Changes in transcripts encoding calcium channel ...

Changes in transcripts encoding calcium channel subunits of rat myometrium during pregnancy NAOHIRO TEZUKA, MARIAM ALI, KRISTOF CHWALISZ, AND ROBERT E. GARFIELD Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77555; and Research Laboratories of Schering, Berlin 65, Germany Tezuka, Naohiro, Mariam Ali, Kristof Chwalisz, and Robert E. Garfield. Changes in transcripts encoding calcium channel subunits of rat myometrium during pregnancy. Am. J. Physiol. 269 (Cell Physiol. 38): C1008-C1017, 1995.Extracellular Ca2+is normally required for myometrial cells to contract. Ca2+ enters muscle cells mainly through voltagedependent Ca2+channels (VDCCs) that open in response to action potentials. The synthesis of myometrial VDCCs may change during pregnancy to alter excitation-contraction coupling. We investigated the mRNA levels for the al- and P-subunits of the L-type VDCC in rat myometrium to determine whether alterations are associated with term or preterm labor. RNA isolated from myometrial tissues was analyzed by reverse transcription-polymerase chain reaction (PCR) using specific primers designed according to the published sequences of the VDCC subunits. From pregnant rat myometrium, two distinct PCR products were obtained for the al-subunit: one of the expected size at 372 bp and a smaller at 339 bp. Sequence analysis of the larger product revealed a 99.5 or 88% sequence homology between rat myometrium and rat aorta or rabbit heart, respectively, and the smaller product had an identical sequence to a 33-bp deletion. The two al-products followed the same trend throughout pregnancy. VDCC al-mRNA levels increased gradually to 6.9-fold just before labor on day 22 but decreased during labor. However, the P-subunit mRNA level increased sharply on day 22 and then also declined during labor. Progesterone treatment from day 19 to day 22 inhibited term delivery and prevented the significant increase in almRNA levels. In contrast, antiprogesterone (onapristone, ZK-98.299) treatment on day 1 7 caused a statistically significant increase in the al- and p-VDCC subunit mRNA after 8 and 15 h, respectively, then a decrease during preterm labor at 24 h. We conclude that mRNA levels for the VDCC subunits increase before term and preterm labor but decline during periods when VDCCs are likely at their peaks. The increase in levels of mRNA for VDCC likely reflects changes in expression of VDCCs during periods of term and preterm labor that may facilitate uterine contractility required for this process. Progesterone withdrawal or blockade appears to be responsible for regulating levels of mRNA for VDCC in the myometrium in preparation for labor. uterus; parturition; preterm labor; voltage-dependent calcium channel; progesterone; onapristone

CALCIUM ION entry through voltage-dependent Ca2+channels (VDCCs) is responsible for excitation-contraction coupling in uterine smooth muscle (1, 19, 22). VDCCs are opened in response to depolarization of the plasma membrane as action potentials propagate over the surface of the muscle cell. The VDCC opening leads to a rapid influx of Ca2+,activation of myosin light-chain kinase, and initiation of muscle contraction (18). Because contractions of the myometrium are dependent on action potentials, VDCCs play a critical role in the regulation of uterine contractility. On the basis of C1008

electrophysiological and pharmacological properties of Ca2+channels, four major types of VDCCs have been described: the T-type channel that generates a lowthreshold current and the other three (L, N, and P) types of channels that generate high-threshold currents. The L-type channels are sensitive to dihydropyridine agents, the N-type channels are blocked by w-conotoxin, and the P-type channels are insensitive to both dihydropyridines and w-conotoxin (3). Most information about VDCC structure, especially the L-type VDCC, comes from research on skeletal muscle. It is now accepted that the VDCC protein is made up of five subunits (al, a2, 6, p, and y) that produce Ca2+currents that are blocked by calcium antagonists such as dihydropyridines (7). The a,-subunit is the main, pore-forming, and minimum component of the L-type VDCC and contains binding sites for calcium antagonists (3, 31, 40). The al-subunit from rat aortic smooth muscle has been cloned (20) and shown to be highly homologous to the channel in rabbit cardiac muscle (24). The p-subunit is believed to be cytoplasmic, and it is joined to the a-subunit between repeat I and repeat I1 a t the cytoplasmic loop (33). Coexpression of the p-subunit with the a-subunit changes voltage dependence, kinetics, and magnitude of Ca2+channel current (4). Neely et al. (28) suggest that the gating current from coexpressed cardiac al- and p-subunits is unchanged in both magnitude and voltage dependence from what is produced by al-subunits alone, but ionic current is enhanced and shifted in voltage dependence. The p-subunits somehow facilitate coupling between movement of the voltage sensor and opening of the channel pore (28). There are some electrophysiological studies with respect to measuring the Ca2+current of the myometrial cells. In the rat myometrium, it has been proposed that Ca2+current density may increase duringgestation (26). On the other hand, Sperelakis and co-workers (17, 43) have reported that the current densities of VDCC may be almost unchanged during pregnancy. The main VDCC existing in the rat myometrium is the L-type VDCC (21, 29). However, little is known of the molecular aspects of VDCC expression in the uterine smooth muscle cells in preparation for contractions that lead to parturition. Recently, a molecular study indicated that the alsubunit of the VDCC of rat myometrium consists of several isoforms that increase during pregnancy but decline during labor (23). However, nothing is known about the hormonal regulation of VDCCs in the myometrium, and there are no data on the p-subunit. In this study, we performed experiments to measure mRNA levels to the a,- and p-subunits of the L-type W C C using the reverse transcription (RT)-polymerase

