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

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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 potentiated the production of diacyl glycerol (DAG) and inositol 1,4,5- platelet inhibitory actions of nitroglycerin by inducing the triphosphate (IP3), which results in a decrease in protein formation of S-nitroso-N-acetylcysteine . A recent study has kinase C activity (Fig . 1) (24). The effects of cyclic GASP are reported that the liberation of nitric oxide from isosorbide terminated by a specific phosphodiesterase (25). dinitrate (ISDN) and nitroglycerin is potentiated by cysteine Several inhibitors of this pathway have been utilized in (40). In contrast, the release of nitric oxide from nitroprus- studies examining the mechanism of action of endothelium- side was reported to be independent of cysteine (41) . How- derived relaxing factor . Because of the natural affinity of ever, thiols can also potentiate the antiplatelet effects of nitric oxide for heme rings, free reduced hemoglobin has nitroprusside, which suggests that they have an important been used as an inhibitor of both endothelium-derived relax- role at other levels (42) . The fact that added thiols potentiate ing factor (8,26) and nitric oxide (27) . Methylene blue has antiplatelet effects does not prove that an important relation also been used to inhibit endothelium-derived relaxing factor exists between thiols and nitrates in vivo in the absence of by the direct inhibition of guanylate cyclase or by the administered thiols. generation of superoxide free radicals (28,29) . The binding of nitric oxide to the home moiety of soluble Does. The nitrate compounds used in the treat- guanylate cyclase and the activation of cyclic GMP follows ment of angina have been termed organic nitrovasodilators the same pathway as that described for endothelium-derived because they possess a nitrate ester bond (R-0--nitric relaxing factor (10) . A recent study in pigs demonstrated

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H2N NH2 H2N

NO synlhase NH- NH

H3- r4 COO- H3+N COO L-arginine ,, L-citruiline

Figure 1 . A, Proposed mechanisms by which nitrouo

compounds are bioconvc,rted to nitric oxide, which binds No to the heme moiety of guanylate cyciase (GC), leading to - RswO increases in intracellular levels of cyclic guanosine mono- RSNO phosphate (CGMP) . Reprinted, with permission, from RSH RSSR RSH, -HSNO NO ' Statnler and Loscalzo (127) . B, Schemata showing some R-ON0 2 NO 2 of the potential sites of action of cyclic guanosine mono- INTG! phosphate-mediating vascular smooth muscle cell relax- ation. Reprinted, with permission, from Ahlner et . al. ONj-eNO -- RSNO (Ndropru5srlea (112) . See text for details. ATPasc = adenosine triphos- 01 GC /GTP phatase ; DAG = diacyl glycerol ; GTI' = guanosine (Gu2nyie,ie mg2"' nahi triphosphate; HONO = nitrous acid: tP- = inositol 1,4,5- rycla ;r) ', ..~ cG10P triphosphate; MIIC = myosic heavy chain; MLC myosin light chain ; htLC1C, = myosin lib ht chain kinase Cal' with active enzyme ; MLCKI = MLCK with inactive P enzyme ; NO = nitric oxide ; NO = nitrite ; NTG = B Contraction Ca"-ATPase 1 PiP2 nitroglycerin ; PIP, = phosphatidyl inositol biphosphate ; / ~ Pk-C = protein kinase C ; R-ON02 = organic nitrate ; cGMP l+l cGMP t-1 RSH = reduced thiol ; RSNO = S-nitrosothiol ; RSSR = ML C--P0 4 disulfide. MHC °® Aclln P spholipase C I+I a --- Pk-C } T DAG + IP3 i+l CGMP [°] - n~rl_cK a _ fca"1 r \ Od0`~r P MLC I MHC MLCK, cGMP i-1 cGMP I+l E

