Faculty Bibliography

Cytochalasin B (CB), a drug that inhibits microfilament polymerization, as well as having other actions, such as inhibition of hexose transport, is reported to block tropic hormone-stimulated steroidogenesis. Cytochalasin D (CD) is a more effective inhibitor of microfilament polymerization than CB, but has no effect on hexose transport. We reevaluated the effects of these inhibitors of microfilament function on steroid formation by freshly isolated granulosa cells from PMSG-primed hamsters. These cells have been shown to produce increased progesterone in response to tropic stimuli using endogenous steroidogenic precursors during short term incubation. In addition, they use exogenous substrate in the form of hydroxysterols for steroid production. Thus, we could examine effects of CB and CD on the metabolism of exogenous and endogenous sterol substrates. To determine the specificity of actions of cytochalasins, the metabolism of other steroid intermediates was examined: the conversion of pregnenolone to progesterone and the conversion of androstenedione to estradiol. Also, the oxidation of [14C]octanoate to 14CO2, a process that occurs in the mitochondria, was examined. Both CB and CD (1-10 micrograms/ml) inhibited LH- and 8-bromo-cAMP-stimulated progesterone production. Neither cytochalasin inhibited the production of progesterone in response to pregnenolone, nor were endogenous pregnenolone levels increased in the presence of CB or CD. Treatment with CB, but not CD, resulted in decreased progesterone production in response to hydroxycholesterol and decreased the side-chain cleavage of [3H]25-hydroxycholesterol. CB completely blocked the conversion of androstenedione to estradiol, whereas the aromatase reaction was unaffected by CD. CB, but not CD, significantly reduced the oxidation of [14C]octanoate to 14CO2 by hamster granulosa cells. Our findings demonstrate significant differences in the effects of CB and CD on granulosa cells. CB, in contrast to CD, has wide-spread effects on granulosa cell function, interfering with the metabolism of hydroxycholesterols, the oxidation of fatty acids, and aromatization. The failure of CD to interfere with these same functions suggests a more specific action of CD on the steroidogenic machinery. We conclude that microfilaments play an important role in the process of steroid formation from endogenous substrate. CD appears to affect a post-cAMP step between the conversion of cholesterol to pregnenolone, presumably the delivery of endogenous cholesterol to the mitochondria

Using isotope-ratio mass spectrometry, newly synthesized 26-hydroxycholesterol and its derivative, 3 beta-hydroxy-5-cholenoic acid, were detected in the liver and bile of animals within 2 h following intravenous administration of [5-13C]mevalonic acid. The findings indicate that 26-hydroxycholesterol merits further consideration as an in vivo modulator of HMG-CoA reductase activity