Sustained pulmonary vasodilation after inhaled nitric oxide for hypoxic pulmonary hypertension in swine

It has been shown that pulmonary vasodilation is sustained after discontinuation of inhaled nitric oxide (INO) during moderate hypoxic pulmonary hypertension (HPH) in swine. The present investigations demonstrated how INO dose, hypoxia duration, and endogenous NO production influence this important phenomenon. Fifteen adolescent Yorkshire swine were randomly assigned to three groups (n = 5 each) and underwent the following phasic experimental protocol: (I) Baseline ventilation (FIO2 = .3); (II) Initiating HPH (FIO2 = .16 to .18, PaO2 = 45 to 55 mm Hg); (III) INO at 10 ppm; (IV) Posttreatment observation; (V) INO of 80 ppm; and (VI) Posttreatment observation. Phase II (pretreatment hypoxia) lasted 30 minutes in group A (short hypoxia) and 120 minutes in group B (long hypoxia). N-nitro-L-arginine methyl ester (NAME) was used to inhibit nitric oxide synthase (NOS) throughout the experiment in group C (short hypoxia + NAME). Hemodynamics and blood gases were monitored by systemic and pulmonary artery catheters placed by femoral cutdown. Analysis of variance with post-hoc adjustment was used to compare groups at each phase, and the paired t test was used for comparisons within a group. With respect to baseline mean pulmonary artery pressure (MPAP) and pulmonary vascular resistance (PVR), there were no significant differences among the three groups. MPAP and PVR were significantly higher in group C than in group A during phase II, (MPAP, 76% +/- 8% v 33% +/- 2%; PVR, 197% +/- 19% v 78% +/- 10%; P < .05). There were no significant differences in MPAP or PVR during phases III through VI. When MPAP was expressed as percent dilation, 80 ppm caused significantly more dilation than did 10 ppm in all three groups. Groups A and C had significantly higher sustained pulmonary artery dilation after 80 ppm than after 10 ppm (A, 82% +/- 31% v 17% +/- 11%; C, 68% +/- 10% v 42% +/- 12%; both P < .05), but group B did not (43% +/- 15% v 30% +/- 9%; P = .25). High dose results in stronger vasodilation than low dose during and after INO for moderate HPH of short duration. Long hypoxia blunts this high-dose advantage. Endogenous NO inhibition augments HPH but does not decrease pulmonary vasodilation during or after INO.     

Endothelial nitric oxide synthase in hypoxic newborn porcine pulmonary vessels

AIMS: To determine if the failure of neonatal pulmonary arteries to dilate is due to a lack of nitric oxide synthase (NOS). METHODS: A monoclonal antibody to endothelial NOS was used to demonstrate the distribution and density of NOS in the developing porcine lung after a period in hypobaric hypoxia. Newborn piglets were made hypertensive by exposure to hypobaric hypoxia (50.8 kPa) from < 5 minutes of age to 2.5 days of age, 3-6 days of age or 14-17 days of age. A semiquantitative scoring system was used to assess the distribution of endothelial NOS by light microscopy. RESULTS: NOS was present in the arteries in all hypoxic animals. However, hypoxia from birth caused a reduction in NOS compared with those lungs normal at birth and those normal at 3 days. Hypoxia from 3-6 days led to a high density of NOS compared with normal lungs at 6 days. Hypoxia from 14-17 days had little effect on the amount of NOS. On recovery in room air after exposure to hypoxia from birth there was a transient increase in endothelial NOS after three days of recovery, mirroring that seen at three days in normal animals. CONCLUSIONS: Suppression of NOS production in the first few days of life may contribute to pulmonary hypertension in neonates.     

The role of neuronal and endothelial nitric oxide synthase in retinal excitotoxicity

PURPOSE: Nitric oxide synthase (NOS) plays an essential role in neuronal function and is critical in the brain for normal and pathologic responses to glutamate. The role of NOS in the retina is less well understood. The retina provides an experimental system in which the intrinsic circuitry is well defined; retinal excitotoxic damage has been well characterized. METHODS: To determine whether neuronal NOS (nNOS) and endothelial NOS (eNOS) are critical in excitotoxic damage in the retina, nNOS- and eNOS-deficient mice were subjected to intravitreal injections of N-methyl-D-aspartate (NMDA) or to arterial occlusions. RESULTS: Retinal ganglion cells in the nNOS-deficient mouse were relatively resistant to NMDA and to arterial occlusion. In contrast, the damage in the eNOS-deficient mouse retina was not distinguishable from that in control animals. Preinjection with an NOS inhibitor was partially protective. CONCLUSIONS: The presence of nNOS is a prerequisite for the full expression of excitotoxicity in the retina; eNOS does not appear to play a significant role.     

