JACC Vol. 24, No. 2 Aug"I 1994: 55 5-66 555 Nitric Oxide and NitrovasodHators : 541 I'laiities,I Differences and Menfi2l 111teY2CHODS TODD J. ANDERSON, MD,* LAN T . MEREDITH, MD, PuD, PETER GANZ, MD, FACC, ANDREW P. SELWYN, MD, FACC, ALAN C. YEUNG, PAD, FACC Bern.. , Manuchusetts Many similarities exist between the exogenous nitrates and nitric oxide. As such, important interactions might be expected endethelium-derived relaxing factor, which is nitric oalde or a between nitrates and endothelium-dependent processes that in- thiot derivative. Both act by way of guany)nte cyclaw, which volve nitric oxide. TIds review explores the mechanisms of action, increases intracellular concentrations of cyclic guanusine mono- biologic effects and potential interactions between nitrates and endo- phosphate, resulting in smooth muscle cell rdantion and anti- thelium-derives) relaxing factor . platelet effects. Thiols may be important in the (j Am Call Cardiol 1994,24.555-66) of exogenous nitrates and other intracellular pnwesses involving Since the first description of the therapeutic use of glyceryl choline stimulated muscarinic receptors on the endothelium, trinitrate appeared in 1879 (1), organic nitrates have re- leading to the release of a nonprostanoid relaxing factor, mained the cornerstone for the treatment of angina pectoris . which they named endothelium-derived relaxing factor . Over the past decade, emphasis has been placed on research A diffusible substance with a half-life of several seconds, to elucidate the subcellular mechanisms of these com- (6,7) endothelium-derived relaxing factor could be inhibited pounds . During the same period, key discoveries have by hence-containing proteins (8), and it was destroyed by shown that the vascular endothelium plays a pivotal role in oxygen free radicals (10). It also activated soluble guanylate the local control of vasomotor tone, primarily through the cyclase and increased the concentration of cyclic guanosine synthesis and release of endothelium-derived relaxing factor monophosphate (cyclic GIMP) (10). Collectively, these char- (2). 'This substance has now been identified as nitric oxide or acteristics led to the simultaneous observation by Ignarro et a related nitrosothiol whose actions parallel those of organic al . (4) and Furchgott and Khan (5) that endothelium-derived nitiovasodilators (3-5). This review discusses the nature of relaxing factor was strikingly similar to nitric oxide . nitric oxide and the mechanism of action of nitric oxide and The first proof that nitric oxide was at least one of the organic nitrates and explores the interactions that might relaxing factors resulted from studies by Paltrier et al . (11) . exist between them . Using a chemiluminescence assay, they demonstrated that cultured endothelial cells release nitric oxide when exposed Chemical Nature of100hytheliumn-Derived. to bradykinin . In this assay, nitric oxide is specifically Relaxing Factor detected by its reaction with ozone . Results of these studies provided strong evidence that endothelium-derived relaxing In 1980, Furchgott and Zawadzki (2) discovered that the factor is nitric oxide or a chemically related compound . aoety[choline-induced relaxation of rabbit aortic strips re- Recent findings suggest that endothelium-derived relaxing quired an intact endothelium. They concluded that acetyl- factor may also be similar to a labile nitroso compound (S-nitroso-L-cysteine) (3,12) . From the Cardiovascular Division, Brigham and Women's Hospital Boston, Massachusetts . This study was supported by a Clinical Fellowship of Mechanism of Action of Endothelium- The Alberta Heritage Foundation for Medical Research (Dr. Anderson), Alberta, Canada ; a National Heart Foundation of Australia Ralph Reader Derived Relaxing Factor and Overseas Research Fellowship (Dr . Meredith), Melbourne, Australia ; and Organic Nitrates Research Career Development Award I K04 HL02566 (Dr . Ganz), Clinician- Investigatnr Development Award I K08 HL 02787 (Dr . Yeung) and R01 Endothelium-derived relaxing factor . The mechanism of HL-38780-c5 (Dr . Selwyn) from the National Heart, Lung, and Blood insti- action of nitric oxide has been examined in considerable tute, National Institutes of Health, Bethesda, Maryland . Manuscript received September 20, 1993 ; revised manuscript received detail (10,13) . Palmer et al . (14) have demonstrated that January 29, 1994, accepted March 25, 1994. nitric oxide is synthesized from the guanidino nitrogen of the "Present and for comsplindente : Cardiovascular Divi- address address amino acid L-arginine . This conversion is controlled by an sion, Foothills Hospital . 1403 29th Street NW, Calgary, Alberta, Canada T2N-2T9. enzyme, nitric oxide synthase, which can be stimulated by a .00 01994 by the American College of Cardiology 0735-1097/94/$7 JACC Vol . 