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Direct Measurement of Nitric Oxide Generation in the Ischemic Heart Using Electron Paramagnetic Resonance Spectroscopy

Direct Measurement of Nitric Oxide Generation in the Ischemic Heart Using Electron Paramagnetic... THE JOURNAL OF BIOLOGICAL CHEMISTRY VoL 270, No. I, Issue of January 6, pp, 304-307, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Direct Measurement of Nitric Oxide Generation in the Ischemic Heart Using Electron Paramagnetic Resonance Spectroscopy* (Received for publication, August 26, 1994, and in revised form, September 27, 1994) Jay L. Zweier:j:, Penghai Wang, and Periannan Kuppusamy From the Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology, and the Electron Paramagnetic Resonance Center, The Johns Hopkins Medical Institutions, Johns Hopkins Bayview Medical Center, Baltimore, Maryland 21224 to the formation of NO' (3-5). Its synthesis was first discovered Nitric oxide, NO', exerts numerous important regula­ tory functions in biological tissues and has been hypoth­ in 1987 in macrophages and endothelial cells, and since that esized to have a role in the pathogenesis of cellular time, NO' has been shown to have effects on target cells in injury in a number of diseases. It has been suggested many tissues (3-6). NO' is now known to play an important that alterations in NO' generation are a critical cause of role in blood pressure regulation, vascular tone, neural signal­ injury in the ischemic heart. However, the precise alter­ ing, and immunological function (7-9). It is known to induce ations in NO' generation which occur are not known, the formation of the second messenger molecule cyclic GMP in and there is considerable controversy regarding both the generating and the target cells (10). It has been whether myocardial ischemia results in increased or demonstrated that there is an enzyme present in macrophages, decreased NO' formation. Therefore, electron paramag­ endothelial cells, and neuronal cells which synthesizes NO' netic resonance studies were performed to directly meas­ from arginine (11, 12). This enzyme, nitric oxide synthase, ure NO' in isolated rat hearts subjected to global ische­ exists in two major forms with the macrophage enzyme differ­ mia, using the direct NO' trap Fe +-N-methyl-n­ ing substantially from the brain and endothelial forms both of glucamine dithiocarbamate, which specifically binds which are quite similar, if not identical. There is evidence that NO' giving rise to a characteristic triplet EPR spectrum NO' may also have an important role as a mediator of tissue with g = 2.04 and aN = 13.2 G. While only a small triplet injury (13). signal was observed in normally perfused hearts, a 10­ It has been suggested that alterations in the generation of fold increase in this triplet EPR spectrum was observed NO' occur in tissues subjected to ischemia, and that these after 30 min of ischemia indicating a marked increase in alterations result in altered endothelial function with altered NO' formation and trapping. Measurements were per­ tissue perfusion on subsequent reperfusion (14, 15). In partic­ formed as a function of the duration of ischemia, and it was determined that with increased duration of ische­ ular, there has been considerable controversy regarding the mia NO' formation and trapping was also increased. NO' effect of ischemia on NO' generation in the heart. Studies of generation was inhibited by the nitric oxide synthase endothelial function have been interpreted to suggest that the blocker, N-nitro-L-arginine methyl ester (L-NAME), sug­ process ofischemia decreases NO' generation, and that a loss of gesting that NO' was generated via nitric oxide syn­ basal NO' production is an important source of injury in hearts thase. Blockade of NO' generation with L-NAME re­ subjected to ischemia (14, 15). Subsequently, other studies sulted in more than a 2-fold increase in the recovery of have been reported which demonstrate that inhibitors of nitric contractile function in hearts reperfused after 30 min of oxide synthase can protect against ischemic injury (16). From global ischemia. Thus, ischemia causes a marked dura­ these later studies it was suggested that NO· may be involved tion-dependent increase of NO' in the heart which may in the process of tissue injury and that the production of NO' in turn mediate postischemic injury. may actually be increased by the process of ischemia, rather than decreased. Thus, indirect assessment of NO' generation from measurements of organ function have resulted in consid­ Over the last several years it has been demonstrated that the erable controversy and uncertainty regarding the effect of is­ gaseous free radical nitric oxide, NO', is generated in biological chemia on NO' generation in the heart. cells and tissues and is of central importance in regulating a While there has been a great need for techniques of directly broad range of important biological functions (1). In 1980 measuring nitric oxide production in biological systems, there Furchgott and Zawadzki (2) demonstrated that the vascular have been few, ifany, techniques with sufficient sensitivity and relaxation induced by acetylcholine was dependent on the pres­ specificity to provide quantitative measurements over the di­ ence of the endothelium and that this effect was mediated by a verse range of physiological and pathophysiological applica­ labile factor termed endothelial-derived relaxing factor. It was tions of interest. Since NO' is a free radical and reacts to form subsequently hypothesized and then demonstrated that this high affinity nitroso complexes with a variety of metal com­ endothelial-derived relaxing factor activity could be attributed plexes and metalloproteins it has been proposed that the dis­ tinctive EPR spectra of these nitroso complexes could be used * This work was supported in part by National Institutes of Health to serve as a quantitative measure