Figure 2. Chemical structure of selected nitrovasodilators inhibition of the antiplatelet effect of nitroglycerin by meth- ; ISDN = isosorbide dinit,ate : IS-5-MN. GTN glyceryl trinitrate = iso- ylene blue, along with a documented decrease in cyclic CMP sorbide 5-mononitrate ; NP = nitroprusside, Reprinted, with permis- level (43). A similar study in humans also demonstrated that sion, from Ahlner et al . (112) . nitroglycerin was capable of increasing platelet cyclic CMP H O-IvO, levels and that the degree of vasodilation correlated with I H-c-O-NO2 these levels ;44). These studies support the view that the I H--C-0-NO, guanylate cyclase system is the final pathway for the action-, I H-C-o-N02 of all nitrovasodilators . I Differences in tolerance between organic nitrates and nitro- H prussiide. A detailed discussion of tolerance is beyond the GTN ISUN F scope of this review . Continuous administration of organic nitrates leads to tolerance to their hemodynamic and clinical effects. Needleman et al . (45,46) demonstrated that arterial strips require the presence of tissue thiol groups to relax to nitroglycerin and nitroprusside . Numerous other studies have demonstrated that thiol group donors, such as cysteine OH and glutathione, can potentiate guanylate cyclase activation Is 5-MN by both nitrates and nitroprusside (41,47-49) . Moreover, I I a

JACC Vol . 24, Nb . 2 558 ANDERSON ET AL . NITRIC OXIDE AND NITRATES August 1994 555--6k,

A 6 - - Prestenotic segment - Normal vessel 150 --- Stenotic segment 0 Figure 3. Recent change in vessel diameter with acetylcholine . a 125 plotted in (A) atherosclerotic and (B) normal coronary arteries U In both the prestenotic and stenotic segments, profound vaso- constriction was seen with acetylcholine, whereas n normal 100 o----i1 n m r+ vessels dilation was seen with acetylcholine . This represents m endothelial dysfunction in atherosclerotic coronary arteries . r= 75 ACh = response to maximal dose of acetylcholine ; Cl. _ control ; C2 = vehicle control, C3 = repeated control ; TNG = 50 nitroglycerin. *p < 0.01 . Reprinted, with permission, from Ludmer et al . (74). 25 I 0 C1 C2 Ach m ~, C3 INC Cl C2 ACh,,, am C3 TNG