0363-6143195 $3.00 Copyright o 1995 the American Physiological Society

CALCIUM CHANNEL EXPRESSION IN MYOMETRIUM

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CYCLE NUMBER Fig. 1. Reverse transcription (RT)-polymerasechain reaction (PCR) signals as a function of amount of total RNA and number of PCR cycles. RT-PCR was performed according to conditions described in MATERIALS AND METHODS. A: photograph of gels stained with ethidium bromide after adding various amounts of total RNA (0.25-2 pg) at 20-24 cycles. Two PCR products (372 and 339 bp) are visible in all groups. B: densitometric scanning of top (372 bp) bands vs. cycle number with various amounts of total RNA (Ao = 0.25-2 pg) from rat myometria at day 19 of gestation. Data shown are normalized relative to value obtained for 2 pg RNA a t 24 PCR cycles. Each data point represents mean value from 2 experiments that differed by < 5%.Similar results were obtained with analysis of 339-bp bands.

chain reaction (PCR) method of the rat myometrium throughout pregnancy and after treatments with progesterone or the antiprogesterone compound onapristone (ZK-98.299). Onapristone is a 13a-configuredprogesterone receptor antagonist showing a reduced antiglucocorticoid activity in common with mifepristone (RU-486) (12,27).The aim of this study was to determine whether alterations of VDCC gene transcripts are associated with term or preterm labor. The study indicates the possibility that the synthesis of myometrial VDCCs may change during pregnancy to alter excitation-contraction coupling.

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Tissue preparation. All experiments were approved by the Animal Care and Use Committee. Wistar rats (SpragueDawley) were used at various times of pregnancy (day 2 2 is day of delivery). To assess gestational changes in VDCC mRNA, animals were killed by asphyxiation with C02 on either days 14, 15, 17, 19-22 (nonlabor), during labor, or 1 or 3 days postpartum (n = 3-5 in each group). Rats during labor were killed once delivew was initiated (usually after the delivew of 1-3 pups). uterine horns were removed, placed in cold phosphate-buffered saline solution on ice, opened along the line of mesometrial attachment, and pinned with endometrial surface upward. After scraping remove the end0metrium and the placental attachment sites, the myometrial tissue was washed thoroughly to remove all traces of blood, immediately frozen in liquid nitrogen, and stored at -70°C for later -

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different of pregnancy' Data points Fig. 2. PCR represent log of densiometric scans of 372-bp product after 23 cycles vs. lo, of various loading amounts of total RNA at 3 different days of pregn-ancy, E ~ ~point-represents - , mean of 2 experiments, similar results were obtained with 339-bp bands. Note that plots are linear andparallel. On

CALCIUM CHANNEL EXPRESSION IN MYOMETRIUM

Fig. 3. RT-PCR products of Ca2+ channel alsubunit mRNA from rat myometrium at different times of pregnancy. A: lanes 1-10, products from myometrial RNAs a t different days of pregnancy (NL, nonlabor; L, labor; pp, postpartum); lane 11: product from rat cardiac RNA; lane 12, molecular size markers (BioMarker Low; BioVentures, Murfreesboro, TN). Two (top and bottom) arrowheads marked on left indicate positions of 372- and 339-bp product bands, respectively. B: gestational changes of Ca2+channel al-subunit mRNA levels in rat myometrium by RT-PCR. Photographs of PCR product bands were scanned, and densities of bands were normalized as described in MATERIALS AND METHODS. For points (n = 3-5 animals) lacking a SE bar, error was smaller than or equal to size of symbol. Values with different lower- or uppercase letters indicate significant differences compared with other time points for 372 or 339 bp, respectively.