of NO' generation (17-19). Grants HL-17655 and HL-38324. The costs ofpublication ofthis article While measurement of nitroso-heme formation serves as an were defrayedin part by the payment ofpagecharges. This article must intrinsic trap providing a measure of NO' generation, these therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. complexes are labile in the presence of oxygen. The Fe + ­ :j:Supported by an Established Investigator Award from the Ameri­ diethyldithiocarbamate complex has been proposed as a more can Heart Association. To whom correspondence should be addressed: stable and oxygen independent trap suitable for measuring Electron Paramagnetic ResonanceCenter, Johns Hopkins Asthma and NO' generation in biological systems (20). This complex has AllergyCenter, 5501 Hopkins BayviewCircle, Johns Hopkins Medical very limited solubility in water; therefore, recently the similar Institutions, Baltimore, MD 21224. This is an Open Access article under the CC BY license. Nitric Oxide Generation in the Ischemic Heart 305 ferrous iron complex of N-methyl-D-glucamine dithiocarbamate 2+·MGD (MGD),1 Fe (Fe·MGD), has been proposed as an oxy­ gen-stable water soluble NO' trap suitable for measuring nitric oxide in living tissues (21). In this study we have applied EPR spectroscopy to directly measure the effect of ischemia on the generation of NO' in the heart. Using the NO' trap Fe·MGD we observed that nitric oxide is markedly increased during ischemia with the forma­ tion of the characteristic Fe·MGD·NO triplet signal. This NO' formation was largely blocked by inhibition of nitric oxide synthase and the resultant decrease in NO' generation was 3216 3240 3265 3290 3315 associated with a marked improvement in the recovery of con­ Magnetic Field (Gauss) tractile function. FIG. 1. EPR spectra of Fe·MGD in the presence and absence of 2+, NO'. A, spectrum of a preparation of 1 mM Fe 5 mMMGD in 50 mM EXPERIMENTAL PROCEDURES HEPES buffer, pH 7.4. B, after incubation with a 2 mMconcentration of Isolated Heart Perfusion-Female Sprague-Dawley rats (250-300 g) the NO' donor S-nitroso-N-acetylpenicillamine. Spectra were recorded were heparinized and anesthetized with intraperitoneal pentobarbital. at 77 K with microwave frequency of 9.316 GHz using 1.0 milliwatt The hearts were excised, the aorta was cannulated, and retrograde microwave power and a modulation amplitude of 4.0 G. Each spectrum perfusion was initiated. Hearts were perfused at a constant pressure of is a 60-s spectral acquisition of 100 G sweep width with a time constant 80 mm Hg using Krebs bicarbonate buffer (17 mM glucose, 120 mx of 0.32 S. sodium chloride, 25 mM sodium bicarbonate, 2.5 mM calcium chloride, 0.5 mM EDTA, 5.9 mM potassium chloride, and 1.2 mM magnesium chloride) bubbled with 95% O and 5% CO gas at 37 "C, as described 2 2 previously (22). A sidearm in the perfusion line allowed direct infusion of the Fe·MGD NO' trap just proximal to the heart. In order to measure contractile function a latex balloon was inserted into the left ventricular cavity and connected to a pressure transducer via a hydraulic line and pressures recorded with a Gould RS4000 recorder. The balloon was initially inflated to achieve an end-diastolic pressure of 8-14 mm Hg. Left ventricular pressures, heart rate, and coronary flow were moni­ tored throughout the period of perfusion. Hearts were rapidly frozen using liquid nitrogen cooled Wollenberger tongs. The frozen tissue was maintained at 77 K in liquid nitrogen and either ground or fractured to 1-2-mm particles and transferred to 3-mm EPR tubes. Alternatively, to 3240 3265 3290 3316 minimize processing the tissue was fractured to 3-4-mm pieces and Magnetic Field (Gauss) placed directly within the EPR finger Dewar. Similar spectra were observed with measurements in 3-mm tubes or directly in the EPR FIG. 2. EPR spectra of NO' trapping in hearts labeled with Dewar. The EPR tube or Dewar was filled to a sufficient height to fill Fe·MGD. A, frozen tissue from a normally perfused heart. B, frozen the critical volume of the EPR cavity. tissue from a heart subjected to 30 min of ischemia. Spectra were recorded with a microwave frequency of 9.323 GHz using 1.0 milliwatt Materials-MGD was synthesized as described previously (23). The microwave power and a modulation amplitude of 4.0 G. Each spectrum N-methyl-D-glucamine and carbon disulfide required for this synthesis was obtained from the sum of 20, 60-s spectral acquisitions of 100-G were purchased from Aldrich. S-Nitroso-N-acetylpenicillamine, was ob­ sweep width with a time constant of 0.32 S. tained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). N-Nitro-L-arginine methyl ester, L-NAME, and all other reagents 2+·MGD were obtained from Sigma. Fresh stock solutions of the Fe seen (Fig. 2A). In hearts subjected to ischemic durations of 30 complex were prepared by addition ferrous ammonium sulfate to aque­ min, however, a prominent triplet NO adduct signal appeared ous solutions of MGD at a ratio of 1:5. The final concentration of the 2+. (Fig. 2B). In these hearts additional intrinsic signals were seen complex used in the heart was 1 mM in Fe EPR Spectroscopy-EPR spectra were recorded at 77 K using a liquid from the 1 electron reduced ubiquinone radical, centered at g = nitrogen Dewar with a Bruker ER 300 spectrometer operating at X­ 2.004 and from ROO' at g = 2.005, as well as from iron sulfur band with 100 kHz modulation frequency and a TMllO cavity, as proteins at g = 1.94, as reported and assigned previously (24, described previously (24). The microwave frequency and magnetic field 25). These radical signals with absorption functions in the g = were precisely measured using an EIP 575 microwave frequency coun­ 2.008-2.000 region are located just up-field from the NO' tri­ ter and Bruker ER 035M NMR gaussmeter. Relative quantitation of the free radical signals was performed by double integration. plet and are seen as the positive deflections