Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells

Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0% O(2)-95% N(2)-5% CO(2)) in the presence and absence of calpain inhibitor I or calpeptin, after which endothelial nitric oxide synthase (eNOS) activity and protein content were assayed. Exposure to hypoxia decreased eNOS activity but not eNOS protein content. Both calpain inhibitor I and calpeptin prevented the hypoxic decrease of eNOS activity. Incubation of calpain with total membrane preparations of PAEC caused dose-dependent decreases in eNOS activity independent of changes in eNOS protein content. Exposure of PAEC to hypoxia also caused time-dependent decreases of heat shock protein 90 (HSP90) that were prevented by calpain inhibitor I and calpeptin. Moreover, the HSP90 content in anti-eNOS antibody-induced immunoprecipitates from hypoxic PAEC lysates was reduced, and repletion of HSP90 reversed the decrease of eNOS activity in these immunoprecipitates. Incubation of PAEC with a specific inhibitor of HSP90 (geldanamycin) mimicked the hypoxic decrease of eNOS activity. These results indicate that the hypoxia-induced reduction in eNOS activity in PAEC is due to a decrease in HSP90 caused by calpain activation.     

Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats

Pulmonary vascular endothelial nitric oxide (NO) synthase (eNOS)-derived NO is the major stimulant of cyclic guanosine 5'-monophosphate (cGMP) production and NO/cGMP-dependent vasorelaxation in the pulmonary circulation. We recently synthesized multiple peptides and reported that an eleven amino acid (SSWRRKRKESS) peptide (P1) but not scrambled P1 stimulated the catalytic activity but not expression of eNOS and causes NO/cGMP-dependent sustained vasorelaxation in isolated pulmonary artery (PA) segments and in lung perfusion models. Since cGMP levels can also be elevated by inhibition of phosphodiesterase type 5 (PDE-5), this study was designed to test the hypothesis that P1-mediated vesorelaxation is due to its unique dual action as NO-releasing PDE-5 inhibitor in the pulmonary circulation. Treatment of porcine PA endothelial cells (PAEC) with P1 caused time-dependent increase in intracellular NO release and inhibition of the catalytic activity of cGMP-specific PDE-5 but not PDE-5 protein expression leading to increased levels of cGMP. Acute hypoxia-induced PA vasoconstriction ex vivo and continuous telemetry monitoring of hypoxia (10% oxygen)-induced elevated PA pressure in freely moving rats were significantly restored by administration of P1. Chronic hypoxia (10% oxygen for 4 weeks)-induced alterations in PA perfusion pressure, right ventricular hypertrophy, and vascular remodeling were attenuated by P1 treatment. These results demonstrate the potential therapeutic effects of P1 to prevent and/or arrest the progression of hypoxia-induced PAH via NO/cGMP-dependent modulation of hemodynamic and vascular remodeling in the pulmonary circulation.     

TNF-alpha augments pulmonary vasoconstriction via the inhibition of nitrovasodilator activity.

We tested the hypothesis that tumor necrosis factor-alpha (TNF-alpha) increases pulmonary vasoconstriction by decreases in nitric oxide- (NO) dependent vasodilation. Lungs were isolated from guinea pigs 18 h after intraperitoneal injection of either TNF-alpha (1.60 x 10(5) U/kg) or control. U-46619 (365 mM/min) caused increases in pulmonary arterial and capillary pressures, pulmonary arterial and venous resistances, and lung weight. TNF-alpha augmented the U-46619-induced increases in pulmonary arterial and capillary pressures, pulmonary arterial and venous resistances, and lung weight. Methylene blue (1 microM), which inhibits the activation of soluble guanylate cyclase by NO, had an effect similar to TNF-alpha on the pulmonary response to U-46619 alone but was not additive to the effect of TNF-alpha. NG-monomethyl-L-arginine (270 microM), an inhibitor of NO generation, also enhanced the response to U-46619. Lung effluent levels of nitrite, the oxidation product of NO, were reduced after treatment with either TNF-alpha or NG-monomethyl-L-arginine compared with U-46619 alone. In addition, lungs isolated after TNF-alpha treatment showed decreased vasodilation in response to acetylcholine (10(-8)-10(-5) M) compared with control; however, vasodilation in response to L-arginine (10 mM) and nitroprusside (10(-6.3) and 10(-6) M), agents that promote NO release, was not decreased in TNF-alpha-treated lungs. The data indicate that TNF-alpha induces an increase in vascular constriction in response to U-46619 and a decrease in vasodilation in response to acetylcholine. The mechanism for the TNF-alpha-induced alteration in pulmonary vascular reactivity may be decreased generation of NO.     

Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC). In isolated porcine pulmonary artery segments (PAS), UA (7.5 mg/dl) inhibits acetylcholine-induced vasodilation. The incubation of PAEC with UA caused a dose-dependent decrease in NO and cGMP production stimulated by bradykinin or Ca(2+)-ionophore A23187. We explored cellular mechanisms by which UA might cause reduced NO production focusing on the effects of UA on the l-arginine-endothelial NO synthase (eNOS) and l-arginine-arginase pathways. Incubation of PAEC with different concentrations of UA (2.5-15 mg/dl) for 24 h did not affect l-[(3)H]arginine uptake or activity/expression of eNOS. However, PAEC incubated with UA (7.5 mg/dl; 24 h) released more urea in culture media than control PAEC, suggesting that arginase activation might be involved in the UA effect. Kinetic analysis of arginase activity in PAEC lysates and rat liver and kidney homogenates demonstrated that UA activated arginase by increasing its affinity for l-arginine. An inhibitor of arginase (S)-(2-boronoethyl)-l-cysteine prevented UA-induced reduction of A23187-stimulated cGMP production by PAEC and abolished UA-induced inhibition of acetylcholine-stimulated vasodilation in PAS. We conclude that UA-induced arginase activation is a potential mechanism for reduction of NO production in PAEC.     

Regulation of endothelial nitric oxide synthase by the actin cytoskeleton

In the present study, the association ofendothelial nitric oxide synthase (eNOS) with the actin cytoskeleton inpulmonary artery endothelial cells (PAEC) was examined. We found thatthe protein contents of eNOS, actin, and caveolin-1 were significantly higher in the caveolar fraction of plasma membranes than in the noncaveolar fraction of plasma membranes in PAEC. Immunoprecipitation of eNOS from lysates of caveolar fractions of plasma membranes in PAECresulted in the coprecipitation of actin, and immunoprecipitation ofactin from lysates of caveolar fractions resulted in thecoprecipitation of eNOS. Confocal microscopy of PAEC, in which eNOS waslabeled with fluorescein, F-actin was labeled with Texasred-phalloidin, and G-actin was labeled with deoxyribonuclease Iconjugated with Texas red, also demonstrated an association betweeneNOS and F-actin or G-actin. Incubation of purified eNOS with purifiedF-actin and G-actin resulted in an increase in eNOS activity. Theincrease in eNOS activity caused by G-actin was much higher than thatcaused by F-actin. Incubation of PAEC with swinholide A, an actinfilament disruptor, resulted in an increase in eNOS activity, eNOSprotein content, and association of eNOS with G-actin and in a decrease in the association of eNOS with F-actin. The increase in eNOS activitywas higher than that in eNOS protein content in swinholide A-treatedcells. In contrast, exposure of PAEC to phalloidin, an actin filamentstabilizer, caused decreases in eNOS activity and association of eNOSwith G-actin and increases in association of eNOS with F-actin. Theseresults suggest that eNOS is associated with actin in PAEC and thatactin and its polymerization state play an important role in theregulation of eNOS activity.

     

In vivo attenuation of endothelium-dependent pulmonary vasodilation by methylene blue.

