Restoration of Endothelium-Dependent Vasodilation After Reperfusion Injury by Tetrahydrobiopterin
Background A deficit in the endothelial production of nitric oxide (NO) is associated with the sequelae of reperfusion injury. Because endothelial NO synthesis depends on the cofactor tetrahydrobiopterin (BH4), we hypothesized that depletion of this cofactor underlies the reduction of endothelium-dependent dilation in reperfusion injury.
Methods and Results After occlusion of the left anterior descending coronary artery of a pig for 60 minutes followed by 90 minutes of reperfusion (ischemia/reperfusion), hearts were removed and the arterioles were isolated, cannulated, pressurized, and placed on an inverted microscope stage. Dose responses to the endothelium-independent dilator sodium nitroprusside and the endothelium-dependent dilators serotonin, A23187, and substance P were obtained under control conditions, after incubation with sepiapterin (intracellularly converted to BH4) or synthetic BH4 6-methyltetrahydropterin (MH4), and again after their washout. After ischemia/reperfusion, sodium nitroprusside maximally dilated arterioles (99±3%), whereas relaxation to serotonin, A23187, and substance P was significantly reduced (19±9%, 44±9%, and 54±8%, respectively). During incubation with sepiapterin (1 μmol/L) or MH4 (10 μmol/L), endothelium-dependent dilation was significantly enhanced (P<.05), whereas the response to sodium nitroprusside was unaltered. After washout, the vasodilatory responses were not significantly different from the initial ischemia/reperfusion responses. Sepiapterin and MH4 did not affect vasodilatory responses in vessels obtained from nonischemic control hearts. As after ischemia/reperfusion, incubation of control vessels with 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, decreased endothelium-dependent vasodilation, which was restored in the presence of sepiapterin or MH4.
Conclusions These data indicate that exogenous administration of sepiapterin or MH4 restores the response to endothelium-dependent vasodilators in pig coronary arterioles after ischemia/reperfusion. We therefore conclude that ischemia/reperfusion alters the availability or production of BH4, which contributes to blunted endothelial nitroxidergic vasodilation.
We1 and others2 have observed that acute ischemia/reperfusion results in coronary microvascular endothelial dysfunction. The methods by which ischemia/reperfusion blunts NO-dependent dilation of coronary arteries3 and microvessels are not completely understood but are thought to include the adhesion of activated neutrophils to endothelial cells,4 an increase in oxygen free radicals,5 6 the actions of various inflammatory mediators,7 8 and a decrease in NO production. A deficit in NO production is also supported by observations that show that l-arginine administration can restore endothelial function after myocardial ischemia and reperfusion.9 A decrease in NO production may induce a positive feedback situation for the promotion of injury because it releases an inhibition to many of the deleterious effects mentioned above.10 11 Although these causes of vascular injury are well established, the underlying mechanism or a possible common denominator of this injury has not been elucidated.
During the formation of NO and citrulline from l-arginine, NO synthase requires several cofactors, including NADPH, Ca2+/calmodulin, flavin nucleotides, and tetrahydrobiopterin (BH4).12 13 14 15 The biosynthesis of BH4 can occur via a de novo pathway in which the enzyme GTP cyclohydrolase I is the rate-limiting step.16 The synthesis of BH4 can also occur via a salvage pathway that utilizes sepiapterin as an intermediate step.14 Our reasons for focusing on BH4 relate to its importance in the regulation of NO synthase and that it reportedly stabilizes the structure of NO synthase.17 Also, BH4 is modulated by the redox state of cells, which is influenced by free radicals and associated oxidant injury.
We therefore proposed that depletion of this cofactor, which is necessary for NO synthase, likely contributes to endothelial dysfunction. More specifically, the purpose of the present study was to test the hypothesis that the altered regulation of BH4 utilization by endothelial cell NO synthase is responsible for the attenuated endothelium-dependent dilation observed in coronary arterioles after ischemia/reperfusion. To test this hypothesis, we compared the vasodilatory response of isolated coronary arterioles from a pig model of coronary occlusion and reperfusion in the absence and presence of sepiapterin. In addition, we tested the ability of a synthetic BH4, 6-methyltetrahydropterin (MH4),15 to increase agonist-stimulated NO production after ischemia/reperfusion.