at the high field end of the spectra. In a series of 5 control and 5 hearts sub­ RESULTS jected to 30 min of ischemia, the intensity of the NO' triplet In the absence of NO', no triplet EPR spectrum is observed was observed to be 10.5 ± 2.1-fold higher after 30 min of from the Fe·MGD complex (Fig. lA). After addition of the NO' ischemia than in controls. donor compound S-nitrosoacetylpenicillamine, a prominent tri­ Further experiments were performed to determine the effect plet EPR spectrum is observed with a central g value of 2.04 of the duration of ischemia on the magnitude of the NO' signal. and hyperfine splitting of 13.2 G (Fig. lB). After 10 min of ischemia only a trace triplet signal was seen After cannulation and a 15-min period of control perfusion and after 20 min this signal increased. With 30 or 60 min of with equilibration of contractile function, developed pressures ischemia, further marked increases in this triplet signal were of 120 ± 10 mm Hg were observed, and hearts were then seen (Fig. 3). Relative quantitation ofthis signal was performed infused and loaded with a 1 mM concentration of the Fe·MGD by double integration, and these results demonstrated that the 2+, complex (1 mM Fe 5 mM MGD) for 5 min. The hearts were magnitude of the triplet spectrum of trapped NO' progressively then immediately frozen or subjected to no flow ischemia and increased as a function of the duration of ischemia (Fig. 4). then frozen at 77 K. In normally perfused control hearts only a In order to determine if the NO' generation observed during very weak spectrum of the triplet NO, Fe + .MGD complex was ischemia was derived from nitric oxide synthase, experiments were performed in hearts treated with the blocker L-NAME. In hearts pretreated with a 1.0 mMconcentration of L-NAME for 1 The abbreviations used are: MGD, N-methyl-D-glucamine dithiocar­ bamate; L-NAME, N-nitro-L-arginine methyl ester. at least 15 min prior to the onset of ischemia, a 70-80% 306 Nitric Oxide Generation in the Ischemic Heart 3216 3240 3265 3290 3315 Magnetic Field (Gauss) FIG. 5. Effect of the nitric oxide synthase blocker L·NAME on 3215 3240 3265 3290 3315 the EPR spectra of hearts labeled with Fe·MGD. A, spectrum of Magnetic Field (Gauss) tissue frozen after 30 min of ischemia in the absence of the blocker. B, FIG. 3. EPR spectra recorded from hearts subjected to varying spectrum of tissue after 30 min of ischemia in the presence of 1.0 mM L-NAME. Spectra were recorded as described in the legend to Fig. 2. duration of ischemia followed by freezing at 77 K. Hearts were labeled with Fe·MGD and spectra recorded as described in the legend to Fig. 2. A, 10 min of ischemia; B, 20 min of ischemia; C, 30 min of 80 ischemia; D, 60 min of ischemia. iii 120 :t t: ::> iii 90 ~ 50 .. 0: E! ~ 60 <Il W '0 I- ~ 30 ..J ~ " -e- Control 00 ~ iii 10 '" 30 60 15 45 _ L-NAME ISCHEMIC DURATION (MIN) 10 15 20 25 30 35 40 45 FIG. 4. Graph of the effect of ischemic duration on the Inten­ Time of Reperfusion (min) sity ofthe observed triplet EPR signal of NO·Fe·+·MGD. FIG. 6. Measurement of the recovery of coronary flow in un­ treated control hearts or hearts pretreated with the nitric oxide decrease in the NO·Fe + ·M GD signal was observed (Fig. 5). synthase blocker L.NAME, 1.0 IDM. Hearts were subjected to 30 min Thus, most of the NO' generated and trapped during ischemia of global ischemia followed by 45 min of reflow. Data are expressed as was generated by nitric oxide synthase. percent recovery of preischemic values. The lower values of coronary Further experiments were performed to determine if block­ flow observed in L-NAME-treated hearts than in the untreated control hearts are consistent with the inhibition of NO' generation by this ing nitric oxide generation in the ischemic heart would amelio­ blocker. rate or exacerbate the functional injury which occurs upon postischemic reperfusion. Hearts were subjected to 30 min of 37 °C global ischemia and 45 min ofreperfusion with continu­ -e- Control ous measurement of contractile function and coronary flow. __ L-NAME Eight hearts were studied, 4 untreated and 4 pretreated for 15 min with 1.0 mM L-NAME. As expected in L-NAME-treated ... hearts, lower coronary flow was observed due to the loss of g ..J NO'-induced vasodilation. Throughout the 45-min period of 20 '0 reflow the coronary flow was decreased by almost 2-fold (Fig. .. 6). This decrease in coronary flow in the L-NAME-treated .. a: hearts was highly significant with p < 0.01. In spite of this decreased coronary flow, much higher recovery of contractile function was observed throughout the period of reperfusion with more than a 2-fold increase in the recovery of left ventric­ 10 15 20 25 30 35 40 45 ular developed pressure (Fig. 7). This increase in left ventric­ Time of Reperfusion (min) ular developed pressure was also highly significant with p < FIG. 7. Measurement ofthe recovery ofcontractile function in 0.01. Thus, inhibition of the marked increase in the NO' formed untreated control hearts or hearts pretreated with the nitric during ischemia resulted in significantly improved recovery of oxide synthase blocker L-NAME, 1.0 IDM. Hearts were subjected to 30 min of global ischemia followed by 45 min of reflow. Left ventricular contractile function in the postischemic heart. developed pressures (LVDP) were measured before ischemia and after DISCUSSION postischemic reflow. Data are expressed as percent recovery of preis­ chemic values. Hearts treated with L-NAME exhibited significantly Since the time that it was first demonstrated that biological higher recovery of LVDP than in untreated control hearts. cells and tissues generate nitric oxide, this free radical has been shown to exert a variety of important functions including modu­ reactive oxidant peroxynitrite (14-16). Based on these opposite lation of vascular tone, neural signaling, and immune response (7,8). In the setting ofischemic injury NO' could exert protective