In vitro evidence suggests that resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation are mediated by changes in vascular smooth muscle concentrations of guanosine 3',5'-cyclic monophosphate (cGMP). We investigated this hypothesis in vivo in 19 mechanically ventilated intact lambs by determining the hemodynamic effects of methylene blue (a guanylate cyclase inhibitor) and then by comparing the hemodynamic response to five vasodilators during pulmonary hypertension induced by the infusion of U-46619 (a thromboxane A2 mimic) or methylene blue. Methylene blue caused a significant time-dependent increase in pulmonary arterial pressure. During U-46619 infusions, acetylcholine, ATP-MgCl2, sodium nitroprusside, isoproterenol, and 8-bromo-cGMP decreased pulmonary arterial pressure. During methylene blue infusions, the decreases in pulmonary arterial pressure caused by acetylcholine and ATP-MgCl2 (endothelium-dependent vasodilators) and sodium nitroprusside (an endothelium-independent guanylate cyclase-dependent vasodilator) were attenuated by greater than 50%. The decreases in pulmonary arterial pressure caused by isoproterenol and 8-bromo-cGMP (endothelium-independent vasodilators) were unchanged. This study in intact lambs supports the in vitro evidence that changes in vascular smooth muscle cell concentrations of cGMP in part mediate resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation.     

Angiotensin IV-mediated pulmonary artery vasorelaxation is due to endothelial intracellular calcium release

Angiotensin (ANG) IV stimulation of pulmonary artery (PA) endothelial cells (PAECs) but not of PA smooth muscle cells (PASMCs) resulted in significant increased production of cGMP in PASMCs. ANG IV receptors are not present in PASMCs, and PASMC nitric oxide synthase activity was not altered by ANG IV. ANG IV caused a dose-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PAs, and this response was blocked by the ANG IV receptor antagonist divalinal ANG IV but not by ANG II type 1 and 2 receptor blockers. ANG IV receptor-mediated increased intracellular Ca(2+) concentration ([Ca(2+)](i)) release from intracellular stores in PAECs was blocked by divalinal ANG IV as well as by the G protein, phospholipase C, and phosphoinositide (PI) 3-kinase inhibitors guanosine 5'-O-(2-thiodiphosphate), U-73122, and LY-294002, respectively, and was regulated by both PI 3-kinase- and ryanodine-sensitive Ca(2+) stores. Basal and ANG IV-mediated vasorelaxation of endothelium-denuded PAs was restored by exogenous PAECs but not by exogenous PAECs pretreated with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. These results demonstrate that ANG IV-mediated vasodilation of PAs is endothelium dependent and regulated by [Ca(2+)](i) release through receptor-coupled G protein-phospholipase C-PI 3-kinase signaling mechanisms.     

Microtubule-active agents modify nitric oxide production in pulmonary artery endothelial cells

The effects of specific microtubule-active agents on nitric oxide (NO) production were examined in pulmonary artery endothelial cells (PAEC). PAEC were incubated with taxol, which stabilizes microtubules, or nocodazole, which disrupts microtubules, or both for 2-4 h. We then examined NO production, endothelial NO synthase (eNOS) activity, and eNOS association with heat shock protein (HSP) 90. Incubation of PAEC with taxol (15 microM) for 2-4 h resulted in an increase in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. Incubation of PAEC with nocodazole (50 microM) for 2-4 h induced a decrease in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. The presence of taxol in the culture medium prevented the effects of nocodazole on NO production and eNOS activity in PAEC. Geldanamycin, a HSP90 inhibitor, prevented the taxol-induced increase in eNOS activity. Taxol and nocodazole did not affect eNOS, HSP90, and tubulin protein contents in PAEC, as detected using Western blot analysis. These results indicate that the polymerization state of the microtubule cytoskeleton regulates NO production and eNOS activity in PAEC. The changes in eNOS activity induced by modification of microtubules are due, at least in part, to the altered binding of HSP90 to eNOS protein.     

