Correlation between Connexin43 Expression, Cell-Cell Communication, and Oxytocin-lnduced Ca2+ Responses in an Immortalized Human Myometrial Cell Line1

Abstract

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 microM 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.

Oxytocin-Induced Ca2+ Responses in Human Myometrial Cells1

“…Significant direct effects of 8-bromo-cAMP on stable oxytocin-induced Ca 2ϩ oscillations in PHM1-41 cells were not detected, although the design of the experiment does not eliminate permeability issues related to the cAMP analogue. However, PHM1-41 cells treated for 12 h with 500 M 8-bromo-cAMP induced suppression of the initial Ca 2ϩ signaling events [14] as well as long-term oscillations. The suppression of Ca 2ϩ responses by cAMP in PHM1-41 may involve a number of mechanisms.…”

“…The suppression of Ca 2ϩ responses by cAMP in PHM1-41 may involve a number of mechanisms. Uterine relaxants and cAMP have been shown to reduce free Ca 2ϩ concentration [14] and phosphatidylinositide turnover in PHM1-41 cells [13] via inhibition of G␣ q / phospholipase C by protein kinase A [17]. These agents also affect intracellular Ca 2ϩ concentrations by stimulating Ca 2ϩ -activated K ϩ channels in PHM1-41 cells via protein kinase A [16].…”

“…Cells were cultured in DME-F12 plus 10% fetal calf serum and used between passages 15 and 23. A number of properties of the cell line have been described [13,14,16,17].…”

“…Human myometrial tissue from premenopausal women was obtained from surplus pieces of surgical specimens after hysterectomies. The primary human myometrial cells, like PHM1-41 cells, are communication competent and exhibit comparable properties including connexin43 gap junctions and estrogen, progesterone, and oxytocin receptors [13,14,[18][19][20]. Myometrial tissue was processed as described by Andersen et al [18].…”

“…Recently, a human myometrial cell line was developed that retains a number of useful morphological, biochemical, and functional properties of human myometrial cells, including communication competence and the presence of oxytocin, relaxin, and estrogen receptors [13,14]. In the present study, we have examined the utility of this cell line to study Ca 2ϩ oscillations in human myometrial cells over an extended period of time (90 min).…”