ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 335, No. 2, November 15, pp. 369–376, 1996 Article No. 0518 Nitric Oxide: A Prostaglandin H Synthase 1 and 2 Reducing Cosubstrate That Does Not Stimulate Cyclooxygenase Activity or Prostaglandin H Synthase Expression in Murine Macrophages John F. Curtis,* Nagi G. Reddy,* Ronald P. Mason,* B. Kalyanaraman,† and Thomas E. Eling* ,1 *National Institutes of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709; and †Medical College of Wisconsin, P.O. Box 26509, Milwaukee, Wisconsin 53226-0509 Received April 5, 1996, and in revised form August 26, 1996 biochemical pathways. • NO has been shown to func- Several recent reports have investigated the possi- tion as a vasodilator, a neurotransmitter, an inhibi- bility that nitric oxide ( • NO) can regulate prostaglan- tor of platelet aggregation, and an antimicrobial din H synthase (PHS) activity. The potential signifi- agent generated by macrophages, to name just a few cance of • NO regulation of PHS is considerable, when of its biological roles (1). One important biological one considers the numerous important biological pro- function of • NO is to increase the activity of guanyl- cesses that are influenced by PHS. In this study, we ate cyclase (2). This amplification of activity is used microsomal and purified PHS to investigate the thought to occur via the reaction of • NO with the direct effect of • NO and • NO-generating compounds naturally occurring ferrous form of the hemoprotein on PHS catalytic activity. We found that • NO neither to form a • NO – heme complex, which, in turn, induces significantly inhibited nor enhanced prostaglandin a conformational change in the protein (3, 4). (PG) formation, despite the fact that • NO stimulated Possible interactions of • NO with other hemoproteins PHS peroxidase activity. We also investigated the ef- is an area currently under investigation. One enzyme fect of • NO and • NO generators on PHS product, pro- that is being investigated for potential interactions tein, and mRNA levels in the RAW264.7 murine macro- with • NO is prostaglandin H synthase (PHS), a protein phage cell line. We found that • NO or • NO generators which has both cyclooxygenase and peroxidase activity. had little or no effect on PG formation, PHS expres- PHS exists in both a constitutive (PHS-1) and inducible sion, or PHS mRNA expression in unstimulated form (PHS-2) (5, 6) and is responsible for the generation RAW264.7 cells. The same results were obtained with of PGH 2 , which is the precursor for both the prosta- macrophages that were stimulated by 18 h pretreat- glandin (PG) and thromboxane pathways (7). These ment with lipopolysaccharide, a known inducer of PGH 2 -derived compounds serve many important func- PHS-2 in macrophages. These data clearly indicate tions, a number of which are similar to the activities that • NO acts as a cosubstrate for PHS peroxidase. associated with • NO, including regulation of vasodila- However, • NO does not enhance or inhibit either cycloxygenase activity or expression of PHS in the tion/vasoconstriction and regulation of platelet aggre- model systems used in this study.  1996 Academic Press, Inc. gation (8, 9). Several conflicting reports have recently been pub- lished on the effect of • NO on PHS and/or the effect of In recent years, nitric oxide ( • NO) 2 has been un- • NO on PG formation in biological systems. Kanner et veiled as a potent agent in a number of important al. reported • NO to be a strong inhibitor of PHS (10). Salvemini et al. (11) and Hajjar et al. (12) showed that 1 To whom correspondence should be addressed. Fax: (919) 541- • NO directly stimulates the activity of purified PHS, 1460. 2 Abbreviations used: • NO, nitric oxide; PHS, prostaglandin H syn- thase; DETA/NO, diethylenetriamine – nitric oxide adduct; DEA/NO, tenyl-hydroperoxide; PPA, 5-phenyl-4-pentenyl-alcohol; AA, arachi- donic acid; SPE, solid-phase extraction; PBS, phosphate-buffered sa- diethylamine – nitric oxide complex; SPER/NO, spermine – nitric ox- ide complex; SNP, sodium nitroprusside; LPS, lipopolysaccharides; line; HBK, Hepes-buffered Kreb’s; 12-HHTrE, 12-hydroxyheptadeca- dienoic acid; PG, prostaglandin. RSVM, ram seminal vesicle microsomes; PPHP, 5-phenyl-4-pen- 369 0003-9861/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.