c, Elena Choobinehe,f,g, Sharon M. Changd, and Albert S. Changd

a Departments of Obstetrics/Gynecology, b Pediatrics, c Biochemistry, and d the Rammelcamp Center for Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109 e Departments of Obstetrics/Gynecology, f Cell Biology, Neurobiology and Anatomy, and g The Cardiovascular Research Institute, Loyola University Medical Center, Maywood, Illinois 60153

ABSTRACT

Pregnancy can influence both the resting membrane potential and the ion channel composition of the uterine myometrium. Calcium flux is essential for excitation-contraction coupling in pregnant uterus. The uterine L-type calcium channel is an important component in mediating calcium flux and is purported to play a role in parturition. This study was undertaken to characterize gestational changes in 1) the uterine contractile response to the L-type calcium channel agonist, Bay K 8644; 2) the mRNA expression of channel subunits by semiquantitative reverse transcriptase-polymerase chain reaction; and 3) estimate channel protein levels by measuring 3H-isradipine binding at the dihydropyridine binding site of the {alpha}1c subunit utilizing saturation binding methods. Sensitivity to Bay K 8644 increases beginning at 0.8 of gestation and persists through term. The change in sensitivity is coincident with an increased mRNA expression of the {alpha}1c and ß2 subunits but with the least detectable amounts of isradipine binding. The expressed {alpha}1c transcript represents a novel structural variant with a 118-amino acid deletion in the III–IV linker and repeats IVS1–S3 of the protein sequence. The guinea pig uterine L-type calcium channel activity is highly regulated through gestation, but the regulation of mRNA expression may be different from regulation of protein levels, estimated by isradipine binding. The up-regulation of function, {alpha}1c subunit mRNA expression, and isradipine binding at term gestation are consistent with a role for this ion channel in parturition.

calcium, parturition, pregnancy, uterus

INTRODUCTION

Calcium flux is an essential component for excitation-contraction coupling in uterine smooth muscle [1–4]. Pregnancy, or more specifically, the hormonal state induced by pregnancy, can influence resting membrane potential and ion channel composition in the myometrium [5–10]. Ion channels in myometrium as well as other gene products such as gap junction proteins [11] and oxytocin receptors [12] may be important constituents that move the myometrium from a state of relative quiescence to a rhythmically contracting organ at the time of parturition. Voltage dependent L-type calcium channels (VDCC) have been identified in uterine myometrium by electrophysiologic [3, 8, 13–18], pharmacologic [19–22], and molecular [23, 24] studies. They are responsible for the majority of the observed calcium current in term human myometrium [17]. Gestational regulation of uterine L-type calcium channels and their role in parturition are not yet completely understood.

The functional VDCC is a multisubunit complex consisting of the {alpha}1 pore-forming subunit and ß, {gamma}, and {alpha}2/{delta} regulatory subunits [25–27]. Known drug binding sites, including the dihydropyridine (DHP) binding site, are located on the {alpha}1 subunit and have been used to help characterize the channel. The {alpha}1 subunit from rat uterine myometrium, a product of the CaCh2 gene [28], has several isoforms resulting from alternative splicing [23] and is DHP sensitive [19–21].

There is evidence that the L-type calcium channel subunits are regulated during gestation in the rat. Mershon et al. [23] found a fourfold increase in the number of DHP binding sites, measured by saturation binding analysis, by 14 days (0.6) of gestation in the rat, a time of supposed uterine quiescence, which was maintained through parturition. There was no further rise immediately prior to or during labor. There was no change in affinity (Kd) throughout gestation. Mershon et al. also studied transcript expression of the {alpha}1 subunit and showed a gradual rise in mRNA beginning at 7 days (0.3) of gestation with a marked decrease and isoform shift at parturition. Similarly, Tezuka et al. [24] showed a gradual increase in the {alpha}1 subunit through gestation, a decrease during labor, but a sharp increase in the ß subunit at parturition. By using progesterone and onapristone, they were able to alter expression of the VDCC in a fashion consistent with a role of this ion channel in parturition. Correlation of uterine contractile function by L-type calcium channel agonists through gestation was not performed in either of these studies.