The pulmonary effect of nitric oxide synthase inhibition following endotoxemia in a swine model

OBJECTIVE: To evaluate the pulmonary effect of treatment with N-nitro-L-arginine methyl ester (NAME) with and without inhaled nitric oxide (NO) in a swine model of endotoxemia. DESIGN: Randomized controlled trial. SETTING: Laboratory. INTERVENTIONS: Following a 20-minute intravenous infusion of Escherichia coli lipopolysaccharide (LPS) (200 micrograms/kg), animals were resuscitated with saline solution (1 mL/kg per minute) and observed for 3 hours while mechanically ventilated (fraction of inspired oxygen [FIO2], 0.6; tidal volume, 12 mL/kg; positive end-expiratory pressure, 5 cm H2O). Group 1 (LPS, n = 6) received no additional treatment; group 2 (NAME, n = 5) received NAME (3 mg/kg per hour) for the last 2 hours; group 3 (NO, n = 6) received NAME (3 mg/kg per hour) and inhaled NO (40 ppm) for the last 2 hours; and group 4 (control, n = 5) received only saline solution without LPS. MAIN OUTCOME MEASURES: Cardiopulmonary variables and blood gases were measured serially. The multiple inert gas elimination technique was performed at 3 hours. The wet-to-dry lung weight ratio was measured following necropsy. RESULTS: Administration of LPS resulted in pulmonary arterial hypertension, pulmonary edema, and hypoxemia with increased ventilation perfusion ratio mismatching. None of these changes were attenuated by NAME treatment alone but all were significantly improved by the simultaneous administration of inhaled NO. CONCLUSIONS: Systemic NO synthase inhibition failed to restore hypoxic pulmonary vasoconstriction following LPS administration. The deleterious effects of endotoxemia on pulmonary function can be improved by inhaled NO but not by systemic inhibition of NO synthase.     

Inhibition of endogenous nitric oxide synthase potentiates nitrovasodilators in experimental pulmonary hypertension

BACKGROUND: The role of endogenous nitric oxide (NO) in the regulation of pulmonary vascular tone is complex. Inhibition of endogenous NO synthase, potentially through upregulation of guanylyl cyclase, results in an increase in potency of nitrovasodilators in the systemic circulation. This study considered whether inhibition of endogenous NO synthase would increase the potency of nitrovasodilators, but not of cyclic adenosine monophosphate-dependent vasodilators, in the pulmonary vasculature. METHODS: We used the isolated buffer-perfused rabbit lung. Preparations were randomized to receive either pretreatment with NG-nitro-L-arginine methyl ester (or L-NAME, an inhibitor of endogenous NO synthase) or no pretreatment. Stable pulmonary hypertension was then produced by infusing the thromboxane A2 analog U46619. The dose-response characteristics of two nitrovasodilators, sodium nitroprusside and nitroglycerin, and two nonnitrovasodilators, prostaglandin E2 and 5'-N-ethylcarboxamidoadenosine, were studied. RESULTS: Inhibition of endogenous NO synthase caused no significant changes in baseline pulmonary artery pressure but did significantly reduce the U46619 infusion rate required to produce pulmonary hypertension. Pretreatment with L-NAME (vs. no L-NAME) resulted in significantly lower values of the log median effective dose with sodium nitroprusside and nitroglycerin. In contrast, pretreatment with L-NAME resulted in no changes in the dose-response characteristics of the cyclic adenosine monophosphate-mediated, NO-independent vasodilators prostaglandin E1 and 5'-N-ethylcarboxamidoadenosine. CONCLUSIONS: These data suggest that endogenous NO synthase is not an important regulator of basal pulmonary tone in this model but is an important modulator of pulmonary vascular responses to vasoconstriction and to nitrovasodilators. The pulmonary vasodilator effects of nitrovasodilators, but not of nonnitrovasodilators, may depend on the level of activity of NO synthase.     

Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis

BACKGROUND: NO regulates vascular tone and structure, platelets, and monocytes. NO is synthesized by endothelial NO synthase (eNOS). Endothelial dysfunction occurs in atherosclerosis. METHODS AND RESULTS: With a porphyrinic microsensor, NO release was measured in atherosclerotic human carotid arteries and normal mammary arteries obtained during surgery. eNOS protein expression was analyzed by immunohistochemistry. In normal arteries, the initial rate of NO release after stimulation with calcium ionophore A23187 (10 micromol/L) was 0.42+/-0.05 (micromol/L)/s (n=10). In contrast, the initial rate of NO release was markedly reduced in atherosclerotic segments, to 0.08+/-0.04 (micromol/L)/s (n=10, P<0.0001). NO peak concentration in normal arteries was 0.9+/-0.09 micromol/L (n=10) and in atherosclerotic segments, 0.1+/-0.03 micromol/L (n=10, P<0.0001). Reduced NO release in atherosclerotic segments was accompanied by marked reduction of immunoreactive eNOS in luminal endothelial cells, although specific endothelial cell markers (CD31) were present (n=13). Endothelial cells of vasa vasorum of atherosclerotic segments, however, remained positive for eNOS, as was the endothelium of normal arteries. CONCLUSIONS: In clinically relevant human atherosclerosis, eNOS protein expression and NO release are markedly reduced. This may be involved in the progression of atherosclerosis.     

Depressed renal and vascular nitric oxide synthase expression in cyclosporine-induced hypertension

With the longer life expectancy of the population, calcific aortic stenosis has become a common cardiac problem in the elderly. When patients with moderate to severe aortic stenosis become symptomatic, the prognosis is usually poor in absence of valve replacement and sudden death is a feared complication. It has been hypothesized that malignant ventricular arrhythmias could be responsible for the high incidence of sudden death in symptomatic patients with aortic stenosis. The purpose of this review is to analyze the prevalence, the electrophysiologic mechanisms, and the possible role of ventricular arrhythmias in the development of symptoms and in the outcome of adult subjects with aortic stenosis.     

Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts

In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA.     

Estrogen upregulates endothelial constitutive nitric oxide synthase expression in human osteoblast-like cells

The protective effects of estrogen on the cardiovascular system are thought to be mediated, in part, by nitric oxide (NO). Estrogen also has protective effects on bone although the mechanisms of action have not been fully established. Since nitric oxide synthase (NOS) inhibitors have been found to abrogate the protective effect of estrogen on bone in ovariectomised rats, we studied the effects of 17beta-estradiol on NOS activity and NOS mRNA levels in cultured human osteoblast-like cells. 17beta-Estradiol stimulated NOS activity by approximately 2.0 fold and this effect was reversed by the calcium chelator, EGTA, and the NOS inhibitor, L-NMMA, implicating activation of a constitutive, calcium-dependent isoform. Further studies using RT/PCR indicated that only the endothelial nitric oxide synthase (ecNOS) isoform was expressed and RNase protection assays showed that 17beta-Estradiol treatment resulted in a 2.2 fold increase in ecNOS mRNA levels. These findings suggest that estrogen stimulates NOS activity in osteoblastic cells by activation of the ecNOS pathway, and taken together with previous data, is consistent with the possibility that NO may act as a mediator of estrogen actions on bone.     

The role of endothelial nitric oxide synthase in the pathogenesis of a rat model of hepatopulmonary syndrome

BACKGROUND & AIMS: The hepatopulmonary syndrome occurs when intrapulmonary vasodilatation causes impaired arterial gas exchange in liver disease. The pathogenesis is poorly understood, although nitric oxide may be involved. Common bile duct ligation in the rat is a model of the hepatopulmonary syndrome, but no studies have evaluated NO in pulmonary vasodilatation in this model. The aim of this study was to determine whether NO contributes to intrapulmonary vasodilatation after bile duct ligation. METHODS: Endothelial and inducible NO synthase (NOS) levels and localization and NO activity in pulmonary artery rings were assessed after bile duct ligation. RESULTS: Pulmonary endothelial NOS levels increased and alveolar vascular staining was enhanced after bile duct ligation. No change in pulmonary inducible NOS levels or localization was detected. Increased endothelial NOS levels correlated with alterations in gas exchange and were accompanied by enhanced NO activity and a blunted response to phenylephrine, reversible by NOS inhibition, in pulmonary artery rings. Portal-vein-ligated animals, which do not develop intrapulmonary vasodilatation, had no changes in pulmonary NOS production or in NO activity in pulmonary artery rings. CONCLUSIONS: NO, derived from pulmonary vascular endothelial NOS, contributes to intrapulmonary vasodilation in animal hepatopulmonary syndrome.     

