Membrane-Initiated Events Account for Progesterone's Ability to Regulate Intracellular Free Calcium Levels and Inhibit Rat Granulosa Cell Mitosis1

It has been proposed that the antimitogenic action of progesterone (P(4)) is mediated through a membrane receptor that has GABA(A) receptor-like characteristics. To test this hypothesis, studies were designed to compare the antimitogenic effects of P(4) with its gamma amino butyric acid(A) (GABA(A)) receptor-activating metabolite, 5alpha-pregnane-3alpha-21-diol-20-one (5alpha3alpha). These studies revealed that P(4) was more effective than 5alpha3alpha in blocking mitogen-dependent mitosis of both small granulosa cells (GCs) and spontaneously immortalized granulosa cells (SIGCs). Ligand binding studies illustrated that P(4) bound to SIGCs with an apparent dissociation constant (K(d)) of 0.32 +/- 0.09 microM, whereas 5alpha3alpha bound with an apparent K(d) of 40 +/- 19 microM. Further, the GABA(A) antagonist, bicuculline, did not attenuate P(4)'s antimitotic action in SIGCs. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that none of the 6 known alpha chains of the GABA(A) receptors to which bicuculline binds were detected in SIGCs. Taken together, these studies suggest that P(4) does not mediate its action via a GABA(A)-like receptor. Additional studies revealed that P(4) regulated intracellular free calcium levels ([Ca(2+)](i)) as part of its antimitotic action. Specifically, P(4) maintained a basal [Ca(2+)](i) level that was slightly lower than normal. Increasing extracellular calcium not only increased basal [Ca(2+)](i) but also attenuated P(4)'s antimitogenic effect. P(4)'s actions appeared to be initiated at the membrane, since horseradish peroxidase conjugated-P(4) (HP-P(4)), which is cell impermeable, was as effective in blocking mitosis as P(4). Progesterone receptor (PR) mRNA was not detected in SIGCs by RT-PCR analysis, which is consistent with the findings in GCs. However, a 60-kDa protein was detected within crude membrane fractions of both GCs and SIGCs using an antibody directed against the ligand binding domain of the PR (C-262). This antibody was also used in immunocytochemical studies to detect a protein that was associated with the plasma membrane of SIGCs. It is proposed that this 60-kDa protein mediates P(4)'s membrane-initiated actions.