beta-adrenergic relaxation in pulmonary arteries: preservation of the endothelial nitric oxide-dependent beta2 component in pulmonary hypertension.

AIMS: beta-adrenoceptor (beta-AR)-mediated relaxation was characterized in pulmonary arteries from normoxic and hypoxic (as model of pulmonary hypertension) mice. The endothelial NO synthase (eNOS) pathway was especially investigated because of its potential vasculoprotective effects. METHODS: Pulmonary arteries from control or hypoxic (0.5 atm for 21 days) wild-type or eNOS-/- mice were used for pharmacological characterization of beta-AR-mediated relaxation in myograph, and for immunohistochemistry using anti-beta-AR antibodies. RESULTS: In pulmonary arteries from normoxic mice, isoproterenol (beta-AR agonist) and procaterol (selective beta2-AR agonist) elicited relaxation, while cyanopindolol and CL316243 (beta3-AR agonists) were ineffective. The effect of isoproterenol was antagonized by CGP20712A and ICI118551 (beta1- or beta2-AR antagonists, respectively) and also partially inhibited by N omega-nitro-L-arginine methylester (L-NAME, a NOS inhibitor), endothelium denudation, or eNOS gene deletion. Relaxation to procaterol was abolished by L-NAME or endothelium removal. In eNOS-/- mice, procaterol-induced relaxation was decreased but was insensitive to L-NAME, this residual effect involving other endothelium-dependent relaxant factors as compensatory mechanisms. Immunostaining for beta2-AR was observed in the endothelial layer, but not the medial layer of pulmonary arteries. Pulmonary arteries from hypoxic mice exhibited decreased endothelial NO-dependent relaxation to acetylcholine. However, in these arteries, relaxation to procaterol was either unaffected (extralobar segments) or even increased (intralobar segments) and was still abolished by L-NAME or endothelium removal. CONCLUSION: beta1- and beta2-AR, but not beta3-AR, mediate relaxation of mice pulmonary arteries. The beta2-AR component is dependent on eNOS activity and is preserved following chronic hypoxia. These data highlight the role of the beta2-AR as a pharmacological target to induce/restore endothelial NO-dependent protective effects in pulmonary circulation.

Impairment of NO-dependent relaxation in intralobar pulmonary arteries: comparison of urban particulate matter and manufactured nanoparticles.

BACKGROUND AND OBJECTIVES: Because pulmonary circulation is the primary vascular target of inhaled particulate matter (PM), and nitric oxide is a major vasculoprotective agent, in this study we investigated the effect of various particles on the NO-cyclic guanosine monophosphate (cGMP) pathway in pulmonary arteries. METHODS: We used intrapulmonary arteries and/or endothelial cells, either exposed in vitro to particles or removed from PM-instilled animals for assessment of vasomotricity, cGMP and reactive oxygen species (ROS) levels, and cytokine/chemokine release. RESULTS: Endothelial NO-dependent relaxation and cGMP accumulation induced by acetylcholine (ACh) were both decreased after 24 hr exposure of rat intrapulmonary arteries to standard reference material 1648 (SRM1648; urban PM). Relaxation due to NO donors was also decreased by SRM1648, whereas responsiveness to cGMP analogue remained unaffected. Unlike SRM1648, ultrafine carbon black and ultrafine and fine titanium dioxide (TiO2) manufactured particles did not impair NO-mediated relaxation. SRM1648-induced decrease in relaxation response to ACh was prevented by dexamethasone (an anti-inflammatory agent) but not by antioxidants. Accordingly, SRM1648 increased the release of proinflammatory mediators (tumor necrosis factor-alpha, interleukin-8) from intrapulmonary arteries or pulmonary artery endothelial cells, but did not elevate ROS levels within intrapulmonary arteries. Decreased relaxation in response to ACh was also evidenced in intrapulmonary arteries removed from rats intratracheally instilled with SRM1648, but not with fine TiO2. CONCLUSION: In contrast to manufactured particles (including nanoparticles), urban PM impairs NO but not cGMP responsiveness in intrapulmonary arteries. We attribute this effect to oxidative-stress-independent inflammatory response, resulting in decreased guanylyl cyclase activation by NO. Such impairment of the NO pathway may contribute to urban-PM-induced cardiovascular dysfunction.

Biopterin metabolism and eNOS expression during hypoxic pulmonary hypertension in mice.