studies have demonstrated that the administration of thiol gregating platelets, thrombin, adenosine diphosphate (ADP), donors, such as N-acetylcysteine, or thiol-containing angio- serotonin, bradykinin, histamine, norepinephrine, vasopres- tensin-converting enzyme inhibitors, such as captopril, can sin, substance P and acetylcholine (64). result in the reversal of nitrate tolerance or the potentiation Experimental evidence . A substantial body of basic re- of nitrate effects in smooth muscle cell preparations (50,51), search has established the importance of endothelium- intact animals (33,52) and clinical studies (53-56) . The sulf- derived relaxing factor in both basal and stimulated control hydryl depletion hypothesis, which has indirect support from of vascular tone in large conduit vessels . Studies have the previous Aata, is now thought to be incomplete, however. demonstrated that basal cyclic GMP levels are higher in Vascular sun lydrl groups are not decreased during develop- aortic strips when the endothelium is intact than when it has ment of nitroglycerin tolerance (57). The recent data by Fung been denuded (8), and bioassay techniques have also dem- (58) that show that nitrates are enzymatically converted in onstrated the basal release of nitric oxide from large conduit smooth muscle cells to nitric oxide have lead to the evolution of vessels. The role of endothelium-derived relaxing factor as the thiol-depletion hypothesis . It may be that tolerance is due the mediator of vasodilation is supported by experiments in part to a decrease in available sulfhydryl groups to interact that used endothelium-dependent agonists in the presence of with enzymes that are necessary for metabolic activation of inhibitors of endothelium-derived relaxing factor (8) . citrates. It is also clear that long-term nitrate administration is Increasing flow is also an important stimulus of endothe- accompanied by a variety of compensatory physiologic effects . lium-dependent vasodilation. It has been shown in the ca- including increased plasma concentrations of renin and cate- nine femoral and coronary artery that removal of the endo- cho] ` s, increased body water and shifts in vascular vol- thelium by balloon injury prevents the flow-mediated ume (59--61) . It is likely that tolerance is a complex process increase in diameter, demonstrating that the response is involving both cellular and physiologic processes . endothelium dependent (65-68) . Cellular tolerance has not been shown to be a major Human studies . In vitro studies using human coronary problem for nitroprusside . Nitric oxide is released from nitro- arteries have shown endothelium-dependent relaxation in prusside by one-electron transfer and may interact with differ- response to acetylcholine in normal arteries and impaired ent pools ofthiols than those required for the transformation of relaxation in atherosclerotic coronary arteries (69-71) . In organic nitrates, but this has not been definitively proved (40). vivo studies of conduit vessel vasomotion have focused on the brachial, femoral and coronary circulation (72-74) . l ud- mer et al. (74) demonstrated coronary vasodilation in re- Biologic Actions of Endothelia - recd sponse to acetyicholine in patients with normal coronary Relaxing Factor arteries and no risk factors for atherosclerosis, but vasocon- Conduit vends. The endothelium modulates vascular striction occurred in patients with atherosclerosis (Fig . 3). tone by releasing various relaxing factors (i.e ., endothelium- This was the first study to recognize that endothelium- derived relaxing factor, endothelium-derived hyperpolariz- dependent vasodilation does exist in vivo in normal human ing factor and prostacyclin) and the contracting factors (i.e., subjects and is impaired in patients with atherosclerosis . A thromboxane-A2, free radicals, endothelium-derived con- number of subsequent studies also showed that other ago- tracting factors and endothelin) (62,63) . Endothelium- nists, including substance P, serotonin and histamine, can derived relaxing factor can be released in response to a increase the coronary artery diameter in normal subjects variety of stimuli, including shear stress and pressure, ag- (75-77). Studies using inhibitors of nitric oxide and nitric

