Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction.

Abstract

HomeHypertensionVol. 31, No. 5Review of Alterations in Endothelial Nitric Oxide Production in Diabetes Free AccessReview ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessReview ArticlePDF/EPUBReview of Alterations in Endothelial Nitric Oxide Production in Diabetes Protective Role of Arginine on Endothelial Dysfunction Galen M. Pieper Galen M. PieperGalen M. Pieper From the Department of Transplant Surgery, Medical College of Wisconsin, Froedtert Memorial Hospital (Milwaukee). Search for more papers by this author Originally published1 May 1998https://doi.org/10.1161/01.HYP.31.5.1047Hypertension. 1998;31:1047–1060Nitric oxide release from the endothelium plays an important role in regulation of vascular tone,1 inhibition of both platelet and leukocyte aggregation and adhesion,12 and inhibition of cell proliferation.3 These properties suggest that the level of NO production by the endothelium may play a pivotal role in the regulation of vascular disease. Analysis using mass spectrometry has revealed that NO is produced by NOS from the terminal guanidino nitrogen of the precursor amino acid l-arginine.4 Thus, utilization of l-arginine and conversion to NO may establish a regulatory site in the development of endothelial dysfunction.Endothelial dysfunction is characterized by defective endothelium-dependent relaxation, and some reviews regarding endothelial dysfunction in diabetes have been published.5678 These reviews have focused on factors that might contribute to defective relaxation, some of which will not be addressed in detail in this review. The purpose of the present review is to summarize evidence that specifically supports either decreased NO production by diabetic vascular endothelium and/or impaired NO-mediated endothelium-dependent relaxation. Second, this review provides reasonable alternatives to explain some of the controversies in this research area. Third, since there is growing evidence that arginine appears to have some benefits for diabetes-associated abnormalities, this review summarizes the current state of knowledge of effects of acute and chronic administration of l-arginine on diabetes-induced endothelial dysfunction and discusses potential NO-dependent and -independent mechanisms whereby therapeutic intervention with l-arginine might benefit the diabetic endothelium.Impaired Endothelium-Dependent RelaxationExperimental Diabetes MellitusDecreases in endothelium-dependent relaxation are a common feature in both conduit91011121314151617181920 and resistance21222324252627282930 arteries of chemically induced experimental diabetic animals (including rats, mice, rabbits, hamsters, and dogs). In two genetic models of IDDM, similar impaired relaxation has been documented in aorta313233 and mesenteric arteries3435 of diabetes-prone BB/Wor or BB/E rats and in aorta36 and mesenteric arteries37 of the diabetes-prone WBN/Kob rat.Almost without exception, studies that have shown impaired endothelium-dependent relaxation have found normal relaxation to nitrovasodilators. These agents relax vascular smooth muscle by activating guanylate cyclase but, unlike NO, do not require the presence of the endothelium. Thus, the intrinsic property to activate vascular smooth muscle guanylate cyclase appears not to be altered by experimental diabetes.Studies in experimental models of NIDDM are few and controversial. In the obese Zucker rat, no alteration in endothelium-dependent relaxation has been observed in intestinal microvessels,38 whereas increased reactivity has been observed in aorta.3940 In contrast, decreased endothelium-dependent relaxation to acetylcholine but not to A23187 was seen in aorta in male (but not female) JCR:LA-corpulent rats.41 Little is known regarding the nature of endothelial dysfunction in these models.Conclusion. Collectively, these studies suggest that endothelial dysfunction (1) is not unique to chemically induced experimental models of IDDM, (2) is found in both conduit and resistance arteries, and (3) cannot be explained by an intrinsic change in reactivity to NO or guanylate cyclase reactivity. Studies conducted in experimental models of NIDDM are few and provide no clear consensus regarding this issue.Human Diabetes MellitusThe first evidence of endothelial dysfunction in humans was reported in penile corpura cavernosa of IDDM and NIDDM patients.42 Impaired endothelium-dependent relaxation of conduit blood vessels has been confirmed in both IDDM4344454647 and NIDDM43484950515253 patients evaluated in vivo or in isolated arteries in vitro.4551 A few studies in IDDM545556 or NIDDM patients57 report no defect. One study of resistance vessels derived from NIDDM patients and evaluated in vitro showed enhanced reactivity to acetylcholine.58 Evaluation of the latter study is compromised because the control patients selected exhibited arterial occlusive disease, hypercholesterolemia, and