Adenylyl Cyclase Messenger Ribonucleic Acid in Myometrium: Splice Variant of Type IV

^a Departments of Anesthesiology and Environmental Health Sciences, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Regulation of uterine tone during pregnancy and parturition involves G protein-coupled receptors that modulate cellular levels of cAMP. Cyclic AMP is synthesized by a family of at least nine adenylyl cyclase enzymes that have unique tissue distributions, basal activities, and modes of regulation. Little is known regarding the expression of adenylyl cyclase isoforms in myometrium. Reverse transcription-polymerase chain reaction (PCR) was performed using RNA extracted from freshly dissected rat uteri and a cell line of homogenous rat myometrial cells. Using PCR primers specific for adenylyl cyclases I–IX, mRNA encoding adenylyl cyclases II–IX were identified in both fresh and cultured myometrial cells. A splice variant of adenylyl cyclase type IV was also identified in RNA from both sources. These data show that multiple isoforms of adenylyl cyclases are expressed at the mRNA level in myometrial cells and suggest that the regulation of cellular levels of cAMP likely involves a complex integration of cellular second messengers acting upon multiple isoforms of adenylyl cyclases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Regulation of uterine tone during pregnancy and parturition involves G protein-coupled receptors that modulate cellular levels of cAMP. The maintenance of uterine quiescence and the initiation of labor are influenced by hormonally induced changes in the expression of both - and ß-adrenergic receptors [1] and the G proteins (G_s and G_i) [2–4] with which these receptors couple. These G proteins in turn regulate the activity of the enzyme adenylyl cyclase, which is responsible for the synthesis of cAMP. Although significant progress has been made in understanding the effects of pregnancy on expression of these receptors and G proteins, little is known about the expression and regulation of adenylyl cyclase in myometrium.

Adenylyl cyclases are now known to consist of a family of enzymes of at least nine isoforms that exhibit unique patterns of tissue distribution, basal activities, and modes of regulation [5, 6]. Examples of type-specific regulation of adenylyl cyclase isoforms include the stimulation of types I, III, and VIII by Ca²⁺/calmodulin [7, 8]; the stimulation of types II and IV by G protein ß subunits [9]; the stimulation of type II by protein kinase (PKC) [10, 11]; and the inhibition of type V by protein kinase A [12]. Thus, the products of multiple signaling pathways can have variable influences over the cellular levels of cAMP depending upon the isoforms of adenylyl cyclase expressed in any given cell type.

The biological function of each of the individual isoforms of adenylyl cyclase is to catalyze the conversion of intracellular ATP to cAMP, but the regulation of their activity differs. Because an increase in intracellular cAMP is one of the major mediators of uterine relaxation, changes in the pattern of adenylyl cyclase isoform expression, with changes in adenylyl cyclase activity, would provide an important mechanism for changes in uterine activity. The current study was designed to determine the expression of mRNA for adenylyl cyclase isoforms in myometrium.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials

Hanks' Balanced Salt Solution, Dulbecco's Modified Eagle's medium (DMEM), antibiotic-antimycotic solution, amphotericin B, Trizol, amplification-grade DNase I, random hexamer primers, Superscript I reverse transcriptase, RNase H, and Taq polymerase were purchased from Gibco Life Technologies (Gaithersburg, MD). Radioisotopes were purchased from New England Nuclear (Boston, MA). Primary antibodies to -actin and desmin were purchased from Sigma (St. Louis, MO).

Isolation of Rat Uterine Tissue and Culture of Myometrial Cells

These studies were approved by the Animal Care and Use Committee of the Johns Hopkins School of Public Health and Hygiene. Adult Sprague-Dawley rats at 19–21 days gestation (considered full-term pregnant) were killed by decapitation during halothane anesthesia. No rats were in labor or postpartum. Rat uterine horns were opened longitudinally via an incision along the mesenteric border. Implantation sites as well as placental and fetal tissues were discarded. The endometrium was removed by scraping the mucosal surface under a dissecting microscope until longitudinal muscle fibers were visible. For RNA extraction from fresh myometrium, muscle tissue was finely minced and then homogenized in Trizol reagent as described below.