Pigs (weight, 6 to 10 kg) were sedated with tiletamine and zolazepam (2 mg/kg IM), anesthetized with sodium pentobarbital (20 to 30 mg/kg IV), intubated, and ventilated with room air. The left femoral artery and vein were cannulated for measurement of arterial pressure and administration of drugs and supplemental anesthetic. ECG tracings (lead II) were obtained with limb electrodes. The heart was exposed via a left thoracotomy, the pericardium incised, and a snare placed around the left anterior descending coronary artery distal to its first diagonal branch. Arterial pressure and ECG tracings were obtained by use of a Gould chart recorder. Arterial blood gases and pH were monitored throughout the study and were maintained within the following ranges: pH 7.35 to 7.45; Pco2, 25 to 40 mm Hg; Po2, 100 to 220 mm Hg.
In Vivo Protocol
The animals were placed into either an ischemia/reperfusion group (n=23) or a control group (n=9). After instrumentation, baseline values of arterial pressure, ECG, and blood gases were obtained. The left anterior descending coronary artery was then occluded for 60 minutes, followed by a 90-minute period of reperfusion. During the ischemia and reperfusion periods, 2 of the animals fibrillated, but only 1 was successfully converted. One animal died because of problems with the occluder. In the remaining animals, the hearts were fibrillated electrically and removed. In control animals, the heart was fibrillated electrically and removed immediately after the thoracotomy was performed.
In Vitro Measurement of Coronary Arteriolar Diameters
After 90 minutes of reperfusion, heparin (1000 IU/kg IV) was administered and the heart was fibrillated electrically, removed, and placed immediately in cold (4°C) saline. The left anterior descending and circumflex coronary arteries were cannulated individually for perfusion with an india ink-gelatin mixture in PSS18 to facilitate microdissection. Coronary arterioles <100 μm in internal diameter were dissected carefully from the myocardial tissue at 4°C and were transferred to an acrylic resin vessel chamber that contained PSS-albumin solution at pH 7.4. Both ends of each arteriole were cannulated with a glass micropipette with an external tip diameter of ≈40 μm and secured with 11-0 ophthalmic suture. The india ink-gelatin PSS solution was flushed out at low pressure (20 cm H2O), and the other end of the microvessel was secured to a second micropipette.
After the vessels were cannulated, the chamber was transferred to the stage of an inverted microscope (IM35, Carl Zeiss; objective ×40, numerical aperture 0.75) fitted with a Cohu TV camera (model 4915) and video micrometer (Texas A&M Microcirculation Research Institute). We pressurized arterioles to 60 cm H2O by adjusting the height of a reservoir connected to each micropipette. This pressure approximates the estimated intraluminal pressures for microvessels of this size in vivo.19 Because both reservoirs were set to the same height, the vessels were pressurized without flow. We detected leaks by closing off the system to the reservoirs and examining for a decline in intraluminal pressure. Vessels with leaks were excluded from further study. Internal diameters were recorded during steady-state conditions after each intervention. The microvessels were set to their in situ length and were bathed in PSS-albumin solution with the temperature maintained at 36°C to 37°C by use of an external heat exchanger. All of the arterioles prepared in this manner developed spontaneous tone of 25% to 30% of maximal diameter. Vessels that did not develop this level of tone were not studied.
Isolated Microvessel Protocol
After the arterioles had been allowed to equilibrate in the bath and develop spontaneous tone, dose responses to the endothelium-independent dilator sodium nitroprusside (10−9 to 10−5 mol/L) and the endothelium-dependent nitroxidergic dilators serotonin (10−9 to 10−5 mol/L), A23187 (10−9 to 10−5 mol/L), and substance P (10−11 to 10−8 mol/L) were measured during steady-state responses. After these dose-response curves were measured, the microvessels were incubated with 1 μmol/L sepiapterin or 10 μmol/L MH4 10 minutes before and during a repeat of dose-response measurements with all drugs. After these measurements, sepiapterin or MH4 was washed from the bath, and the vessels were allowed to reequilibrate. Ten minutes after the vessels redeveloped spontaneous tone, dose-response curves to all drugs were performed again. In control pigs, responses were measured under baseline conditions and during administration of sepiapterin or MH4.
To test the effect of endogenous blockade of BH4 synthesis, vessels were incubated with 2,4-diamino-6-hydroxypyrimidine (DAHP) (10−3 mol/L), an inhibitor of GTP cyclohydrolase I activity, for 3 hours. Thereafter, dose-response curves to serotonin, substance P, and sodium nitroprusside were obtained before and after addition of sepiapterin or MH4.
