Mechanisms responsible for the heterogeneous coronary microvascular response to nitroglycerin. M A Kurz, K G Lamping, J N Bates, C L Eastham, M L Marcus and D G Harrison Circ Res. 1991;68:847-855 doi: 10.1161/01.RES.68.3.847 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1991 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/68/3/847 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. 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Harrison Nitroglycerin dilates large (.100 ,um) but not small coronary arterial microvessels, and a putative metabolite of nitroglycerin, S-nitroso-L-cysteine, has been shown in vitro to dilate both large and small coronary microvessels. Based on this evidence, we tested the hypothesis that the lack of response of small coronary microvessels was due to an inability of small coronary microvessels to convert nitroglycerin into its vasoactive metabolite and examined possible explanations for this phenomenon. We studied left ventricular epicardial microvessels in vivo using video microscopy and stroboscopic epi-illumination in anesthetized, open-chest dogs. Diameters were determined while the epicardium was suffused with nitroglycerin, S-nitroso-Lcysteine, or S-nitroso-D-cysteine (all 10 ,M) and nitroglycerin in the presence of L- or D-cysteine (100 ,uM). None of the agents affected systemic hemodynamics. Nitroglycerin dilated large arterioles (20±2%) but not small arterioles (1±l1%). Both S-nitroso-L-cysteine and S-nitroso-D-cysteine were potent dilators of all size classes of microvessels. Concomitant application of L-cysteine and nitroglycerin evoked dilation in small microvessels (22±4%, p<0.5 versus nitroglycerin alone) and larger microvessels (27±6%, p=NS versus nitroglycerin alone). D-Cysteine did not alter the microvascular response to nitroglycerin in either small (7+4%, p=NS versus nitroglycerin alone) or large (18+3%, p=NS versus nitroglycerin alone) microvessels. Neither L-cysteine nor D-cysteine had a direct effect on microvascular diameter. These findings suggest that 1) sulfhydryl groups are required for the conversion of nitroglycerin to its vasoactive metabolite; 2) the interaction between nitroglycerin and sulfhydryl residues is a stereospecific process, indicating either an intracellular mechanism or a membraneassociated enzymatic reaction; and 3) a lack of available sulfhydryl groups may be responsible for the lack of response of small coronary arterioles to nitroglycerin. (Circulation Research 1991;68:847-855) itroglycerin has been used in the treatment of acute angina for more than 100 years. Despite its acceptance as a useful agent for treatment of acute myocardial ischemia, the mechanism by which nitroglycerin exerts its salutary effect N From the Department of Internal Medicine and the Cardiovascular Center, College of Medicine, University of Iowa, Iowa City, Iowa. Supported by Ischemic Specialized Center of Research grant HL-32295, National Institutes of Health grant HL-39050, and a Veterans Administration merit grant. M.A.K. is supported by an Individual National Research Service Award from the National Institutes of Health (HL-08093-O1A1). D.G.H. is an Established Investigator of the American Heart Association. Address for correspondence: Michael A. Kurz, PhD, Drawer LL, Cardiovascular Division, Department of Internal Medicine, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, GA 30322. tDr. Melvin L. Marcus died on October 19, 1989. Received July 10, 1990; accepted November 19, 1990. only now is becoming well understood. Early studies by Gorlin et all and later by Winbury et al2,3 showed that nitroglycerin was a potent dilator of large epicardial coronary arteries but had little or no effect on total coronary blood flow. These studies suggested that nitroglycerin had minimal effect on small coronary resistance vessels. Indeed, recent in vivo4 and in vitro5 studies in our laboratory using direct visualization of coronary microvessels confirmed that nitroglycerin does not dilate small coronary arterioles. Based on previous reports,5-13 the most plausible explanation for the heterogeneous microvascular response to nitroglycerin is that nitroglycerin is not converted to its vasoactive metabolite in small coronary arterioles. Despite extensive investigation, the mechanism underlying the biotransformation of nitroglycerin to its vasodilator metabolites remains unclear. The most widely accepted hypothesis holds that nitroglycerin must interact with a sulfhydryl- Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014 848 Circulation Research