other nondiabetic diseases. Also, patients using various cardiovascular medications were not excluded from the study.Several investigators have observed normal relaxation to nitrovasodilators in diabetic patients.424344454647515253555657585960 In contrast, other investigators have noted attenuated responses to nitrovasodilators in IDDM diabetic patients54616263 and in NIDDM diabetic patients.48495064Conclusion. The majority of studies have demonstrated impaired endothelium-dependent relaxation. In contrast, there is not sufficient consensus on whether vascular smooth muscle reactivity to NO is compromised in human diabetes mellitus.Importance of Patient Screening and Other Complicating FactorsOne possible explanation of this variability relates to primary nitrate tolerance, since responses to nitroglycerin but not sodium nitroprusside were diminished in diabetic patients.65 Alternatively, one of these studies noted no difference in reactivity to nitroglycerin after correction for differences in baseline diameter.61 Furthermore, diabetic patients with microalbuminuria exhibited impaired dilation to isosorbide dinitrate, while in diabetic patients without microalbuminuria, dilation was unaltered.66 In this regard, dilation in response to carbachol was normal in diabetic patients but was blunted by an NOS inhibitor in both control and diabetic patients without microalbuminuria but not in diabetic patients with microalbuminuria.56 Thus, there may be variable mechanisms of impaired vascular reactivity to nitrovasodilators among different diabetic patient subpopulations.There are several concerns regarding critical evaluation of the data obtained in diabetic patients. Whereas some investigators have carefully eliminated patients taking medications (eg, vasoactive and cardiovascular drugs) and have limited intake of caffeine or alcohol before evaluation, other investigators have either not provided information regarding patient selection or have ignored these potential influences. Some studies have included patients with histories of smoking (an independent risk factor for endothelial dysfunction) or have only limited smoking just before the study.Most studies have used a mixed-gender patient population. Thus, the influence of gender-related differences has not been isolated. Indeed, it is widely known that diabetes-induced complications are enhanced in the female population. A recent study has revealed diminished reactive hyperemia (an endothelium-related phenomenon) in young female IDDM patients compared with age-matched male subjects.67 Furthermore, inclusion of postpuberty female patients using oral contraceptive agents has been acknowledged in some studies,6162 but the significance of inclusion of this subpopulation has not been addressed. In studies using male subjects only, endothelial function was either impaired46474950 or unchanged.545557In studies of vascular reactivity using NIDDM patients, it was acknowledged that many subjects were receiving hypoglycemic drugs or insulin at the time of evaluation.47484951526468 Oral hypoglycemic agents such as glibenclamide can impair dilation mediated by NO-mediated KATP channel activation.69 Thus, the effects of this medication on NO-mediated relaxation have not been uniformly excluded. One study showed no difference in endothelium-dependent relaxation between patients taking oral hypoglycemic agents and those controlling the disease by diet alone.48 In one study in which hypoglycemic medication was removed for a finite period before evaluation, there was diminished reactivity to acetylcholine but not to nitroprusside.53Endothelial function in patients has been determined in vivo using either strain-gauge plethysmography or Doppler techniques, predominately in brachial/forearm protocols. A few have evaluated dilation in the femoral artery536364 or coronary arteries.43 While there is important value in examining reactivity in vivo under ambient conditions, there are several significant complicating factors that make the in vivo delineation of mechanisms difficult. For example, one investigator found that endothelial dysfunction correlated with serum triglyceride and lower HDL cholesterol levels.50 Furthermore, ambient glycated proteins may diminish endothelium-dependent relaxation studied in vitro,7071 although a recent in vitro study in canine conduit and resistance artery preparations could not confirm this effect.72Conclusion. Patient selection and screening have the potential to explain differences in findings obtained in clinical studies. The potential influence of gender, presence of microalbuminuria, and use of medications, including oral contraceptives and hypoglycemic agents, need to be rigorously evaluated in clinical studies. Also, the influence of ambient glucose concentration, glycated proteins, and lipids on endothelial function needs to be considered in clinical evaluations.Confounding Effects of Ambient Glucose