NITRIC-OXIDE INHIBITS AROMATASE-ACTIVITY - MECHANISMS OF ACTION

NO synthase is present in human ovarian granulosa-luteal cells and NO inhibits estradiol secretion by granulosa cells in culture. These find ings suggest that NO is an autocrine regulator of ovarian steroidogene sis. The purpose of this investigation was to explore the mechanisms t hrough which NO exerts an inhibitory effect on cytochrome P450 aromata se activity. To examine the effect of NO on aromatase mRNA levels, hum an granulosa-luteal cells were cultured in the presence or absence of the NO donor SNAP for 16 h. Using a probe for human aromatase, Norther n blots revealed a 26% decrease in aromatase mRNA in cells exposed to SNAP. Because this modest decrease in mRNA is unlikely to explain a ra pid and profound reduction in estradiol secretion that we have observe d, we looked for direct effects of NO on cytochrome P450 aromatase act ivity. Aromatase activity was assayed in placental microsomes and gran ulosa-luteal cells by measuring the release of (H2O)-H-3 from [1 beta- H-3] androstenedione. NO (10(-4)-10(-3)M), added as a saturated saline solution, reduced aromatase activity by as much as 90% in a concentra tion-dependent, non-competitive manner. In contrast, carbon monoxide ( GO), a gas known to bind to the heme iron in aromatase, had no effect on aromatase activity when added alone nor could CO reverse the NO-ind uced inhibition of aromatase. These data suggest that NO binding to th e heme is insufficient to inhibit aromatase activity. NO has been repo rted to alter protein function by reacting with the sulfhydryl group o f cysteines, forming a nitrosothiol group. Because a cysteine sulfhydr yl group is thought to participate in the catalytic mechanism of all P 450 enzymes, experiments were designed to test whether NO might inhibi t aromatase via such a mechanism. Addition of increasing amounts of me rcaptoethanol, a chemical with free sulfhydryl groups, blocked the NO- induced inhibition of aromatase in microsomes. N-Ethylmaleimide, a che mical which covalently modifies sulfhydryl groups, reduced aromatase a ctivity in a concentration-dependent manner. We conclude that NO inhib its aromatase both by decreasing mRNA for the enzyme and by an acute, direct inhibition of enzyme activity. We hypothesize that the direct i nhibition occurs as a result of the formation of a nitrosothiol on the cysteine residue adjacent to the heme in aromatase. Copyright (C) 199 6 Elsevier Science Ltd.