Secretoneurin facilitates endothelium-dependent relaxations in porcine coronary arteries

Secretoneurin enhances the adhesion and transendothelial migration properties of monocytes and is a part of the peptide family encoded by the secretogranin II gene. The expression of the secretogranin II gene is upregulated in senescent endothelium. The present study was designed to examine the effects of secretoneurin on endothelium-dependent responsiveness. Isometric tension was measured in rings (with or without endothelium) of porcine coronary arteries. Secretoneurin did not induce contraction of quiescent or contracted rings. In preparations contracted by U-46619, relaxation was observed with high concentrations of the peptide. This relaxation was endothelium dependent and reduced by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME). It was abolished when the preparations were incubated with l-NAME in combination with the cyclooxygenase inhibitor indomethacin. The relaxation was not affected by the combination of 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-etheno-7H-dibenzo[b,m][1,5,12,16]tetraazacyclotricosine-5,13-diiumditrifluoroacetate hydrate (UCL 1684), which abrogates endothelium-dependent hyperpolarizations. These results indicate that secretoneurin acutely induces relaxation through the activation of endothelial nitric oxide synthase (eNOS) and cyclooxygenase, with nitric oxide playing the dominant role. Prolonged (24 h) incubation with physiological concentrations of secretoneurin enhanced the relaxations to bradykinin and to the calcium ionophore A-23187, but this difference was not observed in preparations incubated with l-NAME or the calmodulin antagonist calmidazolium. Under these conditions, the relaxation to sodium nitroprusside remained unchanged. Incubation with secretoneurin significantly augmented the expression of eNOS and calmodulin as well as the dimerization of eNOS in cultures of porcine coronary arterial endothelial cells. These observations suggest that secretoneurin not only acutely causes but also, upon prolonged exposure, enhances endothelium-dependent relaxations.     

U-46619 but not serotonin increases endocannabinoid content in middle cerebral artery: evidence for functional relevance

Cerebral vascular smooth muscle cells express the CB(1) cannabinoid receptor, and CB(1) receptor agonists produce vasodilation of cerebral arteries. The purpose of this study was to determine whether vasoconstriction of rat middle cerebral artery (MCA) results in the local formation of endocannabinoids (eCBs), which, via activation of CB(1) receptors, oppose the vasoconstriction in a feedback manner. The thromboxane A(2) (TXA(2)) mimetic U-46619 significantly increased N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol (2-AG) content of isolated MCA, whereas 5-hydroxytrypamine (5-HT) decreased AEA and 2-AG content. If eCBs play a feedback role in the regulation of MCA tone, then CB(1) receptor antagonists should enhance the constriction of MCA produced by U-46619 but not 5-HT. U-46619 caused concentration-dependent constrictions of endothelium-denuded MCA. Two CB(1) receptor antagonists SR-141716 and AM-251 decreased the EC(50) value for U-46619 to constrict endothelium-denuded MCA without affecting the maximal effect. A low concentration of CB(1) receptor agonist Win-55212-2 (30 nM) produced vasodilation of MCAs constricted with low but not saturating concentrations of U-46619. SR-141716 had no effect on the 5-HT concentration-contraction relationship. These data suggest that TXA(2) receptor activation increases MCA eCB content, which, via activation of CB(1) receptors, reduces the constriction produced by moderate concentrations of the TXA(2) agonist. Although 5-HT-induced vasoconstriction is reduced by exogenous CB(1) receptor agonist, activation of 5-HT receptors does not increase eCB content. These results suggest that MCA production of eCBs is not regulated by constriction per se but likely via a signaling pathway that is specific for TXA(2) receptors and not 5-HT receptors.     