ConcentrationAmbient plasma glucose concentration could be a confounding factor in vivo as well. Some studies in experimental animal models have noted acute vasodilation (ie, seconds to minutes) in response to concurrent elevation in glucose concentration.737475 In contrast, short-term exposure (ie, minutes to several hours) to elevated glucose concentrations in vitro or topical application in situ impairs basal NO tone76 and agonist-stimulated endothelium-dependent relaxation.7778798081 Interestingly, 24-hour infusion of glucose in normal subjects did not impair relaxation.82 Because multiple agonists were used in the latter study, the significance of masking by crossover effects of individual drugs cannot be excluded in the interpretation of this data. In contrast, evaluation during an acute bolus of glucose administration (ie, during a glucose tolerance test) revealed decreased endothelium-dependent relaxation.83An immediate acute effect of elevated glucose concentration is a marked increase in basal [Ca2+]i and NO production in isolated human endothelial cells.84 [Ca2+]i also increases rapidly (within minutes) in the presence of elevated glucose concentration in isolated bovine aortic endothelial cells (G.M.P., unpublished observation, 1997). This effect may persist for several hours after exposure to elevated glucose concentration.85 In contrast, after 24 hours of exposure of bovine aortic endothelial cells to elevated glucose and subsequent analysis under normal glucose conditions, basal [Ca2+]i and NO production returned to normal, whereas bradykinin-stimulated [Ca2+]i and NO production was reduced.86 Similarly, impaired agonist-stimulated [Ca2+]i and NO production have been observed after exposure of rat or porcine endothelial cells to elevated glucose concentration.8788To circumvent this potential limitation, one study using the euglycemic insulin clamp protocol46 revealed that endothelial function was diminished. Also, analysis in vitro of human gluteal resistance arteries under normoglycemic conditions also revealed diminished endothelium-dependent relaxation.45 The latter study suggested an intrinsic defect in endothelium-dependent relaxation in IDDM that is independent of direct effects of concurrent ambient glucose concentrations in vivo. In contrast, another study showed that relaxation to carbachol was unchanged in IDDM patients under euglycemic conditions produced by insulin infusions.56 Interestingly, a defect in NO synthesis was still suggested in patients with microalbuminuria based on the lack of response to NG-monomethyl-l-arginine (L-NMMA).Conclusion. The confounding effects of ambient glucose concentration on endothelial function in studies performed in patients and in experimental diabetes in vivo should be taken into account when glucose concentration is not normalized. Unfortunately, controlling for this contingency may be difficult, particularly in clinical studies. Clearly, there are important direct stimulatory and inhibitory effects of elevated glucose concentration on vascular reactivity that may be manifested at different times subsequent to rises in glucose concentration.Paradoxical Findings on Endothelial Function: Role of Disease DurationFew studies have examined the temporal nature of the onset of endothelial dysfunction in experimental diabetes. Most have shown a progressive worsening of dysfunction that appears to plateau at some finite point in time. Dysfunction has been reported at as early as 1 week of diabetes in rat intestinal arterioles,30 after 2 weeks in hindquarters but not in mesenteric or renal arteries,89 after 3 weeks in cremaster muscle arterioles,90 after 4 to 6 weeks in mesenteric arterioles,2591 and after 4 weeks in aorta.70 Thus, lack of endothelial dysfunction of diabetic rat aorta at 2 to 3 weeks of disease92 could be explained by the short period of time studied. It is important to note that the onset of endothelial dysfunction may vary widely among individual vascular beds and/or the severity of the diabetic model used in any given study.Interestingly, there are paradoxical reports of normal endothelium-dependent relaxation in aorta after >12 weeks of diabetes939495 and after 15 to 17 weeks of diabetes in perfused mesenteric preparations.96 The reasons for these disparate observations compared with other findings are unclear. Potential explanations might include the use of helical strips,93 diabetes-induced hypersensitivity to phenylephrine and evaluation only in indomethacin-treated preparations,94 use of a low dose of streptozotocin,94 or the potential influence of masking by crossover effects due to multiple drug challenges.95There is clinical and experimental evidence showing augmented blood flow at early stages of diabetes.97 It is not at all clear whether this increased blood flow reflects increased NO-specific endothelium-dependent dilation. One possibility that needs to be examined is whether the diabetes-induced