effects, different physiological studies of the ischemic and postis­ chemic heart have been interpreted to suggest that NO' genera­ effects by increasing coronary flow or harmful effects with cellu­ lar injury resulting from the reaction with superoxide to form the tion may be markedly decreased or markedly increased. Nitric Oxide Generation in the Ischemic Heart 307 In this study we have performed measurements to directly during postischemic reperfusion (29). measure the alterations in NO' generation which occur in the Thus, ischemia triggers both the generation of superoxide as ischemic heart and to determine the functional consequences of well as increased amounts of NO'. This increased production of this NO' generation on the recovery of contractile function. NO' and superoxide in ischemic and postischemic myocardium Measurements of the magnitude of the triplet signal formed on could result in the formation of the more potent oxidant per­ trapping of NO' by Fe·MGD were performed and enabled rel­ oxynitrite which is known to cause cellular injury (13). These ative quantitation of the concentration of NO' in the heart. It observations are consistent with the recent studies which have has recently been reported that the Fe·MGD complex is an reported that treatment with nitric oxide synthase blockers can ideal transition metal complex for the trapping and measure­ prevent postischemic injury (16). While further studies will be ment of NO' in living tissues and in vivo animals (21). In our required to fully understand the role of NO' in the process of studies we also observed that the Fe· MGD complex was highly ischemic heart disease, the present study demonstrates that a soluble in aqueous solution and that the NO·Fe·MGD complex large increase in the formation and accumulation of NO' occurs was stable and observable in aerobic solutions. A characteristic during myocardial ischemia and this NO' in turn contributes to triplet spectrum was observed due to the coupling of the un­ the process of postischemic injury. paired electron to the nitrogen nucleus of NO', which for the natural abundance isotope 14N has nuclear spin, I = 1. This REFERENCES characteristic spectrum is centered at g = 2.04 with a nitrogen 1. Moncada, S., Palmer, R. M., Jr., and Higgs, E. A. (1991) Pharmacol. Rev. 43, hyperfine coupling aN = 13.2 G. In our experiments we ob­ 109-142 2. Furchgott, R. F., and Zawadzki, J. V. (1980) Nature 288, 373-376 served that this trap was non-toxic in the concentrations used 3. Palmer, R. M. J., Ferridge, A. G., and Moncada, S. (1987) Nature 327, 524-526 and could be directly perfused into the heart without adverse 4. Ignarro, L. J., Byrns, R. E., Buga, G. M., and Wood, K. S. (1987) Circ. Res. 61, 866-879 effect. We observed that NO' generation is markedly increased 5. Furchgott, R. F., and Vanhoutte, P. M. (1989) FASEB J. 3,2007-2018 during ischemia as evidenced by the appearance of the charac­ 6. Marietta, M. A., Yoon, P. S., Iyengar, R., Leaf, C. D., and Wishnok, J. S. (1988) teristic triplet EPR signal on direct trapping with Fe·MGD. A Biochemistry 27, 8706-8711 7. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1989) Biochem. Pharmacol. progressive increase in the NO' adduct was seen as a function 38,1709-1715 of the duration of ischemia with more than a lO-fold increase 8. Garthwaite, J. (1991) Trends Neurosci. 14,60-67 after 30 min of ischemia. When hearts were pretreated with the 9. Langreh, J. M., Hoffman, R. A., Lancaster, J. R., Jr., and Simmons, R. L. (1993) Transplantation 55, 1205-1212 specific nitric oxide synthase blocker, L-NAME, the generation 10. Ignarro, L. J., Adams, J. B., Horwitz, P. M., and Woods, K. S. (1986) J. Bioi. of this NO' triplet was decreased by 70-80%, demonstrating Chem. 261,4997-5002 that the generation of NO' was largely derived from nitric oxide 11. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Snyder S. H. (1991) Nature 351, 714-718 synthase. In hearts in which NO' generation was blocked with 12. Bredt, D. S., and Synder, S. H. (1992) Neuron 8, 3-11 L-NAME, a marked cardioprotective effect was observed with 13. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. more than a 2-fold increase in the recovery of contractile func­ (1990) Proc. Nat!. Acad. Sci. U. S. A. 87, 1620-1624 14. Lefer, A. M., Tsao, P. S., Lefer, D. J., and Ma, X. L. (1991) FASEB J. 5, tion. Thus, the marked increase in NO' within the ischemic 2029-2034 heart is associated with the process of postischemic injury. This 15. Tsao, P. S., and Lefer, A. M. (1990) Am. J. Physiol. 259, H1660-1666 16. Matheis, G., Sherman, M. P., Buckberg, G. D., Haybron, D. M., Young, H. H., apparent toxicity may be due to the reaction of NO' with and Ignarro, L. J. (1992) Am. J. Physiol. 262, H616-620 superoxide (13). 17. Lancaster, J. R., Jr., Langreh, J. M., Bergonia, H. A., Murase, N., Simmons, R. There is considerable direct and indirect evidence that su­ L., and Hoffman, R. A. (1992) J. Bioi. Chem. 267, 10994-10998 18. Langreh, J. M., Muller, A. R., Bergonia, H. A., Jacob, T. D., Lee, T. K., Schaut, peroxide and superoxide-derived free radicals are generated in W. H., Lancaster, J. R., Jr., Hoffman, R. A., and Simmons, R. L. (1992) ischemic and reperfused myocardium. It has been demon­ Surgery 112, 395-402 strated that superoxide dismutase treatment during ischemia 19. Greenberg, S. S., Wilcox, D. E., and Rubanyi, G. M. (1990) Circ. Res. 67, 1446-1452 and reperfusion can prevent functional injury and cell death 20. Mordvintcev, P., Mulsch, A., Busse, R., and Vanin, A. (1991) Anal. Biochem. (26, 27). Direct and spin trapping EPR studies have provided 199, 142-146 21. Lai, C. S., and Komarov, A. M. (1994) FEBS Lett. 345, 120-124 direct evidence that oxygen free radicals are generated in the 22. Zweier, J. L. (1988) J. Bioi. Chem. 263, 1353-1357 postischemic heart (24, 25). Further studies have demon­ 23. Shinoby, L. A., Jones, S. G., and Jones, M. M. (1984) Acta Pharmacol. Toxicol. strated that superoxide dismutase can block free radical gen­ 54, 189-194 24. Zweier, J. L., Kuppusamy, P., Williams, R., Rayburn, B. K., Smith, D., Weis­ eration and subsequent contractile dysfunction which occurs feldt, M. L., and Flaherty, J. T. (1989) J. Bioi. Chem. 264, 18890-18895 after postischemic reperfusion (22). It has also been shown that 25. Zweier, J. L., Flaherty, J., and Weisfeldt, M. