Acute hypoxic pulmonary vasoconstriction (HPV) and the development of chronic hypoxic pulmonary hypertension (PHTN) are cardinal features of the pulmonary circulation that differentiate this vascular bed from the systemic circulation. Nitric oxide (NO) produced by pulmonary vascular endothelium is thought to modulate pulmonary vascular responses to a variety of vasoconstrictor stimuli, including hypoxia (1-8). However, despite intensive investigation over the past decade, the role of endotheliumderived nitric oxide (EDNO) in modulating tone and structural remodeling of the chronically hypoxic pulmonary circulation remains controversial (9-13).Simultaneous pharmacologic inhibition of all three isoforms of nitric oxide synthase (NOS) results in acutely increased pulmonary vascular resistance and augmented HPV (1,(14)(15). However, chronic NOS inhibition does not result in PHTN and does not augment the development of hypoxic PHTN (16). These results present a paradox: If EDNO modulates acute HPV, why is pharmacologic inhibition of NOS not associated with either normoxic PHTN or accentuated chronic hypoxic PHTN? One possibility is that HPV is redundantly modulated, and the loss of endothelial nitric oxide synthase (eNOS)-derived NO alone is not sufficient to produce PHTN. Alternatively, the experimental approaches taken in the past to test this question may have been inadequate because of nonspecific effects of pharmacologic inhibitors of NOS, difficulty maintaining NOS inhibition, or confounding effects of simultaneous inhibition of all three isoforms of NOS.Mice with targeted disruption of eNOS (eNOS -/-mice) have recently been constructed and the vascular phenotype explored (17,18). Systemic hypertension and augmented structural remodeling after vascular injury have been reported (19,20). Steudel et al. (21) investigated the pulmonary vascular phenotype of eNOS -/-mice, finding increased pulmonary vascular resistance, but only minimal PHTN, and no evidence of pulmonary vascular remodeling. More recently, this group also found enhanced chronic hypoxic PHTN in eNOS-null mice (22). These studies were limited, however, to mice exposed to severe hypoxia (FiO 2 = 11%) and studied under general anesthesia with hyperoxic (FiO 2 = 80%) mechanical ven- Acute hypoxic vasoconstriction and development of hypoxic pulmonary hypertension (PHTN) are unique properties of the pulmonary circulation. The pulmonary endothelium produces vasoactive factors, including nitric oxide (NO), that modify these phenomena. We tested the hypothesis that NO produced by endothelial nitric oxide synthase (eNOS) modulates pulmonary vascular responses to hypoxia using mice with targeted disruption of the eNOS gene (eNOS -/-). Marked PHTN was found in eNOS -/-mice raised in mild hypoxia when compared with either controls or eNOS -/-mice raised in conditions simulating sea level. We found an approximate twofold increase in partially and fully muscularized distal pulmonary arteries in eNOS -/-mice compared with controls. Consistent with vasoconstriction...

Nitric oxide plays an important role in modulating pulmonary vascular tone. All three isoforms of nitric oxide synthase (NOS), neuronal (nNOS, NOS I), inducible (iNOS, NOS II), and endothelial (eNOS, NOS III), are expressed in the lung. Recent reports have suggested an important role for eNOS in the modulation of pulmonary vascular tone chronically; however, the relative contribution of the three isoforms to acute modulation of pulmonary vascular tone is uncertain. We therefore tested the effect of targeted disruption of each isoform on pulmonary vascular reactivity in transgenic mice. Isolated perfused mouse lungs were used to evaluate the effect of selective loss of pulmonary nNOS, iNOS, and eNOS with respect to hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent and -independent vasodilation. eNOS null mice had augmented HPV (225 ± 65% control, P < 0.02, mean ± SE) and absent endothelium-dependent vasodilation, whereas endothelium-independent vasodilation was preserved. HPV was minimally elevated in iNOS null mice and normal in nNOS null mice. Both nNOS and iNOS null mice had normal endothelium-dependent vasodilation. In wild-type lungs, nonselective NOS inhibition doubled HPV, whereas selective iNOS inhibition had no detectable effect. In intact, lightly sedated mice, right ventricular systolic pressure was elevated in eNOS-deficient (42.3 ± 1.2 mmHg, P< 0.001) and, to a lesser extent, in iNOS-deficient (37.2 ± 0.8 mmHg, P < 0.001) mice, whereas it was normal in nNOS-deficient mice (30.9 ± 0.7 mmHg, P = not significant) compared with wild-type controls (31.3 ± 0.7 mmHg). We conclude that in the normal murine pulmonary circulation 1) nNOS does not modulate tone, 2) eNOS-derived nitric oxide is the principle mediator of endothelium-dependent vasodilation in the pulmonary circulation, and 3) both eNOS and iNOS play a role in modulating basal tone chronically.

Endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein and NO production are increased in hypoxia-induced hypertensive rat lungs, but it is uncertain whether eNOS gene expression and activity are increased in other forms of rat pulmonary hypertension. To investigate these questions, we measured eNOS mRNA and protein, eNOS immunohistochemical localization, perfusate NO product levels, and NO-mediated suppression of resting vascular tone in chronically hypoxic (3–4 wk at barometric pressure of 410 mmHg), monocrotaline-treated (4 wk after 60 mg/kg), and fawn-hooded (6–9 mo old) rats. eNOS mRNA levels (Northern blot) were greater in hypoxic and monocrotaline-treated lungs (130 and 125% of control lungs, respectively; P < 0.05) but not in fawn-hooded lungs. Western blotting indicated that eNOS protein levels increased to 300 ± 46% of control levels in hypoxic lungs ( P < 0.05) but were decreased by 50 ± 5 and 60 ± 11%, respectively, in monocrotaline-treated and fawn-hooded lungs ( P < 0.05). Immunostaining showed prominent eNOS expression in small neomuscularized arterioles in all groups, whereas perfusate NO product levels increased in chronically hypoxic lungs (3.4 ± 1.4 μM; P < 0.05) but not in either monocrotaline-treated (0.7 ± 0.3 μM) or fawn-hooded (0.45 ± 0.1 μM) lungs vs. normotensive lungs (0.12 ± 0.07 μM). All hypertensive lungs had increased baseline perfusion pressure in response to nitro-l-arginine but not to the inducible NOS inhibitor aminoguanidine. These results indicate that even though NO activity suppresses resting vascular tone in pulmonary hypertension, there are differences among the groups regarding eNOS gene expression and NO production. A better understanding of eNOS gene expression and activity in these models may provide insights into the regulation of this vasodilator system in various forms of human pulmonary hypertension.

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Mechanisms that regulate endothelial nitric oxide synthase (eNOS) expression in normal and hypoxic pulmonary circulation are poorly understood. Lung eNOS expression is increased after chronic hypoxic pulmonary hypertension in rats, but whether this increase is due to altered hemodynamics or to hypoxia is unknown. Therefore, to determine the effect of blood flow changes on eNOS expression in the normal pulmonary circulation, and to determine whether the increase in eNOS expression after chronic hypoxia is caused by hemodynamic changes or low oxygen tension, we compared eNOS expression in the left and right lungs of normoxic and chronically hypoxic rats with surgical stenosis of the left pulmonary artery (LPA). LPA stenosis in normoxic rats reduced blood flow to the left lung from 9.8+/-0.9 to 0.8+/-0.4 ml/100 mg/min (sham surgery controls vs. LPA stenosis, P < 0.05), but there was not a significant increase in right lung blood flow. When compared with the right lung, eNOS protein and mRNA content in the left lung was decreased by 32+/-7 and 54+/-13%, respectively (P < 0.05), and right lung eNOS protein content was unchanged. After 3 wk of hypoxia, LPA stenosis reduced blood flow to the left lung from 5.8+/-0.6 to 1.5+/-0.4 ml/100 mg/min, and increased blood flow to the right lung from 5.8+/-0.5 to 10.0+/-1.4 ml/ 100 mg/min (sham surgery controls vs. LPA stenosis, P < 0.05). Despite reduced flow and pressure to the left lung and increased flow and pressure to the right lung, left and right lung eNOS protein and mRNA contents were not different. There were also no differences in lung eNOS protein levels when compared with chronically hypoxic sham surgery controls (P > 0.05). We conclude that reduction of pulmonary blood flow decreases eNOS mRNA and protein expression in normoxic adult rat lungs, and that hypoxia increases eNOS expression independently of changes in hemodynamics. These findings demonstrate that hemodynamic forces maintain eNOS content in the normoxic pulmonary circulation of the adult rat, and suggest that chronic hypoxia increases eNOS expression independently of changes in hemodynamics.