decreases in 2,3-diphosphoglyceric acid levels in red blood cells,98 which regulate oxygen release from hemoglobin, lead to a “hypoxic-like” environment.99 Decreases in tissue ATP concentration could result in increased blood flow due to hypoxic-induced dilation in compensation for reduced oxygen delivery to tissue. Indeed, myocardial ATP concentration is reduced in diabetes but is rapidly replenished within minutes of perfusion in vitro with oxygenated blood-free salt solutions.100 Furthermore, in situ analysis using microelectrodes reveals diminished oxygen tension in aorta of alloxan-diabetic rabbits.101 Thus, increase in tissue blood flow at the early stages of diabetes may be due to hypoxic vasodilation. Alternatively, increased blood flow may be a direct response to acute or short-term hyperglycemia, since coronary blood flow is increased with glucose infusions in isolated hearts in the presence of indomethacin.75At least one study has noted augmented endothelium-dependent relaxation in indomethacin-treated rat renal arteries at an early stage of diabetes.102 Because endothelium-dependent relaxation is reduced in renal arteries of diabetic rats of longer disease duration,103 this observation raises the possibility that diabetes induces biphasic effects on endothelium-dependent relaxation. Thus, an early increase in blood flow may be followed by a transition state to impaired relaxation. In support of this hypothesis, one study noted an increase in endothelium-dependent relaxation of mesenteric arteries to both acetylcholine and bradykinin at 6 weeks of diabetes that reverts back to normal after 12 weeks.104 This enhanced relaxation could not be accounted for by intrinsic changes in smooth muscle reactivity because responses to sodium nitroprusside were normal.It remains to be resolved in temporal studies using various preparations whether the enhanced endothelium-dependent relaxation at early stages might be due to diabetes-induced increases in synthesis of vasodilator prostaglandins, EDHF, or NO, or a combination of any of these endothelium-derived factors. In this regard, studies conducted in mesenteric104 and renal102 arteries in the presence of indomethacin suggest that vasoactive prostanoids do not contribute to enhanced endothelium-dependent relaxation, leaving EDHF and NO as candidate factors.Conclusion. Time of evaluation after onset of disease may be critical to demonstrating endothelial dysfunction. Indeed, evidence exists for enhanced endothelial function at early stages followed by dysfunction at later stages. Whether these opposing actions are causally linked is not yet known. Further studies are warranted to understand this temporal dichotomy.Role of Prostanoids in Endothelium-Dependent Dilation in DiabetesImpaired endothelium-dependent relaxation in diabetes cannot always be assumed to be mediated by a reduction in NO activity or synthesis, since some vasodilators also release prostaglandins. Changes in prostaglandin synthesis may alter NO production or reactivity to NO. Because diabetes alters the reactivity to prostanoids and may either increase or decrease prostacyclin production depending on the artery chosen,105 this issue needs to be resolved, especially at various stages of disease and in individual blood vessel preparations.Simultaneous enhancement and release of vasoconstrictor prostaglandins may explain some instances of impaired endothelium-dependent relaxation. Indeed, in aorta, pial artery, and mesenteric artery, relaxation is either normal94 or is normalized in the presence of indomethacin or thromboxane receptor antagonists.1321103106 In contrast, endothelial function was normal in coronary arteries of alloxan-diabetic dogs, but dysfunction was unmasked in the presence of inhibitors of cyclooxygenase,11107 suggesting enhanced compensatory increases in vasodilator prostanoid release. Furthermore, increases in production of the vasodilator prostacyclin have been reported in perfused mesenteric beds of 3-week diabetic rats.108 While not directly examined, this might provide an alternative explanation for one of the early reports showing augmented relaxation to the agonist histamine in mesenteric arteries of diabetic rats109 or the observation of normal relaxation seen in perfused mesenteric beds of long-term diabetic rats.94 In contrast, this may not be adequate to explain the increased endothelium-dependent relaxation seen in perfused kidney at early stages of disease, since this increase was seen in indomethacin-treated preparations.102One investigator has shown that thromboxane receptor antagonism does not alter relaxation in basilar artery110 but restores relaxation of pial arteries taken from the streptozotocin-diabetic rat model.21 These observations suggest important regional differences, although one cannot exclude the possible contribution of the superimposition of the variable in the duration of disease, which was 4 to 5 months in