For the establishment of cultured cells lines, myometrial cells were dispersed from dissected uteri by incubation for 90 min at 37°C in Hanks' Balanced Salt Solution containing 640 µg/ml collagenase type IV, 10 U/ml elastase type IV, 1 mg/ml soybean trypsin inhibitor, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Quantity and viability of dispersed cells were established by trypan blue staining and viewed in a hemocytometer. Cells were plated at a density of 10⁵ cells/cm² in high-glucose DMEM containing 10% heat-inactivated fetal bovine serum and the antibiotics listed above. Because of the short life span of these myometrial cells in primary culture (4 wk), cells were transformed to allow unlimited growth and thus adequate numbers of cultured myometrial cells for RNA analysis. An amphotropic recombinant retroviral construct containing the E7 open reading frame of human papillomavirus type 16 was used [13]. Supernatants from CRIP cells were incubated with primary cultures of rat myometrial cells for 24 h. After infection with this immortalizing vector, the transformed cells have grown for 18 mo.

Phenotypic Characterization of Transformed Cells

Morphologically, immortalized cells demonstrate characteristics consistent with smooth muscle, including elongation to ends that fan out for broad areas of attachment. To confirm that these cells were of smooth muscle lineage, confluent cells were analyzed for the expression of smooth muscle specific -actin and desmin proteins by immunoblotting. Briefly, confluent cells were harvested, suspended in cold Hanks' buffer, and homogenized by 20 strokes with a motor-driven pestle in a prechilled glass homogenizer. Then 100 µg of membrane protein was subjected to PAGE as previously described [14] except that 3% milk protein was used during blocking and 1% milk protein was used during primary antibody incubations. A mouse monoclonal antibody (clone DE-U–10), which recognizes a 50- to 55-kDa desmin protein, was used at a 1:200 dilution; a monoclonal antibody directed against smooth muscle -actin (clone 1A4) was used at a 1:1000 dilution in antibody buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 0.05% Tween 20, 1% milk protein) rocking overnight at room temperature. After three 10-min washes in wash buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 0.05% Tween 20), polyvinylidene fluoride (PVDF) membranes were incubated for 2 h at room temperature with goat anti-mouse secondary antibody conjugated to alkaline phosphatase and then developed with chemiluminescence enhancement according to the manufacturer's protocol (ImmunoLite II; Bio-Rad, Hercules, CA). PVDF was then exposed to film for 30 sec to 5 min.

To further characterize the phenotypic expression of cell surface receptors that are typical of myometrial cells, transformed cells were characterized for the expression of ß-adrenergic and oxytocin receptors. The presence of ß-adrenergic receptors was confirmed by the measurement of adenylyl cyclase activity in response to isoproterenol, and the presence of oxytocin receptors was confirmed by the measurement of an increase in inositol phosphate formation in response to oxytocin. Adenylyl cyclase activity was measured as previously described [15]. Twenty-five micrograms of membrane protein (prepared as described above) was incubated in a final volume of 100 µl for 10 min at 30°C. Incubation buffer included 50 mM Hepes, pH 8.0, 50 mM NaCl, 7 mM MgCl₂, 0.4 mM EGTA, 0.25 mg/ml BSA, 0.1 mM [-³²P]ATP (0.1–0.2 mCi/mmol), 1 mM cAMP, 5 mM creatine phosphate, 50 U/ml creatine phosphokinase, and 10 µM GTP. Reactions were terminated, and [³²P]cAMP was quantitated as previously described [15]. Adenylyl cyclase activity was measured under basal state (GTP alone) and in the presence of GTP plus 100 µM isoproterenol, and activities were expressed as pmol cAMP/mg protein per 10 min.