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analysis. The removal of the endometrium was verified by microscopic examination of tissue sections frozen at the time of collection. Rat cardiac tissues were obtained from the ventricles of the heart and were handled in the same manner as uterine tissues. Antiprogesterone treatment. Rats at 17 days of gestation were injected subcutaneously under the back skin with 10 mg of the progesterone receptor antagonist onapristone (ZK98.299) (Schering, Berlin, Germany) suspended in castor oil and benzyl benzoate. Animals were killed before or 3,8, 15, or 24 h after treatment with onapristone, and the uteri were processed as above. Onapristone induced partial to complete preterm labor in all rats by 24 h as judged by uterine bleeding and delivery of some or all of the fetuses. Control rats were injected with the vehicle only and were subsequently handled the same as the treated animals. Progesterone treatment. Rats were injected subcutaneously with either 2.5 mgprogesterone (4-pregnene-3,20-dione;Sigma, St. Louis, MO) suspended in sesame oil or the vehicle alone twice a day from day 19 to day 22 of gestation. Animals were killed on days 20-22 (before or during delivery for the control group) or on day 23 for the progesterone-treated group. The uteri were prepared as above. R N A isolation. Total cellular RNA from the collected tissue was isolated and purified by the acid-guanidinium thiocyanatephenol-chloroform method (8). We routinely test all our RNA preparations for 28s and 18s RNA on agarose/formaldehyde gels. DNA contamination was eliminated by treating the RNA with ribonuclease (RNase)-free deoxyribonuclease (Stratagene, La Jolla, CA). RNA concentrations in individual samples from separate rats were measured by ultraviolet absorbance at

260 nm using a spectrophotometer (DU-64; Beckman Instruments, Palo Alto, CA) and then stored as an ethanol precipitation at - 70°C until it was used. Primer design. Sets of specific amplimers were designed to correspond to regions of low degeneracy that are conserved between the rat aortic smooth muscle and rabbit cardiac muscle isoforms of the L-type VDCC a l - and P-subunits (16, 20, 24, 30) using Oligo Primer Analysis Software (National Biosciences, Plymouth, MN). The oligonucleotide sequences used for the cil-subunit were 5'-ATC CCC AAG AAC CAG CAC CAG-3' (sense primer) and 5'-GGT GAT GGA GAT GCG GGA GTT-3' (antisense primer). These primers flanked a 330-base fragment in the region of the cDNA. This product spanned a region between segment 6 in domain I11 (IIIS6) and the IVS4, with an expected product size of 372 bp. The amplimers used for the P-subunit were 5'-TGG TGG ATA GGA CGG CTG GTT-3' (sense primer) and 5'-TGC GTG CIT ACT GGG GTT GTT-3' (antisense primer). These primers flanked a 507-base fragment in the region of the cDNA. This product represents a region between the a-helical domain I and domain I11 with an expected size of 549 bp (37). The oligonucleotide primers were purchased from Genosys Biotechnologies (Woodlands, TX). RT-PCR. To analyze different samples of RNA quantitatively for mRNA transcripts of the L-type VDCC al- and P-subunits, we performed RT-PCR using the GeneAmp RNA PCR kit (Perkin-Elmer Cetus, Nonvalk, CT). For the firststrand cDNA synthesis, 1 ~g total cellular RNA was reversetranscribed in 20 ~1 of a solution containing 10 mM tris(hydroxymethy1)aminomethane (Tris) .HCl (pH 8.3), 50 mM KCl, 5 mM MgC12, 1mM of each deoxyribonucleotide triphosphate, 20 U RNase inhibitor (recombinant RNase inhibitor from

CALCIUM CHANNEL EXPRESSION I N MYOMETRIUM

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product (arrowhead) is visible in ail lanes at different days ofgestation (days 14-22; NL, nonlabor; L, labor) and postpartum (pp; days 1 and 3). B: quantitative data based on densiometric scanning of bands a t various times of gestation and postpartum. Values (n = 3-5 animals) with different lowercase letters indicate significant differences a t different times of pregnancy.

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placenta; Perkin-Elmer), 50 U Moloney murine leukemia virus reverse transcriptase, and 0.75 KM of the specific antisense primer. After 15 min of incubation at 42"C, the reaction mixture was heated to 99°C for 5 min and cooled to 5°C. The first-strand cDNA was subsequently amplified in the same tube with a GeneAmp PCR System 9600 (Perkin-Elmer Cetus). The cDNA mixture was supplemented with 2.5 U AmpliTaq DNA polymerase, 15 KMof the sense primer, 8 ~1of 100 mM Tris. HCl (pH 8.3),500 mM KCl, 4 ~1of 25 mM MgC12, and water to bring the final volume to 100 ~ 1 PCR . was performed with the followingconditions: denaturation at 94°C for 15 s, annealing at 53°C for 30 s, and polymerization at 72°C for 45 s. In preliminary studies, we showed that the RT-PCR was carried out reproducibly in a quantitative manner for 20-24 PCR cycles for the a,-subunit and 32-36 cycles for the p-subunit, as shown in Fig. 1. To control for possible DNA carryover and contamination, we routinely included water blanks and control RNA samples not exposed to reverse transcriptase. Quantitative analysis of RT-PCRproducts. A portion (15 ~ 1 of the PCR mixture was electrophoresed on 8%polyacrylamide gel. The gel was stained with ethidium bromide and photographed using Polaroid type 55 positive/negative film. The density of bands for both myometrial and cardiac mRNA was determined by a scanning densitometry system (model DNA 35; PDI, Huntington Station, NY), then the levels of PCR products from myometrial mRNA were normalized to a standard cardiac PCR product (from laboring rats) within each gel. To quantitate mRNA levels in the exponential range of PCR

