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 findings suggest that NO is an autocrine regulator of ovarian steroidogenesis. The purpose of this investigation was to explore the mechanisms through which NO exerts an inhibitory effect on cytochrome P450 aromatase activity. To examine the effect of NO on aromatase mRNA levels, human granulosa-luteal cells were cultured in the presence or absence of the NO donor SNAP for 16 h. Using a probe for human aromatase, Northern blots revealed a 26% decrease in aromatase mRNA in cells exposed to SNAP. Because this modest decrease in mRNA is unlikely to explain a rapid and profound reduction in estradiol secretion that we have observed, we looked for direct effects of NO on cytochrome P450 aromatase activity. Aromatase activity was assayed in placental microsomes and granulosa-luteal cells by measuring the release of 3H2O from [1 beta-3H] androstenedione. NO (10(-4)-10(-3)M), added as a saturated saline solution, reduced aromatase activity by as much as 90% in a concentration-dependent, non-competitive manner. In contrast, carbon monoxide (CO), 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-induced inhibition of aromatase. These data suggest that NO binding to the heme is insufficient to inhibit aromatase activity. NO has been reported to alter protein function by reacting with the sulfhydryl group of cysteines, forming a nitrosothiol group. Because a cysteine sulfhydryl group is thought to participate in the catalytic mechanism of all P450 enzymes, experiments were designed to test whether NO might inhibit aromatase via such a mechanism. Addition of increasing amounts of mercaptoethanol, a chemical with free sulfhydryl groups, blocked the NO-induced inhibition of aromatase in microsomes. N-Ethylmaleimide, a chemical which covalently modifies sulfhydryl groups, reduced aromatase activity in a concentration-dependent manner. We conclude that NO inhibits aromatase both by decreasing mRNA for the enzyme and by an acute, direct inhibition of enzyme activity. We hypothesize that the direct inhibition occurs as a result of the formation of a nitrosothiol on the cysteine residue adjacent to the heme in aromatase.
Snyder
G D
GD
Department of Obstetrics and Gynecology, University of Iowa College of Medicine, Iowa City 52242, USA. gsnyder@obgyn-po.weeg.uiowa-edu
Holmes
R W
RW
Bates
J N
JN
Van Voorhis
B J
BJ
D000735:Androstenedione / Q000494:pharmacology; D047072:Aromatase Inhibitors; D002248:Carbon Monoxide / Q000494:pharmacology; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006107:Granulosa Cells / Q000201:enzymology; D006801:Humans; D008184:Luteal Cells / Q000201:enzymology; D008623:Mercaptoethanol / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis; D026423:S-Nitroso-N-Acetylpenicillamine; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D047072:Aromatase Inhibitors; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D013439:Sulfhydryl Reagents; D009569:Nitric Oxide; D000735:Androstenedione; D009249:NADP; D008623:Mercaptoethanol; D026423:S-Nitroso-N-Acetylpenicillamine; D002248:Carbon Monoxide; D010396:Penicillamine; D003545:Cysteine; D005033:Ethylmaleimide