Increased inositol phosphate formation in response to 1 µM oxytocin was measured in confluent transformed myometrial cells grown in 24-well plates. Confluent cells were washed three times in serum-free and inositol-free DMEM medium and incubated overnight, with each well containing 0.5 ml of this medium with 5 µl [³H]myoinositol (22 Ci/mmol, 1 mCi/ml). The following day, cells were rinsed three times with 37°C Hanks' buffer (calcium- and magnesium-free) containing 10 mM LiCl and incubated for 15 min in 270 µl of this buffer. Oxytocin was added for 30 min to achieve a final 1 µM concentration; the reaction was terminated by the addition of 330 µl cold methanol and 660 µl chloroform to each well, after which samples were immediately transferred to Eppendorf tubes. After centrifugation at 820 x g for 10 min at 4°C, 450 µl of the upper aqueous phase was mixed with 300 µl of cold 50 mM formic acid and 100 µl 3% ammonium hydroxide in a separate tube. This 850-µl sample was then placed on preequilibrated 8 x 20-mm Dowex columns (AG1-X8 [100–200 mesh] formate form; Bio-Rad), and the eluent was collected in a 21-ml scintillation vial. The column was washed with 1 ml of 0.18% ammonium hydroxide, and this eluent was collected in the same vial. This 1.85 ml represented the total [³H]inositol fraction. The columns were rinsed with 10 ml 40 mM ammonium formate/0.1 M formate, which was discarded. Total [³H]inositol phosphates were eluted with 1 ml 4 M ammonium formate/0.2 M formic acid. Inositol phosphate formation was expressed as [³H]inositol phosphate/[³H]inositol phosphate + [³H]inositol.

Protein Assay

Protein was assayed with the Pierce Chemical Co. (Rockford, IL) BCA protein assay reagent, consisting of a bicinchoninic acid and copper sulfate solution [16]. BSA was used as a standard.

Isolation of RNA

RNA was isolated using Trizol reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer's protocol. One milliliter of this phenol/guanidine isothiocyanate reagent was added for each 100 mg of freshly dissected uterine muscle or for each 10 cm² of confluent primary cultures of uterine smooth muscle cells. Typically, 1 g of fresh tissue or cells from a confluent T–175 flask was used. After extraction and ethanol precipitation, total RNA was dissolved in 50–100 µl of diethyl pyrocarbonate (DEPC)-treated water. Aliquots were assessed for quantity and purity of RNA by 260/280-nm absorbance readings, and RNA was stored at -70°C until used for reverse transcription-polymerase chain reaction (RT-PCR).

RT-PCR

These RT-PCR experiments were performed at least three times for each adenylyl cyclase isoform, using RNA samples obtained from the uteri from at least three different rats and RNA samples obtained from at least three different clones of immortalized rat myometrial cells. These clones arose from a single rat uterus immortalized under different conditions of viral dose and time. Total RNA was first treated with DNase I to remove residual genomic DNA that might yield false-positive results during RT-PCR amplification of mRNA. Four micrograms of total RNA was incubated with 4 units of amplification-grade ribonuclease (RNase)-free DNase I (Gibco BRL) in buffer (final concentration: 20 mM Tris pH 8.3, 50 mM KCl, 2.5 mM MgCl₂) in a final volume of 40 µl for 15 min at room temperature. DNase I was inactivated by the addition of 4 µl of 25 mM EDTA and heated to 65°C for 10 min. RNA was precipitated by the addition of 1/10 volume of 3 M NaOAc, pH 5.2, and 2 volumes of cold 100% ethanol. After 15 min at -20°C, the samples were centrifuged at 17 000 x g for 15 min at 4°C, and the pellet was washed with 70% ethanol before partial air drying and resuspension in 20 µl of DEPC-treated water. Ten microliters of this final suspension was then used for cDNA synthesis.