amplification, the relative yield was estimated for different numbers of PCR cycles using a variable amount of total RNA isolated from myometrium (depicted in Fig. 1).The amount of heart mRNA for the subunits did not change during pregnancy. Direct sequencing of PCR products. To prepare for sequencing of the a,-subunit, each band was excised from the polyacrylamide gel and reamplified for 30 cycles of PCR (above conditions). Amplified products were run on 1.2% agarose gel, excised, then purified using a Geneclean I1 kit (Bio 101, La Jolla, CA). Direct sequencing of the purified PCR products was performed using a Tag dideoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CAI by the dideoxynucleotide chain-termination method (39). Restriction enzyme analysis. For verification of the RT-PCR product of the p-subunit, restriction enzyme analysis of the amplified products was carried out (35). PCR samples were incubated with the restriction endonucleaseApa I according to the manufacturer's instructions (Life Technologies, Gaithers) burg, MD). After digestion with the enzyme Apa I, DNA fragments were ethanol precipitated, redissolved in sterile water, and analyzed by electrophoresis on 10%polyacrylamide gels. Statistical analysis. Data obtained were subjected to a one-way analysis of variance. Fisher's least significant difference test was used to assess the statistical significance of the difference between mean values. P < 0.05 was considered significant. All results are expressed as means SE from three to five rats.

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CALCIUM CHANNEL EXPRESSION I N MYOMETRIUM

Fig. 5. RT-PCR products of Ca2+ channel al-subnit mRNA from rat myometrium after ZK-98.299 treatment. A: lanes 1-6, products from myometrial RNAs with or without ZK-98.299 treatment a t different hours after injection; lane 7, product from rat cardiac RNA; lane 8, molecular size markers (BioMarker Low). Two (top and bottom) arrowheads marked on left indicate positions of 372- and 339-bp product bands, respectively. B: effects of ZK-98.299 on Ca2+channel al-subunit mRNA levels (left, 372-bp product; right, 339-bp product) in rat myometrium by RT-PCR. Line graph represents density of PCR products from control and ZK-98.299-treated rats. Mean values of 372-bp products showing significant differences a t different times of gestation and postpartum. Onapristone induced partial or complete delivery by 24 h . Values (n = 3-5 animals) with different lowercase letters indicate significant differences a t different times of pregnancy. *Significant differences between mean values of control tissues vs. tissues from treated animals. There were no significant changes in 339-bp product (right) when mean values were compared a t different times or between treated and control means.

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of RNA from myometrium. The quantity present after adding various amounts of RNA vs. cycle number yielded Two distinct PCR products were detected for the parallel lines, indicating that the efficiency of amplificaa,-subunit of myometrial samples; one was 372 bp l9 of gestation). In

tion was constant (Fig. l B , (RtMyl), the expected fragment, and the other was PCR product was P ~ smaller a t 339 bp (RtMy2), whereas only the 372-bp addition, the RNA for a given number to the product was present in cardiac samples (Figs. 1 , 3 , 5 ,and 7). RT-PCR amplification of RNA samples for the pmsub- of cycles. Similar data were obtained on days 15and 22 unit from myornetrial and brain tissues a (nonlabor) of gestation (Fig. 2). These results indicate that the efficiency of amplification is constant across the single expected product of 549 bp (Figs. 4 and 6). To determine the levels of mRNA transcripts for the different days of gestation. Therefore, in this case, L-typeVDCC and P-subunit m~~~ transcripts in RT-PCR can be carried out in a quantitative manner. pregnant rat myometrium, RNA samples were prepared Subsequently, PCR for the ai-subunit was performed a t different stages of gestation with or without steroid for 23 cycles. Similar control studies were carried out on hormone treatments and analyzed by RT-PCR. We the P-subunit and indicated exponential amplification initially analyzed serial amounts of total cellular RNA over the range of 32-36 cycles (data not shown). Gestational changes in m~ometrialm R N A levels. The on either days 15, 19, or 22 (nonlabor) of gestation a t different PCR cycles to determine whether the RT-PCR two PCR products of the a,-subunit followed almost the could be carried out reproducibly in a quantitative same increasing and decreasing trends throughout pregmanner. Figure 1 shows the relationship between opti- nancy (Fig. 3). Levels of mRNA for the myornetrial cal density and PCR cycle number with different amounts a,-subunit were very low on days 14 and 15 of pregof RNA used in the RT-PCR reaction for the a,-subunit. nancy. Statistically significant increases in levels were Quantitative analysis of the a,-subunit PCR products found between days 15and 1 7 of pregnancy ( P < 0.005). indicated exponential amplification over the range of Myometrial mRNA levels for the subunit then gradually 20-24 cycles, as shown by the linearity of the semi- increased to 6.9-fold just before labor on day 22 (relative logarithmic plot in all groups regardless of the amount to those on day 14) of gestation (Fig. 3). During labor on