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oxide synthase have confirmed that nitric oxide mediates may be involved, or the contribution of nitric oxide is small acety1choline-induced coronary artery dilation (69,78,79) . and was not detected because of the inherent variance in a Flow-mediated vasodilation has also been demonstrated biologic system . in human femoral, brachial and coronary arteries (72,80,81) . Basal effects of endothelium-derived relaxing factor on Physiologic stimuli that lead to an increase in coronary blood the microvasculature in humans have been addressed by flow, including the use of papaverine or adenosine (82,83), studies using endothelium-derived relaxing factor inhibitors . bicycle exercise testing (84), cold pressor testing (85,86) and Valiance et al . (98) demonstrated a 50% decrease in forearm mental arithmetic (87), have all been shown to increase blood flow to locally infused N °-monomethyl-L-arginine, coronary artery diameter in patients without atherosclerosis which suggests a role for endothelium-derived relaxing fac- or risk factors . An impaired response is seen in patients with tor in the maintenance of resistance vessel tone . Lefroy et al . atherosclerosis . The parallel response of coronary arteries to (79) showed that NG-monomethyl-L-arginine produced only the physiologic stimuli and to acetylcholine led investigators a small, nonsignificant decrease in basal flow . Thus, it is to the conclusion that these stimuli were endothelium depen- generally believed that endothelium-derived relaxing factor dent. However, studies using nitric oxide inhibitors have not plays a role in the stimulated and basal maintenance of tone been done to confirm the role of nitric oxide in flow-mediated in the resistance vessels, but the story is far from complete . vasodilation in the coronary circulation . Platelet function. The antiplatelet effects of nitric oxide Resistance vessels . Experimental studies . In vitro studies and endothelium-derived relaxing factor have been known that used microscopy have demonstrated dilation of resis- for some time (42,101) . The role of endothelium-derived tance vessels in response to acetylcholine from rabbit ear relaxing factor as an antiadhesive agent was Aiovin by (88), rat cremaster muscle (89) and canine coronary resis- Radomski et al . (102) and may be an important action of tance vessels (90). In vivo studies using rabbit hind limbs or endothelium-derived relaxing factor because prostacyclin isolated buffer-perfused rabbit heart and primate studies has potent antiaggregatory properties but only mildly using nitric oxide inhibitors have confirmed that nitric oxide antiadbcsive function. There is additional evidence that mediates vasodilation in the microcirculation (91-94) . .P.1ot'helium-derived relaxing factor can disaggregate plate- Studies that used systemic doses of 0-monomethyl-L- lets (42), and prostacyclin has been shown to potentiate arginine have shown an increase in blood pressure and reflex this andaggregating and disaggregating activity (102,103) . bradycardia in the rabbit (95), rat (96) and guinea pig (97), Endothelium-derived relaxing factor is also known to in- suggesting that the basal release of endothelium-derived crease cyclic guanosine monophosphate level in platelets, relaxing factor is important at the resistance vessel level in but the mechanism by which this inhibits platelet aggregation regulating systemic blood pressure . and adhesion is poorly understood . Studies have suggested Human studies . Vasodilation in resistance vessels in vivo that a reduction in calcium influx may inhibit the phospho- is assessed by measuring blood flow . For a constant pressure, lipase C stimulation of protein kinase C (104,105) . "tone" in the resistance vessels is the major determinant of Nitric oxide subserves the antithrombotic process by mech- flow. Importantly, any stimulus that relaxes resistance vessels anisms other than its antiaggregatory and antiadhesive proper- and increases flow will result in a mild increase in conduit ties. The release products of "activated platelets" mediate vessel diameter as a consequence of flow-mediated vasodila- changes in vasomotor tone in part through endothelium- tion. Resistance vessel dilation therefore effects changes in dependent mechanisms . These factors, which in general result conduit vessels through flow-mediated dilation . in vasodilatation if the endothelium is intact, have been shown Cholinergic agonists infused into the brachial artery are to p--oduce constriction in patients with atherosclerosis known to increase flow as measured by impedance plethys- (76,106,107). Thus, the released platelet products can amplify mography . Valiance et al . (98) demonstrated that this the ischemic effects by causing constriction at sites of endo- response could be attenuated by the infusion of 0- thelial dysfunction . This adverse platelet-endothelium interac- monomethyl-L-arginine, which suggests that the dilator ac- tion is an important cause of vasoconstriction at sites of platelet tion of endothelium-derived relaxing factor contributes to aggregation. the stimulated regional blood flow response to acetylcholine . Other actions. Although the major actions of eradothelium- In the coronary circulation, the blood flow response to derived relaxing factor are related to its effect on vasomotion acetylcholine and substance P is impaired in the microvas- and platelet function, a number of other actions have been culature of patients with nonobstructive coronary artery reported . Several studies have reported that endothelium- disease (99,100). Lefroy et al . (79) used coronary sinus derived relaxing factor is cytoprotective and has an antipro- oxygen saturation as a measure of coronary blood flow to liferative effect on smooth muscle cells (108-111). New demonstrate that NG-menomethyl-L-arginine did not abolish interest has now been focused on the ability of endothelium- the blood flow increase in response to acetylcholine . They derived relaxing factor released from the myocardial cells to suggested that endothelium-derived relaxing factor may not regulate local inotropy . Studies of septic shock suggest that be the only mediator of acetylcholine-induced increase in vasodilation and decreased inotropy may resu!t in hypoten- blood flow in the coronary microvasculature . Other media- sion because of the systemic release of large quantities of tors, such as endothelium-derived hyperpolarizing factor, endothelium-derived relaxing factor . As a result of the

