It has been proposed that the antimitogenic action of progesterone (P4) is mediated through a membrane receptor that has GABAA receptor-like characteristics. To test this hypothesis, studies were designed to compare the antimitogenic effects of P4 with its gamma amino butyric acidA (GABAA) receptor-activating metabolite, 5α-pregnane-3α–21-diol-20-one (5α3α). These studies revealed that P4 was more effective than 5α3α in blocking mitogen-dependent mitosis of both small granulosa cells (GCs) and spontaneously immortalized granulosa cells (SIGCs). Ligand binding studies illustrated that P4 bound to SIGCs with an apparent dissociation constant (Kd) of 0.32 ± 0.09 μM, whereas 5α3α bound with an apparent Kd of 40 ± 19 μM. Further, the GABAA antagonist, bicuculline, did not attenuate P4's antimitotic action in SIGCs. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that none of the 6 known α chains of the GABAA receptors to which bicuculline binds were detected in SIGCs. Taken together, these studies suggest that P4 does not mediate its action via a GABAA-like receptor. Additional studies revealed that P4 regulated intracellular free calcium levels ([Ca2 ]i) as part of its antimitotic action. Specifically, P4 maintained a basal [Ca2 ]i level that was slightly lower than normal. Increasing extracellular calcium not only increased basal [Ca2 ]i but also attenuated P4's antimitogenic effect. P4's actions appeared to be initiated at the membrane, since horseradish peroxidase conjugated-P4 (HP-P4), which is cell impermeable, was as effective in blocking mitosis as P4. 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 P4's membrane-initiated actions.
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