The pregnant guinea pig is a well-established model for the study of uterine contractility and parturition because of the similarity to humans in placentation and sex steroid profiles [29]. We are interested in the hormonal regulation of the VDCC and regulation by an endogenous L-type calcium channel inhibitor [19, 30, 31]. This study was undertaken to provide a gestational profile of the VDCC agonist mediated uterine contractile response in relationship to L-type calcium channel mRNA expression and to isradipine binding level in pregnant guinea pigs.

MATERIALS AND METHODS

Animals

Timed-pregnant Hartley guinea pigs (Camm Research Lab, Wayne, NJ, or Hilltop, Scottdale, PA) were maintained in a 12-h day/night cycle with free access to food and water. Animals were killed at 33–36, 44–47, 54–56, and 63–65 days of gestation by injection of pentobarbital (75 mg/kg) intraperitoneally followed by exsanguination/thoracotomy according to a protocol approved by the Case Western Reserve University and Loyola University Medical Center Institutional Animal Care and Use Committees.

Uterine Muscle Strips

The pups and placentas were rapidly removed and the uterus placed in a warmed buffer (110 mmol/L sodium chloride; 4 mmol/L potassium chloride; 20 mmol/L N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], pH 7.4 at 33°C; 0.5 mmol/L magnesium chloride; 2.0 mmol/L calcium chloride; 15 mmol/L sodium bicarbonate; 1.8 mmol/L glucose; 0.45 mmol/L sodium phosphate) to which was added 100 units/ml penicillin G and 100 µg/ml streptomycin sulfate (Sigma, St. Louis, MO). A template was used to cut 0.5-cm-long strips that were then anchored into a Plexiglas chamber and attached with a suture to a strain gauge (FORT-10 Transducer, World Precision Instruments, New Haven, CT) in order to measure the force generated by the longitudinal muscle [19, 30, 31]. The muscle was preset to a resting tension of 10 millinewtons (mN) during an initial 30-min equilibration period. Ten millinewtons was previously determined to be optimal tension necessary to obtain maximal contractions for the 0.5-cm uterine strips. Contiguous strips of uterine muscle from the same animal were used in six individual chambers. After an initial 30-min equilibration period the muscle was washed and the uterine tension was reset to 10 mN. The S(-)-Bay K 8644 (Research Biochemicals Inc., Natick, MA) was added to the maternal side of the chamber in 10-min cumulative doses from 100 pM to 10 µM. At the end of the experiment, all uterine muscle strips were maximally stimulated with 20 mM KCl.

Data and Statistical Analysis

Uterine contraction data were collected as voltage change using FORT-10 transducers and Transbridge amplifiers (World Precision Instruments), and processed through an A/D board (WB-800, Omega Engineering, Stamford, CT). Data were originally collected at 10 Hz, averaged over 1 sec, and converted from volts to newtons using Labtech Notebook (Omega Engineering) software.

Data points were integrated over 10-min intervals for each dose of Bay K 8644. The 10-min period before the start of the dose response was considered the basal period, and this integral was set equal to one. Each of the other 10-min intervals in that chamber was expressed as a fold change from basal. Thus, dose response curves are displayed as fold increase from basal versus -log dose (molar) of Bay K 8644. The data points represent the mean ± SEM of the number of individual experiments expressed as the n (each n represents a separate animal). Data were curve fit to the equation for a sigmoidal curve Y = bottom + [(top - bottom)/(1 + 10logEC50-X)] where Y was the response and X was the logarithm of the concentration (Prism software package; Graphpad Inc., San Diego, CA).

Dose response curves for the different gestations were compared using between-group ANOVA. Fisher protected least significant differences was used as the posthoc test. Two-tailed tests were used in all cases and P values <=0.05 were considered significant.