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ubiquity of endothelium-derived relaxing factor, many new relieves angina, however, remains controversial, although it roles are likely to be discovered from future research . probably plays some role . One of the interesting aspects about organic nitrates is their relative inability to dilate small coronary arterioles in Biologic E ..sects of Org ic. Nitrates vitro. The response to nitroglycerin in vessels veins and more recently on their and has been nicely reviewed by Harrison and Bates (38) . ability to inhibit platelet aggregation . Despite this, however, nitrates may increase coronary flow V r smooth m _ . The major action of organic by 25% to 50%, suggesting a mild effect on larger coronary nitrates is the dilation of vascular smooth muscle in arteries resistance vessels (54,56,126) . This effect is transient only and veins (112). The magnitude of this effect depends on the and avoids the potentially deleterious coronary steal phe- type of preparation studied and the nitrate preparation used . nomenon that may be seen with potent arteriolar vaso- Nitroglycerin tends to exhibit a biphasic relaxation pattern, dilators. whereas isosorbide dinitrate and isosorbide-5-mononitrate Platelet effects. The effect of organic nitrates on platelet (IS-5-MN) have no such biphasic response (113) . Nitroglyc- function has recently been reviewed (127). In vitro studies erin has a greater sensitivity for veins than arteries (114) . An beginning in the late i s uniformly demonstrated an an- in vitro study comparing the vasorelaxant effects of nitro- ti regatory effect of nitroglycerin on platelets (118, 129) . It glycerin, isosorbide dinitrate and isosorbide-5-mononitrate was also known that nitroprusside can directly inhibit plate- on isolated renal arteries and veins from rabbits suggested let aggregation and promote disc gction (130,131) . that the latter two were even more venoselective than As is the case for endothelium-derived relaxing factor, nitroglycerin (115,116) . Most in vitro studies have demon- several groups have reported synergistic actions between the strated that therapeutic doses of nitrates have only a small antiplatelet effects of nitroglycerin and nitroprusside with relaxing effect on peripheral arteries (117) . prostacyclin (132,133) . In vitro studies using supraphysio- In vi" studies in animals, normal volunteers and patients logic concentrations have demonstrated that prostaglandin- with coronary artery disease, both with and without conges- mediated pathways may be important for the antiplatelet tive heart failure have consistently shown that venous ca- effects of nitrates (129,134,135) . pacitance vessels have the highest sensitivity to nitrates Folts et al. (136) used an in vivo canine coronary model to (112) . This venodilation leads to a reduction in cardiac demonstrate that platelet aggregation and dislodgment can preload with a decrease iri left ventricular volume and filling be inhibited by nitroglycerin and that this effect may be pressure that results in r1 decrease in oxygen consumption . mediated by cyclic GMP. Lam et al . (137) demonstrated that The magnitude of the decrease in these variables is in the nitroglycerin can decrease platelet deposition in the common map 2 to 40%. carotid arteries of balloon-injured pigs . Both studies demon- In patients with heart failure, sufficient arterial unloading strated that the antiplatelet effect paralleled the hemody- occurs to actually increase cardiac output. The in vivo effect namic changes induced by nitroglycerin . However, initial in on compliance changes in large arteries may be greater than vivo human studies failed to demonstrate an antiplatelet that predicted from in vitro studies (118) . effect for the organic nitrates at physiologic concentrations Cdr artery dila . A number of studies demon- (138). Stamler et al. (139) investigated the effect of intrave- strate that nitrates are potent dilators of both normal and nous nitroglycerin on platelets ex vivo. They found that in atherosclerotic coronary arteries, a mechanism that may vivo infusions of nitroglycerin titrated to the hemodynamic potentially increase blood flow to the myocardium . Feldman response did not alter ADP-induced platelet a regation . et al. (119) have shown that intracoronary doses of nitroglyc- However, after they added N-acetylcysteine (a thiol donor) erin can increase coronary artery diameter by 10% to 30%o . ex vivo, platelet inhibition was seen . Diodati et al . (140) used Low doses of intravenous or sublingual nitroglycerin have bedside impedance aggregometry before and after 45 min of also been shown to dilate coronary stenoses compared with nitroglycerin infusion and demonstrated a decrease in the placebo infusions (120,121) . Brown et al . (122) subsequently area under the a . .regation curve in response to ADP and demonstrated that -75 of stenoses will dilate to some thrombin, suggesting an antiplatelet effect . They also re- extent in response to nitroglycerin and suggested that this ported that if blood was left to stand for 30 min before resulted from dilation of the normal portion of the artery in analysis, no difference was noted between the control and eccentric stenoses. Feldman et al. (123) also showed that nitroglycerin infusion (140) . They believed that prolonged dilation of collateral vessels was possible after nitrate ad- ex vivo experiments usually led to depletion of thiol ministration. Reversal of distal or collateral vessel constric- groups and was a possible explanation for some of the tion, which may occur during exercise, may be another previously negative results from studies using nitroglycerin important mechanism by which nitroglycerin works (131,139,141). Another recently reported in vivo study (44) (124,125). The extent to which epicardial coronary dilation was able to demonstrate a good relation between platelet

