Exhaled NO is reduced at an early stage of hypoxia-induced pulmonary hypertension in newborn piglets

Altered nitric oxide (NO) production could contribute to the pathogenesis of hypoxia-induced pulmonary hypertension. To determine whether parameters of lung NO are altered at an early stage of hypoxia-induced pulmonary hypertension, newborn piglets were exposed to room air (control, n = 21) or 10% O(2) (hypoxia, n = 19) for 3-4 days. Some lungs were isolated and perfused for measurement of exhaled NO output and the perfusate accumulation of nitrite and nitrate (NOx-), the stable metabolites of NO. Pulmonary arteries (20-600-microm diameter) and their accompanying airways were dissected from other lungs and incubated for NOx- determination. Abundances of the nitric oxide synthase (NOS) isoforms endothelial NOS and neural NOS were assessed in homogenates of PAs and airways. The perfusate NOx- accumulation was similar, whereas exhaled NO output was lower for isolated lungs of hypoxic, compared with control, piglets. The incubation solution NOx- did not differ between pulmonary arteries (PAs) of the two groups but was lower for airways of hypoxic, compared with control, piglets. Abundances of both eNOS and nNOS proteins were similar for PA homogenates from the two groups of piglets but were increased in airway homogenates of hypoxic compared with controls. The NO pathway is altered in airways, but not in PAs, at an early stage of hypoxia-induced pulmonary hypertension in newborn piglets.     

Nitric oxide exerts protective effects against bleomycin-induced pulmonary fibrosis in mice

Background

Increased expression of nitric oxide synthase (NOS) and an increase in plasma nitrite plus nitrate (NOx) have been reported in patients with pulmonary fibrosis, suggesting that nitric oxide (NO) plays an important role in its development. However, the roles of the entire NO and NOS system in the pathogenesis of pulmonary fibrosis still remain to be fully elucidated. The aim of the present study is to clarify the roles of NO and the NOS system in pulmonary fibrosis by using the mice lacking all three NOS isoforms.

Methods

Wild-type, single NOS knockout and triple NOS knockout (n/i/eNOS^−/−) mice were administered bleomycin (BLM) intraperitoneally at a dose of 8.0 mg/kg/day for 10 consecutive days. Two weeks after the end of the procedure, the fibrotic and inflammatory changes of the lung were evaluated. In addition, we evaluated the effects of long-term treatment with isosorbide dinitrate, a NO donor, on the n/i/eNOS^−/− mice with BLM-induced pulmonary fibrosis.

Results

The histopathological findings, collagen content and the total cell number in bronchoalveolar lavage fluid were the most severe/highest in the n/i/eNOS^−/− mice. Long-term treatment with the supplemental NO donor in n/i/eNOS^−/− mice significantly prevented the progression of the histopathological findings and the increase of the collagen content in the lungs.

Conclusions

These results provide the first direct evidence that a lack of all three NOS isoforms led to a deterioration of pulmonary fibrosis in a BLM-treated murine model. We speculate that the entire endogenous NO and NOS system plays an important protective role in the pathogenesis of pulmonary fibrosis.     

Nitric oxide synthase in pulmonary hypertension: lessons from knockout mice

Nitric oxide (NO) is implicated in a wide variety of biological roles. NO is generated from three nitric oxide synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) all of which are found in the lung. While there are no isoform-specific inhibitors of NOS, the recent development and characterization of mice deficient in each of the NOS isoforms has allowed for more comprehensive study of the importance of NO in the lung circulation. Studies in the mouse have identified the role of NO from eNOS in modulating pulmonary vascular tone and in attenuating the development of chronic hypoxic pulmonary hypertension.     

