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.     

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.     

Role of nitric oxide in heparin-induced attenuation of hypoxic pulmonary vascular remodeling.

Heparin and nitric oxide (NO) attenuate changes to the pulmonary vasculature caused by prolonged hypoxia. Heparin may increase NO; therefore, we hypothesized that heparin may attenuate hypoxia-induced pulmonary vascular remodeling via a NO-mediated mechanism. In vivo, rats were exposed to normoxia (N) or hypoxia (H; 10% O(2)) with or without heparin (1,200 U x kg-1 x day-1) and/or the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME; 20 mg x kg-1 x day-1) for 3 days or 3 wk. Heparin attenuated increases in pulmonary arterial pressure, the percentage of muscular pulmonary vessels, and their medial thickness induced by 3 wk of H. Importantly, although L-NAME alone had no effect, it prevented these effects of heparin on vascular remodeling. In H lungs, heparin increased NOS activity and cGMP levels at 3 days and 3 wk and endothelial NOS protein expression at 3 days but not at 3 wk. In vitro, heparin (10 and 100 U x kg-1 x ml-1) increased cGMP levels after 10 min and 24 h in N and anoxic (0% O2) endothelial cell-smooth muscle cell (SMC) coculture. SMC proliferation, assessed by 5-bromo-2'-deoxyuridine incorporation during a 3-h incubation period, was decreased by heparin under N, but not anoxic, conditions. The antiproliferative effects of heparin were not altered by L-NAME. In conclusion, the in vivo results suggest that attenuation of hypoxia-induced pulmonary vascular remodeling by heparin is NO mediated. Heparin increases cGMP in vitro; however, the heparin-induced decrease in SMC proliferation in the coculture model appears to be NO independent.     

Nitric oxide scavenging by hemoglobin regulates hypoxic pulmonary vasoconstriction

Although the importance of red blood cells in augmenting hypoxic pulmonary vasoconstriction has been recognized for decades, only recently has it become clear that this occurs primarily because of the inactivation of nitric oxide (NO) by hemoglobin. This interaction between red blood cells, NO, and the pulmonary circulation is critical in understanding the effects of anemia and polycythemia on pulmonary blood flow distribution, gas exchange, and global O2 delivery and in understanding the development of hemoglobin-based oxygen carriers. This review will discuss the proposed mechanisms for initiation of hypoxic pulmonary vasoconstriction and regulation of hypoxic pulmonary vasoconstriction by red blood cells with an emphasis on hemoglobin-NO interactions. In addition, the review will discuss how biologic (S-nitrosation) or pharmacologic (cross-linking) modification of hemoglobin may affect pulmonary circulatory-hemoglobin interactions.     

Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice

This study investigated the role of endogenous nitric oxide (NO) in the progression of atherosclerosis in apolipoprotein E-deficient [apoE-knockout (KO)] mice. Mice were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME) an inhibitor of nitric oxide synthase (NOS) or with the NOS substrate L-arginine for 8 wk. L-NAME treatment resulted in a significant inhibition of NO-mediated vascular responses and a significant increase in the atherosclerotic plaque/surface area in the aorta of apoE-KO mice. L-arginine treatment had no influence on endothelial function and did not alter lesion size. Mean arterial blood pressure and serum lipid levels were not altered by the treatments. At the beginning of the study impairment in endothelial function was only apparent in the case of N(G)-nitro-L-arginine-induced, NO-mediated contraction, whereas ACh-induced, NO-mediated relaxation was not different between age-matched apoE-KO and C57Bl/6J mice. After the 8-wk treatment with the NOS inhibitor, both NO-mediated responses were significantly inhibited. The acceleration in lesion size concomitant to the severely impaired NO-mediated responses indicates that lack of endogenous NO is an important progression factor of atherosclerosis in the apoE-KO mouse.     

Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.     

Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction

PKC contributes to regulation of pulmonary vascular reactivity in response to hypoxia. The role of individual PKC isozymes is less clear. We used a knockout (null, -/-) mouse to test the hypothesis that PKC-epsilon is important in acute hypoxic pulmonary vasoconstriction (HPV). We asked whether deletion of PKC-epsilon would decrease acute HPV in adult C57BL6xSV129 mice. In isolated, salt solution-perfused lung, reactivity to acute hypoxic challenges (0% and 3% O(2)) was compared with responses to angiotensin II (ANG II) and KCl. PKC-epsilon -/- mice had decreased HPV, whereas responses to ANG II and KCl were preserved. Inhibition of nitric oxide synthase (NOS) with nitro-l-arginine augmented HPV in PKC-epsilon +/+ but not -/- mice. Inhibition of Ca(2+)-gated K(+) channels (K(Ca)) with charybdotoxin and apamin did not enhance HPV in -/- mice relative to wild-type (+/+) controls. In contrast, the voltage-gated K(+) channel (K(V)) antagonist 4-aminopyridine increased the response of -/- mice beyond that of +/+ mice. This suggested that increased K(V) channel expression could contribute to blunted HPV in PKC-epsilon -/- mice. Therefore, expression of the O(2)-sensitive K(V) channel subunit Kv3.1b (100-kDa glycosylated form and 70-kDa core protein) was compared in whole lung and pulmonary artery smooth muscle cell (PASMC) lysates from +/+ and -/- mice. A subtle increase in Kv3.1b was detected in -/- vs. +/+ whole lung lysates. A much greater rise in Kv3.1b expression was found in -/- vs. +/+ PASMC. Thus deletion of PKC-epsilon blunts murine HPV. The decreased response could not be attributed to a general loss in vasoreactivity or derangements in NOS or K(Ca) channel activity. Instead, the absence of PKC-epsilon allows increased expression of K(V) channels (like Kv3.1b) to occur in PASMC, which likely contributes to decreased HPV.     

Activation of Toll-like receptor 2 impairs hypoxic pulmonary vasoconstriction in mice

Toll-like receptors (TLRs) mediate inflammation in sepsis, but their role in sepsis-induced respiratory failure is unknown. Hypoxic pulmonary vasoconstriction (HPV) is a unique vasoconstrictor response that diverts blood flow away from poorly ventilated lung regions. HPV is impaired in sepsis and after challenge with the TLR4 agonist lipopolysaccharide (LPS). Unlike TLR4 agonists, which are present only in Gram-negative bacteria, TLR2 agonists are ubiquitously expressed in all of the major classes of microorganisms that cause sepsis, including both Gram-positive and Gram-negative bacteria and fungi. We tested the hypothesis that (S)-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys(4)-OH, trihydrochloride (Pam3Cys), a TLR2 agonist, impairs HPV and compared selected pulmonary and systemic effects of Pam3Cys vs. LPS. HPV was assessed 22 h after challenge with saline, Pam3Cys, or LPS by measuring the increase in the pulmonary vascular resistance of the left lung before and during left lung alveolar hypoxia produced by left mainstem bronchus occlusion (LMBO). Additional endpoints included arterial blood gases during LMBO, hemodynamic parameters, weight loss, temperature, physical appearance, and several markers of lung inflammation. Compared with saline, challenge with Pam3Cys caused profound impairment of HPV, reduced systemic arterial oxygenation during LMBO, weight loss, leukopenia, and lung inflammation. In addition to these effects, LPS-challenged mice had lower rectal temperatures, metabolic acidosis, and were more ill appearing than Pam3Cys-challenged mice. These data indicate that TLR2 activation impairs HPV and induces deleterious systemic effects in mice and suggest that TLR2 pathways may be important in sepsis-induced respiratory failure.     

Downregulation of hypoxic vasoconstriction by chronic hypoxia in rabbits: effects of nitric oxide

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange. Chronic alveolar hypoxia results in vascular remodeling and pulmonary hypertension. Previous studies have reported conflicting results of the effect of chronic alveolar hypoxia on pulmonary vasoreactivity and the contribution of nitric oxide (NO), which may be related to species and strain differences as well as to the duration of chronic hypoxia. Therefore, we investigated the impact of chronic hypoxia on HPV in rabbits, with a focus on lung NO synthesis. After exposure of the animals to normobaric hypoxia (10% O(2)) for 1 day to 10 wk, vascular reactivity was investigated in ex vivo perfused normoxic ventilated lungs. Chronic hypoxia induced right heart hypertrophy and increased normoxic vascular tone within weeks. The vasoconstrictor response to an acute hypoxic challenge was strongly downregulated within 5 days, whereas the vasoconstrictor response to the thromboxane mimetic U-46619 was maintained. The rapid downregulation of HPV was apparently not linked to changes in the lung vascular NO system, detectable in the exhaled gas and by pharmacological blockage of NO synthesis. Treatment of the animals with long-term inhaled NO reduced right heart hypertrophy and partially maintained the reactivity to acute hypoxia, without any impact on the endogenous NO system being noted. We conclude that chronic hypoxia causes rapid downregulation of acute HPV as a specific event, preceding the development of major pulmonary hypertension and being independent of the lung vascular NO system. Long-term NO inhalation partially maintains the strength of the hypoxic vasoconstrictor response.     