Reverse transcriptase-polymerase chain reaction (RT-PCR) cloning Uterine muscle was dissected from 65-day (term, not in labor) timed-pregnant guinea pigs. Total cellular RNAs were isolated (Qiagen RNA isolation kit, Santa Clarita, CA), and mRNAs were enriched from the total RNAs (Pharmacia QuickPrep Micro mRNA Purification kit, Piscataway, NJ). One microgram of mRNA was used to direct first-strand cDNA synthesis (Pharmacia First Strand cDNA Synthesis kit) initiated by random hexameric primers. The resulting cDNAs were used directly as templates for PCR amplification. Each PCR reaction incorporated 0.1 µM of primers, 40 µM of each deoxynucleotide, 1.25 mM magnesium chloride, and 1 unit of AmpliTaq-Gold polymerase (Perkin-Elmer, Norwalk, CT) per 20 µl of reaction volume. Thermocycling conditions were 40 cycles of 1 min at 95°C, 1 min at 50°C, 2 min at 72°C. An additional 5 min of extension at 72°C was used at the end of each thermocycling protocol. Oligonucleotide primers used in these reactions were: VDCC {alpha}1 subunit, 5'-tcgtgggtttcgtcattgtcacc-3' and 5'-caaagcatgtagaagctgatgaag-3'; VDCC ß2 subunit, 5'-atgactggtggatcgggcggctrg-3' and 5'-gtttagtaccgagcgtttggc-3'; cyclophilin, 5'-gtctccttcgagctgtttgcag-3' and 5'-ccagcatttgccatggac-3'. Amplifications of ß-actin transcripts (using primers 5'-tcatgtttgagaccttcaac-3' and 5'-cacgtcacacttcatgatgg-3') were also routinely performed. Because these primers span a known intron in the ß-actin gene, this PCR reaction was designed to yield products of discernibly distinct sizes depending upon whether the starting template was derived from cellular RNA transcripts or contaminating genomic DNA. These control reactions consistently indicated that the observed PCR products were indeed derived from uterine transcripts. The VDCC subunit-specific PCR products were directly ligated into the TA cloning vector, pCRII (Invitrogen, Carlsbad, CA), and transformed into DH5{alpha} bacterial cells (Life Technologies, Gaithersburg, MD). Resulting clones were broth-amplified and corresponding plasmids were purified using the Wizard MiniPrep Kit (Promega, Madison, WI), and sequenced by the dideoxy chain-termination method using Sequenase II in the presence of 7-deaza-dGTP (US Biochemicals, Cleveland, OH). Obtained nucleotide sequences were compared with those of known calcium channel subunits in the GenBank database. Novel guinea pig cDNA sequences were electronically deposited into GenBank using Bankit (accession no. AF005938 for VDCC {alpha}1 fragment; accession no. AF005939 for VDCC ß2 fragment).

Semiquantitative PCR analyses The RT-PCR was performed for uterine cyclophilin, {alpha}1 and ß2 VDCC subunits using total RNAs purified from uteri at 34, 47, 54, and 64 days of guinea pig pregnancy. In order to compare the relative abundances of mRNA transcripts for these genes at the indicated gestational times, each gene transcript was enzymatically amplified such that its PCR yield was a linear function of cycle number. The following cycle numbers were empirically determined to provide reproducible amplifications for the present purpose: Cyclophilin, 20 cycles; {alpha}1, 30 cycles; ß2, 25 cycles. The amplified products were electrophoretically fractionated through a 1% agarose gel and Southern-blotted onto HyBond-N membrane (Amersham, Arlington Heights, IL). Transferred DNA fragments were immobilized onto the membrane by UV crosslinking (Stratagene, La Jolla, CA), then solution-hybridized at 65°C, with 6x SSC (standard saline citrate: 0.9 M NaCl, 0.09 M sodium citrate), 5x Denhardts (0.1% [w/v] each of polyvinylpyrrolidone, BSA, and Ficoll 400), 0.5% SDS, and radiolabeled probe. Each probe contained incorporated [{alpha}-32P]dCTP (3000 Ci/mmol; New England Nuclear, Boston, MA) using the Prime-It Labeling Kit (Stratagene). Posthybridization washes were conducted at 65°C in 0.2x SSC, 0.1% SDS. The membrane was subjected to autoradiography in the presence of intensifying screens (DuPont, Boston, MA) and, upon visualization of hybridization results, the membrane region corresponding to each radioactive signal was excised and directly quantitated by liquid scintillation spectrometry.

