Phospholipase C-beta and ovarian sex steroids in pig granulosa cells

We compared the membrane effects of estradiol, progesterone, and androstene dione in a single experimental model, the ovarian granulosa cells collected from immature Large While sows. We measured changes in cytosolic free calc ium concentration ([Ca2+]i) in confluent Fura-2 loaded cells. We used pharm acological tools and polyclonal phospholipase C-beta (PLC-beta) antibodies. Each steroid (0.1 pM to 1 nM) transiently increased intracellular calcium concentration ([Ca2+]i) within 5 sec. They mobilized Ca2+ from the endoplas mic reticulum as shown by using two phospholipase C inhibitors, neomycin an d U-73122. Ca2+ mobilization involved PLC-beta 1 for progesterone, PLC-beta 2 for estradiol and PLC-beta 4 for androstenedione. A pertussis toxin-inse nsitive C protein was involved in the effects of progesterone on Ca2+ mobil ization whereas estradiol and androstenedione effects were mediated via a p ertussis toxin-sensitive G-protein. Ca2+ influx from the extracellular mili eu was involved in the increase in [Ca2+]i induced by progesterone and estr adiol, but not by androstenedione. Influx of Ca2+ was independent of Ca2+ m obilization from calcium stores, and it was suggested that L-type Ca2+ chan nels for estradiol and T-type Ca2+ channels for progesterone were involved. The three steroids had no effect on cAMP. Rapid effects of progesterone, e stradiol, and androstenedione involved a direct action on cell membrane ele ments such as PLC-beta, G-proteins, and calcium channels, and these mechani sms were hormone-specific. J Cell. Biochem. 74.50-60, 1999. (C) 1999 Wiley- Liss, Inc.