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1404-1407 reoxygenated endothelial cells give rise to a burst offree radical 26. Shlafer, M., Kane, P. F., and Kirsch, M. M. (1982) J. Thorac. Cardiovasc. Surg. generation and suggested that the endothelial cell is an impor­ 83,830-839 tant site of free radical generation in the heart (28, 29). In 27. Jolly, S. R., Kane, W. J., Bailie, M. B., Abrams, G. D., and Lucchesi, B. R. (1984) Circ. Res. 54,277-285 particular, it has been demonstrated that the superoxide gen­ 28. Zweier, J. L., Kuppusamy, P., and Lutty, G. A. (1988) Proc. Nat!. Acad. Sci. erating enzyme xanthine oxidase is present within vascular U. S. A. 85,4046-4050 29. Thompson-Gorman, S. L., and Zweier, J. L. (1990) J. BioI. Chem. 265,6656­ endothelium of human, bovine, and rat aortic endothelial cells (28-30). This enzyme is present in the rat heart and is respon­ 30. Zweier, J. L., Broderick, R., Kuppusamy, P., Thompson-Gorman, S., and Lutty, sible in part for the burst of radical generation which occurs G. A. (1994) J. Bioi. Chem. 269, 24156-24162 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Direct Measurement of Nitric Oxide Generation in the Ischemic Heart Using Electron Paramagnetic Resonance Spectroscopy

Zweier, Jay L.; Wang, Penghai; Kuppusamy, Periannan
Journal of Biological Chemistry – Jan 1, 1995
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VoL 270, No. I, Issue of January 6, pp, 304-307, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Direct Measurement of Nitric Oxide Generation in the Ischemic Heart Using Electron Paramagnetic Resonance Spectroscopy* (Received for publication, August 26, 1994, and in revised form, September 27, 1994) Jay L. Zweier:j:, Penghai Wang, and Periannan Kuppusamy From the Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology, and the Electron Paramagnetic Resonance Center, The Johns Hopkins Medical Institutions, Johns Hopkins Bayview Medical Center, Baltimore, Maryland 21224 to the formation of NO' (3-5). Its synthesis was first discovered Nitric oxide, NO', exerts numerous important regula­ tory functions in biological tissues and has been hypoth­ in 1987 in macrophages and endothelial cells, and since that esized to have a role in the pathogenesis of cellular time, NO' has been shown to have effects on target cells in injury in a number of diseases. It has been suggested many tissues (3-6). NO' is now known to play an important that alterations in NO' generation are a critical cause of role in blood pressure regulation, vascular tone, neural signal­ injury in the ischemic heart. However, the precise alter­ ing, and immunological function (7-9). It is known to induce ations in NO' generation which occur are not known, the formation of the second messenger molecule cyclic GMP in and there is considerable controversy regarding both the generating and the target cells (10). It has been whether myocardial ischemia results in increased or demonstrated that there is an enzyme present in macrophages, decreased NO' formation. Therefore, electron paramag­ endothelial cells, and neuronal cells which synthesizes NO' netic resonance studies were performed to directly meas­ from arginine (11, 12). This enzyme, nitric oxide synthase, ure NO' in isolated rat hearts subjected to global ische­ exists in two major forms with the macrophage enzyme differ­ mia, using the direct NO' trap Fe +-N-methyl-n­ ing substantially from the brain and endothelial forms both of glucamine dithiocarbamate, which specifically binds which are quite similar, if not identical. There is evidence that NO' giving rise to a characteristic triplet EPR spectrum NO' may also have an important role as a mediator of tissue with g = 2.04 and aN = 13.2 G. While only a small triplet injury (13). signal was observed in normally perfused hearts, a 10­ It has been suggested that alterations in the generation of fold increase in this triplet EPR spectrum was observed NO' occur in tissues subjected to ischemia, and that these after 30 min of ischemia indicating a marked increase in alterations result in altered endothelial function with altered NO' formation and trapping. Measurements were per­ tissue perfusion on subsequent reperfusion (14, 15). In partic­ formed as a function of the duration of ischemia, and it was determined that with increased duration of ische­ ular, there has been considerable controversy regarding the mia NO' formation and trapping was also increased. NO' effect of ischemia on NO' generation in the heart. Studies of generation was inhibited by the nitric oxide synthase endothelial function have been interpreted to suggest that the blocker, N-nitro-L-arginine methyl ester (L-NAME), sug­ process ofischemia decreases NO' generation, and that a loss of gesting that NO' was generated via nitric oxide syn­ basal NO' production is an important source of injury in hearts thase. Blockade of NO' generation with L-NAME re­ subjected to ischemia (14, 15). Subsequently, other studies sulted in more than a 2-fold increase in the recovery of have been reported which demonstrate that inhibitors of nitric contractile function in hearts reperfused after 30 min of oxide synthase can protect against ischemic injury (16). From global ischemia. Thus, ischemia causes a marked dura­ these later studies it was suggested that NO· may be involved tion-dependent increase of NO' in the heart which may in the process of tissue injury and that the production of NO' in turn mediate postischemic injury. may actually be increased by the process of ischemia, rather than