Dihydropyridine Binding

Uterine cell membrane fragments from the various gestations were prepared as previously described [32]. Protein was determined by the Bradford method utilizing BSA as the standard [33]. The reaction consisted of 100 µg cell membrane protein and 3H-isradipine (NEN, Boston, MA) at 79.7 Ci/mmol in concentrations from 62.5 pM to 5 nM in 50 mM Tris buffer at pH 7.4 at 25°C. Nonspecific binding was determined by including 1 µM unlabeled isradipine at each concentration of labeled isradipine. Binding was performed at 25°C for 90 min. Incubation time was chosen based on the plateau phase of an association binding experiment (data not shown). Bound isradipine was separated from free by filtration through glass fiber filters (Schleicher & Schuell, Keene, NH) and counted in a liquid scintillation counter. All samples were run in triplicate. Bmax and Kd were determined by nonlinear least-squares curve fit utilizing the ligand depletion method to determine free concentration (Graphpad Prism Software, San Diego, CA). Bmax and Kd are displayed as mean ± SD with each n representing a separate animal. Values were compared by ANOVA with Fishers protected least significant differences as the posthoc test. Two-tailed tests were used in all cases, and P values <=0.05 were considered significant.

RESULTS

Gestational Profile of Uterine Contractile Response to Bay K 8644

The gestational changes in the pattern of the basal uterine contractions (shaded region, Fig. 1) were consistent with those seen in prior studies in sheep and monkey, i.e., a change from a slow, long-lasting preterm contraction to a rapid, spiking type of term contraction [34]. There was an increasing responsiveness of the guinea pig uterine muscle through gestation to the L-type calcium channel agonist, Bay K 8644. At 35 days (~0.5) and 45 days (~0.67) of gestation, little response to all doses of Bay K 8644 (Figs. 1 and 2) was appreciated. Although still 10–12 days prior to term, by 55 days (~0.8 of gestation), there was a marked change in VDCC activation compared to the earlier gestational ages. This was seen as both an increase in the sensitivity (Fig. 2) and as an increase in maximal uterine contractile response to Bay K 8644 (contraction data, Fig. 1). The 55-day dose response curve was significantly increased over those at 35 and 45 days gestation (ANOVA; P <= 0.01 for 35 days, 45 days vs. 55 days) (Fig. 2). By Day 65 (term, 0.97 of gestation, not in labor), there was a small, further increase in response of guinea pig uterus to calcium channel activation (Figs. 1 and 2) that was significantly different from either the 35- or 45-day uterus (Fig. 2) (ANOVA; P <= 0.005 for 35 days, 45 days cf. 65 days) but not discernibly different from the 55-day uterus (ANOVA; P = 0.15). The maximal Bay K 8644 response, represented by the top of the fitted dose response curve also increased with gestational age. The maximal responses at 35 and 45 days were 1.66- and 1.38-fold over basal, respectively. By 55 days gestation, the maximal response was 3.0-fold greater than basal activity (ANOVA; P <= 0.05 for 35 days, 45 days cf. 55 days). It increased further to 3.9-fold by term gestation (ANOVA; P <= 0.05 for 35 days, 45 days cf. 65 days). Again, the 55- and 65-day maximal responses were not significantly different from each other. With progressive gestation, the uterine muscle also became increasingly sensitive to Bay K 8644 based on EC50 analysis. Because there was minimal to no response at 35 and 45 days, an EC50 was not calculated. At 55 days, the EC50 was 37.4 nM and at 65 days, the EC50 decreased to 3.8 nM (t-test; P = 0.04, compared as log EC50 (-7.43 ± 0.38 for 55 days and -8.42 ± 0.37 for 65 days).



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  		FIG. 1. Gestational profile of uterine contractile response to Bay K 8644. The uterine contractile patterns from representative experiments for the basal state (shaded region) and in response to stimulation with the L-type Ca2+ channel agonist, Bay K 8644, are shown. Maximal stimulation is with 20 mM KCl



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  		FIG. 2. Dose response curves. Cumulative dose response curves to Bay K 8644 through gestation are shown. At 35 and 45 days of gestation, there is very little uterine response to Bay K 8644 in uterine muscle. Although still preterm, at 55 days, there is a marked increase in VDCC function, which continues to increase to term (65–67 days) (ANOVA P <= 0.005 for 35 days, 45 days cf. 65 days; P <= 0.01 for 35 days, 45 days cf. 55 days; P = 0.77 for 35 days cf. 45 days; P = 0.15 for 55 days cf. 65 days, mean ± SEM)