MH4 and DAHP (Sigma Chemical Co) were dissolved in distilled water. Sepiapterin (Research Biochemicals Intl) was dissolved in DMSO. Two microliters of the sepiapterin stock solution was added to the bath to obtain a final concentration of 1 μmol/L. Administration of 2 μL DMSO alone to the bath had no vasoactive effect. The concentrations of sepiapterin, BH4, and DAHP used were similar to those reported in previous studies.14 15
Measurements of microvascular diameters during interventions were expressed as the mean normalized to the maximal diameter (±SEM). Comparisons of dose-response curves to different interventions, baseline diameters before each intervention, and hemodynamics were made by use of ANOVA. All statistics were computed with use of Statview 4.1 software on a Macintosh 8100 computer. A probability level of 95% was used in all studies as the criterion of statistical significance.
In Vivo Hemodynamics
Mean arterial pressure and heart rate were measured during the initial control period, at the end of the 60-minute coronary occlusion, and at the end of the 90-minute reperfusion period. Mean arterial pressure averaged 68±2, 53±4, and 52±3 mm Hg during control, occlusion, and reperfusion, respectively. Pressure was significantly depressed during occlusion and reperfusion compared with control (P<.05). Heart rate during occlusion and reperfusion did not differ from control (147±8, 144±6, and 152±6 bpm during control, occlusion, and reperfusion, respectively).
Ischemia/Reperfusion: Effects of Sepiapterin
Dose-response curves to the endothelium-independent vasodilator sodium nitroprusside and the endothelium-dependent vasodilators serotonin, A23187, and substance P under baseline conditions (ischemia/reperfusion), during incubation with 1 μmol/L sepiapterin, and after washout of the sepiapterin are shown in Fig 1⇓. After ischemia/reperfusion, sodium nitroprusside induced maximal relaxation of arterioles (107±1%), whereas relaxation to the endothelium-dependent dilators was considerably less (25±6%, 28±9%, and 48±10% relaxation for serotonin, A23187, and substance P, respectively; Fig 1⇓). During incubation with 1 μmol/L sepiapterin, the response of arterioles to all endothelium-dependent dilators was significantly enhanced (Fig 1⇓; P<.05). There was no difference in the response to sodium nitroprusside, however (Fig 1⇓). Importantly, incubation with sepiapterin did not alter the baseline diameter (70.4±4.3 versus 70.3±4.3 μm for ischemia/reperfusion and sepiapterin, respectively) of these vessels. To demonstrate that the enhanced relaxation in the presence of sepiapterin was not a time-dependent phenomenon, responses were measured again after washout and restoration of spontaneous tone. At that time, the responses to the endothelium-dependent dilators were not significantly different from the initial ischemia/reperfusion responses (Fig 1⇓).
Ischemia/Reperfusion: Effects of MH4
Fig 2⇓ shows the responses of arterioles during initial conditions (ischemia/reperfusion), during incubation with the synthetic BH4 analog MH4 (10 μmol/L), and after washout of MH4. There was a significant increase in relaxation to all endothelium-dependent dilators in the presence of MH4 (P<.05; Fig 2⇓), although it was not as pronounced as with sepiapterin. In contrast to the results obtained with sepiapterin, after washout of MH4, the responses to serotonin and A23187 were still significantly enhanced compared with the baseline ischemia/reperfusion responses (P<.05), whereas the response to substance P did not remain enhanced (Fig 2⇓). Also, the response to nitroprusside was not altered either during or after incubation with MH4. As with sepiapterin incubation, there was no change in baseline diameter during incubation with the BH4 analog (77.3±6.1 versus 77.4±6.0 μm for ischemia/reperfusion and MH4, respectively).
Control Vessels: Effects of Sepiapterin and MH4
The response of coronary arterioles isolated from control animals to sodium nitroprusside, serotonin, A23187, and substance P in the presence and absence of sepiapterin and in the presence of MH4 are presented in Figs 3 and 4⇓⇓, respectively. In both cases, there was no significant enhancement of the relaxation to either the endothelium-dependent or endothelium-independent agents. Indeed, there was a slight but statistically significant attenuation of the response to sodium nitroprusside and serotonin in the presence of MH4. Also, similar to the ischemia/reperfusion groups, neither sepiapterin (67.3±3.7 versus 70.0±2.7 μm for control and sepiapterin, respectively) nor MH4 (67.6±2.9 versus 67.6±2.7 μm for control and MH4, respectively) affected the basal microvascular diameter.
