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 ad-enylyl 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. ventolin inhaler
To confirm that the transformed myometrial cells exhibited phenotypic similarity to the myometrial cell of origin, two smooth muscle-specific proteins were analyzed by im-munoblotting, 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 a-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 p-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).
FIG. 5. Representative KI-PCK results for DNase-treated total KNAtrom cultured rat myometrial cells (Cx Uterus) or rat whole brain amplified with primers specific tor adenylyl cyclase (AC) isotorms I—IV. Lane 1, ФХ174КР DNA/Hae III molecular weight standards; lanes 2 and 3, total KNA from whole rat brain; lanes 4-11, total KNA from cultured rat myometrial cells. PCK products tor 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 ot type IV (lane 10). No PCK products were obtained when reverse transcriptase was omitted from the cDNA synthesis reaction (lanes 3, 5, 7, 9, and 11), indicating a lack ot genomic DNA contamination atter DNase I treatment.
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, фX174RF 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.
FIG. 7. Kepresentative immunoblot analysis ot cultured transformed rat myometrial cells. Mouse monoclonal antibodies directed against smooth muscle-specific a-actin (lett lane) and desmin (right lane) identified im-munoreactive proteins ot predicted molecular mass (42 and 52 kDa, respectively), indicating that these cells express proteins typical ot cells ot smooth muscle lineage.