decreased. Thus, indirect assessment of NO' generation from measurements of organ function have resulted in consid­ Over the last several years it has been demonstrated that the erable controversy and uncertainty regarding the effect of is­ gaseous free radical nitric oxide, NO', is generated in biological chemia on NO' generation in the heart. cells and tissues and is of central importance in regulating a While there has been a great need for techniques of directly broad range of important biological functions (1). In 1980 measuring nitric oxide production in biological systems, there Furchgott and Zawadzki (2) demonstrated that the vascular have been few, ifany, techniques with sufficient sensitivity and relaxation induced by acetylcholine was dependent on the pres­ specificity to provide quantitative measurements over the di­ ence of the endothelium and that this effect was mediated by a verse range of physiological and pathophysiological applica­ labile factor termed endothelial-derived relaxing factor. It was tions of interest. Since NO' is a free radical and reacts to form subsequently hypothesized and then demonstrated that this high affinity nitroso complexes with a variety of metal com­ endothelial-derived relaxing factor activity could be attributed plexes and metalloproteins it has been proposed that the dis­ tinctive EPR spectra of these nitroso complexes could be used * This work was supported in part by National Institutes of Health to serve as a quantitative measure of NO' generation (17-19). Grants HL-17655 and HL-38324. The costs ofpublication ofthis article While measurement of nitroso-heme formation serves as an were defrayedin part by the payment ofpagecharges. This article must intrinsic trap providing a measure of NO' generation, these therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. complexes are labile in the presence of oxygen. The Fe + ­ :j:Supported by an Established Investigator Award from the Ameri­ diethyldithiocarbamate complex has been proposed as a more can Heart Association. To whom correspondence should be addressed: stable and oxygen independent trap suitable for measuring Electron Paramagnetic ResonanceCenter, Johns Hopkins Asthma and NO' generation in biological systems (20). This complex has AllergyCenter, 5501 Hopkins BayviewCircle, Johns Hopkins Medical very limited solubility in water; therefore, recently the similar Institutions, Baltimore, MD 21224. This is an Open Access article under the CC BY license. Nitric Oxide Generation in the Ischemic Heart 305 ferrous iron complex of N-methyl-D-glucamine dithiocarbamate 2+·MGD (MGD),1 Fe (Fe·MGD), has been proposed as an oxy­ gen-stable water soluble NO' trap suitable for measuring nitric oxide in living tissues (21). In this study we have applied EPR spectroscopy to directly measure the effect of ischemia on the generation of NO' in the heart. Using the NO' trap Fe·MGD we observed that nitric oxide is markedly increased during ischemia with the forma­ tion of the characteristic Fe·MGD·NO triplet signal. This NO' formation was largely blocked by inhibition of nitric oxide synthase and the resultant decrease in NO' generation was 3216 3240 3265 3290 3315 associated with a marked improvement in the recovery of con­ Magnetic Field (Gauss) tractile function. FIG. 1. EPR spectra of Fe·MGD in the presence and absence of 2+, NO'. A, spectrum of a preparation of 1 mM Fe 5 mMMGD in 50 mM EXPERIMENTAL PROCEDURES HEPES buffer, pH 7.4. B, after incubation with a 2 mMconcentration of Isolated Heart Perfusion-Female Sprague-Dawley rats (250-300 g) the NO' donor S-nitroso-N-acetylpenicillamine. Spectra were recorded were heparinized and anesthetized with intraperitoneal pentobarbital. at 77 K with microwave frequency of 9.316 GHz using 1.0 milliwatt The hearts were excised, the aorta was cannulated, and retrograde microwave power and a modulation amplitude of 4.0 G. Each spectrum perfusion was initiated. Hearts were perfused at a constant pressure of is a 60-s spectral acquisition of 100 G sweep width with a time constant 80 mm Hg using Krebs bicarbonate buffer (17 mM glucose, 120 mx of 0.32 S. sodium chloride, 25 mM sodium bicarbonate, 2.5 mM calcium chloride, 0.5 mM EDTA, 5.9 mM potassium chloride, and 1.2 mM magnesium chloride) bubbled with 95% O and 5% CO gas at 37 "C, as described 2 2 previously (22). A sidearm in the perfusion line allowed direct infusion of the Fe·MGD NO' trap just proximal to the heart. In order to measure contractile function a latex balloon was inserted into the left ventricular cavity and connected to a pressure transducer via a hydraulic line and pressures recorded with a Gould RS4000 recorder. The balloon was initially inflated to achieve an end-diastolic pressure of 8-14 mm Hg. Left ventricular pressures, heart rate, and coronary flow were moni­ tored throughout the period of perfusion. Hearts were rapidly frozen using liquid nitrogen cooled Wollenberger tongs. The frozen tissue was maintained at 77 K in liquid nitrogen and either ground or fractured to 1-2-mm particles and transferred to 3-mm EPR tubes. Alternatively, to 3240 3265 3290 3316 minimize processing the tissue was fractured to 3-4-mm pieces and Magnetic Field (Gauss) placed directly within the EPR finger Dewar. Similar spectra were observed with measurements in 3-mm tubes or directly in the EPR FIG. 2. EPR spectra of NO' trapping in hearts labeled with Dewar. The EPR tube or Dewar was filled to a sufficient height to fill Fe·MGD. A, frozen tissue from a normally perfused heart. B, frozen the critical volume of the EPR cavity. tissue from a heart subjected to 30 min of ischemia. Spectra were recorded with a microwave frequency of 9.323 GHz using 1.0 milliwatt Materials-MGD was synthesized as described previously (23). The microwave power and a modulation amplitude of 4.0 G. Each spectrum N-methyl-D-glucamine and carbon disulfide required for this synthesis was obtained from the sum of 20, 60-s spectral acquisitions of 