Gestational Profile of L-Type Calcium Channel mRNA

In order to correlate the functional response of the uterus with mRNA expression for L-type calcium channel subunits, an RT-PCR approach was used to delineate first the cDNA sequence of the calcium channel {alpha}1 subunit expressed in term-pregnant guinea pig uterus. Initially, the oligonucleotide primers used in a previous study of PCR amplification of rat uterine VDCC {alpha}1 subunit cDNA were employed [23]. These primers bracket the fourth repeat domain. However, they failed to provide successful amplification with guinea pig gestational mRNA from a 65-day uterus. Therefore, two primers were designed that were based upon conserved nucleotide sequences of the known {alpha}1 subunit cDNA that bracket the protein-coding region for the cytoplasmic linker between the third and fourth repeat domains of the {alpha}1 subunit. This protein region overlaps that targeted by Tezuka et al. [24] in their studies of rat uterine {alpha}1 subunit expression during gestation. These latter primers provided specific amplification of a product of approximately 550 base pairs (bp) that, upon nucleotide sequencing analyses, was revealed to encode a fragment of the guinea pig uterine {alpha}1 subunit cDNA (Fig. 3).



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  		FIG. 3. The VDCC {alpha}1 subunit of guinea pig uterus. Nucleotide and deduced protein sequence of the cDNA fragment attained from 65-day-pregnant uterus by RT-PCR. This sequence has been deposited in GenBank (accession no. AF005938)

The size of this product was significantly smaller than expected. The same primers, when used in PCR amplification of rat cardiac {alpha}1 cDNA, amplified a fragment about 900 bp in length (data not shown). This variation in size was due to an apparent deletion of 354 bp that encoded 118 amino acid residues (Fig. 4). The deletion occurred exactly in frame between two codons, thereby incurring no change in the reading frame of the nucleotide sequence downstream of the deletion. Both the nucleotide sequence and the deduced primary protein sequence of this guinea pig cDNA are highly homologous with known {alpha}1c subunits of VDCCs [35, 36]. The guinea pig cDNA is 85% homologous with the rat aortic smooth muscle {alpha}1 at the cDNA level and at the nucleotide level and is over 90% homologous at the protein level.



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  		FIG. 4. Deduced protein sequence for VDCC {alpha}1 subunit of guinea pig uterus. Alignment of the deduced protein sequence for the guinea pig subunit with {alpha}1c from rabbit [35] and rat [36]. Conservation of amino acid residue between aligned sequences is indicated by |. Underlines beneath the rabbit sequence denote positions of presumptive transmembrane segments IIIS6 through IVS6. Note the deletion in the guinea pig sequence that begins in the distal portion of the III–IV linker and extends to just before IVS4

The deletion in the guinea pig sequence encompassed the majority of the III–IV linker and all of transmembrane segments IVS1 through IVS3. Structural variations in this region of the channel protein have been reported previously, including two splice variants that are coexpressed in gestational rat uteri [23, 24]. We searched for other {alpha}1c variants in gravid guinea pig uterus that might also manifest structural variations in this protein region. We designed two additional PCR primers (one on each duplex strand) within the region of rat {alpha}1c sequence that is absent in the guinea pig sequence and paired them appropriately with the primers that provided successful amplification of the guinea pig {alpha}1 sequence. Following PCR reactions with extended cycles, amplicon presence was assessed by Southern blotting analysis using, as radiolabeled probe, the 900-bp fragment amplified from the rat {alpha}1c cDNA (which contains the region deleted in the guinea pig sequence). This approach, however, failed to provide specific amplification of any discernible product. Moreover, combined RT-PCR (using the productive primers) and Southern blotting analyses of guinea pig uteri at other gestational ages have yet to provide amplification of an {alpha}1c fragment that differed in size from that described above (despite extended PCR amplifications and posthybridization autoradiographies).