In vessels from the nonoccluded left circumflex coronary artery region of the ischemia/reperfusion animals, all endothelium-dependent vasodilators produced maximal relaxation (Fig 5⇓). Sodium nitroprusside also produced a maximal relaxation in these vessels. These responses are similar to those from control animals, as described above.
Control Vessels: Effects of DAHP
The responses of control vessels to the GTP cyclohydrolase I inhibitor DAHP are presented in Fig 6⇓. DAHP did not alter the response to sodium nitroprusside alone or in the presence of sepiapterin or MH4. DAHP, however, significantly depressed endothelium-dependent dilation to serotonin and substance P (Fig 6⇓; P<.05), a response that was reversed in the presence of sepiapterin or MH4. Although these changes were not significant at all doses, there was a significant improvement in the DAHP-induced inhibition in the presence of sepiapterin and MH4 at the highest doses of both serotonin and substance P (Fig 6⇓; P<.05).
In this study, we have confirmed our1 observations and those of others2 that coronary microvessels have a depressed endothelium-dependent, NO-mediated vasodilator function after ischemia/reperfusion. However, we have expanded this finding to demonstrate that arteriolar endothelial dysfunction produced by reperfusion injury can be reversed when the levels of BH4 are increased either by an endogenous salvage pathway with the use of sepiapterin or by administration of the synthetic BH4 MH4. Specifically, isolated arterioles that were subjected to reperfusion injury and then incubated with sepiapterin or MH4 demonstrated restored vasodilation to the endothelium-dependent dilators serotonin, A23187, and substance P, whereas endothelium-independent dilation to sodium nitroprusside was not affected. Moreover, reactivity of control vessels was not altered by either compound. Our conclusions depend critically on issues regarding the methodology and other observations relating to modulation of NO synthase activity.
Critique of Experimental Methods
To evaluate the effect of ischemia/reperfusion on endothelium-dependent dilation, we used the receptor-mediated agonists serotonin and substance P and the nonreceptor-mediated endothelium-dependent vasodilator A23187. The actions of each of these agonists are mediated by the production of NO; therefore, diminished dilation after ischemia/reperfusion was interpreted as decreased endothelial production of NO. Because it also was important to demonstrate that vascular smooth muscle responsiveness to NO was not affected by ischemia/reperfusion, we used the NO donor sodium nitroprusside. Maintenance of vasodilatory responses to the NO donor is also consistent with the view that altered vasoactivity is not due to impaired vascular smooth muscle responsiveness.
We also examined responses to endothelium-dependent and -independent vasodilators in arterioles isolated from the control region of the ischemia/reperfusion animals. These responses were not different from those of the control animals, which demonstrates that microvascular reperfusion injury in our model was restricted to the ischemic region. This also addresses the concern we had with the relatively low arterial pressures observed in this model of ischemia/reperfusion, demonstrating that the reduced vasodilatory response observed after ischemia/reperfusion was not the result of systemic hypotension.
Our hypothesis was that a reduction in BH4 during ischemia/reperfusion contributes to decreased endothelium-dependent vasodilation. Since we cannot measure BH4 levels in isolated arterioles because of the small mass of tissue (100 to 200 μg), we targeted this cofactor using substances that influence BH4 levels or serve as synthetic analogs. To increase the levels of BH4 in the coronary microvessels, we incubated the vessels with 1 μmol/L sepiapterin. Sepiapterin is a dihydropterin that can be used as a substrate to form BH4 via a salvage pathway.13 14 16 This salvage pathway, which utilizes the enzyme dihydrofolate reductase, is independent of GTP cyclohydrolase I, the rate-limiting reaction in the normal biosynthetic pathway. Other investigators13 have demonstrated that increasing levels of exogenously applied sepiapterin in confluent monolayers of human umbilical vein endothelial cells produced dose-dependent increases in intracellular levels of BH4. Sepiapterin has also been used to enhance NO synthesis in activated macrophages20 and cultured vascular smooth muscle cells.14
Although incubation with either sepiapterin or MH4 resulted in an enhanced relaxation to all endothelium-dependent vasodilators, the synthetic BH4 MH4 was less effective. Similar results were obtained by other investigators who demonstrated that MH4 was less effective than native BH4 for NO synthase-induced citrulline formation in murine macrophages20 or NO synthase activity in rat cerebellum.21 Because we used only a single dose of each agent, we cannot directly address the issue of relative efficacies. Also, as observed in Fig 2,⇑ the effect of MH4 appeared to last longer than that of sepiapterin. This again may be due to differences in the pharmacological dynamics of the two compounds.