Tetrahydrobiopterin (BH4), which fosters the formation of and stabilizes endothelial NO synthase (eNOS) as an active dimer, tightly regulates eNOS coupling / uncoupling. Moreover, studies conducted in genetically-modified models demonstrate that BH4 pulmonary deficiency is a key determinant in the pathogenesis of pulmonary hypertension. The present study thus investigates biopterin metabolism and eNOS expression, as well as the effect of sepiapterin (a precursor of BH4) and eNOS gene deletion, in a mice model of hypoxic pulmonary hypertension. In lungs, chronic hypoxia increased BH4 levels and eNOS expression, without modifying dihydrobiopterin (BH2, the oxidation product of BH4) levels, GTP cyclohydrolase-1 or dihydrofolate reductase expression (two key enzymes regulating BH4 availability). In intrapulmonary arteries, chronic hypoxia also increased expression of eNOS, but did not induce destabilisation of eNOS dimers into monomers. In hypoxic mice, sepiapterin prevented increase in right ventricular systolic pressure and right ventricular hypertrophy, whereas it modified neither remodelling nor alteration in vasomotor responses (hyper-responsiveness to phenylephrine, decrease in endothelium-dependent relaxation to acetylcholine) in intrapulmonary arteries. Finally, deletion of eNOS gene partially prevented hypoxia-induced increase in right ventricular systolic pressure, right ventricular hypertrophy and remodelling of intrapulmonary arteries. Collectively, these data demonstrate the absence of BH4/BH2 changes and eNOS dimer destabilisation, which may induce eNOS uncoupling during hypoxia-induced pulmonary hypertension. Thus, even though eNOS gene deletion and sepiapterin treatment exert protective effects on hypoxia-induced pulmonary vascular remodelling, increase on right ventricular pressure and / or right ventricular hypertrophy, these effects appear unrelated to biopterin-dependent eNOS uncoupling within pulmonary vasculature of hypoxic wild-type mice.

Role of G(i/o)-Src kinase-PI3K/Akt pathway and caveolin-1 in ß2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery.

Activation of the ß2-adrenoceptor (ß2-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the ß2-AR-mediated eNOS activation, with special focus on G(i/o) proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective ß2-AR agonist procaterol was reduced by inhibitors of G(i/o) proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-ß-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser(1177), which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr(14), which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-ß-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial ß2-AR is coupled to a G(i/o)-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser(1177) leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr(14), through a G(i/o)-Src kinase pathway. Since pulmonary ß2-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.

Hypoxia-induced hyperreactivity of pulmonary arteries: role of cyclooxygenase-2, isoprostanes, and thromboxane receptors.

AIMS: This study investigates the role of the cyclooxygenase (COX)/prostanoid pathway in chronic hypoxia-induced hyperreactivity of pulmonary arteries. METHODS AND RESULTS: Pulmonary arteries were removed from normoxic or hypoxic (0.5 atm for 21 days) mice and studied for protein expression/localization of COX-1, COX-2, and thromboxane A2 (TXA2)-synthase, release of TXA2, prostacyclin (PGI2) and the isoprostane 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), and vasomotor responses. COX-2 expression was increased in all layers of pulmonary arteries from hypoxic mice. In contrast, COX-1 expression was not significantly modified following chronic hypoxia, whereas TXA2-synthase was decreased. Chronic hypoxia differentially affected prostanoid release from pulmonary arteries: TXA2 secretion was not significantly modified; PGI2 secretion was decreased, whereas 8-iso-PGF2alpha secretion was increased. A selective COX-2 inhibitor decreased 8-iso-PGF2alpha release. Arachidonic acid elicited an endothelium- and COX-1-dependent relaxation in pulmonary arteries from normoxic mice. In contrast, arachidonic acid induced an endothelium-independent contraction in pulmonary arteries from hypoxic mice that was partially reduced by catalase, COX-1, COX-2, or TXA2-synthase inhibitors and was totally abolished by blockade of the thromboxane (TP) receptor. Hyperresponsiveness to phenylephrine (PE) of pulmonary arteries from hypoxic mice was also decreased by COX-2 inhibitors, TP receptor antagonists or catalase, but not by TXA2-synthase inhibitors. Finally, 8-iso-PGF2alpha induced a TP receptor-dependent contraction in pulmonary arteries and markedly potentiated the contractile response to PE. CONCLUSION: Chronic hypoxia up-regulates COX-2 expression, increases 8-iso-PGF2alpha release, and shifts arachidonic acid-induced, endothelium-dependent relaxation to an endothelium-independent and TP receptor-dependent contraction in pulmonary arteries. COX-2-dependent production of 8-iso-PGF2alpha, by activating TP receptors, participates in hypoxia-induced hyperreactivity of pulmonary arteries.