24, No. 2 556 ANDERSON ET AL. NITRIC OXIDE AND NITRATES August 1594 :555-66 number of physical and chemical stimuli and is inhibited by oxides) (Fig. 2). This distinguishes them from nitric oxide- several arginine analogs, including N °-monomethyl-L- containing compounds without an ester bond, such as nitro- arginine . Receptors on' he endothelial cell membrane mod- prusside. Important similarities and differences exist in the ulate this interaction in association with signal transduction biotransformation of these organic and inorganic nitrates, but it proteins (the G protein family) (15) . Serotonin receptors, for is generally accepted that they share a final cnnunon pathway example, interact with the pertussis-sensitive inhibitory pro- wish endothelium-derived relaxing factor . tein (G;) to modulate release of nitric oxide (16). After The mechanism by which nitroprusside releases nitric release from the endothelium, endothelium-derived relaxing oxide has recently been reported . It has been previously factor diffuses across the extracellular space into the smooth thought that nitric oxide release occurred spontaneously muscle cell and utilizes the guanylate cyclase second- (30). However, more recently Bates et al . (31) have reported messenger system to implement its biologic actions . Both that a one-electron reduction with accompanying cyanide nitric oxide and endothelium-derived relaxing factor have loss was required before nitric oxide could be released . been shown to activate soluble guanylate cyclase, leading to The biotramtsformation of organic nitrates is probably an increase in the intracellular concentration of cyclic GMP more complex . Although there is much controversy about (10,17). the role of thiols, they may be important at several levels in Cycic guanoalne 3'-,5'-momphosphate system: intraccellu- the nitrate/nitric oxide pathway including 1) the formation of lar d r. Cyclic GMP is formed from guanosine S-nitrosothiol intermediaries, and 2) the activation of soluble 5'-triphosphate by the activation of guanylate cyclase, of guanylate cyclase by nitric oxide . The formation of nitro- which there are at least three distinct types: 1) soluble sothiol intermediaries from organic nitrates seems to depend guanylate cyclase, which is activated by nitric oxide and on the denitrification of the R-0-nitric oxide, to a reactive organic nitrates ; 2) membrane-bound (particulate) guanylate thiol intermediary (32,33) . Using a rat model of tolerance, cyclase, which is induced by atrial natriuretic peptide ; and 3) Fung et al. (34) showed that the beneficial effect of the thiol cytoskeleton guanylate cyclase, which is sensitive to E. coil donor N-acetylcysteine on nitroglycerin might be mediated heat-stable enterotoxin (18) . Purified soluble guanylate cy- by extracellular S-nitrosothiol formation in addition to intra- clase has been shown to contain heme, which is now thought cellular thiol repletion . A recent study in rats, which used to be the receptor site for nitric oxide on soluble guanylate various thiol donors and depleting agents, demonstrated that cyclase (19-21) . Electron paramagnetic resonance studies both intracellular and extracellular thiols are important in have demonstrated that the binding of nitric oxide to heme modulating the effect of nitroglycerin in vivo (35) . Although results in a structural change of the hence molecule in which suggestive, it has not been shown conclusively that nitro- the iron atom protrudes out of the plane of the protoporphy- sothiols are intermediaries in the biotransfcrmation process . rin ring (22). Local thiol groups are necessary to reduce the Recent data by Chung and Fang (36) have demonstrated heme iron atom to the ferrous state, thus facilitating the that nitrates are enzymatically converted to yield nitric oxide nitric oxide-heme interaction . by a surface membrane enzyme system that is not identical The net result of the activation of soluble guanylate to that provided by glutathione-S-transferases (37). It has cyclase is the formation of cyclic GMP, which then activates been suggested by these investigators and others (38) that a serinelthreonine protein kinase (cG-Pk) (23) . Several pro- sulfhydryl.donating compounds may act instead as cofactors teins may be phosphorylated by cG-Pk, resulting in smooth in the enzymatic transformation of organic nitrates . muscle relaxation. Cyclic GMP also stimulates calcium Thiols have also been shown to be potentially important efflux and uptake by intracellular stores . It may also inhibit for the antiplatelet effect of the nitrovasodilators . Loscalzo calcium influx across the plasma membrane by decreasing (39) demonstrated that N-acetylcysteine