Our conclusion that the levels of the cofactor are reduced in ischemia/reperfusion is based on extrapolation from our results showing that administration of sepiapterin or MH4 restored endothelium-dependent vasodilation. Also supporting this conclusion were results from experiments that used the inhibitor of GTP cyclohydrolase I, DAHP, which reportedly reduces the levels of BH4.14 15 Specifically, vasodilation to the endothelium-dependent agonists was impaired after administration of this inhibitor, and either of the BH4 mimetics could restore vasodilation to the nitroxidergic vasodilators. These results mimicked those from the ischemia/reperfusion experiments, ie, restoration of nitroxidergic vasodilation after administration of the BH4 mimetics. In the aggregate, these results are complementary and support our contention that impaired NO-mediated vasodilation of coronary arterioles is attributed to reduced levels of BH4.
To rule out nonspecific effects of sepiapterin and MH4, coronary arterioles were dissected from nonischemic control hearts and incubated with either compound. As in the ischemic vessels, after 10 minutes of incubation, dose-response curves to sodium nitroprusside, serotonin, A23187, and substance P were obtained. As shown in Figs 3 and 4,⇑⇑ there was no statistically significant enhancement in the relaxation to the different vasodilators after incubation with sepiapterin or MH4 compared with nonincubated vessels.
Physiological and Pathophysiological Implications
Many investigators have reported endothelial dysfunction after ischemia/reperfusion in large coronary arteries3 4 6 7 8 and coronary microvessels.1 2 Results from the current study are similar to those from a previous report2 that demonstrated depressed endothelium-dependent dilation in isolated coronary arterioles after ischemia and reperfusion in vivo. It is not clear whether the attenuated endothelium-dependent relaxation associated with ischemia/reperfusion is the result of a decrease in the amount of NO produced by endothelial cells or the scavenging of NO. Studies5 6 have implicated the scavenging of NO by oxygen radicals by demonstrating restored endothelial function after treatment during reperfusion with oxygen radical scavengers. In either case, however, reperfusion injury can be prevented via an increase in NO levels during reperfusion. This has been accomplished in experimental models of myocardial ischemia/reperfusion either by the addition of NO donors11 or by exogenous administration of large amounts of l-arginine9 22 during reperfusion. Therefore, these studies suggest that a decrease in NO is responsible for reperfusion injury. Furthermore, it has been proposed that decreased levels of NO precede the enhanced neutrophil adherence to endothelial cells,23 which provides evidence for a protective role of NO. The results from the current study provide a mechanism by which ischemia/reperfusion may alter the production and release of NO from endothelial cells and thereby not only reduce endothelium-dependent vasodilation but also reduce an inhibition on other mediators of reperfusion injury.
Previous reports9 22 demonstrated that administration of l-arginine can restore NO-mediated endothelial dilation after reperfusion injury. Generally, these results have been used to support the hypothesis that in this pathology, arginine availability becomes rate limiting in the production of NO, and therefore, supplementation with the amino acid will restore function. Our results, however, argue for a different mechanism: a decrease in the availability of BH4 is responsible for the decreased nitroxidergic vasodilation. We believe these observations can be reconciled along two different lines of reasoning. First, l-arginine does more in a cell than just serve as a precursor for the synthesis of NO. Specifically, it is metabolized as an energetic substrate, and its degradation to glutamic acid results in the production of NADPH,24 a cofactor for the biosynthesis of NO and BH4. Second, the binding of BH4 to NO synthase increases the affinity of the enzyme for arginine, and equally important, the binding of arginine to NO synthase also increases the affinity for BH4.25 Thus, addition of either substance may affect the generation of NO via alterations in the affinity of one enzyme for the other. Taken together, these observations address complexities involved in the regulation of NO synthesis by cofactors and substrates, which likely render interpretations of the enhancement of NO production by an increase in substrate availability through arginine supplementation as overly simplistic.