Growth-related oncogene-alpha induces endothelial dysfunction through oxidative stress and downregulation of eNOS in porcine coronary arteries

Growth-related oncogene-alpha (GRO-alpha) is a member of the CXC chemokine family, which is involved in the inflammatory process including atherosclerosis. We hypothesized that GRO-alpha may affect endothelial functions in both porcine coronary arteries and human coronary artery endothelial cells (HCAECs). Vasomotor function was analyzed in response to thromboxane A2 analog U-46619 for contraction, bradykinin for endothelium-dependent vasorelaxation, and sodium nitroprusside (SNP) for endothelium-independent vasorelaxation. In response to 10(-6) M bradykinin, GRO-alpha (50 and 100 ng/ml) significantly reduced endothelium-dependent vasorelaxation by 34.73 and 48.8%, respectively, compared with controls (P < 0.05). There were no changes in response to U-46619 or SNP between treated and control groups. With the lucigenin-enhanced chemiluminescence assay, superoxide anion production in GRO-alpha-treated vessels (50 and 100 ng/ml) was significantly increased by 50 and 86%, respectively, compared with controls (P < 0.05). With real-time PCR analysis, endothelial nitric oxide synthase (eNOS) mRNA levels in porcine coronary arteries and HCAECs after GRO-alpha treatment were significantly decreased compared with controls (P < 0.05). The eNOS protein levels by both immunohistochemistry and Western blot analyses were also decreased in GRO-alpha-treated vessels. Antioxidant seleno-l-methionine and anti-GRO-alpha antibody effectively blocked these effects of GRO-alpha on both porcine coronary arteries and HCAECs. In addition, GRO-alpha immunoreactivity was substantially increased in the atherosclerotic regions compared with nonatherosclerotic regions in human coronary arteries. Thus GRO-alpha impairs endothelium-dependent vasorelaxation in porcine coronary arteries through a mechanism of overproduction of superoxide anion and downregulation of eNOS. GRO-alpha may contribute to human coronary artery disease.     

Increased pulmonary vascular resistance and defective pulmonary artery filling in caveolin-1-/- mice

Caveolin-1, the structural and signaling protein of caveolae, is an important negative regulator of endothelial nitric oxide synthase (eNOS). We observed that mice lacking caveolin-1 (Cav1(-/-)) had twofold increased plasma NO levels but developed pulmonary hypertension. We measured pulmonary vascular resistance (PVR) and assessed alterations in small pulmonary arteries to determine the basis of the hypertension. PVR was 46% greater in Cav1(-/-) mice than wild-type (WT), and increased PVR in Cav1(-/-) mice was attributed to precapillary sites. Treatment with NG-nitro-l-arginine methyl ester (l-NAME) to inhibit NOS activity raised PVR by 42% in WT but 82% in Cav1(-/-) mice, indicating greater NO-mediated pulmonary vasodilation in Cav1(-/-) mice compared with WT. Pulmonary vasculature of Cav1(-/-) mice was also less reactive to the vasoconstrictor thromboxane A2 mimetic (U-46619) compared with WT. We observed redistribution of type I collagen and expression of smooth muscle alpha-actin in lung parenchyma of Cav1(-/-) mice compared with WT suggestive of vascular remodeling. Fluorescent agarose casting also showed markedly decreased density of pulmonary arteries and artery filling defects in Cav1(-/-) mice. Scanning electron microscopy showed severely distorted and tortuous pulmonary precapillary vessels. Thus caveolin-1 null mice have elevated PVR that is attributed to remodeling of pulmonary precapillary vessels. The elevated basal plasma NO level in Cav1(-/-) mice compensates partly for the vascular structural abnormalities by promoting pulmonary vasodilation.     

Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress

Tempol catalyzes the formation of H(2)O(2) from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H(2)O(2) by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H(2)O(2). Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-46619, or norepinephrine. During U-46619, tempol caused a transient dilation (22 +/- 2%), whereas NBT was ineffective (2 +/- 1%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K(+) channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 +/- 7%. Tempol, but not NBT, increased H(2)O(2) in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (184 +/- 20%) of mouse cremaster arterioles exposed to angiotensin II for 30 min, which was not seen with NBT (9 +/- 4%). The vasodilation to tempol was reduced by 68 +/- 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-46619 or angiotensin II is mediated in part via production of H(2)O(2) and is largely independent of the endothelium and potassium channels.     