the basilar artery study versus 2.5 to 3.5 months for the pial artery study. In another model of alloxan-diabetic dogs in which duration of disease was held constant, inclusion of indomethacin or ibuprofen unmasked a defect of endothelium-dependent relaxation in coronary artery11 but not renal artery.111 Collectively, these studies suggest important regional differences in the mechanism of endothelial dysfunction.Many studies using either rat conduit arteries914161718112113114 or rat resistance arteries252691103115116117 have indicated no improvement in endothelial function after evaluation under conditions of cyclooxygenase blockade or thromboxane receptor antagonism. Similar interventions have failed to modify defective relaxation in aorta of the genetic diabetic BB rat33 and in coronary arteries of alloxan-diabetic dogs.20The effect of prostanoids on endothelial function in human diabetes has not been routinely evaluated. Prior treatment with indomethacin in vitro normalized impaired endothelium-dependent relaxation in cutaneous arteries of gestational diabetes.118 In contrast, in one human study in which all patients received aspirin before evaluation,44 the authors concluded that exogenous prostanoid synthesis cannot account for the endothelial dysfunction in IDDM.Conclusion. There exist some potentially important regional differences in the role of prostanoids in contributing to altered endothelium-dependent dilation in diabetes mellitus, although this cannot be easily predicted among various conduit versus resistance vessels. Nevertheless, there is clear and ample evidence to suggest that alterations in prostanoid production may not always account for and/or may not be obligatory for impaired endothelial function in diabetes.NO-Dependent or -Independent Endothelial DysfunctionImplicit in all of these studies is the assumption that endothelium-dependent relaxation in both control and diabetic blood vessels is exclusively mediated via NO. This assumption is hazardous because endothelium-dependent relaxation to certain agonists and in certain arteries appears to be mediated in part by vasodilator prostanoids or by an EDHF.119 The entity of EDHF is not known with complete certainty, but at least one EDHF is believed to be an epoxide of arachidonic acid that is formed by a cytochrome P450–derived monooxygenase.120EDHF appears to activate K+ channels, especially calcium-activated K+ channels.119 The contribution of KATP channels to relaxation in diabetes is uncertain and should also be considered. A few studies have noted diminished responses to KATP channel openers in diabetic rat aorta15121122 and basilar artery.123 One study in the aorta of WBN/Kob rat showed no alteration in relaxation to the KATP channel agonist cromakalim.124 In contrast, others have observed a paradoxically enhanced response in dog coronaries to aprikalim, albeit in short-term diabetes.125 In normal arteries, it is generally believed that the component of EDHF versus NO that contributes to total relaxation increases with decreasing vessel size. The observation that diabetes-induced endothelial dysfunction can occur in both conduit and resistance arteries despite cyclooxygenase blockade suggests that this dysfunction could be explained by defects in EDHF or by deficits in NO synthesis unrelated to or in addition to changes in EDHF or prostanoids.Two studies in rat aorta reveal that endothelial dysfunction persists despite pretreatment with TEA (to inhibit calcium-activated K+ channels), suggesting that defects in EDHF may not be operative.126127 Alternatively, a recent study in perfused kidney indicates that endothelium-dependent relaxation in control kidney arises from both NO and EDHF, whereas relaxation in diabetic kidney arises from NO, EDHF, and prostanoids.128Few studies have examined perturbations in membrane polarization in diabetic vascular tissue. Membrane hyperpolarization is known to occur in gastric gland of 2- to 3-day diabetic rabbits129 and in endothelial cells from human subjects with gestational diabetes.130 One study showed normal resting membrane potential but diminished hyperpolarization in the response of diabetic mesenteric artery to acetylcholine131 despite unaltered hyperpolarization in response to the K+ channel agonist pinacidil. This report conducted in the presence of NOS and cyclooxygenase inhibitors suggested diminished endothelium-dependent, TEA-sensitive hyperpolarization and relaxation.Conclusion. Endothelial dysfunction in some cases of diabetes and in certain blood vessel types may arise from deficits in EDHF, but it is also clear that endothelial dysfunction can also occur despite blockade of EDHF and prostanoid synthesis and action, suggesting a role for deficits of endothelium-derived NO. It is possible that previous studies may need to be carefully reevaluated, since alternative compensatory pathways including cytochrome P450–derived EDHF may be activated or