100-G were purchased from Aldrich. S-Nitroso-N-acetylpenicillamine, was ob­ sweep width with a time constant of 0.32 S. tained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). N-Nitro-L-arginine methyl ester, L-NAME, and all other reagents 2+·MGD were obtained from Sigma. Fresh stock solutions of the Fe seen (Fig. 2A). In hearts subjected to ischemic durations of 30 complex were prepared by addition ferrous ammonium sulfate to aque­ min, however, a prominent triplet NO adduct signal appeared ous solutions of MGD at a ratio of 1:5. The final concentration of the 2+. (Fig. 2B). In these hearts additional intrinsic signals were seen complex used in the heart was 1 mM in Fe EPR Spectroscopy-EPR spectra were recorded at 77 K using a liquid from the 1 electron reduced ubiquinone radical, centered at g = nitrogen Dewar with a Bruker ER 300 spectrometer operating at X­ 2.004 and from ROO' at g = 2.005, as well as from iron sulfur band with 100 kHz modulation frequency and a TMllO cavity, as proteins at g = 1.94, as reported and assigned previously (24, described previously (24). The microwave frequency and magnetic field 25). These radical signals with absorption functions in the g = were precisely measured using an EIP 575 microwave frequency coun­ 2.008-2.000 region are located just up-field from the NO' tri­ ter and Bruker ER 035M NMR gaussmeter. Relative quantitation of the free radical signals was performed by double integration. plet and are seen as the positive deflections at the high field end of the spectra. In a series of 5 control and 5 hearts sub­ RESULTS jected to 30 min of ischemia, the intensity of the NO' triplet In the absence of NO', no triplet EPR spectrum is observed was observed to be 10.5 ± 2.1-fold higher after 30 min of from the Fe·MGD complex (Fig. lA). After addition of the NO' ischemia than in controls. donor compound S-nitrosoacetylpenicillamine, a prominent tri­ Further experiments were performed to determine the effect plet EPR spectrum is observed with a central g value of 2.04 of the duration of ischemia on the magnitude of the NO' signal. and hyperfine splitting of 13.2 G (Fig. lB). After 10 min of ischemia only a trace triplet signal was seen After cannulation and a 15-min period of control perfusion and after 20 min this signal increased. With 30 or 60 min of with equilibration of contractile function, developed pressures ischemia, further marked increases in this triplet signal were of 120 ± 10 mm Hg were observed, and hearts were then seen (Fig. 3). Relative quantitation ofthis signal was performed infused and loaded with a 1 mM concentration of the Fe·MGD by double integration, and these results demonstrated that the 2+, complex (1 mM Fe 5 mM MGD) for 5 min. The hearts were magnitude of the triplet spectrum of trapped NO' progressively then immediately frozen or subjected to no flow ischemia and increased as a function of the duration of ischemia (Fig. 4). then frozen at 77 K. In normally perfused control hearts only a In order to determine if the NO' generation observed during very weak spectrum of the triplet NO, Fe + .MGD complex was ischemia was derived from nitric oxide synthase, experiments were performed in hearts treated with the blocker L-NAME. In hearts pretreated with a 1.0 mMconcentration of L-NAME for 1 The abbreviations used are: MGD, N-methyl-D-glucamine dithiocar­ bamate; L-NAME, N-nitro-L-arginine methyl ester. at least 15 min prior to the onset of ischemia, a 70-80% 306 Nitric Oxide Generation in the Ischemic Heart 3216 3240 3265 3290 3315 Magnetic Field (Gauss) FIG. 5. Effect of the nitric oxide synthase blocker L·NAME on 3215 3240 3265 3290 3315 the EPR spectra of hearts labeled with Fe·MGD. A, spectrum of Magnetic Field (Gauss) tissue frozen after 30 min of ischemia in the absence of the blocker. B, FIG. 3. EPR spectra recorded from hearts subjected to varying spectrum of tissue after 30 min of ischemia in the presence of 1.0 mM L-NAME. Spectra were recorded as described in the legend to Fig. 2. duration of ischemia followed by freezing at 77 K. Hearts were labeled with Fe·MGD and spectra recorded as described in the legend to Fig. 2. A, 10 min of ischemia; B, 20 min of ischemia; C, 30 min of 80 ischemia; D, 60 min of ischemia. iii 120 :t t: ::> iii 90 ~ 50 .. 0: E! ~ 60 <Il W '0 I- ~ 30 ..J ~ " -e- Control 00 ~ iii 10 '" 30 60 15 45 _ L-NAME ISCHEMIC DURATION (MIN) 10 15 20 25 30 35 40 45 FIG. 4. Graph of the effect of ischemic duration on the Inten­ Time of Reperfusion (min) sity ofthe observed triplet EPR signal of NO·Fe·+·MGD. FIG. 6. Measurement of the recovery of coronary flow in un­ treated control hearts or hearts pretreated with the nitric oxide decrease in the NO·Fe + ·M GD signal was observed (Fig. 5). synthase blocker L.NAME, 1.0 IDM. Hearts were subjected to 30 min Thus, most of the NO' generated and trapped during ischemia of global ischemia followed by 45 min of reflow. Data are expressed as was generated by nitric oxide synthase. percent recovery of preischemic values. The lower values of coronary Further experiments were performed to determine if block­ flow observed in L-NAME-treated hearts than in the untreated control hearts are consistent with the inhibition of NO' generation by this ing nitric oxide generation in the ischemic heart would amelio­ blocker. rate or exacerbate the functional injury which occurs upon postischemic reperfusion. Hearts were subjected to 30 min of 37 °C global ischemia and 45 min ofreperfusion with continu­ -e- Control ous measurement of contractile function and coronary flow. __ L-NAME Eight hearts were studied, 4 untreated and 4 pretreated for 15 min with 1.0 mM L-NAME. As expected in L-NAME-treated ... hearts, lower coronary flow was observed due to the loss of g ..J NO'-induced vasodilation. Throughout the 45-min period of 20 '0 reflow the coronary flow was decreased by almost 2-fold (Fig. .. 