Reverse transcriptase-PCR was also utilized to characterize VDCC ß subunit(s) expressed in gestational guinea pig uteri. Known cDNA sequences for VDCC ß subunits were aligned to reveal conserved sequence motifs that were then used to design ß-specific PCR primers. These primers bracketed a protein region that overlaps with that targeted in a recent study on rat uterine ß expression [24] and amplified a cDNA fragment from 65-day guinea pig uterus of approximately 550 bp (Fig. 5) that is highly homologous with rat and rabbit ß2A [37, 38]. The guinea pig and rat sequences share over 93% nucleotide homology and 100% amino acid homology; sequence variations between these sequences reside entirely in the codon-wobble positions, hence the lack of change in the deduced protein sequences (Fig. 6). The high level of sequence homologies between these sequences clearly indicate that the guinea pig cDNA is the species homolog of VDCC ß2A.



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  		FIG. 5. The VDCC ß2 subunit of guinea pig uterus. Nucleotide and deduced protein sequence of the cDNA fragment attained from 65-day-pregnant uterus by RT-PCR. This sequence has been deposited in GenBank (accession no. AF005939)



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  		FIG. 6. Deduced protein sequence for ß2 subunit of guinea pig uterus. Alignment of the protein sequence for the guinea pig subunit with those of rat [42] and rabbit [37]. Conservation of amino acid residue between aligned sequences is indicated by |

Following structural analysis of the {alpha} and ß subunits of the L-type calcium channel in the guinea pig uterus, we assessed the relative changes in their gestational expression at the mRNA level. Semiquantitative PCR was used to compare relative transcript levels for both {alpha}1 and ß2 subunits at 34, 47, 54, and 64 days gestational age. Because of concerns over different efficiencies of PCR amplification when comparing different gestational ages, the following were done to address this: 1) the PCR conditions most appropriate for each transcript were determined such that the PCR-derived product yields were reproducibly and linearly related to the PCR cycle and 2) parallel expression of cyclophilin (a housekeeping gene invariant with gestational age) was assessed and used to normalize the expression levels of the two VDCC subunits. However, under these PCR conditions, the products were often present at levels beneath visual detectability and therefore required Southern blotting analysis for visualization and quantification. Both {alpha}1 and ß2 mRNA expression displayed distinct gestational age profiles (Fig. 7, top panel) while there was no discernible change in cyclophilin mRNA expression during these same gestational stages. Specifically, {alpha}1 mRNA levels were relatively constant between 34 and 47 days, increased by approximately threefold (relative to the expression level of 34 days) by 54 days and persisted at the elevated levels through term (Fig. 7, middle panel). In contrast, the ß2 mRNA level remained low at 34 and 47 days, increased by almost 20-fold by 54 days, and then fell to 7-fold (relative to the expression level of 34 days) by 64 days (Fig. 7, bottom panel). These findings clearly indicated dynamic changes in the expression of L-type VDCC subunits in gestational guinea pig uteri.



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  		FIG. 7. Expression profile of mRNA VDCC subunits in the gestational uterus of guinea pig. Relative levels of uterine transcripts encoding {alpha}1 and ß2 subunits at 34, 47, 54, and 64 days of guinea pig gestation were compared by semiquantitative PCR analyses. Subunit-specific PCR was conducted at each gestational age to provide linear amplification, then subjected to Southern blotting analyses with the corresponding radiolabeled probe. Cyclophilin transcript levels were also assessed in order to provide a normalizing basis for comparing the various time points. A representative autoradiograph is shown (top panel). Note that {alpha}1 expression was detectable at all time points and seemed to elevate at 54 and 64 days, relative to 34 and 47 days. Expression of ß2 was barely detectable at 34 and 47 days and subsequently escalated dramatically by 54 days; by 64 days, expression appeared to have declined. Hybridizing signals observed on autoradiographs were excised from the Southern blots and each quantified by liquid scintillation spectrometry. Each result was first normalized by cyclophilin expression level and then normalized by the corresponding subunit's expression level observed at 34 days of gestation. Approaching term, {alpha}1 expression steadily increased to about 3-fold that of the 34-day level (middle panel), while ß2 expression exhibited ~20-fold elevation (during late gestation) followed by a decline (prior to term) to about 7-fold that of the 34-day level (bottom panel). Each bar represents mean ± SD of eight experiments

Gestational Profile of Isradipine Binding at the DHP Binding Site of the {alpha}1c Subunit

In order to correlate the uterine functional response and the channel subunit transcript profiles with the amount of isradipine binding, saturation binding analysis specific for VDCC was utilized. Representative binding isotherms are shown in Figure 8. There was a single, high affinity, saturable site at all gestations. The number of binding sites, Bmax (fmoles/mg protein), and affinity of the binding site for isradipine, Kd (pM), are shown in Table 1. Specific binding was present throughout gestation, but levels were highest in early gestation (35 and 45 days), nadired at 55 days, and rose again at term (65 days) (ANOVA; P <= 0.02 for 55 days vs. 35 and 65 days gestation). The gestational profile of isradipine binding correlated with neither VDCC function nor with subunit mRNA expression. The affinity, Kd, did not change significantly through gestation.