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cyclic GMP levels and coronary vasodilatien to sublinguall response to acetylcholine after L-arginine infusion but did nitroglycerin in patients with and without nitrate tolerance . not demonstrate any effect on large-vessel vasomotion, In It is likely, but not conclusive, that nitrates have a beneficial contrast, Dubois-Rande et al. (149) showed an improvement antiplatelet effect in in vivo situations . in large-vessel response to acetylcholine after L-Rrghqir1e infusion . Therefore, in certain situations, depletion or impaired utilization of L-arginine may contribute to the Endothelial Dysfunction : dysfunction seen in hypercholesterolemia ; however, a defi- ciency of the substrate has not been proved by measure- NO% hiechanisims ments of intracellular concentrations . Endothelial vasodilator dysfunction has been demon- Rubanyi and Vanhoutte (9) have shown that superoxide strated in vitro and in vivo in animal and human studies in a anions can inactivate endothelium-derived relaxing factor . wide range of conditions, including hypercholesterolealia, In states in which excessive free radicals are generated, an atherosclerosis, hypertension, diabetes mellitus, heart fail- increase in oxidation of low density lipoproteins (LDLs) ure and postischemic reperfusion. A detailed discussion is would also result. Oxidized LDL itself has been shown to be beyond the scope of this article, but several reviews are a potent inhibitor of endothelium-dependent relaxation available (63). The mechanism of this vasodilator dysfunc- through mechanisms that are transduction dependent tion is unclear, and because the actions of endothelium- (150,151) . Still others have suggested that free radicals derived relaxing factor are complex, abnormalities could interfere with the production of potent vasodilators and may occur at many sites of action: 1) impairment of endothelial lead to increased destruction of endothelium-derived relax- membrane receptors that interact with agonists or physio- ing factor (152) . A recent study has shown that superoxide logic stimuli to release nitric oxide ; 2) diminished levels or dismutase can attenuate the vasoconstricting effect of ace- impaired utilization of L-arginine, the substrate for nitric tylcholine in the coronary arteries of patients with athero- oxide synthesis ; 3) reductions in nitric oxide synthase, the sclerosis (153), suggesting a role for oxygen free radicals in enzyme responsible for the conversion of L-arginine to nitric the inactivation of nitric oxide in vivo . oxide; 4) impaired release of nitric oxide from the endothe- A defect in the vascular smooth muscle of resistance vessels lium in its most active form (i.e., nitrosothiols) ; 5) enhanced in relation to the action of endothelium-derived relaxing factor degradation of nitric oxide by oxygen free radicals ; 6) has been suggested by two recent forearm studies . Creager et impaired transport from endothelium to smooth muscle cell al. (146) demonstrated a decrease in the stimulated increase in (functional barrier) ; 7) impaired interaction of nitric oxide flow in response to methacholine and nitroprusside in patients with guanylate cyclas,,. and subsequent increase in cyclic with hypercholesterolemia compared with control subjects . GMP; and 8) decrease in generalized smooth muscle cell Calver and Valiance (154) also demonstrated a decrease in flow sensitivity to vasodilators . response to nilroprusside in patients with type I diabetes The best studied conditions associated with endothelial mellitus compared with control subjects . These studies did not dysfunction are hypercholesterolemia and atherosclerosis . determine whether the attenuated responses seen with nitro- Hypercholesterolemia inhibits endothelium-dependent re- prusside are the result of augmented inactivation of nitric oxide laxation evoked by receptor-dependent stimuli, but this from nitroprusside or are a generalized defect in smooth generally does not inhibit the direct relaxing effect of nitro- muscle cell function. glycerin. Studies in hypercholesterolemic porcine coronary Other mechanisms of endothelial dysfunction include arteries have demonstrated that the activity of the pertussis intimal thickening, which acts as a barrier to the diffusion of toxin-sensitive Gi protein-dependent receptor pathway is endothelium-derived relaxing factor (155) and the increased reduced in hypercholesterolemic endothelial cells (142,143). production of vasoconstrictor substances that offset the This may be one of the mechanisms of endothelial dysfunc- balance toward vasoconstriction . Endothelin may be one tion in the early stages of the atherosclerotic process (144) . such factor. However, the role of these vasoconstrictors has The synthesis of nitric oxide involves the oxidation of a not been well defined . Thus, even though the normal phys- guanidino nitrogen on L-arginine by means of specific nitric iology of endothelium-derived relaxing factor has been ex- oxide synthases. Several groups have suggested that a tensively studied, the mechanisms involved in its failure are depletion of L-arginine is responsible for this endothelial incompletely understood at this time . dysfunction in hypercholesterolemia . It was shown in rabbit hind limbs that L-arginine administration potentiated the acetylcholine-induced increase in hind limb flow in rabbits Interactions Between Exogenous with dietary hypercholesterolemia (145) . Creager et al . Vitro- asodilators and (146,147) also demonstrated that an infusion of L-arginine Endothelia erived improved the forearm blood flow response to methacholine Relaxing Factor in hypercholesterolemic patients . Two similar studies have been performed in human coronary arteries . Drexler et al. Extensive evidence is available to show that the chemical (148) were able to demonstrate an improvement in the flow nature, mechanism of action and biologic actions of