左旋精氨酸对低氧性肺动脉高压治疗作用的实验研究

目的：探讨结构型一氧化氮合酶（ｃＮＯＳ）,内皮素－１（ＥＴ－１）在低氧性肺动脉高压（ＨＰＨ）发病中的机制及左旋精氢酸（Ｌ－Ａｒｇ）对ＨＰＨ的治疗作用。方法：３０只健康雄性ＳＤ大鼠平均分为三组：正常对照组（ＮＣ组）、低氧组（ＨＰ组）、低氧左旋精氨酸治疗组（ＬＴ组）。后组每日低氧前给予２００ｍｇ／ｋｇ Ｌ－Ａｒｇ。于低氧２１ｄ检测运动血流动力学,肺组织ＮＯ、ＥＴ－１含量,肺动脉内皮ｃＮＯＳ含量的改变,     

Increased nitric oxide production accompanies blunted hypoxic pulmonary vasoconstriction in hyperoxic rat lung

Hyperoxia may affect lung physiology in different ways. We investigated the effect of hyperoxia on the protein expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, and hypoxic pulmonary vasoconstriction (HPV) in rat lung. Twenty-four male rats were divided into hyperoxic and normoxic groups. Hyperoxic rats were placed in > 90% F1O2 for 60 h prior to experiments. After baseline in vitro analysis, the rats underwent isolated, perfused lung experiments. Two consecutive hypoxic challenges (10 min each) were administered with the administration of a non-specific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), in between. We measured intravascular NO production, pulmonary arterial pressure, and protein expression of eNOS and iNOS by immunohistochemistry. We found that hyperoxia rats exhibited increased baseline NO production (P < 0.001) and blunted HPV response (P < 0.001) during hypoxic challenges compared to normoxia rats. We also detected a temporal association between the attenuation in HPV and increased NO production level with a negative pre-L-NAME correlation between HPV and NO (R = 0.52, P < 0.05). After L-NAME administration, a second hypoxic challenge restored the HPV response in the hyperoxic group. There were increased protein expression of eNOS (12.6 +/- 3.1-fold, n = 3) (X200) and iNOS (8.1 +/- 2.6-fold, n = 3) (X200) in the hyperoxia group. We conclude that hyperoxia increases the protein expression of eNOS and iNOS with a subsequent increased release of endogenous NO, which attenuates the HPV response.     

Contribution of oxygen radicals to altered NO-dependent pulmonary vasodilation in acute and chronic hypoxia

Chronic hypoxia (CH) increases pulmonary arterial endothelial nitric oxide (NO) synthase (NOS) expression and augments endothelium-derived nitric oxide (EDNO)-dependent vasodilation, whereas vasodilatory responses to exogenous NO are attenuated in CH rat lungs. We hypothesized that reactive oxygen species (ROS) inhibit NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined responses to the EDNO-dependent vasodilator endothelin-1 (ET-1) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP) in isolated lungs from control and CH rats in the presence or absence of ROS scavengers under normoxic or hypoxic ventilation. NOS was inhibited in lungs used for SNAP experiments to eliminate influences of endogenously produced NO. Additionally, dichlorofluorescein (DCF) fluorescence was measured as an index of ROS levels in isolated pressurized small pulmonary arteries from each group. We found that acute hypoxia increased DCF fluorescence and attenuated vasodilatory responses to ET-1 in lungs from control rats. The addition of ROS scavengers augmented ET-1-induced vasodilation in lungs from both groups during hypoxic ventilation. In contrast, upon NOS inhibition, DCF fluorescence was elevated and SNAP-induced vasodilation diminished in arteries from CH rats during normoxia, whereas acute hypoxia decreased DCF fluorescence, which correlated with augmented reactivity to SNAP in both groups. ROS scavengers enhanced SNAP-induced vasodilation in normoxia-ventilated lungs from CH rats similar to effects of hypoxic ventilation. We conclude that inhibition of NOS during normoxia leads to greater ROS generation in lungs from both control and CH rats. Furthermore, NOS inhibition reveals an effect of acute hypoxia to diminish ROS levels and augment NO-mediated pulmonary vasodilation.     

Nitric oxide-driven hypoxia initiates synovial angiogenesis, hyperplasia and inflammatory lesions in mice

Background

Rheumatoid arthritis (RA) is an inflammatory articular disease with cartilage and bone damage due to hyperplasic synoviocyte invasion and subsequent matrix protease digestion. Although monoclonal antibodies against tumor necrosis factor alpha (TNFα) have been approved for clinical use in patients with RA, desired therapeutic regimens suitable for non-responders are still unavailable because etiological initiators leading to RA remain enigmatic and unidentified.