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  		FIG. 8. Saturation binding. Representative experiments of gestational changes in saturation binding with 3H-isradipine in guinea pig uterus are shown above. Each point is the average of triplicate determinations. Note the change in scale on the ordinates. The inset shows the Scatchard analysis. There was a single, high affinity, saturable site at all gestations


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  		TABLE 1. The number of binding sites and affinity of the binding site for isradipine

DISCUSSION

This study was undertaken to characterize the functional state, mRNA expression, and protein levels of the uterine L-type calcium channel through gestation in the guinea pig. The guinea pig VDCC appears to be highly regulated through gestation. The regulation of contractile function may be different from the regulation of the subunit mRNA expression and isradipine binding, as these are not completely parallel throughout gestation. There is a concomitant up-regulation of VDCC function, {alpha}1c subunit mRNA expression, and detectable isradipine binding amount at term (65 days), supporting a role of this ion channel in the onset of parturition [2, 3].

There are no other known reports on the gestational change in sensitivity of the guinea pig uterus to Bay K 8644 stimulation. The marked increase in channel sensitivity to Bay K 8644 at term is consistent with a role of this ion channel in parturition. It is unclear why the change in sensitivity occurs in preterm uterus (55 days) at 80% of gestation. In human gestation, 32 wk corresponds to 80% of gestation and preterm labor occurs in women anywhere from 24 to 37 wk. This argues for the concept that there is normally active inhibition of uterine contractions during preterm gestation. There may also be active inhibition during the time near term gestation when the prerequisite molecular and cellular changes necessary for labor (e.g., formation of gap junctions, oxytocin receptors, prostaglandin receptors) are taking place [19, 30, 31].

The change in sensitivity to Bay K 8644 stimulation at 55 days gestation occurs coincident with a rise in the {alpha}1c subunit transcript expression but at a time when the level of discernible channel protein, as estimated by 3H-isradipine binding, is minimal. This discrepancy between function and mRNA expression and isradipine binding may simply reflect the lag time from mRNA expression to protein production and subsequently to membrane channel complex assembly. It is possible that Bmax, as assessed by saturation binding, does not accurately reflect protein amount. This issue might be resolved via Western analysis using specific antibodies to the guinea pig {alpha}1c subunit. However, lack of commercially available {alpha}1c subunit antibody and the low copy number for this channel are limiting for performing Western analysis at this time. Therefore, the more sensitive method of saturation binding with a radiolabeled, high affinity DHP was chosen to estimate channel protein amounts. The change in sensitivity is not due to a change in affinity at the DHP site because the Kd does not change through gestation. It is intriguing that the ß2 subunit transcript is up-regulated at 55 days coinciding temporally with the change in sensitivity to Bay K 8644 stimulation. In other systems, the ß2 subunit confers properties to the VDCC that enhance its function [39]. Additionally, other regulatory mechanisms such as changes in phosphorylation or G-protein interactions or changes in downstream contractile elements may enhance VDCC function [1, 40, 41]. The current study is unable to distinguish the exact mechanism(s) responsible for the gestational changes.