The biochemical role for BH4 in the generation of NO is not known exactly. Whereas BH4 has been proposed to be essential for cytokine-induced NO synthesis in vascular smooth muscle cells14 and human umbilical vein endothelial cells,13 other studies21 26 suggest that BH4 was rather a catalytic agent that does not influence basal NO synthase activity. Because BH4 plays a role in the hydroxylation of aromatic amino acids, it has been proposed that it may contribute to the hydroxylation of l-arginine, the first step in the synthesis of NO from l-arginine. However, other studies21 suggest that BH4 only stabilizes NO synthases and therefore catalytically increases NO production. Thus far, it is also unknown how and to what extent BH4 can be recycled, which indicates that in conditions such as ischemia/reperfusion, a loss of BH4 could be compensated only by de novo synthesis.
Recently, it has been demonstrated in canine coronary artery rings that after inhibition of BH4 synthesis with a GTP cyclohydrolase I inhibitor, endothelium-dependent relaxation may be mediated by hydrogen peroxide formed by a dysfunctional NO synthase instead of NO, because the endothelium-dependent vasodilation could be inhibited with radical scavengers. These findings are supported by studies demonstrating that hydrogen peroxide causes vasodilation in coronary arteries27 and that brain NO synthase is able to produce hydrogen peroxide at suboptimal levels of BH4.28 29 In the present study, endothelium-dependent relaxation was significantly attenuated after ischemia/reperfusion. This may indicate that the vascular effects of oxygen radicals may be different after ischemia. In addition, there may be a difference in the mechanism that leads to impairment of BH4 availability after ischemia compared with a chemically induced inhibition of BH4 synthesis with a GTP cyclohydrolase I inhibitor. Furthermore, there may be a difference in the effects of oxygen radicals on coronary arterioles compared with large arteries.
An important question that must be considered is why should ischemia/reperfusion affect levels of BH4? Reperfusion injury is associated with the production of free radicals, which leads to oxidant stress in many cells. In particular, endothelial cells are thought to produce free radicals during reperfusion; thus, these cells may be prone to oxidant injury associated with free radicals. Oxidant injury alters the redox state in cells, and the biosynthesis of BH4 depends on a normal cellular redox state.16 Increased levels of hydrogen peroxides and oxygen radicals may either alter the involvement of BH4 in the oxidation process of NO synthesis from l-arginine, affect BH4 biosynthesis by depletion of NADPH, or prevent recycling of BH4, which may occur through flavin nucleotides via NADPH as suggested by Scott-Burden.17 The first two proposed mechanisms appear unlikely because we can restore endothelium-dependent vasodilation with substitution of BH4 via sepiapterin or MH4. Taken together, our results support the idea that reperfusion injury depletes the levels of BH4, making this cofactor unavailable for many critical enzymatic reactions, such as enzymatic oxidation of arginine to NO by NO synthase.
From these data, we conclude that decreased availability of BH4 is responsible for coronary microvascular endothelial dysfunction associated with reperfusion injury.
Selected Abbreviations and Acronyms
|PSS||=||physiological salt solution|
This work was supported in part by a Grant-In-Aid (94G-018) from the American Heart Association, Texas Affiliate (Dr DeFily) and by grants from the American Heart Association (94-1505 to Dr DeFily), the NHLBI (HL-32788 to Dr Chilian), and the Deutsche Forschungsgemeinschaft (Dr Tiefenbacher). We would like to acknowledge the technical assistance of Jan Patterson in these studies.
- Received January 4, 1996.
- Revision received February 16, 1996.
- Accepted March 26, 1996.
- Copyright © 1996 by American Heart Association
DeFily DV, Chilian WM. Preconditioning protects coronary arteriolar endothelium from ischemia-reperfusion injury. Am J Physiol. 1993;265(Heart Circ Physiol 34):H700-H706.
Quillen JE, Sellke FW, Brooks LA, Harrison DG. Ischemia-reperfusion impairs endothelium-dependent relaxation of coronary microvessels but does not affect large arteries. Circulation. 1990;82:586-594.
Ku DD. Coronary vascular reactivity after acute myocardial ischemia. Science. 1982;218:576-578.
Dreyer WJ, Michael LH, West MS, Smith CW, Rothlein R, Rossen RD, Anderson DC, Entman ML. Neutrophil accumulation in ischemic canine myocardium: insights into time course, distribution, and mechanism of localization during early reperfusion. Circulation. 1991;84:400-411.