Ten microliters DNase-treated RNA was incubated in a final volume of 20 µl containing 10 units of placental RNase inhibitor, 10 mM dithiothreitol, 0.5 mM each dNTP, 50 mM Tris pH 8.3, 15 mM KCl, 3 mM MgCl₂, and either 20 pmol of random hexamer primers or 500 ng of oligo-T(dT)_12–18 primer. The components listed above were mixed and incubated at 42°C for 2 min before the addition of 200 units of Superscript I reverse transcriptase (Gibco BRL) and further incubated for 50 min at 42°C. Parallel reactions were performed as outlined above except that reverse transcriptase was not added to the reactions. These "-RT controls" were then carried through to the PCR reaction to ensure that no PCR products were generated from sample tubes lacking reverse transcriptase, indicating a lack of genomic DNA contamination. The reaction was inactivated by heating to 70°C for 15 min. Complementary RNA was removed from the newly synthesized cDNA by the addition of 2 units of RNase H and incubation at 37°C for 20 min. Aliquots (1 µl) of cDNA were either used immediately in 25-µl PCR reactions or stored at -20°C for later use.

PCR primers were used that would specifically amplify cDNA encoding adenylyl cyclase isoforms. For adenylyl cyclase isoforms II–VI, primers were used that have previously been shown to selectively amplify adenylyl cyclase isoforms from rat tissues [17]. Adenylyl cyclase isoform I primers were designed on the basis of published bovine cDNA sequence [18], and rat brain total RNA was used as a positive control to ensure cross-species annealing of the PCR primers with rat adenylyl cyclase I. Adenylyl cyclase isoform VIII primers were designed on the basis of published rat cDNA sequence, and primers for isoforms VII and IX were designed based on published murine sequences. Type I primers were sense 5'-AGCACTTCCTAATGTCCAACCCT and antisense 5'-CTAAGAAGTGCATCTCCTCCCAC, which correspond to bases 2651–2952 of the bovine type I adenylyl cyclase sequence [18]. Type VII primers were sense 5'-CCAGTTATTTAGAGAGAGACCTG and antisense 5'-CTTGCTCATCAGGGCCATGCTAA, which correspond to bases 3098–3657 of the murine type VII adenylyl cyclase sequence [19]. Type VIII primers were sense 5'-GGACAGCAGCTGGAGTACACAGC and antisense 5'-CCTGATCCTTCAGGATGAGATAG, which correspond to bases 3513–4196 of the rat type VIII adenylyl cyclase sequence [20]. Type IX primers were sense 5'-AGCTTATCCTCACCTTCTTCTTCCTC and antisense 5'-AGGACACGGTAGCACTCCTTGCC, which correspond to bases 3015–3327 of the murine type IX adenylyl cyclase sequence [21].

PCR reactions (25 µl) using 1 µl of cDNA were assembled with the following (final concentrations) per reaction: 20 mM Tris, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 0.1 mM each dNTP, 1 µM each primer, and 1.25 U Taq polymerase. PCR was carried out for 40 cycles as follows: 1) 94°C for 4 min, 2) 94°C for 1 min, 3) annealing temperature for 1 min, 4) 72°C for 2 min, 5) 39 cycles to step 2, 6) 72°C for 10 min. Annealing temperatures were 50°C for type I, 55°C for types II, VII, and VIII, and 65°C for types III, IV, V, VI, and IX. Two controls were included with each PCR experiment. The "-RT controls" were RNA sample tubes that lacked reverse transcriptase during the RT reaction to ensure that no PCR products were generated from contaminating genomic DNA despite DNase digestion. Secondly, PCR tubes were assembled that included all components except input cDNA to demonstrate the lack of nonspecific contamination during PCR. PCR products were analyzed by electrophoresis through 7% polyacrylamide gels with DNA molecular weight markers and were visualized by ethidium bromide staining with UV transillumination. To confirm that PCR products represented the cDNA encoding specific adenylyl cyclase isoforms, representative PCR products were gel purified on 2–3% Nusieve (FMC Bioproducts, Rockland, ME) agarose gels, excised, electroeluted, and commercially sequenced using the same primers employed for PCR.