Endothelial monocyte-activating polypeptide II causes NOS-dependent pulmonary artery vasodilation: a novel effect for a proinflammatory cytokine

Endothelial monocyte-activating polypeptide (EMAP) II is a novel proinflammatory cytokine that is released from apoptotic and hypoxic cells. The purpose of this study was to determine the effect of EMAP II on the pulmonary artery (PA) and to characterize its mechanism of action. To study this, isolated PA rings from adult male Sprague-Dawley rats were suspended on steel hooks connected to force transducers and immersed in 37 degrees C organ baths containing modified Krebs-Henseleit solution. After equilibration, force displacement of phenylephrine-preconstricted PA was measured in response to EMAP II. Experiments were performed in endothelium-intact rings, endothelium-denuded rings, and in the presence of the NOS inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME). Pulmonary artery rings were then subjected to quantitative PCR analysis for inducible NOS (iNOS) mRNA. EMAP II caused a maximal vasodilation of 251 +/- 30.7 mg in endothelium-intact PA. EMAP II caused no vasodilation in endothelium-denuded and l-NAME-treated PA (20 +/- 14.0 mg and 17.5 +/- 7.5 mg, respectively, P < 0.001 vs. endothelium intact). In addition to its vasoactive properties, EMAP II increased PA iNOS mRNA twofold compared with controls. These results demonstrate that 1) EMAP II causes PA vasodilation; 2) EMAP II-mediated PA vasodilation is endothelium dependent and NOS dependent; and 3) EMAP II upregulates iNOS mRNA expression in PA. This report constitutes the first demonstration of EMAP II's effects on the pulmonary artery, its mechanism of action, and represents the identification of the first proinflammatory cytokine to cause PA vasodilation.     

Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased NO release and impaired pulmonary vasodilation. We investigated the hypothesis that increased superoxide (O(2)(*-)) release by an uncoupled endothelial nitric oxide synthase (eNOS) contributes to impaired pulmonary vasodilation in PPHN. We investigated the response of isolated pulmonary arteries to the NOS agonist ATP and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus and in sham-ligated controls in the presence or absence of the NOS antagonist nitro-L-arginine methyl ester (L-NAME) or the O(2)(*-) scavenger 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron). ATP caused dose-dependent relaxation of pulmonary artery rings in control lambs but induced constriction of the rings in PPHN lambs. L-NAME, the NO precursor L-arginine, and Tiron restored the relaxation response of pulmonary artery rings to ATP in PPHN. Relaxation to NO was attenuated in arteries from PPHN lambs, and the response was improved by L-NAME and by Tiron. We also investigated the alteration in heat shock protein (HSP)90-eNOS interactions and release of NO and O(2)(*-) in response to ATP in the pulmonary artery endothelial cells (PAEC) from these lambs. Cultured PAEC and endothelium of freshly isolated pulmonary arteries from PPHN lambs released O(2)(*-) in response to ATP, and this was attenuated by the NOS antagonist L-NAME and superoxide dismutase (SOD). ATP stimulated HSP90-eNOS interactions in PAEC from control but not PPHN lambs. HSP90 immunoprecipitated from PPHN pulmonary arteries had increased nitrotyrosine signal. Oxidant stress from uncoupled eNOS contributes to impaired pulmonary vasodilation in PPHN induced by ductal ligation in fetal lambs.     