Stewart DJ, Pohl U, Bassenge E. Free radicals inhibit endothelium-dependent dilation in the coronary resistance bed. Am J Physiol. 1988;255(Heart Circ Physiol 24):H765-H769.
Tsao PS, Aoki N, Lefer DJ, Johnson GI, Lefer AM. Time course of endothelial dysfunction and myocardial injury during myocardial ischemia and reperfusion in the cat. Circulation. 1990;82:1402-1412.
Dreyer WJ, Michael LH, Nguyen T, Smith CW, Anderson DC, Entman ML, Rossen RD. Kinetics of C5a release in cardiac lymph of dogs experiencing coronary artery ischemia-reperfusion injury. Circ Res. 1992;71:1518-1524.
Shandelya SML, Kuppusamy P, Weisfeldt ML, Zweier JL. Evaluation of the role of polymorphonuclear leukocytes on contractile function in myocardial reperfusion injury: evidence for plasma-mediated leukocyte activation. Circulation. 1993;87:536-546.
Weyrich AS, Ma X, Lefer AM. The role of l-arginine in ameliorating reperfusion injury after myocardial ischemia in the cat. Circulation. 1992;86:279-288.
Kurose I, Wolf R, Grisham MB, Granger DN. Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide. Circ Res. 1994;74:376-382.
Lefer DJ, Nakanishi K, Johnston WE, Vinten-Johansen J. Antineutrophil and myocardial protecting actions of a novel nitric oxide donor after acute myocardial ischemia and reperfusion in dogs. Circulation. 1993;88:2337-2350.
Werner-Felmayer G, Werner ER, Weiss G, Wachter H. Modulation of nitric oxide synthase activity in intact cells by intracellular tetrahydrobiopterin levels. In: Ayling JE, ed. Chemistry and Biology of Pteridines and Folates. New York, NY: Plenum Press; 1993:309-312.
Werner-Felmayer G, Werner ER, Fuchs D, Hausen A, Reibnegger G, Schmidt K, Weiss G, Wachter H. Pteridine biosynthesis in human endothelial cells. J Biol Chem. 1993;268:1842-1846.
Gross SS, Levi R. Tetrahydrobiopterin synthesis: an absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem. 1992;267:25722-25729.
Cosentino F, Katusic ZS. Tetrahydrobiopterin and dysfunction of endothelial nitric oxide synthase in coronary arteries. Circulation. 1995;91:139-144.
Scott-Burden T. Regulation of nitric oxide production by tetrahydrobiopterin. Circulation. 1995;91:248-250.
Kuo L, Davis MJ, Chilian WM. Endothelium-dependent, flow-induced dilation of isolated coronary arterioles. Am J Physiol. 1990; 259(Heart Circ Physiol 28):H1063-H1070.
Chilian WM, Eastham CL, Marcus ML. Microvascular distribution of coronary vascular resistance in beating left ventricle. Am J Physiol (Heart Circ Physiol 20). 1986;251:H779-H788.
Gioavnelli J, Campos KL, Kaufman S. Tetrahydrobiopterin, a cofactor for rat cerebellar nitric oxide synthase, does not function as a reactant in the oxygenation of arginine. Proc Natl Acad Sci U S A. 1991;88:7091-7095.
Nakanishi K, Vinten-Johansen J, Lefer DJ, Zhao Z, Fowler WC III, McGee DS, Johnston WE. Intracoronary l-arginine during reperfusion improves endothelial function and reduces infarct size. Am J Physiol. 1992;263(Heart Circ Physiol 32):H1650-H1658.
Ma X, Weyrich AS, Lefer DJ, Lefer AM. Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium. Circ Res. 1993;72:403-412.
Mathews CK, van Holde KE. Biochemistry. Redwood City, Calif: The Benjamin/Cummings Publishing Co Inc; 1990.
Klatt P, Schmid M, Leopold E, Schmidt K, Werner ER, Mayer B. The pteridine binding site of brain nitric oxide synthase: tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain. J Biol Chem. 1994;269:13861-13866.
Rubanyi GM, Vanhoutte PM. Oxygen-derived free radicals, endothelium, and responsiveness of vascular smooth muscle. Am J Physiol. 1986;250(Heart Circ Physiol 19):H815-H821.
Heinzel B, John M, Klatt P, Bo¨hme E, Mayer B. Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase. Biochem J. 1992;281:627-630.