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CALCIUM CHANNEL EXPRESSION IN MYOMETRIUM

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Fig. 6. Effects of ZK-98.299 on Ca2+ channel P-subunit mRNA levels in rat myometrium by RT-PCR. A: photograph of PCR product bands. Lanes 1-6, products from myometrial RNAs with or without ZK-98.299 treatment a t different hours after injection; lane 7, product from rat brain RNA, lane 8, molecular size markers (BioMarker Low). Arrowhead on left indicates position of 549-bp product band. B: normalized densitometric data of RT-PCR products of P-subunit after treatment with ZK-98.299 compared with control. Data are from 3-5 rats a t each time point. Significant differences a t various times in tissues from treated rats are indicated by different lowercase letters. *Significant differences between tissues from control vs. treated rats.

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day 22 (5-12 h after day 22 nonlabor) and postpartum, a continuous decrease was observed. There was a rapid significant decrease in the levels of the 372-bp product between day 22 nonlabor and labor (P < 0.05), whereas no significant change was found in the level of 339-bp product. Unlike the a,-subunit, the P-subunit in the myometrium increased rapidly a t term (Fig. 4). Levels of mRNA for myometrial VDCC @-subunitwere very low on days 14-19 of pregnancy, rose rapidly to 6.3-fold just before labor a t term on day 22, but decreased during labor (Fig. 4). Effects of antiprogesterone on VDCC transcripts. In animals treated with onapristone on day 1 7 of gestation, deliveries occurred after 24 h of treatment. There were no obvious deliveries or signs (vaginal bleeding) of labor in the onapristone-treated rats after 3, 8, and 15 h and in control animals. The two a,-subunits PCR products followed the same trends after onapristone treatment (Fig. 5). Administration of onapristone resulted in a rapid increase in myometrial a,-subunit mRNA levels of the 372-bp product by 8 h postinjection to cause maximal values 1.5-fold higher than those seen both before onapristone exposure and after 8 h in control rats (both

P < 0.05). These mRNA levels of 372-bp product then fell to statistically lower levels by 24 h during preterm labor in comparison with other groups (all P < 0.05: Fig. 5). Although in onapristone-treated rats the same qualitative changes were exhibited in the levels of mRNA in both 372- and 339-bp products, there was no significant change in those of 339-bp product (Fig. 5). Treatment with onapristone also resulted in a rapid increase in myometrial P-subunit VDCC mRNA levels by 15 h postinjection to cause maximal values 4.3-fold higher than those seen both before onapristone exposure and after 15 h in control rats. The mRNA levels then fell to levels lower than control values by 24 h during preterm labor (Fig. 6). Effects of progesterone on VDCC transcripts at term. None of the rats injected with progesterone from day 1 9 to 22 of gestation delivered or showed vaginal bleeding as a sign of labor by day 23 of pregnancy. Control rats receiving injections of vehicle exhibited a significant increase in the levels of VDCC mRNA of the a-subunit on day 22 nonlabor (relative to those on day 19) and a significant decrease between day 22 nonlabor and labor (both P < 0.05: Fig. 7) as noted in other control rats (Fig. 3). In contrast, animals treated with progesterone

CALCIUM CHANNEL EXPRESSION IN MYOMETRIUM

Fig. 7. RT-PCR products of Ca2+ channel alsubunit mRNA from rat myometrium after progesterone treatment. A: lanes 1-7, products from myometrial RNAs with or without progesterone treatment at different days of pregnancy (NL, nonlabor; L, labor); lane 8, product from rat cardiac RNA; lane 9, molecular size markers (BioMarker Low). Two (top and bottom) arrowheads marked on left indicate positions of 372- and 339-bp product bands, respectively. B: line graph indicates mean 2 SE density of PCR products of control and progesterone-treated rats ( n = 3-5 at each time). Significant differences at various times after ZK-98.299 are indicated by different lower case letters. *Significant differences between values from tissues of control vs. ZK-98.299treated rats.