Sphingosine 1-phosphate and control of vascular tone

Sphingosine-1-phosphate (S1P) is a platelet-derived lipid mediator that activates the endothelial isoform of nitric oxide synthase (eNOS) in endothelial cells. However, the role of S1P in endothelium-dependent vasodilation and the signaling pathways elicited by S1P in intact vessels are largely unknown. We found that S1P induces dose-dependent transient relaxation of isolated pressurized mesenteric arterioles (EC(50) 10 +/- 3 nM); maximal vasodilation (55 +/- 8%) is seen approximately 2 min after S1P addition and returns to baseline by 5 min. S1P promotes comparable responses in arterioles from wild-type but not eNOS(null) mice. S1P-induced vasodilation is abrogated by removal of endothelium or by the addition of the NOS inhibitor N(omega)-monomethyl-l-arginine but is not affected by the cyclooxygenase inhibitor indomethacin, nor by the blockade of K(+) channels by using 4-aminopyridine. S1P-induced vasodilation is attenuated by pertussis toxin, by the phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin, and by the calcium chelator BAPTA. With the use of high-sensitivity protein immunoblots in extracts from single pressurized vessels, we found that S1P, but not bradykinin, promotes the phosphorylation of eNOS at Ser(1179). Maximum S1P-induced eNOS Ser(1179) phosphorylation was reached at the time of maximum vasorelaxation, but enzyme phosphorylation persisted for several minutes after vasodilation had resolved. Thus regulatory pathways distinct from eNOS Ser(1179) dephosphorylation serve to terminate agonist-promoted vasorelaxation. Taken together, our findings demonstrate that S1P, an important intercellular mediator of platelet-vessel wall interactions, is a effective arteriolar vasodilator that acts via G protein-dependent, calcium-sensitive, and PI3-kinase-modulated signaling pathways.     

Differential effects of chronic hypoxia and intermittent hypocapnic and eucapnic hypoxia on pulmonary vasoreactivity.

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO(2), however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO(2) inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O(2)/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O(2), 5% CO(2)/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO(2).     

Stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to globular adiponectin-induced NO production: vasorelaxation in response to globular adiponectin

The present study examined potential interactions between endothelial NO synthase (eNOS), heat shock protein (HSP)90, and Akt in vascular endothelial cells stimulated with globular adiponectin to produce nitric oxide (NO). Globular adiponectin-induced eNOS phosphorylation was accompanied by eNOS-HSP90-Akt complex formation, resulting in a dose-dependent increase in NO release. Globular adiponectin stimulated binding of HSP90 to eNOS, and inhibition of HSP90 significantly suppressed globular adiponectin-stimulated NO release. Globular adiponectin also caused Akt phosphorylation, and inhibition of PI3 kinase significantly suppressed globular adiponectin-stimulated NO release. This study also examined whether globular adiponectin really induces endothelial-dependent vasodilation using rings from rat thoracic aorta. It was observed that globular adiponectin caused dose-dependent vasorelaxation in the aorta. These results indicate that stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to globular adiponectin-induced eNOS phosphorylation and NO production, and to endothelium-dependent vasorelaxation.     

Impaired pulmonary artery contractile responses in a rat model of microgravity: role of nitric oxide.

Vascular contractile hyporesponsiveness is an important mechanism underlying orthostatic intolerance after microgravity. Baroreceptor reflexes can modulate both pulmonary resistance and capacitance function and thus cardiac output. We hypothesized, therefore, that pulmonary vasoreactivity is impaired in the hindlimb-unweighted (HLU) rat model of microgravity. Pulmonary artery (PA) contractile responses to phenylephrine (PE) and U-46619 (U4) were significantly decreased in the PAs from HLU vs. control (C) animals. N(G)-nitro-L-arginine methyl ester (10(-5) M) enhanced the contractile responses in the PA rings from both C and HLU animals and completely abolished the differential responses to PE and U4 in HLU vs. C animals. Vasorelaxant responses to ACh were significantly enhanced in PA rings from HLU rats compared with C. Moreover, vasorelaxant responses to sodium nitroprusside were also significantly enhanced. Endothelial nitric oxide synthase (eNOS) and soluble guanlyl cyclase expression were significantly enhanced in PA and lung tissue from HLU rats. In marked contrast, the expression of inducible nitric oxide synthase was unchanged in lung tissue. These data support the hypothesis that vascular contractile responsiveness is attenuated in PAs from HLU rats and that this hyporesponsiveness is due at least in part to increased nitric oxide synthase activity resulting from enhanced eNOS expression. These findings may have important implications for blood volume distribution and attenuated stroke volume responses to orthostatic stress after microgravity exposure.