Vol 68, No 3 March 1991 containing compound to be converted to a vasoactive intermediate.8,14-21 The most likely candidate for this sulfhydryl agent is cysteine, because nitroglycerin is more rapidly converted to active metabolites in the presence of cysteine than other naturally occurring thiols.16,20 This interaction probably results in the production of a nitrosothiol, such as S-nitrosocysteine.13,1617 It is not clear whether this nitrosothiol directly activates guanylate cyclase or must be further reduced to release free nitric oxide (NO), which then activates guanylate cyclase. In the present study, we sought to determine whether a putative metabolite of nitroglycerin, S-nitroso-L-cysteine, could dilate small coronary microvessels in vivo, as it was previously shown to do in vitro.5 We then sought to determine whether small coronary microvessels could convert nitroglycerin to its vasoactive metabolites if supplemented with cysteine. To accomplish these objectives, we used an in vivo preparation to study coronary microvascular responses to S-nitroso-L-cysteine and to concurrent application of L-cysteine and nitroglycerin. To determine whether the interaction between nitroglycerin and cysteine was a stereospecific process (i.e., required activity of enzyme), studies also were performed using the nonphysiological enantiomers S-nitroso-D-cysteine and D-cysteine. Materials and Methods General Preparation Adult mongrel dogs (3-11 kg) of either sex were anesthetized with ketamine (25 mg/kg i.m.) and acepromazine (0.2 mg/kg i.m.) followed by a-chloralose (80 mg/kg i.v.). Cannulas were introduced into both femoral arteries and advanced into the aorta for the monitoring of aortic pressure, for withdrawal of blood samples for determination of arterial blood gases and pH, and for withdrawal of reference blood flow samples for measurement of myocardial perfusion. A femoral vein was cannulated for administration of drugs and fluids. The chest was opened in the left fifth intercostal space, and the heart was suspended in a pericardial cradle. A solid-state pressure transducer (Millar Instruments, Houston) was inserted into the left ventricle via the left atrial appendage and was used to measure left ventricular pressure. The left atrium also was cannulated for injection of the radiolabeled microspheres used to determine myocardial perfusion. The epicardial surface of the heart was bathed continuously with Krebs-Hensleit bicarbonate buffer (37°C, bubbled with 20% 02-5% C02, balance N2) to maintain the physiological environment of the epicardium. Body temperature was maintained at 37°C with a servocontrolled thermal blanket. High-Frequency Ventilation Respiratory-induced motion of the heart was minimized by using high-frequency jet ventilation synchronized to the cardiac cycle. After tracheal intubation with a cuffed endotracheal tube, a catheter was passed through the lumen of the endotracheal tube and advanced to the pulmonary carina. Compressed air (5-15 psi) was injected through the catheter for 25-90 msec by a solenoid valve triggered by the left ventricular dP/dt signal. The catheter was small enough to allow expired gases to freely pass out of the endotracheal tube. Positive end-expiratory pressure was provided by placing the end of the expiration tube under 1-3 cm water. Arterial blood gases and pH were maintained within the physiological range by varying the duration of inflation, the pressure of the air injected, the position of the air injection catheter relative to the pulmonary carina, and the end-expiratory pressure. Microscope and Illumination System The heart was positioned under a vertically mounted incident illumination system (Carl Zeiss, Inc., Thornwood, N.Y.). The table beneath the animal could be moved in an x-y plane to allow visualization of multiple vascular fields in a single heart. The epicardial surface was epi-illuminated at a single point of each cardiac cycle with a xenon arc stroboscopic light source (Chadwick Helmuth, El Monte, Calif.) and the Zeiss incident illumination system. Zeiss x 6.3 and x 10 (0.20 and 0.25 numerical aperatures) objectives were used with a x 10 eyepiece. A PDP 11/73 computer (Digital Equipment Corp., Chelmsford, Mass.) controlled the triggering of the strobe (15-25msec duration) using the first positive slope of the left ventricular dP/dt signal as the initial trigger point. The delay between the initial trigger point and the strobe flash was adjusted so that the microvasculature was visible only at a single point during mid-to-late diastole. Microvascular arteries and