Calcium channel blockade enhances nitric oxide synthase expression by cultured endothelial cells

In a recent study, we found marked increases in nitric oxide (NO) production and endothelial and inducible NO synthase (eNOS and iNOS) expressions with calcium channel blockade in rats with chronic renal failure. This study was undertaken to determine whether enhanced NO production with calcium channel blockade is a direct effect of this therapy or a consequence of the associated hemodynamic and humoral changes. We tested the effects of a calcium channel blocker, felodipine (10(-5), 10(-6), and 10(-7) mol/L), on nitrate and nitrite (NOx) generation, Ca2+-dependent and -independent NOS activity, and eNOS and iNOS protein masses in proliferating and quiescent rat aortic endothelial cells in culture. Compared with vehicle alone, felodipine significantly increased NOx generation, Ca2+-dependent NOS activity, and eNOS protein mass in proliferating and quiescent endothelial cells. Felodipine did not modify the stimulatory action of 10% fetal calf serum on DNA synthesis (thymidine incorporation) and cell proliferation. Ca2+-independent NOS activity and iNOS protein expression were negligible and unaffected by calcium channel blockade. NOx production and NOS expression were greater in proliferating cells than in quiescent cells. Thus, calcium channel blockade upregulates endothelial NO production in vitro, confirming our previous in vivo study. This observation indicates that the reductions in cytosolic [Ca2+] and vasodilation with calcium channel blockade are not only due to inhibition of Ca2+ entry but also to an NO-cGMP mediated mechanism.     

Genetic contribution of the endothelial constitutive nitric oxide synthase gene to plasma nitric oxide levels

Nitric oxide (NO) has an important physiological role in regulating vascular tone and is also relevant to many pathological processes including hypertension and atherosclerosis. Endothelial constitutive nitric oxide synthase (ecNOS) is the key enzyme in determining basal vascular wall NO production. We used a combination of maximum-likelihood-based statistical genetic methods to explore the contributions of the ecNOS gene and other unmeasured genes to basal NO production measured by its metabolites (NOx: nitrite and nitrate) in 428 members of 108 nuclear families. Our initial quantitative genetic analysis estimated that approximately 30% of the variance in fasting NOx levels is due to genes (chi 2(1) = 16.04, P = .000062). Complex segregation analysis detected the effects of both a single locus and residual polygenes on NOx levels, and measured genotype analysis showed that plasma NOx levels in those homozygous for the rare allele (64.9 +/- 7.8 mumol/L) were significantly higher (P = .000242) than those homozygous for the common allele (30.2 +/- 3.1 mumol/L). The results of the variance component linkage analysis were consistent with linkage of a quantitative trait locus in or near the ecNOS gene to variation in plasma NOx levels (P = .0066). While many environmental factors have been shown to alter transiently plasma NOx levels, our study is the first to identify a substantial effect of the ecNOS locus on the variance of plasma NOx, i.e. basal NO production. This finding may be relevant to atherogenesis and NO-related disorders.     

The role of inducible nitric oxide synthase in cardiac allograft rejection

Rats were exposed to either a footshock stimulus (FS) or emotional stimulus (ES, forced perception of another rat receiving footshocks) during a daily 10-min session for 5 consecutive days. The consequences of FS and ES on their behavioural responsiveness were assessed at different post-stress intervals using a small open-field. FS induced a decrease in ambulation, rearing and sniffing and an increased immobility in the small open field. These effects were present in rats tested immediately after the last session and remained present for at least 15 days. In contrast, ES induced a transient decrease in ambulation and rearing immediately after the last session, but in the period from half an hour until at least 15 days after the stimulus experience, an increase in ambulation, rearing and sniffing was observed. Exposure to one footshock per session for 5 consecutive days or to 10 footshocks in a single session also resulted in a long-lasting reduction in ambulation and sniffing and an increase in immobility. The former regime did not influence the behavioural response of ES rats, but the latter resulted in an increase in ambulation, rearing and sniffing in ES rats. Naloxone (1 mg/kg s.c.) pretreatment antagonized the increased behavioural activity of the ES rats whereas the activity of control and FS animals was not affected, suggesting an involvement of endogenous opioid systems in the behavioural responses observed in ES rats. It is suggested that the behavioural responses of the ES and FS animals are regulated by different mechanisms.     

