Mechanisms subserving calcium’s modulation of luteinizing hormone action in isolated swine granulosa cells

We studied the mechanism(s) by which calcium ions modulate progesterone biosynthesis by isolated swine granulosa cells incubated in chemically defined medium in vitro. In selectively calcium-deficient incubations, the capacity of 8- bromo-cAMP to stimulate pregnenolone synthesis from endogenous sterol substrate was significantly impeded. This effect of calcium ions was specific, because calcium ions did not influence basal pregnenolone production or alter progesterone production in response to exogenously supplied cholesterol substrate. Moreover, calcium ions did not modify other biosynthetic processes in granulosa cells, such as de novo synthesis of cholesterol from [¹⁴C] acetate or the aromatization of testosterone to 17β-estradiol. The possible role of calmodulin in mediating calcium’s actions in pig granulosa cells was tested by measuring the calmodulin content of these cells and assessing the functional responses to classical calmodulin antagonists. By immunoassay, swine granulosa cells contained high concentrations of calmodulin, viz. 4.21-4.88 µg calmodulin/mg protein. Moreover, calmodulin antagonists inhibited LH-stimulated progesterone production with the following rank order of potencies [estimated by half-maximally inhibitory concentrations (ID₅₀)]: penfluridol (1 µM), trifluoroperazine (9 µM), chlorpromazine (95 µM), and trifluoperazine sulfoxide (>300 µM). In addition, the nonphenothiazine calmodulin antagonist W7 inhibited stimulated progesterone production with an ID₅₀ of 16.7 µM. W5 was less active. None of these antagonists significantly suppressed LH-stimulated cAMP generation at the low concentrations capable of inhibiting progesterone production. The effects of calcium ions seemed to depend upon the availability of intracellular pools of calcium, because TMB-8, an inhibitor of intracellular calcium mobilization, effectively suppressed LH-stimulated progesterone production (ID₅₀, 18 µM). However, even 100 fiM TMB-8 failed to alter basal progesterone production or suppress LH-stimulated cAMP generation in these cells. In summary, the present studies indicate that calcium ions significantly modulate LH’s stimulation of pregnenolone biosynthesis from endogenous cholesterol substrate in swine ovarian cells. Calcium does not influence basal pregnenolone production, estrogen synthesis from androgen substrate, de novo biosynthesis of cholesterol from [¹⁴C]acetate, or progesterone production from exogenously supplied sterol substrate. Further studies indicate that these actions of calcium may be mediated in part via calmodulin, since: 1) pig granulosa cells contain high concentrations of calmodulin, and 2) both phenothiazine and naphthalenesulfonamide antagonists of camodulin significantly impair the maximal stimulatory effects of LH on progesterone production. These antagonists also impede the stimulatory actions of exogenously supplied cAMP, but do not suppress LH’s ability to augment intracellular cAMP generation. Thus, we suggest that calcium ions modulate LH action in ovarian cells in part by calcium-calmodulin-dependent mechanisms. Such mechanisms seem to regulate steroidogenesis at a site(s) distal to LHstimulated cAMP production, probably at the level of delivery or utilization of endogenous sterol substrate in the side-chain cleavage reaction.