缺氧性肺血管收缩的细胞机制

缺氧直接作用于肺血管平滑肌细胞而使肺血管收缩。缺氧使细胞膜Ca~(2 )通透性增加,K~ 电导降低、膜电位下降,产生Ca~(2 )依赖性动作电位,导致肺血管张力增加和肺血管收缩。缺氧还能使平滑肌细胞内能量代谢发生改变,抑制氧化磷酸化和三羧酸循环作用,降低磷酸势能,引起肺血管收缩。缺氧减少细胞内氧自由基的产生而使细胞内氧化还原状态发生改变,GSH/GSSG和NADPH/NADP~ 比值增高,导致肺血管阻力增高。     

Role of endogenous nitric oxide in classic preconditioning in rat hearts

Ischemic preconditioning (IPC) protects the heart against subsequent sustained ischemia reperfusion (RP). Despite many triggers and signaling pathways, which seem to be involved in IPC, the IPC-mechanisms remain a controversial issue. One of them is endogenous production of nitric oxide (NO). To assess the role of NO in IPC and its relation with glycogen and glycolysis, the effects of inhibiting NO synthase with L-NAME (50 microM) were examined in IPC rat hearts perfused with medium containing 10 mM glucose. Left ventricular developed pressure-rate product (RPP) and end diastolic pressure (EDP), lactate and glycogen contents, and cell viability were measured. Global ischemia (25 min) was followed by 30 min RP. IPC consisted in one cycle of 3 min ischemia-5 min RP. IPC reduced EDP and improved RP recovery of RPP. L-NAME had no effects on the non-IPC group but abolished these effects of IPC. IPC reduced ischemic decrease of glycogen and the acceleration of glycolysis, and improved cell viability. L-NAME did not affect these effects of IPC. The results suggest that NO is ineffective on the noxious effects of ischemia-RP in non-IPC hearts and on the effects of IPC on cell viability, glycogenolysis and glycolysis whereas it is only involved in functional protection.     

Role of endogenous nitric oxide synthase inhibitor in gastric mucosal injury

To explore the role of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in gastric mucosal injury, 3 models of gastric mucosal injury induced by ethanol, indomethacin, or cold stress were used in rats. The cultured human gastric mucosal epithelial cell line GES-1 infected by Helicobacter pylori (Hp) was selected to mimic human gastric mucosal injury. Gastric mucosal ulcer index (UI), levels of ADMA and NO, and activity of dimethylarginine dimethylaminohydrolase (DDAH) were determined in the mucosal injury models; in Hp-infected or ADMA-treated GES-1 cells, levels of ADMA, NO, and TNF-alpha and activity of DDAH were measured. The results showed that UI and levels of ADMA were markedly increased and accompanied by significantly decreased DDAH activity in the mucosal injury models. Incubation of GES-1 cells with Hp increased levels of TNF-alpha and ADMA and decreased activity of DDAH. Administration of ADMA also increased levels of TNF-alpha. The results suggest that ADMA plays an important role in facilitating gastric mucosal injury, an effect which is associated with inhibiting NO synthesis and inducing inflammatory reaction.     

Regional hypoxic pulmonary vasoconstriction in prone pigs.