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endothelium-derived relaxing factor and the organic nitrates response to exercise in patients with coronary stenoses in parallei each other. However, it is not known whether the vessels presumably deficient in endogenous nitric oxide . The administration of exogenous nitrates has an advantageous or vasomotor response of exercise at sites of stenoses has been potentially deleterious effect on the endothelium-dependent shown to be vasoconstriction (84). This vasoconstriction nitric oxide pathway or whether exogenous nitrates can paralleled changes seen in response to acetylcholine and replace the decreased activity of endogenous nitric oxide in have been interpreted to be due to endothelial dysfunction. various diseases . Moreover, it is unclear whether endothelial Cage et al . (164) also studied the effect of exercise on dysfunction affects the response of exogenous nitrates . vasomotion at stenotic sites in patients with angina . They Effect of n y on mm nt demonstrated a decrease in area of 71% at stenotic sates in v . Potential adverse effects could result from response to exercise, nitroglycerin subsequently increased prolonged nitrate therapy sufficient to induce nitrate toler- the area to 12% above control levels . In patients given ance. Thiol depletion could theoretically have an adverse nitroglycerin before exercise, there was no decrease in area effect on the intracellular activation of guanylate cyclase and at the stenotic site as a result of exercise . Thus, prevention the subsequent production of cyclic OMP. Some investiga- of sympathetically mediated, endothelium-dependent vaso- tors have found cross-tolerance between nitroglycerin and constriction may be an important anti-ischemic mechanism endothelium-derived relaxing factor with regard to both of nitroglycerin . Other evidence of nitroglycerin as a re- relaxation and cyclic GMP increase (156,157) . Rapoport et placement for endogenous nitric oxide is lacking in humans al. (157) for example, pretreated rat thoracic aortas to induce at this time. but the use of nitrates as surrogates for nitrate tolerance. They then demonstrated that there was diminished intrinsic nitric oxide would be very appealing . attenuation of relaxation in response to nitroglycerin, nitro- However, improper tinting of nitrate administration might prusside and acetylcholine, with an associated decrease in deplete thiol stores, negating any potential beneficial effects the formation of cyclic GMP. Other investigators have found of administered nitrates . no cross-tolerance (158-160) . Lawson et al. (161) studied rat aortic strips rendered nitrate tolerant and were able to Conclusions demonstrate normal relaxation in response to acetylcholine . Thus, at this time there is inconclusive evidence to show The organic and inorganic nitrovasodilators share many whether nitrate tolerance has a significant physiologically common features with the reccntiv discovered endothelium- adverse effect on endothelium-mediated events . The ques- derived relaxing factor. The one major difference may be the tion, nonetheless, has importatt clinical ramifications . lack of effect of organic