6). This decrease in coronary flow in the L-NAME-treated .. a: hearts was highly significant with p < 0.01. In spite of this decreased coronary flow, much higher recovery of contractile function was observed throughout the period of reperfusion with more than a 2-fold increase in the recovery of left ventric­ 10 15 20 25 30 35 40 45 ular developed pressure (Fig. 7). This increase in left ventric­ Time of Reperfusion (min) ular developed pressure was also highly significant with p < FIG. 7. Measurement ofthe recovery ofcontractile function in 0.01. Thus, inhibition of the marked increase in the NO' formed untreated control hearts or hearts pretreated with the nitric during ischemia resulted in significantly improved recovery of oxide synthase blocker L-NAME, 1.0 IDM. Hearts were subjected to 30 min of global ischemia followed by 45 min of reflow. Left ventricular contractile function in the postischemic heart. developed pressures (LVDP) were measured before ischemia and after DISCUSSION postischemic reflow. Data are expressed as percent recovery of preis­ chemic values. Hearts treated with L-NAME exhibited significantly Since the time that it was first demonstrated that biological higher recovery of LVDP than in untreated control hearts. cells and tissues generate nitric oxide, this free radical has been shown to exert a variety of important functions including modu­ reactive oxidant peroxynitrite (14-16). Based on these opposite lation of vascular tone, neural signaling, and immune response (7,8). In the setting ofischemic injury NO' could exert protective effects, different physiological studies of the ischemic and postis­ chemic heart have been interpreted to suggest that NO' genera­ effects by increasing coronary flow or harmful effects with cellu­ lar injury resulting from the reaction with superoxide to form the tion may be markedly decreased or markedly increased. Nitric Oxide Generation in the Ischemic Heart 307 In this study we have performed measurements to directly during postischemic reperfusion (29). measure the alterations in NO' generation which occur in the Thus, ischemia triggers both the generation of superoxide as ischemic heart and to determine the functional consequences of well as increased amounts of NO'. This increased production of this NO' generation on the recovery of contractile function. NO' and superoxide in ischemic and postischemic myocardium Measurements of the magnitude of the triplet signal formed on could result in the formation of the more potent oxidant per­ trapping of NO' by Fe·MGD were performed and enabled rel­ oxynitrite which is known to cause cellular injury (13). These ative quantitation of the concentration of NO' in the heart. It observations are consistent with the recent studies which have has recently been reported that the Fe·MGD complex is an reported that treatment with nitric oxide synthase blockers can ideal transition metal complex for the trapping and measure­ prevent postischemic injury (16). While further studies will be ment of NO' in living tissues and in vivo animals (21). In our required to fully understand the role of NO' in the process of studies we also observed that the Fe· MGD complex was highly ischemic heart disease, the present study demonstrates that a soluble in aqueous solution and that the NO·Fe·MGD complex large increase in the formation and accumulation of NO' occurs was stable and observable in aerobic solutions. A characteristic during myocardial ischemia and this NO' in turn contributes to triplet spectrum was observed due to the coupling of the un­ the process of postischemic injury. paired electron to the nitrogen nucleus of NO', which for the natural abundance isotope 14N has nuclear spin, I = 1. This REFERENCES characteristic spectrum is centered at g = 2.04 with a nitrogen 1. Moncada, S., Palmer, R. M., Jr., and Higgs, E. A. (1991) Pharmacol. Rev. 43, hyperfine coupling aN = 13.2 G. In our experiments we ob­ 109-142 2. Furchgott, R. F., and Zawadzki, J. V. (1980) Nature 288, 373-376 served that this trap was non-toxic in the concentrations used 3. Palmer, R. M. J., Ferridge, A. G., and Moncada, S. (1987) Nature 327, 524-526 and could be directly perfused into the heart without adverse 4. Ignarro, L. J., Byrns, R. E., Buga, G. M., and Wood, K. S. (1987) Circ. Res. 61, 866-879 effect. We observed that NO' generation is markedly increased 5. Furchgott, R. F., and Vanhoutte, P. M. (1989) FASEB J. 3,2007-2018 during ischemia as evidenced by the appearance of the charac­ 6. Marietta, M. A., Yoon, P. S., Iyengar, R., Leaf, C. D., and Wishnok, J. S. (1988) teristic triplet EPR signal on direct trapping with Fe·MGD. A Biochemistry 27, 8706-8711 7. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1989) Biochem. Pharmacol. progressive increase in the NO' adduct was seen as a function 38,1709-1715 of the duration of ischemia with more than a lO-fold increase 8. Garthwaite, J. (1991) Trends Neurosci. 14,60-67 after 30 min of ischemia. When hearts were pretreated with the 9. Langreh, J. M., Hoffman, R. A., Lancaster, J. R., Jr., and Simmons, R. L. (1993) Transplantation 55, 1205-1212 specific nitric oxide synthase blocker, L-NAME, the generation 10. Ignarro, L. J., Adams, J. B., Horwitz, P. M., and Woods, K. S. (1986) J. Bioi. of this NO' triplet was decreased by 70-80%, demonstrating Chem. 261,4997-5002 that the generation of NO' was largely derived from nitric oxide 11. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Snyder S. H. (1991) Nature 351, 714-718 synthase. In hearts in which NO' generation was blocked with 12. Bredt, D. S., and Synder, S. H. (1992) Neuron 8, 3-11 L-NAME, a marked cardioprotective effect was observed with 13. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. more than a 2-fold increase in the recovery of contractile func­ (1990) Proc. Nat!. Acad. Sci. U. S. A. 87, 1620-1624 14. Lefer, A. M., Tsao, P. S., Lefer, D. J., and Ma, X. L. 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Published: Jan 1, 1995

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