Brain nitric oxide synthase messenger RNA in central mineralocorticoid hypertension

The mechanism underlying the central hypertensinogenic effects of mineralocorticoids remains unclear. Given that nitric oxide (NO) is thought to act at autonomic sites in the brain to regulate arterial blood pressure, the effects of the potent mineralocorticoids aldosterone and 19-noraldosterone on the abundance of neuronal NO synthase (nNOS) mRNA in the brain were investigated. Wistar-Kyoto rats received a continuous intracerebroventricular infusion of aldosterone or 19-noraldosterone (5 ng/h) from an implanted osmotic minipump for 4 weeks. Total RNA was purified from microdissected tissue blocks containing the hypothalamus, dorsal medulla, rostral ventrolateral medulla, or caudal ventrolateral medulla, and changes in the abundance of nNOS mRNA were determined with a semiquantitative competitive polymerase chain reaction method. Blood pressure was significantly increased in rats 2, 3, and 4 weeks after the onset of intracerebroventricular aldosterone or 19-noraldosterone infusion compared with that in animals receiving vehicle. Subcutaneous infusion of either mineralocorticoid had no effect on blood pressure. Compared with controls, rats treated with aldosterone or 19-noraldosterone for 4 weeks showed significant decreases in the amount of nNOS mRNA in the hypothalamus and rostral and caudal ventrolateral medulla. These data suggest that reduced nNOS activity may contribute to the increase in blood pressure in rats with central mineralocorticoid-induced hypertension.     

Non-anticoagulant heparin increases endothelial nitric oxide synthase activity: role of inhibitory guanine nucleotide proteins

Heparin, which is widely used clinically, has recently been shown to have specific properties affecting the vascular endothelium. We hypothesized that heparin stimulates endothelial nitric oxide synthase (eNOS) activity by a mechanism independent of its anticoagulant properties and dependent on an inhibitory guanine nucleotide regulatory protein (Gi). We determined the effect of both heparin and N-acetyl heparin (Non-Hep), a heparin derivative without anticoagulant properties, on eNOS activity in cultured bovine aortic endothelial cells and on endothelium-dependent relaxation in isolated vascular rings. The eNOS activity was determined by measuring both citrulline and nitric oxide (NO) metabolite formation. Heparin and Non-Hep dose-dependently increased basal eNOS activity (ED50 1.0 microgram/ml or 0.15 U/ml), an effect that was significantly inhibited by pertussis toxin (100 ng/ml), a Gi-protein inhibitor. Agonist-stimulated (acetylcholine, 10 microM) eNOS activity was potentiated following pre-treatment with both heparin and Non-Hep and reversed by pertussis toxin. Heparin and Non-Hep induced a dose-dependent relaxation in preconstricted thoracic aortic rings, an effect that was significantly inhibited by pertussis toxin, endothelial inactivation (following treatment with sodium deoxycholate) and NG-nitro-L-arginine-methyl ester (L-NAME). We conclude that heparin and non-anticoagulant heparin induce endothelium-dependent relaxation following activation of eNOS by a mechanism involving a Gi-protein. Administration of heparin derivatives without anticoagulant properties may have therapeutic implications for the preservation of eNOS in conditions characterized by endothelial dysfunction.     

Rebound pulmonary hypertension after inhalation of nitric oxide

BACKGROUND: We describe the hemodynamic response to initiation and withdrawal of inhaled nitric oxide (NO) in infants with pulmonary hypertension after surgical repair of total anomalous pulmonary venous connection. METHODS: Between January 1, 1992, and January 1, 1995, 20 patients underwent repair of total anomalous pulmonary venous connection. Nine patients had postoperative pulmonary hypertension and received a 15-minute trial of inhaled NO at 80 parts per million. Five of these patients received prolonged treatment with NO at 20 parts per million or less. RESULTS: Mean pulmonary artery pressure decreased from 35.6 +/- 2.4 to 23.7 +/- 2.0 mm Hg (mean +/- standard error of the mean) (p = 0.008), and pulmonary vascular resistance decreased from 11.5 +/- 2.0 to 6.4 +/- 1.0 U.m2 (p = 0.03). After prolonged treatment with NO, pulmonary artery pressure increased transiently in all patients when NO was discontinued. CONCLUSIONS: After operative repair of total anomalous pulmonary venous connection, inhaled NO selectively vasodilated all patients with pulmonary hypertension. Withdrawal of NO after prolonged inhalation was associated with transient rebound pulmonary hypertension that dissipated within 60 minutes. Appreciation of rebound pulmonary hypertension may have important implications for patients with pulmonary hypertensive disorders when interruption of NO inhalation is necessary or when withdrawal of NO is planned.     

