Correlation between connexin43 expression, cell-cell communication, and oxytocin-induced Ca2+ responses in an immortalized human myometrial cell line

The effects of 8-bromo-cAMP on gap junction expression and intracellular Ca2+ ([Ca2+](i)) were examined in an immortalized human myometrial cell line (PHM1-41) generated from myometrial cells obtained from term-pregnant myometrium. PHM1-41 cells express the gap junction protein connexin43 (Cx43). Gap junction-mediated intercellular communication, monitored with a method using fluorescence recovery after photobleaching, revealed low communication rates between cells. Addition of 1.0 mM 8-bromo-cAMP to culture medium resulted in an increase in junctional communication within 5 min, while 30 mu M forskolin treatment resulted in increased communication within 10 min. Cells treated with 0.1, 0.5, or 1.0 mM 8-bromo-cAMP for 24 h revealed a dose-dependent increase in communication and an increase in immunostained gap junction plaques at cell-cell contacts. A direct correlation between the rate of dye transfer and the number of plaques was detected. No effect on communication or immunoreactive Cx43 was detected in cells exposed to 10 or 50 nM estradiol-17 beta for 24 h. In experiments analyzing [Ca2+](i), a concentration-dependent increase was induced by oxytocin (1-100 nM) with half-maximal stimulation at approximately 5 nM. Treatment of PHM1-41 cells for 24 h with 0.1, 0.5, or 1.0 mM 8-bromo-cAMP reduced the magnitude of the oxytocin-induced [Ca2+](i) response. Thus, 8-bromo-cAMP increased Cx43-containing cell surface gap junctional plaques and up-regulated junctional communication but attenuated the oxytocin-induced [Ca2+](i) response. These data indicate that the action of 8-bromo-cAMP in PHM1-41 cells has potentially opposite effects on the contractile and conductive properties of cells, and suggest that up-regulation of myometrial conductivity by agents that increase cAMP could diminish the tocolytic benefits of these agents.