veins were differentiated by observing the sequence of illumination of vessels after a left atrial injection of fluoresceinlabeled dextran (fluorescein isothiocyanate-dextran, 497,000 Da [Sigma Chemical Co., St. Louis], 50 mg/ml in 0.9% saline). With this technique, arterioles fluoresced first, followed by venules. Measurements of Microvascular Diameters Images of microvessels were obtained with a silicone-intensified tube video camera (Dage-MTI, Inc., Michigan City, Ind.) and a video digitizer (Image Technology, Woburn, Mass.). The video camera was optically coupled to the intravital microscope. Images were digitized and displayed on a high-resolution monitor (Panasonic, Secaucus, N.J.). Selected images during an intervention were stored in the computer for later analysis using a digitizing tablet (Summagraphics Corp., Cambridge, Mass.). Microvascular diameters were measured as the average distance between two lines drawn on the luminal edges of the vessel wall. The monitor screen was calibrated by projecting a micrometer grid in the microscope field and by determining the ratio of micrometer length to video length. The pixel size of the digitized images was 4 ttm with a x 6.3 objective and 2 gm with a x 10 objective. We have shown previously that the spatial Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014 Kurz et al Heterogeneous Microvascular Responses to Nitroglycerin resolution of the digitized images is 5 ,um for the x 10 objective and that the interobserver variability in the measurement of arteriolar diameters is negligible using this technique.22 Measurement of Myocardial Perfusion Measurements of myocardial perfusion (radioactive microsphere technique) were used to establish that the experimental preparation was within the normal physiological range and remained stable throughout each procedure. Because drugs were applied topically, changes in myocardial perfusion likely occurred only very near the epicardial surface, and because the microsphere technique may not be accurate with very small tissue samples, no attempt was made to determine changes in myocardial flow evoked by these vasoactive agents. Microspheres diameter) labeled with 46Sc, 85Sr, (lx106, or 114in were 1l3Sn1n 141Ce,15-gm 103Ru, 51Cr,and57co,95 19Cd injected into the left atrial vortexed (5 minutes) appendage, followed by a saline flush (2 ml). Before and 90 seconds after microsphere injection, a reference blood flow sample was withdrawn at a constant rate from the femoral artery. Tissue and reference blood flow samples were counted in a germanium crystal gamma counter (Canberra Industries, Inc., Meriden, Conn.), and myocardial blood flow (MBF) was calculated using the equation MBF (ml/minx 100 g)=[CMxW]/CR, where CM is sample counts (counts per time per gram), W is withdrawal rate of reference sample (milliliters per minute), and CR is reference blood flow counts (counts per time). Preparation of Drugs Our method for synthesizing S-nitroso-L-cysteine has been described previously.23 Briefly, L-cysteine (1 mmol HCl salt, Sigma) was dissolved in 1 ml methanol to produce a 1 M solution. Twenty-five milliliters of oxygen and 50 ml NO were mixed in a gas-tight syringe at room temperature to form NO2 gas. This gaseous mixture was injected into a vacutainer tube in dry ice until frozen (several seconds). The frozen NO2 then was dissolved into the cysteine methanol solution in a vial surrounded by dry ice to produce a 1 M stock solution of S-nitroso-L-cysteine. The purity of the S-nitroso-L-cysteine solutions prepared in this manner has been previously verified with high-performance liquid chromatography. The stock solution was stored at -20°C in the absence of light. Immediately before application, S-nitroso-L-cysteine stock solutions were diluted to 10 times the dose to be applied in deoxygenated, ultrapure water at room temperature. This solution then was infused into the warmed, oxygenated Krebs' suffusate and dripped onto the epicardial surface over the vessels being observed. The rate of superfusion was calibrated so that the S-nitroso-Lcysteine solution was diluted by a factor of 10, warmed, oxygenated, and delivered to the epicardial surface in Krebs' buffer at a rate of 2 ml/min. S-Nitroso-D-cysteine was prepared using an identi- 849 cal technique, except that D-cysteine was substituted for L-cysteine. L-Cysteine and D-cysteine stock solutions were prepared each day by dissolving in ultrapure water to a 1 mM concentration. To determine whether the physiological half-life of S-nitroso-D-cysteine was identical to that of