Cytoplasmic localization of endothelial constitutive nitric oxide synthase in endometrial carcinomas

To understand the structural features that dictate the selectivity of diverse nonsteroidal antiinflammatory drugs for the two isoforms of the human prostaglandin H2 synthase (PGHS), the three-dimensional (3D) structure of human COX-2 was assessed by means of sequence homology modeling. The ovine COX-1 structure, solved by X-ray diffraction methods and sharing a 61% sequence identity with human COX-2, was used as template. Both structures were energy minimized using the AMBER 4.0 force field with a dielectric constant of 4r. (S)-Flurbiprofen, a nonselective COX inhibitor, and SC-558, a COX-2-selective ligand, were docked at the cyclooxygenase binding site in both isozymes, evidencing the role of different residues in the ligand-protein interaction. The 3D structures of the constructed four ligand-enzyme complexes were refined by energy minimization. Molecular dynamics simulations were also carried out, to understand more deeply the structural origins of the selectivity. Distances calculated during the dynamics process between the different ligands and the interacting residues of the two PGHS isozymes provided evidence of the flexible nature of the cyclooxygenase active site, permitting the identification of different conserved and nonconserved residues as responsible for ligand selectivity.     

Smoking impairs the activity of endothelial nitric oxide synthase in saphenous vein

Smoking impairs the endothelium-dependent relaxation of arteries and veins, with the maximum relaxation in response to the calcium ionophore A23187 of saphenous vein rings being reduced from 53 +/- 4% in nonsmokers to 27 +/- 5% in smokers. We have investigated whether this endothelial dysfunction was attributable to altered activity or concentration of nitric oxide synthase (NOS). The concentration of NOS in saphenous vein endothelium, determined by Western blotting and immunohistochemistry, was not different in nonsmokers and smokers. Nitrite production from vein strips stimulated with A23187 was higher in nonsmokers (median 23.6 nmol.cm-2.h-1) than smokers (median 3.3 nmol.cm-2.h-1), P=.001, this difference being abolished when vein strips were preincubated in the presence of NG-monomethyl-L-arginine. Organ chamber studies to monitor the endothelium-dependent relaxation of vein rings in response to A23187 showed that preincubation of rings from smokers with either L-arginine (3mmol/L) or superoxide dismutase (250 U/mL) did not improve the maximum relaxation. In contrast, preincubation of vein rings from smokers with 20 micromol/L tetrahydrobiopterin increased the maximum relaxation from 27 +/- 5% to 51 +/- 6%, P=.01. Preincubation of vein from smokers with tetrahydrobiopterin also significantly increased nitrite and cGMP production in response to stimulation with A23187. The impaired endothelium-dependent relaxation of saphenous vein rings from smokers appears to be caused by a reduction in the activity of endothelial NOS that is attributable to an inadequate supply of the coenzyme tetrahydrobiopterin.     

Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis

The presence of nitric oxide (NO) in the kidney has been implicated in the pathogenesis of human glomerulonephritis. However, the exact type of glomerular cells that express NO synthase (NOS) and the NOS isoform involved in the local production of NO has not been identified in the human diseased kidney. We examined the expression of three isoforms of NOS, inducible NOS (iNOS), endothelial NOS (eNOS) and brain NOS (bNOS) in the renal tissue of patients with IgA nephropathy (IgAN, N = 10), lupus nephritis (LN, N = 5), membranous nephropathy (MN, N = 5) and minimal change nephrotic syndrome (MCNS, N = 5). Sections were immunostained and the correlation between the expression of each NOS and the degree of glomerular injury in that section was also examined. Normal portions of surgically resected kidneys served as controls. eNOS was present in glomerular endothelial cells and endothelium of cortical vessels in the control and diseased kidneys. iNOS was localized in mesangial cells, glomerular epithelial cells and infiltrating cells in the diseased glomeruli, whereas immunostaining for iNOS was hardly detected in control kidneys. In addition, the expression pattern of eNOS in each glomerulus was the reverse of that of iNOS. In IgAN and LN, the extent of staining for eNOS correlated negatively with the degree of glomerular injury, while the extent of staining for iNOS correlated positively with the degree of glomerular injury in the same tissues. bNOS was not detected in normal or nephritic glomeruli. Our results indicate the presence of a NO pathway in human diseased kidney, and suggest that NO derived from eNOS and iNOS may be involved in the progression of renal diseases and that NO derived from each NOS may play an important role in different way in human inflamed glomeruli.