Statistics

Adenylyl cyclase activity increases by isoproterenol and inositol phosphate formation by oxytocin were analyzed by two-tailed, paired t-test. Values are expressed as mean ± SE; p values of less than 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We found expression of eight of nine subtypes of adenylyl cyclase in freshly isolated rat myometrial cells. Isoforms II–IX were identified by appropriately sized PCR products after RT-PCR of rat total myometrial RNA (Figs. 1 and 2). These products were identified in at least three experiments using RNA from different rat uteri. In all cases, the controls lacking reverse transcriptase or lacking cDNA did not exhibit PCR products, confirming that PCR products were arising from newly synthesized cDNA and not residual genomic DNA or contaminating cDNA. We did not identify adenylyl cyclase type I mRNA in fresh rat myometrial cells despite obtaining an expected PCR product from rat brain RNA (Fig. 1).

View larger version (43K): [in this window] [in a new window]   FIG. 1. Representative RT-PCR results for DNase-treated total RNA from fresh rat uterine muscle or rat whole brain amplified with primers specific for adenylyl cyclase (AC) isoforms I–IV. Lane 1, X174RF DNA/Hae III molecular weight standards; lanes 2 and 3, total RNA from whole rat brain; lanes 4–11, total RNA from rat uterine muscle. Reverse transcriptase (R.T.) was present (+) (lanes 2, 4, 6, 8, and 10) or absent (-) (lanes 3, 5, 7, 9, and 11) during cDNA synthesis. PCR products for adenylyl cyclase type I were detected in rat brain (lane 2) but not rat uteri (lane 4). Adenylyl cyclase types II–IV were detected in rat uteri (lanes 6, 8, and 10), including two splice variants of type IV (lane 10). No PCR products were obtained when reverse transcriptase was omitted from the cDNA synthesis reaction (lanes 3, 5, 7, 9, and 11), indicating a lack of genomic DNA contamination after DNase I treatment.

View larger version (50K): [in this window] [in a new window]   FIG. 2. Representative RT-PCR results for DNase-treated total RNA from fresh rat uterine muscle amplified with primers specific for adenylyl cyclase (AC) isoforms V–IX. Lane 1, X174RF DNA/Hae III molecular weight standards; lanes 2–11, total RNA from rat uterine muscle. Adenylyl cyclase types V–IX were detected in rat uteri (lanes 2, 4, 6, 8, and 10). No PCR products were obtained when reverse transcriptase was omitted from the cDNA synthesis reaction (lanes 3, 5, 7, 9, and 11), indicating a lack of genomic DNA contamination after DNase I treatment.

To confirm that the PCR products obtained represented amplified adenylyl cyclase cDNA, products were gel purified, excised, and sequenced. For isoforms II, III, V, VI, and VIII, PCR products corresponded to the published sequences for these rat isoforms. For adenylyl cyclases VII and IX, the sequenced PCR products corresponded to the published sequences for mouse types VII and IX.

Two PCR products were obtained with type IV primers (Fig. 3) corresponding to the published sequence for rat type IV and a newly identified splice variant of type IV containing 24 nucleotides with two possible splice sites. It is possible that the splice site is either between nucleotides 2600 and 2601 or between nucleotides 2607 and 2608 of the published rat sequence [22]. These two splice insertion sites arise due to a seven-nucleotide repeat (TGGAGAA) that could be the first or last seven nucleotides of the insert (Fig. 4a). Either splice insertion site results in an identical predicted amino acid insertion of eight amino acids (LTRLLLEN) between amino acids 833 and 834 of the initially described sequence of type IV adenylyl cyclase [22]. Both splice variants of adenylyl cyclase type IV were coexpressed in uterine smooth muscle cells (Fig. 3). This region of the newly identified splice variant for type IV has a higher putative amino acid homology to types II and VII adenylyl cyclase than the previously published type IV sequence (Fig. 4b) [22].