Hypoxic pulmonary vasoconstriction (HPV) is known to affect regional pulmonary blood flow distribution. It is unknown whether lungs with well-matched ventilation (V)/perfusion (Q) have regional differences in the HPV response. Five prone pigs were anesthetized and mechanically ventilated (positive end-expiratory pressure = 2 cmH2O). Two hypoxic preconditions [inspired oxygen fraction (FI(O2)) = 0.13] were completed to stabilize the animal's hypoxic response. Regional pulmonary blood Q and V distribution was determined at various FI(O2) (0.21, 0.15, 0.13, 0.11, 0.09) using the fluorescent microsphere technique. Q and V in the lungs were quantified within 2-cm3 lung pieces. Pieces were grouped, or clustered, based on the changes in blood flow when subjected to increasing hypoxia. Unique patterns of Q response to hypoxia were seen within and across animals. The three main patterns (clusters) showed little initial difference in V/Q matching at room air where the mean V/Q range was 0.92-1.06. The clusters were spatially located in cranial, central, and caudal portions of the lung. With decreasing FI(O2), blood flow shifted from the cranial to caudal regions. We determined that pulmonary blood flow changes, caused by HPV, produced distinct response patterns that were seen in similar regions across our prone porcine model.     

Role of endogenous nitric oxide on PAF-induced vascular and respiratory effects

The role of endogenous nitric oxide (NO) on vascular and respiratory smooth muscle basal tone was evaluated in six anaesthetized, paralysed, mechanically ventilated pigs. The involvement of endogenous NO in PAF-induced shock and airway hyperresponsiveness was also studied. PAF (50 ng/kg, i.v.) was administered before and after pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v.), an NO synthesis inhibitor. PAF was also administered to three of these pigs after indomethacin infusion (3 mg/kg, i.v.). In normal pigs, L-NAME increased systemic and pulmonary vascular resistances, caused pulmonary hypertension and reduced cardiac output and stroke volume. The pulmonary vascular responses were correlated with the increase in static and dynamic lung elastances, without changing lung resistance. Inhibition of NO synthesis enhanced the PAF-dependent increase in total, intrinsic and viscoelastic lung resistances, without affecting lung elastances or cardiac activity. The systemic hypotensive effect of PAF was not abolished by pretreatment with L-NAME or indomethacin. This indicates that systemic hypotension is not correlated with the release of endogenous NO or prostacyclines. Indomethacin completely abolished the PAF-dependent respiratory effects.     

Acetazolamide prevents hypoxic pulmonary vasoconstriction in conscious dogs.

Acute hypoxia increases pulmonary arterial pressure and vascular resistance. Previous studies in isolated smooth muscle and perfused lungs have shown that carbonic anhydrase (CA) inhibition reduces the speed and magnitude of hypoxic pulmonary vasoconstriction (HPV). We studied whether CA inhibition by acetazolamide (Acz) is able to prevent HPV in the unanesthetized animal. Ten chronically tracheotomized, conscious dogs were investigated in three protocols. In all protocols, the dogs breathed 21% O(2) for the first hour and then 8 or 10% O(2) for the next 4 h spontaneously via a ventilator circuit. The protocols were as follows: protocol 1: controls given no Acz, inspired O(2) fraction (Fi(O(2))) = 0.10; protocol 2: Acz infused intravenously (250-mg bolus, followed by 167 microg.kg(-1).min(-1) continuously), Fi(O(2)) = 0.10; protocol 3: Acz given as above, but with Fi(O(2)) reduced to 0.08 to match the arterial Po(2) (Pa(O(2))) observed during hypoxia in controls. Pa(O(2)) was 37 Torr during hypoxia in controls, mean pulmonary arterial pressure increased from 17 +/- 1 to 23 +/- 1 mmHg, and pulmonary vascular resistance increased from 464 +/- 26 to 679 +/- 40 dyn.s(-1).cm(-5) (P < 0.05). In both Acz groups, mean pulmonary arterial pressure was 15 +/- 1 mmHg, and pulmonary vascular resistance ranged between 420 and 440 dyn.s(-1).cm(-5). These values did not change during hypoxia. In dogs given Acz at 10% O(2), the arterial Pa(O(2)) was 50 Torr owing to hyperventilation, whereas in those breathing 8% O(2) the Pa(O(2)) was 37 Torr, equivalent to controls. In conclusion, Acz prevents HPV in conscious spontaneously breathing dogs. The effect is not due to Acz-induced hyperventilation and higher alveolar Po(2), nor to changes in plasma endothelin-1, angiotensin-II, or potassium, and HPV suppression occurs despite the systemic acidosis with CA inhibition.