S-nitroso-L-cysteine, left circumflex coronary artery rings were studied using a superfusion bioassay preparation. Vessels were preconstricted with prostaglandin F2a, and relaxations to either S-nitroso-D-cysteine or S-nitroso-L-cysteine were examined. The transit time of the nitrosothiols from infusion into oxygenated Krebs' buffer to the vascular ring was varied (12, 27, or 40 seconds). Using this technique, the physiological half-life of S-nitroso-L-cysteine in oxygenated aqueous solution was found to be 34±13 seconds, which is similar to the half-life described by previous investigators.24 The physiological half-life of S-nitroso-D-cysteine was identical to that of the L-isomer (35±9 seconds, p=NS versus S-nitroso-L-cysteine). To be certain that the rates of oxidation of L- and D-cysteine were the same in our preparation, additional experiments were performed with Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid]),25 a colorimetric test specific for reduced thiol compounds. These studies indicated that the compounds were identical when prepared and the rates of oxidation were identical over 5 hours. Experimental Protocols To examine the effects of nitroglycerin on microvascular diameters, control diameters and myocardial perfusion were measured, and nitroglycerin (10 ,M in warmed Krebs' buffer) was suffused over the epicardial surface directly under the microscope objective. Microvascular diameters then were recorded at steady state (after 15-20 minutes of nitroglycerin superfusion). Local superfusion of nitroglycerin at this dose did not affect systemic arterial blood pressure or heart rate. After vessel responses were recorded, nitroglycerin suffusion was discontinued, and 30-60 minutes was allowed for vessels to return to control diameters. Diameters and myocardial perfusion again were determined to establish that the preparation had remained stable. Microvascular responses to nitroglycerin were studied in 30 vessels in 10 dogs (one to four vessels per dog). Control vessel diameters ranged from 29 to 267 ,um. To determine whether a putative metabolite of nitroglycerin could dilate coronary microvessels in vivo, responses to S-nitroso-L-cysteine were measured in 13 dogs (36 vessels, one to four vessels per dog; control diameters, 26-300 gm). After control diameters and myocardial perfusion were recorded, S-nitroso-L-cysteine was added to the Krebs' superfusate to provide 2 ml/min of 1 nM to 100 ,M S-nitroso-Lcysteine. To minimize decomposition of the S-nitrosoL-cysteine before it reached the epicardial surface, S-nitroso-L-cysteine was added to the warmed, oxygenated Krebs' so that exposure to oxygen was limited to approximately 1.5 seconds. Responses were deter- Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014 850 Circulation Research Vol 68, No 3 March 1991 mined after 15-20 minutes of S-nitroso-L-cysteine suffusion. In eight dogs, S-nitroso-L-cysteine suffusion was stopped for 30-60 minutes to allow vessels to return to baseline diameter before repeating applications of S-nitroso-L-cysteine. In these dogs, S-nitrosoL-cysteine was applied in randomly selected doses (1 nM-100 ,uM), with control diameters recorded before and after each application. In five other dogs, S-nitroso-L-cysteine was applied in increasing concentrations without control diameters recorded between applications. Final control diameters and myocardial blood flows were determined in these experiments to establish that the preparation had remained stable. No more than three applications of S-nitroso-L-cysteine were performed in any one experiment to minimize any potential for development of tolerance or tachyphylaxis. To determine whether response to S-nitroso-Lcysteine might be attributable to extracellular mechanisms, responses to S-nitroso-D-cysteine also were measured (16 vessels in four dogs). Because S-nitroso-D-cysteine is a highly polar molecule (and thus would not diffuse freely across the sarcolemma) and sarcolemmal transport mechanisms are generally stereospecific for levorotary isomers, it would not be expected to cross the sarcolemma. Because 10 ,M S-nitroso-L-cysteine caused a maximum dilatory response in all size classes of coronary microvessels, this concentration was used to determine microvascular responses to S-nitroso-D-cysteine. The protocol for applying S-nitroso-D-cysteine was the same as that used for application of S-nitroso-L-cysteine. To determine the role of thiol groups in the heterogeneous response to nitroglycerin, nitroglycerin was applied to the epicardial surface of six dogs after 30 minutes