View larger version (35K): [in this window] [in a new window]   FIG. 3. Representative RT-PCR results for DNase-treated total RNA from fresh rat uterine muscle amplified with primers specific for adenylyl cyclase type IV. Two PCR products were obtained in each experiment, representing a fragment of type IV adenylyl cyclase (426 base pairs [bp]) [22] and a newly identified splice variant (450 bp) putatively encoding eight additional amino acids within the C2a region of type IV adenylyl cyclase [22].

View larger version (15K): [in this window] [in a new window]   FIG. 4. a) Nucleotide and amino acid sequence of the larger adenylyl cyclase type IV splice variant within the C2a region. Removal of either of the 24 nucleotides indicated by lines above or below the nucleotide sequence yields the adenylyl cyclase type IV sequence previously published [22]. Either of these two splicing alternatives yields the same putative eight-amino acid addition (indicated by italics). Nucleotide 2591 and amino acid 828 refer to previously described adenylyl cyclase type IV sequence [22]. b) Putative amino acid alignment of the 5' end of the C2a region of related adenylyl cyclase types II, IV, and VII. Identical residues among sequences are indicated by -. Numbers in parentheses are references to previously published sequences. The newly identified adenylyl cyclase type IV splice variant contains eight additional amino acids that retain higher homology to adenylyl cyclase types II and VII than the previously published shorter version of adenylyl cyclase type IV [22].

These results obtained in fresh myometrial cells relied on total RNA extracted from freshly dissected uteri. Although this tissue consisted of primarily myometrial cells, it also contained multiple other cell types of vascular, stromal, and hematopoietic origin. Because of the high sensitivity of RT-PCR, it was possible that some or all of the PCR products obtained were amplified from RNA from non-myometrial cells. To confirm that these adenylyl cyclase isoforms were expressed in a pure population of myometrial cells, RT-PCR was performed using RNA from a cell line derived from rat myometrial cells. These cells, which were transformed by the E7 opening reading frame of human papilloma virus type 16, exhibited morphologic and phenotypic similarities to the myometrial cell of origin. At least three experiments were performed on three separate clones of these cultured myometrial cells for each adenylyl cyclase isoform. The results were identical to those obtained in fresh myometrial cells in which adenylyl cyclase types II–IX were expressed and no mRNA encoding adenylyl cyclase type I was identified (Figs. 5 and 6). No PCR products were obtained in control samples lacking input reverse transcriptase or lacking input cDNA.

View larger version (51K): [in this window] [in a new window]   FIG. 5. Representative RT-PCR results for DNase-treated total RNA from cultured rat myometrial cells (Cx Uterus) or rat whole brain amplified with primers specific for adenylyl cyclase (AC) isoforms I–IV. Lane 1, X174RF DNA/Hae III molecular weight standards; lanes 2 and 3, total RNA from whole rat brain; lanes 4–11, total RNA from cultured rat myometrial cells. PCR products for adenylyl cyclase type I were detected in rat brain (lane 2) but not in cultured myometrial cells (lane 4). Adenylyl cyclase types II–IV were detected in rat uteri (lanes 6, 8, and 10), including two splice variants of type IV (lane 10). No PCR products were obtained when reverse transcriptase was omitted from the cDNA synthesis reaction (lanes 3, 5, 7, 9, and 11), indicating a lack of genomic DNA contamination after DNase I treatment.

View larger version (38K): [in this window] [in a new window]   FIG. 6. Representative RT-PCR results for DNase-treated total RNA from cultured rat myometrial (Cx Uterus) cells amplified with primers specific for adenylyl cyclase types V–IX. Lane 1, X174RF DNA/Hae III molecular weight standards; lanes 2–11, total RNA from cultured rat myometrial cells. Adenylyl cyclase types V–IX were detected in cultured rat myometrial cells (lanes 2, 4, 6, 8, and 10). No PCR products were obtained when reverse transcriptase was omitted from the cDNA synthesis reaction (lanes 3, 5, 7, 9, and 11), indicating a lack of genomic DNA contamination after DNase I treatment.