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failed to exhibit the expected significant changes in mRNA levels from day 20 to day 23. Although there were no significant differences for the 372-bp product between the control and progesterone-treated groups on days 20 or 21, al-subunit mRNA levels in rats treated with progesterone were significantly lower than those in control rats on day 22 (nonlabor) (P < 0.05: Fig. 7). Identity of subunit products. Figure 8 shows the nucleotide and deduced amino acid sequences of the al-subunit of the RT-PCR products from rat myometrial (RtMyl and RtMy2) mRNA. Sequence comparisons with the published rat aortic (RtAo) (20) or rabbit cardiac (RbHt) (24) VDCC cDNA have revealed that the RtMyl and RtMy2 cDNAs correspond to the nucleotides 3694-4065 or 3697-4068, respectively (Fig. 8). The nucleotide sequences of the RtMyl and RtMy2 are identical, except the RtMy2 has a 33-bp deletion between the IVS3 and IVS4 regions. The RtMyl nucleotide sequence is 99.5 or 88% homologous to that of the RtAo or RbHt, respectively. There is a 96% nucleotide identity between the RtMyl and RbHt, except for an 84-bp segment including the IVS3 region corresponding to the RbHt nucleotides 3919-4002, where they are only 60% identical. Rat brain RNA was used as a positive control for the VDCC @-subunit.The identity of this product from

myometrial and brain RNA was confirmed by restriction enzyme analysis. The 549-bp product was digested into the expected fragments of 389 and 160 bp by the restriction endonuclease Apa I. Thus each of the products contained the predicted restriction site a t the correct location. Therefore, digestion of both myometrial and brain products confirms that the PCRamplified products encode the VDCC @-subunit. DISCUSSION

This study describes the changes in mRNA levels to the L-type VDCC subunits that occur in the myometrium throughout pregnancy and after treatment with progesterone or antiprogesterone. The observed changes indicate that the mRNA levels for the al-and @-subunits increase gradually for the al-subunit and abruptly for the @-subunitin late pregnancy to reach peaks before term labor, then decrease sharply during labor and postpartum. In addition, antiprogesterone and progesterone treatments, which either stimulate preterm labor or inhibit term labor, respectively, increased or decreased the mRNA for the subunits of the VDCCs. These results show that increases in the VDCC subunit gene transcripts are associated with either term or preterm labor. Furthermore, when transcript levels are prevented from

CALCIUM CHANNEL EXPRESSION IN MYOMETRIUM

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tion is constant for different days of pregnancy and indicates that, using appropriate cycles of PCR, the

I P K Y Q E Q Y K V I Y V V Y S linearity with respect to input RNA is closely similar for

- - - - - - - - - - - - - - =aYa M UtAo M 1232 - - - - - - - - - - . - - days 15, 19, and 22 of gestation. Therefore, our results llbst M 1233 - - - - - . - - - - . - - - - indicate that the differences in product yields at different days of gestation are only due to the mRNA copy t a c t t c gag t a t c t g a t g t t c g t c c t c a t c c t g c t c aac acc a t c t g c c t g gcc .....................................................

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . number rather than some nonspecific function in the . . . . ..t ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t . . ..

samples. These results are also supported by observaT Y P E Y L Y P V L I L L Y T I C L A

tions showing that diluting the input RNA does not change the slope of the relationship. We observed and sequenced two PCR products for the aaa a t c atg aat a t a a t g cag cac t a c aac a t g agc tgc ...... . . . . . ...... . . . . . . . . . . . . . . . . . . . . . . . . . al-subunits in the myometrium. It has been shown in ...... ...... ...... ...... ...... ...... . . . . . . . . ...... ...... . . . . . . . . . . . . - .C . .C . . . . . . other tissues that there are several isoforms of the m I I L I * 8 C K I A *. Q. E- Y- - - - - - - - al-subunit (11, 20, 23, 24). The exact functional signifi- - - - - - - - - - - - - - - - - - - - - - cance of the isoforms in the myometrium is not known, - but there are changes in isoform expression with matuc t t t t c a c t ggc c t c t t c acg g t g gag a t g a t c c t g aag c t c a t t gcc t t c aaa ccc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ration of heart cells (11). In this study, there was a ....................................................... gradual increase in both products during the last one..c . . . ..c . . . . . . . . . . . c . . . ..a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L P T a L P T V E * I L K L I A r x P third of pregnancy with levels of the larger 372-bp product (designated RtMyl) product higher than the smaller 339-bp product (RtMy2) (Figs. 2 and 3). Both products decreased in tissues taken during labor. These sag gac t a t t t c t g t g a t gca tgg a a t aca t t t gac gcc t t g a t t g t t 9 t g ggt agc ...... . . . . . . . . . ............... . . . . . . . . . . . . . . . ............