of L-cysteine suffusion (n=23 vessels). Control diameters and flow were recorded, and L-cysteine was added to the superfusate to produce an epicardial suffusion of 100 1uM L-cysteine at 2 ml/min. After 30 minutes, control diameters again were recorded, and nitroglycerin was added to the L-cysteine-Krebs' suffusate. Response diameters were recorded after 15-20 minutes. The nitroglycerin-L-cysteine superfusion then was stopped, and final control diameters and myocardial flows were recorded. This same protocol was performed with D-cysteine substituted for L-cysteine (22 vessels in six dogs) to determine whether vasoactive responses to cysteine-nitroglycerin coinfusion could be attributed to extracellular, nonstereospecific mechanisms. Criteria for an Acceptable Experiment Experiments were not conducted in animals in which mean arterial blood pressure was less than 60 mm Hg or if arterial blood gases or myocardial perfusion were out of the normal physiological ranges (pH 7.35-7.45, Pco2 25-40 mm Hg, Po2>70 mm Hg, and transmural flow <200 ml/minx 100 g). In addition, experiments were excluded from the study if mean arterial blood pressure or heart rate changed by more than 10% during the protocol (n =4) or if vessel diameters did not return to within 10% of the original control diameters after application of drugs (n=7). Statistical Analysis All data are expressed as mean±SEM. For evaluation of responses to topical application of nitroglycerin and nitrosothiols, response diameters were compared with the control diameter recorded immediately before the application of the compound. Responses evoked by application of nitroglycerin and L- or D-CYSteine were compared with the diameter recorded after a 30-minute preincubation with cysteine and immediately before nitroglycerin was added to the superfusate. Because responses to nitroglycerin differed markedly depending on vessel size, microvessels were divided into two groups (control diameter <100 ,um and >100 ,uM) for analysis. These size classes also were used for comparison of heterogeneous responses to all other drugs studied. Student's paired t test was used in comparing response diameters to control diameters.26 Analysis of variance and the Newman-Keuls multiple comparison procedure were used to compare responses between groups.26 Responses were considered significant atp<0.05. Results Systemic arterial blood pressure and heart rate were within the normal range for all groups and were not affected by topical application of any of the agents studied (Table 1). Transmural myocardial perfusion also was within the normal range and did not change during the course of the study (beginning, 114±8 ml/minx 100 g; termination, 110±9 ml/ minx 100 g, p=NS). Responses evoked by epicardial application of nitroglycerin (10 ,uM, 2 ml/min) varied with vessel size (Figure 1). Small vessels (control diameter< 100 ,um) did not dilate (1 ± 1%), but large vessels dilated 20+2% (p<0.05 versus small vessels). The maximum dilatory responses to S-nitroso-Lcysteine occurred at a superfusion concentration of 10 ,uM (Figure 2). S-Nitroso-L-cysteine dilated both small and large epicardial coronary arterioles (39±7% and 19±2%, respectively, Figure 3). S-Nitroso-D-cysteine (10 ,M) elicited similar responses in small and large vessels (+32±5% and 23±4%, respectively, Figure 4). These responses were comparable to those evoked by S-nitroso-L-cysteine. Incubation with either L-cysteine or D-cysteine alone (100 KM) had no direct effect on large or small microvessels (Table 2). Coronary microvascular responses to nitroglycerin after preincubation with L-cysteine or D-cysteine are shown in Figure 5. D-Cysteine did not alter the response to nitroglycerin in either small or large coronary arteries (+7±4% and +18+±3%, respectively, p=NS versus nitroglycerin alone). L-Cysteine also did not change the largevessel response to nitroglycerin (+27±6%, p=NS versus nitroglycerin alone), but after preincubation with L-cysteine, nitroglycerin produced dilation in small coronary arteries comparable to that of large Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014 Kurz et al Heterogeneous Microvascular Responses to Nitroglycerin 851 TABLE 1. Systemic Hemodynamic Responses to Topical Application of Various Treatments to Left Ventricular Epicardium MABP Treatment 100+3 104±5 88±4 97±4 94±6 Control 99+3 105±5 87±5 96±4 96±5 86±7 88±7 Treatment p NS NS NS NS NS NS NS Heart rate Treatment 132+6 141±7 151±14 133±9 141±8 155±5 155 ±5 Control 135+7 140±5 153±15 128±7 135±11 155±5 155 ±5 p NS NS NS NS NS NS NS GTN (n=10) L-cysNO (n=13) D-cysNO (n=4) L-cys (n=8) L-cys+GTN (n=6) 89±8 D-cys (n=6) 87±7 D-cys+GTN (n=6) Values are mean±SEM. MABP, mean arterial blood pressure; GTN, nitroglycerin; L-cysNO, S-nitroso-L-cysteine; D-cysNO, S-nitroso-D-cysteine; L-cys, L-cysteine; D-cys, D-cysteine. vessels (+22+4%,p =NS versus large vessels,p<0.05 versus nitroglycerin alone). Data are summarized and expressed as percent change from control diameter in Figure 6. Analysis of variance and multiple comparison procedures revealed that the data fell into two groups. In one group, responses were similar to the response evoked by nitroglycerin in small microvessels (i.e., no effect). Included in this group are large- and small-microvessel responses to L-cysteine and D-cysteine alone, as well as small-vessel responses to D-cysteine plus nitroglycerin. The second group is composed of small- and large-microvessel responses to S-nitrosoL-cysteine, S-nitroso-D-cysteine, and L-cysteine plus nitroglycerin, as well as large-microvessel responses to D-cysteine plus nitroglycerin. Responses to these agents were found to be similar to the response evoked by nitroglycerin in large coronary arterioles. Discussion The present experiments provide important new information regarding the biotransformation of nitroglycerin to its active vasodilator metabolite. By studying vessels in vivo that do not normally relax in response to nitroglycerin, we have tried to clarify the 350 biochemical intermediates in this pathway. We have further tried to provide insight into why nitroglycerin does not dilate small coronary microvessels. Our findings clearly show that coronary arterioles with diameters less than approximately 100 gm do not dilate in response to nitroglycerin, thus confirming the reports of previous investigators.45 We also demonstrated for the first time that a putative metabolite of nitroglycerin, S-nitroso-L-cysteine, could relax small coronary resistance vessels in vivo, and we were able to show that nitroglycerin evokes relaxation in small coronary microvessels in the presence of exogenous L-cysteine. Mechanism of Nitroglycerin-Induced Dilation NO, or an NO-containing compound, generally has been accepted as the active vasodilator metabolite of nitroglycerin, but the mechanism by which nitroglycerin is metabolized to NO has not been determined 45 r A O0 CONTROL Dc 100 pm ,10 WOOm CONTROL D cc 30 W r 0 E z =L 15 CM 100 oL 0 CONTROL GTH FIGURE 1. Coronary arteriolar responses to 10 pMnitroglycerin (GTN). Large symbols represent means for groups with control diameters less than 100 ,an (open circles) and control diameters greater than 100 tam (filled circles) ±SEM. Nitroglycerin dilated large vessels from 183±12 to 218 +15 ,um but had no effect on small vessels (58.5+5 to 58.9+5 pm). *Different from control value, p <0. 05. -9 -8 -7 -6 -5 -4 log [L-cysNO] FIGURE 2. Dose-response curve for S-nitroso-L-cysteine (L-cysNO). Significant dilation occurred with 1 pML-cysNO. Maximum response in large vessels occurred with 1 ttM L-cysNO; maximum response in small vessels occurred at 10 pM. *Significant response, p<O.05. tDifferent from largevessel response, p <0. 05. D, diameter. Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014 852 Circulation Research Vol 68, No 3 March 1991 300 250 r r 0 0~~~~~~~ E 200 W0 =L ~~~~~0 0 Ul =L c- W ~~~~0 OD a 150 0 100 . * 50 L CYSTEINE 0 CONTROL L-cysNO FIGURE 3. Changes in coronaty microvascular diameter evoked by 10 pM S-nitroso-L-cysteine (L-cysNO). Large vessels dilated from 180±12 to 211±12 pum; small vessels dilated from 74+6 to 102±9 pum. *Different from control value, p <0.05. Large symbols represent means for groups with control diameters less than 100 pum (open circles) and control diameters greater than 100 pum (filled circles) ±SEM. precisely. A few recent reports have suggested that hepatic cytochrome P-450 is capable of denitrating nitroglycerin and releasing NO.27'28 However, because this reaction is strongly inhibited by oxygen, its importance in the vasodilatory response to nitroglyc300 [ 200~ =L c- a 100 Q 0 T. GOTO oyN CYSTEINE + GTN FIGURE 5. Effects of L-cysteine (open symbols) and D-cysteine (filled symbols) on the coronary microvascular response to nitroglycerin (GTN). Group with control diameters greater than 100 pm is shown with boxes; group with control diameter less than 100 tun is shown with circles. In the presence of exogenous L-cysteine, nitroglycenn evoked dilation in small vessels (61±5 to 75±8 pm, 22±4%o) similar to that evoked in large vessels (159±13 to 198±14 pm, 27±6%). D-Cysteine did not alter the response to nitroglycerin in eithergroup (small vessels: 55±3 to 59±6 pm, 7±4%o, p=NS; large vessels: 147±19 to 174±22 pm, 18±3%). *Significant response, p < 0. 