To confirm that the transformed myometrial cells exhibited phenotypic similarity to the myometrial cell of origin, two smooth muscle-specific proteins were analyzed by immunoblotting, and the expression of cell surface receptors typical of myometrial cells was confirmed biochemically. We found an immunoreactive 42-kDa protein in cultured cell lysates using a primary antibody directed against a smooth muscle-specific -actin, and an immunoreactive protein of approximately 52 kDa using a primary antibody directed against the protein desmin (Fig. 7). Adenylyl cyclase activity increased in response to the ß-adrenergic agonist isoproterenol. One hundred micromolar isoproterenol increased activity to 137 ± 4.5% above basal (GTP alone) levels (n = 4, p = 0.017). One micromolar oxytocin increased inositol phosphate formation to 173 ± 13.5% of control levels (n = 5, p = 0.01).

View larger version (56K): [in this window] [in a new window]   FIG. 7. Representative immunoblot analysis of cultured transformed rat myometrial cells. Mouse monoclonal antibodies directed against smooth muscle-specific -actin (left lane) and desmin (right lane) identified immunoreactive proteins of predicted molecular mass (42 and 52 kDa, respectively), indicating that these cells express proteins typical of cells of smooth muscle lineage.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Results of this study show that late-gestation rat myometrium expresses mRNA for multiple isoforms of adenylyl cyclase. Messenger RNA for each of the isoforms (II–IX) was expressed both in fresh uterine muscle and in cultured, immortalized rat myometrial cells, while mRNA for adenylyl cyclase isoform I was not identified.

The mRNA for adenylyl cyclase isoforms II–IX identified in fresh late-gestation rat uteri was likely to have originated from muscle cells rather than from neural, vascular, or stromal cells, since we identified an identical pattern of expression in a homogeneous population of cultured myometrial cells derived from the same source. Because of this similarity in the pattern of mRNA expression and because the cultured cell line is composed of only smooth muscle cells, it is likely that smooth muscle cells were the source of the mRNA identified in the fresh uterus. Furthermore, the presence of an identical pattern of expression of the adenylyl cyclase isoforms in the cultured cell line supports the use of these cells for further investigation of hormonal regulation of adenylyl cyclase isoform expression.

The pattern of expression of mRNA for all adenylyl cyclase isoforms in myometrium has not been previously described. One study [1], using the relatively insensitive technique of Northern blotting, identified mRNA for adenylyl cyclase isoforms II and IV in uteri from late-gestation rats. Messenger RNA for the other isoforms of adenylyl cyclase were not identified, nor was the cell of origin of mRNA for isoforms II and IV. Results of the present study, using the more sensitive technique of RT-PCR, show that myometrium expresses mRNA for adenylyl cyclase isoforms II–IX. We did not identify mRNA for adenylyl cyclase isoform I despite obtaining PCR products from the rat brain using primers based on bovine sequence. This suggests that these adenylyl cyclase type I primers, which were designed on the basis of bovine sequence, do anneal to rat adenylyl cyclase cDNA. The lack of a PCR product with the use of identical primers in rat myometrial cells suggests that this isoform is not expressed in myometrial cells.