data may indicate that the potential for excitation of the ........ ............... .............. ............ ... . . . .gt . . t a.. . - . . . . . . . g t t . . . . . . t t . C." ..C . . a a . t . . c . . . myometrial cells slowly develops during the last few Y P C V V O B

K D D A I Y T P D A L I - - - - - - - - - - - - - - - - - - - days of pregnancy, and it probably reaches its highest -- aE - - - - - - - - -- -- -p -- -- -- -I -- -S P v level as VDCC density peaks during labor when the VDCC mRNA levels are declining. This also implies that a t t g t t gat a t a gca c t c acc gag gta cac cca g c t gaa c a t acc caa t g c t c t ccc .............................

some step in the labor process may partially inactivate . . . . . . . . . . . . . . . a,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . a a , . ..c g . c a t t . . . . g t . . . a c t a . t . . . . . . . . . . . . . . . . . . . . . . . . . .

further transcription of VDCCs. Similarly, mRNA levels I V D I A L T E V E P A E E T Q C S P

for VDCCs were elevated during preterm labor after onapristone (Figs. 4 and 5), indicating that VDCCs may be important for contractility that begins early labor. t c t a t g a g t gca t c c cgc a t c t c c a t c acc The results with the al-subunit in this study are very ...... ..................

............ . . . . . . . . . . . . . . . . . . 4065

similar to observations recently obtained by Mershon et . . . . . . .ac . . . . . . . . . . . . . . . . . . . . . 4068

S W S A 8 1 1 1 1 1 T

al. (23). Their study indicated that the al-subunit - - - - - - - - - - - - - - - 1355 mRNA in the myometrium increased to the highest . - 1356 I -

levels on days 20 and 21 of gestation and decreased Fig. 8. Nucleotide and deduced amino acid sequence of a*-subunit. during labor when binding of dihydropyridine to VDCC Nucleotide sequences from rat myometrium (RtMyl and RtMy2) are was elevated (3- to 10-fold). Thus our findings confirm shown in 1st and 2nd rows of each line and are aligned with sequences of rat aortic (3rd row; RtAo) and rabbit cardiac (4th row; RbHt) cDNA. that the al-subunits mRNA of the VDCC, although Deduced amino acid sequences are shown in single-letter code in 5th to increased during pregnancy, decline considerably with 8th rows of each line. Sequence identity to RtMyl is shown a s dots (for the onset of myometrial contractions associated with nucleotide) or dashes (for amino acid), and gaps required to align term labor. The fact that our data are very similar to sequences are denoted by spaces. those of Mershon et al. (23) suggests that our methodology is appropriate. increasing further by progesterone treatment late in Although the a1-subunit increases slowly during gespregnancy, labor fails to occur. Therefore, these studies tation, the p-subunit mRNA of the VDCC, on the other suggest that these events (labor and rise in mRNA for hand, increases dramatically on the last day of pregVDCC) are associated and that a rise in VDCC subunits nancy (Fig. 4). The levels of the p-subunit mRNA are may be required for labor. low during periods when the al-subunit mRNA is increasThe RT-PCR method has been shown to be many ing (Fig. 3), day 1 7 to day 21, but increase significantly orders of magnitude more sensitive than the traditional on the day of parturition, day 22. Because the p-subunit Northern blot techniques (6,44). However, it is critical is known to change the voltage dependence and magnito determine the optimal conditions for detection and tude of Ca2+ channel current of the al-subunit of the quantitation of the target mRNA expression. We per- VDCC (4, 33, 40), an abrupt increase in p-subunit formed kinetic and titration analysis of VDCC-specific expression in the myometrium may be required for the amplification in RNA preparations from myometrium onset of labor to control the al-subunit function. (Figs. 1 and 2) and determined the number of PCR Several lines of evidence from this study suggest that cycles at which the amount of product was synthesized progesterone may suppress the mRNA levels for the before the plateau phase was reached. This series of VDCC subunits. 1) The final rise in subunits in the experiments shows that the efficiency of the amplifica- myometrium at term (Figs. 3 and 4) occurs during RtMyl RtMy2 RtAo 3694 RbHt 3697 ntyl M

a t c ccc aag aac cag cac cag t a c aaa g t g tgg Lac g t g g t c a a c t c c

... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

act

. . C

~ C C

C..