nitrates in the coronary microcircu- Enhanced sensitivity to nitroglycerin in the setting of lation. Both endothelium-derived relaxing factor and nitro- endothelial dysfunction . Most in vitro and in vivo studies vasodilators have similar biologic effects as a result of their have demonstrated retained or even augmented relaxant similar, final:,. commapi 'P v y predominantly relax properties of nitrates when endothelial dysfunction is vascular smooth muscle in most species and tissue beds present (155). Moncada et al . (162) recently suggested that tested. Recent data have demonstrated the potent antiplate- an enhanced sensitivity to nitrates may be present after let effect of endothelium-derived relaxing factor and exoge- removal of endothelial nitric oxide . They performed in vitro nous nitrates, which may act in concert with prostacyclin . studies on isolated rat aortic rings and demonstrated that The action of the exogenous nitrovasodilators may be en- after endothelial denudation or inhibition of endothelium- hanced in patients with dysfunctional endothelium, and . derived relaxing factor, the potency of nitroglycerin and conversely, nitrate tolerance may impair endothelium- nitroprusside as relaxing ents was significantly enhanced . dependent processes . Much needs to be learned about the They also demonstrated an enhanced hypotensive response potentially important interaction between exogenous ni- to nitroglycerin in rats given nitric oxide synthase inhibitors trates and the "endogenons nitroglycerin," endothelium- compared with rats without endothelium-derived relaxing derived relaxing factor . Suet, insights might enable a more factor inhibition. These experiments suggested that a super- logical use of our oldest and most prescribed antianginal sensitivity to nitrovasodilators at the level of soluble guany- medication . late cyclase was induced by the removal or inhibition of endothelial influences . Holland et al . (163) suggested that the endothelium is known to produce reactive oxidative metab- References olites that can scavenge nitric oxide derived from exogenous 1. Murrml W . Nitro-glycerine as a remedy for angina pectoris . Lancet vasodilator drugs and that its removal or inhibition might 1579;1:80-1.113-5,151 225-7. 2. Furchgett RF, Zawadzki JV. The obligatory role of endothelial cells in result in an enhanced response to exogenous nitrates as an the relaxation of arterial smooth muscle by ayatylcholine. Nature alternate mechanism . Either way, this has important clinical 1980;21 :373-6. implications . 3. Myers PR, Minor RL Jr., Guerra R Jr., Bates JN, Harrison 0G. Vasore- Nitroglycerin as t for endogenous nitric oxide. laxant properties of the endothelium-derived relaxing factor more closely resemble S-nitmsocysteine than nitric oxide . Nature 1990;345:161-3. The short-term administration of nitroglycerin has been 4. Ignarm LJ, Byrns RE, Buga GM, Wood KS. Endothelium-derived shown to prevent endothelium-dependent constriction in relaxing factor from pulmonary artery and vein possess pharmacologic JACC Vol. 24, No . 2 ANDERSON ET AL, 563 August 1994 :555-66 NITRIC OXIDE AND NITRATES

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