While the expression of adenylyl cyclase type I is generally limited to neural tissues [5, 6], the mRNA encoding several adenylyl cyclase isoforms is widely expressed in peripheral tissues. The ability to detect the mRNA expression of a given isoform is dependent upon the sensitivity of the technique used. While RT-PCR may detect the expression of a particular isoform, it is unknown whether or not this very sensitive technique may detect the expression of low-abundance mRNAs that do not result in the synthesis of a biologically significant amount of protein. Adenylyl cyclases type IV [22], V, VI [23], and VII [19] showed wide tissue expression as detected by RT-PCR. Adenylyl cyclases type V [24] and IX [25] were detected by RNase protection in a wide variety of peripheral tissues, while Northern blotting showed expression of types VI [26] and VII [19] in many peripheral tissues. Adenylyl cyclase types II–VI were detected in endothelial cells from a wide variety of peripheral tissues [17]. In the current study, the very sensitive technique of RT-PCR demonstrated expression of multiple adenylyl cyclase isoforms in uterine smooth muscle cells, consistent with the demonstration here of a generally widespread expression of adenylyl cyclase isoforms with the exception of type I. This suggests that the regulation of cellular levels of cAMP in many cells is the result of the integrated regulation of multiple isoforms of adenylyl cyclase by inputs (e.g., Ca²⁺/calmodulin, ß subunits, PKC) from multiple other signaling pathways.

In the current study we detected the expression of a splice variant of adenylyl cyclase type IV in both fresh and cultured rat uterine muscle cells. A splice variant for this isoform has not been previously reported, although splice variants for adenylyl cyclase type V [27] and functionally different splice variants for type VIII [7] have been described. In the current study we did not attempt to evaluate the potential functional significance of the type IV splice variant that encodes an additional eight amino acids in the 5' end of the C2a region of the protein (Fig. 4b) [5, 22]. A large portion of the C2a region is highly conserved among all adenylyl cyclases [5], and even greater homology of this region is seen among the adenylyl cyclase subfamily that includes types II, IV, and VII. The splice variant of type IV identified in the present study occurs in the 5' end of the C2a region, which is not highly homologous among all adenylyl cyclases but which is highly conserved within the type II, IV, and VII subfamily. The eight-amino acid addition in this newly identified splice variant of type IV results in even greater homology of this new splice variant with types II and VII than does the previously published type IV sequence (Fig. 4b) [22].

The rat uterus is composed of two separate smooth muscle layers: an outer longitudinal and an inner circular layer. Responses to a variety of pharmacological agents differ between the two layers. The longitudinal layer demonstrates greater sensitivity to isoproterenol [1], which results, in part, from a threefold [28] greater maximum increase in isoproterenol-stimulated adenylyl cyclase activity. Because of the greater importance of the ß-adrenergic/cAMP relaxation pathway in the longitudinal muscle layer, and because of the very low basal, GTP-, or isoproterenol-stimulated adenylyl cyclase activity in the inner circular layer [28], we limited our investigation of adenylyl cyclase isoforms to the longitudinal muscle layer.

A potential mechanism for postreceptor changes in adenylyl cyclase activity during pregnancy and parturition is an alteration in expression of adenylyl cyclase isoforms. Because regulation of activity of these isoforms differs, an increase or decrease in the expression of a single isoform could result in a global alteration of adenylyl cyclase activity. For example, ß subunits liberated from heterotrimeric G proteins inhibit adenylyl cyclase type I, stimulate type II, and are without effects on type III [9]. If expression of adenylyl cyclase isoform II, but not type III, decreased at the end of pregnancy, and if adenylyl cyclase isoform II is a predominant isoform in myometrium, then activation of G proteins and release of ß subunits would produce less stimulation of total adenylyl cyclase, leading to decreased cAMP production, decreased uterine relaxation, loss of uterine quiescence, and initiation of labor.

In summary, we found that fresh uterus and cultured, immortalized myometrial cells from late-gestation rats express an identical pattern of mRNA for adenylyl cyclase isoforms. The presence of these multiple isoforms of adenylyl cyclase may provide myometrial cells with flexibility to alter uterine contractility in response to external influences.

	FOOTNOTES

 
¹ Correspondence: Charles W. Emala, Johns Hopkins School of Public Health and Hygiene, Dept. of Environmental Health Sciences, Div. of Physiology, 615 N. Wolfe St., Room 7006, Baltimore, MD 21205. FAX: (410) 955–0299; cemala{at}welchlink.welch.jhu.edu

Accepted: March 2, 1998.

Received: September 22, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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