. . C

C.C

C ~ C

C1016

CALCIUM CHANNEL EXPRESSION IN MYOMETRIUM

periods when progesterone levels are known to decline (29,34,45). 2 ) Onapristone, a n antiprogesterone, treatment elevated VDCC levels for both the a- and P-subunits above control values (Figs. 5 and 6). 3) Progesterone treatment from day 19 to term suppressed further increases in the al-subunit VDCC transcripts (Fig. 7). However, the changes that could be attributed to progesterone withdrawal or blockade are relatively small for the a-subunit. Furthermore, estrogens increase during pregnancy and parallel the rise in VDCCs. Also, progesterone is known to downregulate the estrogen receptor, and there is usually an increase in estrogen receptors after antiprogesterone treatment (10, 15). Thus it is possible that tissue levels of both estrogen and progesterone regulate VDCCs. Estrogen is known to regulate other steps likely also to be important in the excitation of the myometrium, including gap junctions (14,38) and Kc channels (5). Progesterone has an inhibitory effect on myometrial gap junction channels, and treatment with an antiprogesterone results in the elevation in gap junctions (9, 13). In contrast, progesterone treatment of nonpregnant rats appears to decrease myometrial Ca2+ currents (36). This result may indicate that the Ca2+ channels in immature rat myometrium may be different from those found in mature pregnant rats. Perhaps the channels present in immature rats are related to developmental changes as in the heart (11). Sequence analysis of the a,-subunit (Fig. 8) revealed two PCR products (RtMyl and RtMy2) that were coded by the sequence of L-type VDCC cDNA (20,24). Variant isoforms with or without the 33-bp deletion are reported in neonatal and fetal cardiac muscles (11,321. However, in our experiments, these isoforms were detected only in myometrium (RtMyl and RtMy2; Fig. 81, not in adult heart (Figs. 2, 4, and 6). These data indicate that the isoform with the 33-bp deletion is more rare in adult cardiac muscles than in myometrium. A ratio of RtMy2 to RtMyl in the myometrium did not change significantly throughout pregnancy and after treatments with progesterone or the antiprogesterone onapristone (data not shown). This may mean that progesterone has no effects on the ratio, that is, the deletion of the 33-bp exon splicing (11) does not depend on progesterone levels. The IVS3 region is believed to be a hypervariable portion (11,32, 42). Therefore, we designed the primers with which PCR products were made to include the TVS3 region. Other studies also support the concept that the rise in VDCCs in the myometrium may increase at the end of gestation and facilitate parturition. These include an increase in dihydropyridine binding (23), a rise in Ca2+ current density (26), and enhanced Ca2+influx through VDCCs stimulated by oxytocin (2,25,26),which accompanies a dramatic increase in oxytocin receptor number (42). However, there are electrophysiological studies of the myometrium that show little change in Ca2+current during pregnancy (17, 43). The reason for this discrepancy may be because we use the entire myometrium, whereas electrophysiological studies generally use only the longitudinal muscles.

In summary, we optimized RT-PCR conditions for detection and quantitation of VDCC al- and P-subunit mRNA expression in rat myometrium. Two PCR products for the VDCC al-subunit were distinguished during pregnancy. VDCC al-subunit mRNA levels increased gradually to 6.9-fold before labor on day 22 but decreased during labor and postpartum. The P-subunit also increased but only on the last day of pregnancy. Antiprogesterone treatment on day 17 caused a significant increase in the VDCC subunit mRNA, then a decrease during preterm labor. Progesterone treatment from day 19 to day 22 inhibited term delivery and blocked the rise in the levels of VDCC al-subunit mRNA. Sequence analysis revealed that the rat myometrial a-subunits, with or without a 33-bp deletion, were highly homologous to the published sequences (rat aorta, rabbit heart) of the VDCC. We conclude that the nucleotide sequences of rat myometrial PCR products are highly homologous to that of rat aorta or rabbit heart VDCC al-subunit. Therefore, our PCR products are likely to represent the myometrial VDCC al-subunit. The increase in mRNA levels of VDCC subunits may reflect a rise in VDCCs. Because the mRNA levels of VDCC are highest immediately before term or preterm labor, the expression of VDCCs is likely to be greatest during labor. The increase in VDCCs during periods of term or preterm labor may facilitate uterine contractility required for this process. The rapid increase in the P-subunit just before labor may activate the al-subunit for maximum contractility during labor. Progesterone withdrawal appears to be responsible in part for regulating VDCCs in preparation for the myometrium to contract. However, the development of VDCCs and their mRNA transcripts may be only one of many changes that occur in the muscle cells in preparation for forceful contractility to facilitate parturition. This work was supported in part by the Ernst Schering Research Foundation and The March of Dimes Birth Defects Foundation. Address for reprint requests: R. E. Garfield, Dept. of Obstetrics and Gynecology, Univ. of Texas Medical Branch, 301 University Blvd. 5-62, Galveston, TX 77555-1062. Received 29 August 1994; accepted in final form 21 April 1995.

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