Methodology/Principal Findings

Bacteria-induced arthritis (BIA) that simulates collagen-induced arthritis (CIA) is developed in mice upon daily live bacterial feeding. The morphological lesions of paw erythema and edema together with the histological alterations of synovial hyperplasia and lymphocytic infiltration emerge as the early-phase manifestations of BIA and CIA. Bacteria- or collagen-mediated global upregulation of pro-inflammatory cytokines is accompanied by the burst of nitric oxide (NO). Elevation of the serum NO level is correlated with decline of the blood oxygen saturation percentage (SpO2), reflecting a hypoxic consequence during development towards arthritis. NO-driven hypoxia is further evident from a positive relationship between NO and lactic acid (LA), an end product from glycolysis. Upregulation of hypoxia inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) validates hypoxia-induced angiogenesis in the inflamed synovium of modeling mice. Administration of the NO donor compound sodium nitroprusside (SNP) causes articular inflammation by inducing synovial hypoxia. Anti-bacteria by the antibiotic cefotaxime and/or the immunosuppressant rapamycin or artesunate that also inhibits nitric oxide synthase (NOS) can abrogate NO production, mitigate hypoxia, and considerably ameliorate or even completely abort synovitis, hence highlighting that NO may serve as an initiator of inflammatory arthritis.

Conclusions/Significance

Like collagen, bacteria also enable synovial lesions via upregulating pro-inflammatory cytokines, triggering NO production, driving hypoxic responses, and inducing synovial angiogenesis and hyperplasia, suggesting that sustained infection might be, in part, responsible for the onset of synovitis and arthritis in mice.     

Interaction of caveolin-1, nitric oxide, and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells

Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells.     

Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway

The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. Objective To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. Methods Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO₂ incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. Results (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. Conclusions Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.     

Sodium-Coupled Neutral Amino Acid Transporter 1 (SNAT1) Modulates L-Citrulline Transport and Nitric Oxide (NO) Signaling in Piglet Pulmonary Arterial Endothelial Cells

Rationale

There is evidence that impairments in nitric oxide (NO) signaling contribute to chronic hypoxia-induced pulmonary hypertension. The L-arginine-NO precursor, L-citrulline, has been shown to ameliorate pulmonary hypertension. Sodium-coupled neutral amino acid transporters (SNATs) are involved in the transport of L-citrulline into pulmonary arterial endothelial cells (PAECs). The functional link between the SNATs, L-citrulline, and NO signaling has not yet been explored.

Objective

We tested the hypothesis that changes in SNAT1 expression and transport function regulate NO production by modulating eNOS coupling in newborn piglet PAECs.

Methods and Results

A silencing RNA (siRNA) technique was used to assess the contribution of SNAT1 to NO production and eNOS coupling (eNOS dimer-to-monomer ratios) in PAECs from newborn piglets cultured under normoxic and hypoxic conditions in the presence and absence of L-citrulline. SNAT1 siRNA reduced basal NO production in normoxic PAECs and prevented L-citrulline-induced elevations in NO production in both normoxic and hypoxic PAECs. SNAT1 siRNA reduced basal eNOS dimer-to-monomer ratios in normoxic PAECs and prevented L-citrulline-induced increases in eNOS dimer-to-monomer ratios in hypoxic PAECs.

Conclusions

SNAT1 mediated L-citrulline transport modulates eNOS coupling and thus regulates NO production in hypoxic PAECs from newborn piglets. Strategies that increase SNAT1-mediated transport and supply of L-citrulline may serve as novel therapeutic approaches to enhance NO production in patients with pulmonary vascular disease.     

Decreased synthesis and vasodilation to nitric oxide in piglets with hypoxia-induced pulmonary hypertension