The present findings indicate the structural expression of two subunits of the L-type VDCC in pregnant guinea pig uteri. The ß2 subunit is extremely homologous with other known ß2 subunits. The {alpha}1c subunit, while highly homologous to known {alpha}1c subunits, bears a significant in-frame deletion that has not been described elsewhere. This deletion encompasses the distal majority of the III–IV linker and all of transmembrane segments IVS1 through IVS3. Two previous studies have described a splicing variant of {alpha}1c in gestational rat uteri that introduced an 11-residue deletion in the linker between segments IVS3 and IVS4 [23, 24]. Studies in other systems have also demonstrated the existence of several splicing variants of this channel protein [42]. The presently described variant in guinea pig, however, manifests a large deletion in the protein structure and, according to the current model of {alpha}1c's transmembrane topology, may induce an inversion in the topology of the rest of the protein distal to the deletion (by deleting an odd number of transmembrane segments in domain IV). These considerations initially suggested that an {alpha}1c protein bearing such a deletion would be functionally altered or compromised and that such structural variation may not exist in native mRNA transcripts but instead may represent an artifact arising from PCR or cloning manipulations. Several lines of evidence collectively suggest that this variant is not an artifact. First, this form is the only one to be detected (by combined RT-PCR and Southern blotting analyses) in guinea pig uteri of several gestational ages. Additionally, the same PCR primers, when used in amplification of rat cardiac {alpha}1 cDNA, provided a fragment about 900 bp in length exactly in accord with expectations. Secondly, deliberate attempts to detect the uterine presence of the deleted {alpha}1 sequence, by using primers that both reside within and perimetrically bracket the deleted region, failed to support productive PCR amplification. Thirdly, the reading frame of the guinea pig sequence was preserved across the deletion, an aspect of the nucleotide sequence not expected to be preserved in PCR- or cloning-generated artifacts. Fourthly, this deletion actually helps to rationalize our initial failure to identify {alpha}1 cDNA by using a primer pair employed in a previous study [23], as the said upstream primer resided within the sequence region that is absent in the guinea pig sequence. Finally, the Bay K 8644 stimulation studies and the 3H-isradipine binding studies reported here support a functional VDCC in uterus as well as preservation of the DHP binding site. Thus, these independent lines of evidence are mutually consistent in suggesting that the observed deletion may be endogenous to the guinea pig uterine {alpha}1c. Clearly this issue will be better resolved in the future following the elucidation of the entire primary sequence of this channel protein and in vitro expression of the full-length cDNAs for electrophysiological and pharmacological analysis.

The gestational profile of the VDCC {alpha}1 subunit indicated that its uterine expression increased with progression toward term. The relatively higher expression of this subunit during Days 54 and 64, relative to that of Days 34 and 47, exactly paralleled a previous study of expression in gestational rat uteri [24]. Another study in rat uteri, however, indicated that {alpha}1c transcript levels remained invariant during gestation and became elevated only at labor [23]. Despite variations in gestational age, it seems that the present findings support a generalized trend of increasing uterine {alpha}1c expression with gestational progression toward term. Expression of ß2 exhibited very dramatic and abundant elevation by Day 54 but began to decline prior to term. This differed from the expression profile discerned from rat uteri, wherein ß2 expression began to increase just prior to term and reached its peak at term [24]. The two gestational uterine models are in agreement in that the expression of this subunit undergoes an increase prior to term, but in guinea pig this occurred earlier in preterm gestation.

In summary, this study shows that gestational regulation of the guinea pig uterine L-type calcium channel is complex. Overall, increased transcript presence of both VDCC subunits in gestational uteri collectively indicate that progression toward term is accompanied by increased VDCC activity. The changes toward term of increased VDCC-mediated contractile function, mRNA expression, and isradipine binding levels support a role for the VDCC in the process of parturition.

ACKNOWLEDGMENTS

We are grateful to Dr. A.M. Brown for providing us with the cloned rat {alpha}1c and ß2A cDNAs. We thank Edo Idriss and Teresa Kulp for their technical assistance with the uterine contraction experiments and Susanne Rovansek for her administrative aid in the preparation of the manuscript and figures.

FOOTNOTES

First decision: 2 March 2000.

1 This study was funded in part by March of Dimes grants 6-1070 and 6-0247 (to P.L.C.) and 6-0649 and 6-0609 (to P.L.C. and A.S.C.) and by the NIH/NICHD HD-28433 (to P.L.C.). This work was presented in part in abstract form at The Society for Gynecologic Investigation 43rd annual meetings in Philadelphia, Pennsylvania. Back

2 Correspondence: Patricia L. Collins, Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Loyola University Medical Center, 2160 South 1st Ave., Maywood, IL 60153. FAX: 708 216 5669; pcollin{at}luc.edu Back

Accepted: June 5, 2000.

Received: January 12, 2000.

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