05. tDifferent from small-vessel response, p < 0. 05. erin under normal physiological conditions is not clear. A majority of the literature suggests that interaction with specific sulfhydryl-containing groups, such as cysteine, is necessary for nitroglycerin to activate guanylate cyclase and produce vasodilation. Based on this literature and the present findings, we suggest the following mechanism of action for nitroglycerin (Figure 7). Nitroglycerin interacts with a thiol, probably cysteine, to produce a nitrosothiol (S-nitroso-Lcysteine). Previous investigators have suggested that this biotransformation involves two separate sulffhydryl-requiring steps.'6,17 It has been suggested that the first step is an oxidation of cysteine to cystine, which results in the production of nitrite ion (NO2 ). NO2 then is protonated to nitrous acid (HONO), 0 FIGURE 4. Changes in coronary microvascular diameter evoked by 10 MriM S-nitroso-D-cysteine (D-cysNO). Large vessels dilated from 148+16 to 189±19 pum; small vessels dilated from 67±6 to 88±9 pum. *Different from control value, p <0.05. Large symbols represent means for groups with control diameters less than 100 pm (open circles) and control diameters greater than 100 (filled circles) ±SEM. pm which then interacts with a second sulfhiydryl group to produce a nitrosothiol.29 This mechanism seems improbable, however, because NO2 is a weak vasodilator compared with nitroglycerin. If nitroglycerin had to be converted to NO2 to be activated, then its potency could not exceed that of NO2J. Furthermore, the pK of NO2- does not favor the formation of HONO under physiological conditions. A more likely TABLE 2. Left Ventricular Epicardial Coronary Microvascular Responses to L-Cysteine or D-Cysteine Baseline Thiol L-cys, D<100 ,m (n=17) 63±5 61+5 185+14 L-cys, D>100 gm (n=15) 175±14 54±4 D-cys, D<100 gm (n=14) 55+3 155+±20 D-cys, D.100 gm (n=8) Values are mean+SEM. L-cys, L-Cysteine; D-cys, D-cysteine; D, diameter. 147±+19 Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014 p NS NS NS NS Kurz et al Heterogeneous Microvascular Responses to Nitroglycerin 50 r t El CTRDc100O±m E CTR D> 100pmn lr t T LW -J 0 25 853 f U 0 IL 0 FIGURE 6. Summary of responses to nitroglycerin (GTN), S-nitrosocysteine, and cysteine. Responses fall into two groups: those similar to the small-vessel nitroglycerin response (L-cysteine [L-CYS], D-cysteine [D-CYS], and small-vessel D-CYS+GTN); and those similar to the largevessel nitroglycerin response (S-nitroso-L-cysteine [L-cYsNO], S-nitroso-D-cysteine [D-cYsNO], L-CYS+GTN, and large-vessel D-CYS+GTN). *Different from small-vessel response, p<0.05. tDifferent from large-vessel GTN response, p<O.OS. CTR D, control diameter. -10 L GTN L-CYS L-CYS + GTN D-CYS D-CYS CYSNO D-CYSNO + GTN mechanism would involve a direct conversion of nitroglycerin to a nitrosothiol, probably accomplished by an enzymatic mechanism. It is not clear whether the initial interaction between nitroglycerin and cysteine occurs inside the vascular smooth muscle cell or in the extracellular space. In the present study, S-nitroso-D-cysteine was found to be as potent a dilator of small microvessels as S-nitroso-L-cysteine, indicating that nitrosothiol intermediates can evoke relaxation without entering the cell, presumably by releasing NO at the cell membrane. Although this does not directly support previous evidence of an extracellular pathway for the interaction of nitroglycerin and cysteine,20,30 it does suggest that a metabolite of nitroglycerin can evoke vascular relaxation from the extracellular space. The most likely mechanism by which NO release would occur is by a one-electron reduction. This reaction would result in the release of a thiol ion (SH) and the NO radical. Because this reduction is not likely to FIGURE 7. Proposed mechanism of nitroglycerin (GTN) -induced activation ofguanylate cyclase (GC). L-Cysteine (orpossibly another thiol-containing group, L-R-SH) potentiates the effect of nitroglycerin in small vessels either by interacting with nitroglycerin at the cell surface to form an S-nitroso-L-thiol (L-R'-S-NO) or by being transported into the cell and interacting with nitroglycerin there to produce L-RK-S-NO. S-Nitrosothiols containing dextrorotary R'-groups (D-R'-S-NO) such as S-nitroso-D-cysteine also are potent dilators of all size classes of coronary arterioles, presumably via reduction at the cell