Nitric oxide (NO) is thought to play an important role in the regulation of neonatal pulmonary vasculature. It has been suggested that neonates with pulmonary hypertension have a defective NO pathway. Therefore, we measured in 1-day-old piglets exposed to hypoxia (fraction of inspired O(2) = 0.10) for 3 or 14 days to induce pulmonary hypertension 1) the activity of NO synthase (NOS) via conversion of L-arginine to L-citrulline and the concentration of the NO precursor L-arginine in isolated pulmonary vessels, 2) the vasodilator response to the NO donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) and the cGMP analog 8-bromo-cGMP in isolated perfused lungs, and 3) the production of cGMP in response to SIN-1 in isolated perfused lungs. After 3 days of exposure to hypoxia, endothelial NOS (eNOS) activity was unaffected, whereas, after 14 days of hypoxia, eNOS activity was decreased in the cytosolic fraction of pulmonary artery (P < 0.05) but not of pulmonary vein homogenates. Inducible NOS activity was decreased in the cytosolic fraction of pulmonary artery homogenates after both 3 (P < 0.05) and 14 (P < 0.05) days of hypoxia but was unchanged in pulmonary veins. Pulmonary artery levels of L-arginine were unaffected by hypoxic exposure. After 3 days of exposure to hypoxia, the reduction in the dilator response to SIN-1 (P < 0.05) coincided with a decrease in cGMP production (P < 0.005), suggesting that soluble guanylate cyclase activity may be altered. When the exposure was prolonged to 14 days, dilation to SIN-1 remained decreased (P < 0.05) and, although cGMP production normalized, the dilator response to 8-bromo-cGMP decreased (P < 0.05), suggesting that, after prolonged exposure to hypoxia, cGMP-dependent mechanisms may also be impaired. In conclusion, neonatal hypoxia-induced pulmonary hypertension is associated with multiple disruptions in the NO pathway.     

Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia

Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.     

Impaired NO signaling in small pulmonary arteries of chronically hypoxic newborn piglets

We performed studies to determine whether chronic hypoxia impairs nitric oxide (NO) signaling in resistance level pulmonary arteries (PAs) of newborn piglets. Piglets were maintained in room air (control) or hypoxia (11% O(2)) for either 3 (shorter exposure) or 10 (longer exposure) days. Responses of PAs to a nonselective NO synthase (NOS) antagonist, N(omega)-nitro-L-arginine methylester (L-NAME), a NOS-2-selective antagonist, aminoguanidine, and 7-nitroindazole, a NOS-1-selective antagonist, were measured. Levels of NOS isoforms and of two proteins involved in NOS signaling, heat shock protein (HSP) 90 and caveolin-1, were assessed in PA homogenates. PAs from all groups constricted to L-NAME but not to aminoguanidine or 7-nitroindazole. The magnitude of constriction to L-NAME was similar for PAs from control and hypoxic piglets of the shorter exposure period but was diminished for PAs from hypoxic compared with control piglets of the longer exposure period. NOS-3, HSP90, and caveolin-1 levels were similar in hypoxic and control PAs. These findings indicate that NOS-3, but not-NOS 2 or NOS-1, is involved with basal NO production in PAs from both control and hypoxic piglets. After 10 days of hypoxia, NO function is impaired in PAs despite preserved levels of NOS-3, HSP90, and caveolin-1. The development of NOS-3 dysfunction in resistance level PAs may contribute to the progression of chronic hypoxia-induced pulmonary hypertension in newborn piglets.     

Nitric oxide production in the hypoxic lung

Nitric oxide (NO) is a potent vasodilator and inhibitor of vascular remodeling. Reduced NO production has been implicated in the pathophysiology of pulmonary hypertension, with endothelial NO synthase (NOS) knockout mice showing an increased risk for pulmonary hypertension. Because molecular oxygen (O2) is an essential substrate for NO synthesis by the NOSs and biochemical studies using purified NOS isoforms have estimated the Michaelis-Menten constant values for O2 to be in the physiological range, it has been suggested that O2 substrate limitation may limit NO production in various pathophysiological conditions including hypoxia. This review summarizes numerous studies of the effects of acute and chronic hypoxia on NO production in the lungs of humans and animals as well as in cultured vascular cells. In addition, the effects of hypoxia on NOS expression and posttranslational regulation of NOS activity by other proteins are also discussed. Most studies found that hypoxia limits NO synthesis even when NOS expression is increased.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs

We investigated the effects of the nitric oxide (NO) donor molsidomine and the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) on pulmonary endothelin (ET)-1 gene expression and ET-1 plasma levels in chronic hypoxic rats. Two and four weeks of hypoxia (10% O2) significantly increased right ventricular systolic pressure, the medial cross-sectional vascular wall area of the pulmonary arteries, and pulmonary ET-1 mRNA expression (2-fold and 3.2-fold, respectively). ET-1 plasma levels were elevated after 4 wk of hypoxia. In rats exposed to 4 wk of hypoxia, molsidomine (15 mg x kg(-1) x day(-1)) given either from the beginning or after 2 wk of hypoxia significantly reduced pulmonary hypertension, pulmonary vascular remodeling, pulmonary ET-1 gene expression, and ET-1 plasma levels. L-NAME administration (45 mg x kg(-1) x day(-1)) in rats subjected to 2 wk of hypoxia did not modify these parameters. Our findings suggest that in chronic hypoxic rats, exogenously administered NO acts in part by suppressing the formation of ET-1. In contrast, inhibition of endogenous NO production exerts only minor effects on the pulmonary circulation and pulmonary ET-1 synthesis in these animals.     

Impaired iNOS–sGC–cGMP signalling contributes to chronic hypoxic and hypercapnic pulmonary hypertension in rat

Nitric oxide (NO) is an important vascular modulator in the development of pulmonary hypertension. NO exerts its regulatory effect mainly by activating soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP). Exposure to hypoxia causes pulmonary hypertension. But in lung disease, hypoxia is commonly accompanied by hypercapnia. The aim of this study was to examine the changes of sGC enzyme activity and cGMP content in lung tissue, as well as the expression of inducible nitric oxide synthase (iNOS) and sGC in rat pulmonary artery after exposure to hypoxia and hypercapnia, and assess the role of iNOS–sGC–cGMP signal pathway in the development of hypoxic and hypercapnic pulmonary hypertension. Male Sprague–Dawley rats were exposed to hypoxia and hypercapnia for 4 weeks to establish model of chronic pulmonary hypertension. Weight‐matched rats exposed to normoxia served as control. After exposure to hypoxia and hypercapnia, mean pulmonary artery pressure, the ratio of right ventricle/left ventricle + septum, and the ratio of right ventricle/body weight were significantly increased. iNOS mRNA and protein levels were significantly increased, but sGC α₁ mRNA and protein levels were significantly decreased in small pulmonary arteries of hypoxic and hypercapnic exposed rat. In addition, basal and stimulated sGC enzyme activity and cGMP content in lung tissue were significantly lower after exposure to hypoxia and hypercapnia. These results demonstrate that hypoxia and hypercapnia lead to the upregulation of iNOS expression, downregulation of sGC expression and activity, which then contribute to the development of pulmonary hypertension. Copyright © 2012 John Wiley & Sons, Ltd.     

NOS3 deficiency augments hypoxic pulmonary vasoconstriction and enhances systemic oxygenation during one-lung ventilation in mice.

Nitric oxide (NO), synthesized by NO synthases (NOS), plays a pivotal role in regulation of pulmonary vascular tone. To examine the role of endothelial NOS (NOS3) in hypoxic pulmonary vasoconstriction (HPV), we measured left lung pulmonary vascular resistance (LPVR), intrapulmonary shunting, and arterial PO2 (PaO2) before and during left mainstem bronchus occlusion (LMBO) in mice with and without a deletion of the gene encoding NOS3. The increase of LPVR induced by LMBO was greater in NOS3-deficient mice than in wild-type mice (151 +/- 39% vs. 109 +/- 36%, mean +/- SD; P < 0.05). NOS3-deficient mice had a lower intrapulmonary shunt fraction than wild-type mice (17.1 +/- 3.6% vs. 21.7 +/- 2.4%, P < 0.05) during LMBO. Both real-time PaO2 monitoring with an intra-arterial probe and arterial blood-gas analysis during LMBO showed higher PaO2 in NOS3-deficient mice than in wild-type mice (P < 0.05). Inhibition of all three NOS isoforms with Nomega-nitro-L-arginine methyl ester (L-NAME) augmented the increase of LPVR induced by LMBO in wild-type mice (183 +/- 67% in L-NAME treated vs. 109 +/- 36% in saline treated, P < 0.01) but not in NOS3-deficient mice. Similarly, systemic oxygenation during one-lung ventilation was augmented by L-NAME in wild-type mice but not in NOS3-deficient mice. These findings indicate that NO derived from NOS3 modulates HPV in vivo and that inhibition of NOS3 improves systemic oxygenation during acute unilateral lung hypoxia.