inactivated, which could mask impaired endothelium-dependent relaxation or compensate for defective NO synthesis. It should be emphasized that parallel compensatory pathways may be important at certain stages but perhaps not at all stages of the disease.Evidence Supporting Altered NO Production From Diabetic EndotheliumFunctional Studies in the Presence of SODNO activity is known to be reduced by chemical interaction and destruction by superoxide anion radicals. Several investigators have noted improved relaxation after acute incubation with SOD.1425263377117132 This issue has been discussed in detail in a previous review.7 Nevertheless, these studies are consistent with increased destruction of NO and decreased bioactivity of NO in diabetes, but they do not give adequate direct information regarding perturbations in NO production.Currently, there have been no studies that have directly determined NO synthesis or release from diabetic endothelium. Only one laboratory has examined intraluminal release of NO activity from perfused diabetic rat aorta donor segments using the bioassay technique.133 Accordingly, luminally released basal NO activity in perfused diabetic rat aorta was normal, but addition of SOD caused a larger incremental increase in relaxation of the bioassay detector when the diabetic donor segment was used. This suggested the increased release of superoxide anion radicals from diabetic rat endothelium. In contrast, acetylcholine-stimulated endothelium-derived relaxing factor/NO bioactivity from perfused diabetic rat aorta was diminished but also normalized by perfusion with SOD.114Conclusion. Several studies support the concept that IDDM decreases NO bioactivity. This may be due in part to enhanced destruction of NO by increased superoxide synthesis.Indirect Evidence Using Guanylate Cyclase InhibitorsUse of inhibitors of NOS or NO reactivity (eg, hemoglobin or methylene blue) has given indirect information regarding the contribution of NO to basal tone and agonist-induced relaxation in control versus diseased blood vessels. Many studies that have used removal of endothelium or NOS inhibitors or hemoglobin, which increase the precontracted tone of arteries, reveal decreased basal NO activity in both diabetic conduit1694134135136137138139140 and resistance242535132140141142143144145 arteries under both in vitro and in vivo conditions.Two studies noted enhanced contraction to methylene blue in control versus diabetic rat aorta ring preparations, suggesting diminished basal NO in diabetic arteries.16133 The use of methylene blue alone is inadequate because of the known effects of methylene blue to stimulate superoxide anion radical or inhibit NOS.146147 One study using a new highly selective inhibitor of guanylate cyclase, ODQ, revealed that ODQ completely inhibits acetylcholine-induced relaxation in control rat aorta and inhibits relaxation in diabetic rat aorta by 80%.148 This suggests that almost all of the relaxation in both groups is mediated by cGMP activation, implicating NO as the EDRF. Also, the small ODQ-resistant component of relaxation in diabetic rat aorta leaves open the possibility of some other unknown factor that appears unrelated to vasodilator prostanoids, H2O2, or a TEA-sensitive EDHF.Conclusion. Overall, these studies suggest diminished basal NO production in diabetes.Evidence Using NO Trapping AgentsBasal NO activity in rat aorta has also been assessed using either a small-molecular-weight, iron-thiol-based NO scavenger149 or a nitronyl nitroxide trapping agent.148 These agents are different in that they do not alter NOS activity but react with NO after its release. These probes were effective in scavenging all basal NO based on similar tension responses after removal of endothelium or by using NOS inhibitors and confirm a smaller basal NO activity in diabetic rat aorta. This NO scavenger–sensitive basal NO activity was augmented by l-arginine in diabetic but not control aorta.149In contrast, despite nearly complete inhibition of acetylcholine-mediated relaxation by both control and diabetic rat aorta by NOS inhibitors, use of a nitronyl nitroxide revealed an NO scavenger–resistant component of agonist-stimulated relaxation that is greater in diabetic than control arteries.148 This resistant component of endothelium-dependent relaxation was sensitive to NOS inhibitor but was insensitive to indomethacin, TEA, or catalase, suggesting that this additional EDRF is likely not EDHF, prostanoids, or H2O2. A significant portion of this resistant component was eliminated using ODQ, suggesting that this EDRF is an activator of guanylate cyclase. Current research continues to attempt to identify whether diabetic arteries produce an additional vasoactive product that is derived from the NOS reaction but that may not necessarily be a free NO radical.Measurement of NO by cGMP GenerationInformation regarding potential deficits in NO synthesis have also been derived f