membrane and release offree nitric oxide (NO). D-oriented R-SH groups such as D-cysteine, however, do not interact with nitroglycerin to form vasodilator compounds. For further description, see "Discussion. " Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014 854 Circulation Research Vol 68, No 3 March 1991 occur in the oxygen-rich interstitium, we propose that an electron is donated by the vascular smooth muscle sarcolemma to facilitate the reduction (see Figure 7). Preliminary evidence in our laboratory indicates that vascular smooth muscle cells possess the capacity to donate electrons in this manner. Another significant finding of the present study was that nitroglycerin relaxed small coronary microvessels in the presence of exogenous L-cysteine, but not D-cysteine, indicating that a stereospecific process was involved in the biotransformation. It is interesting to speculate that the biotransformation of nitroglycerin requires an enzyme, likely associated with the plasma membrane, which produces a nitrosothiol from nitroglycerin and cysteine. Indeed, a very recent report by Chung and Fung31 indicates that the biotransformation of nitroglycerin occurs primarily in the plasma membrane fragment of vascular smooth muscle cells. Mechanism Responsible for Heterogeneity of Nitroglycerin Response Many investigators have demonstrated that thiols are required for nitroglycerin to evoke vasodilation.8 13,14-20 The present findings suggest that small coronary resistance vessels are unresponsive to nitroglycerin because these thiol groups are not available in sufficient quantity for the biotransformation process. Although it is clearly implausible that thiolcontaining compounds are absent in the smooth muscle cells of small coronary resistance vessels, it is conceivable that specific pools of sulfhydryl groups, which are necessary for the biotransformation of nitroglycerin and are present in large vessels, are lacking in very small vessels. Exogenous cysteine had no effect on larger microvessels in the present study, possibly because these vessels already possessed a sufficient supply of available sulfhydryl compounds. This essential store of sulfhydryl groups would most likely be associated with the interior of the vascular smooth muscle cell, because if it were a plasma or interstitial pool, it would be available to all size classes of coronary microvessels. The "pool" also apparently is not located on the outside of the smooth muscle membrane, because Moffat et a132 have reported that inactivation of membrane-bound sulfhydryl groups does not affect the response to nitroglycerin in canine saphenous vein. The concept that a sulthydryl "pool" exists is new. Depletion of "available sulfhydryl" groups has long been implicated in the development of tolerance to nitroglycerin and other nitrovasodilators.13,14,20,33,34 The preponderance of evidence suggests that nitrate tolerance can be reduced or reversed by the administration of cysteine either directly or via a "delivery system" such as N-acetylcysteine20,33,34 or L-2-oxothiazolidine-4-carboxylate,35 although a few recent studies have presented evidence to the contrary.30,3236 Most of these studies used large-vessel preparations such as aortic strips or epicardial coronary artery rings, and so their implications for small arteriole mechanisms are limited. Winniford et a137 showed, however, that intracoronary infusion of N-acetylcysteine in humans greatly potentiated the coronary blood flow response to nitroglycerin. Horowitz et a138 also have shown that N-acetylcysteine potentiates systemic vascular responses to nitroglycerin. These studies strongly suggest that the addition of cysteine residues potentiates the vasodilatory response to nitroglycerin in small arterial resistance vessels. The present study provides the first direct in vivo evidence to support this suggestion. The finding that nitroglycerin is a potent dilator of small coronary resistance vessels in the presence of exogenous cysteine may have important clinical implications. A clinical strategy that has been used to try to potentiate the vasodilatory effects of nitrates or reverse nitrate tolerance involves concurrent administration of a sulfhydryl-containing agent, most often N-acetylcysteine, with nitroglycerin. Our data suggest that the improvement in flow caused by concurrent administration of cysteine and nitroglycerin is likely due to dilation of small coronary resistance vessels rather than to an increased dilation of larger epicardial vessels. 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