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Recent advances have identified an important role of bone morphogenetic protein 4 (BMP4) in pulmonary vascular remodeling, yet the underlying mechanisms remain largely unexplored. We have previously found that [Ca.sup.2+] influx through store-operated calcium channels (SOCC), which are mainly thought to be composed of canonical transient receptor potential (TRPC) proteins, likely contribute to the pathogenic development of chronic hypoxic pulmonary hypertension. In this study, we investigated the effect of BMP4 on expression of TRPC and store-operated [Ca.sup.2+] entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs). Real-time quantitative PCR and Western blotting revealed that treatment with BMP4 (50 ng/ml, 60 h) increased TRPC1, TRPC4, and TRPC6 mRNA and protein expression in growth-arrested rat distal PASMCs. Moreover, in comparison to vehicle control, cells treated with BMP4 also exhibited enhanced SOCE, and elevated basal intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) as determined by fluorescent microscopy using the [Ca.sup.2+] indicator Fura-2 AM. Perfusing cells with [Ca.sup.2+]-free Krebs-Ringer bicarbonate solution (KRBS) or KRBS containing SOCC antagonists SKF-96365 or Ni[Cl.sub.2] attenuated the increases in basal [[[Ca.sup.2+]].sub.i] caused by BMP4. Specific knockdown of BMP4 by small interference RNA significantly decreased the mRNA and protein expression of TRPC1, TRPC4, and TRPC6 and reduced SOCE and basal [[[Ca.sup.2+]].sub.i] in serum-stimulated PASMCs. We conclude that BMP4 regulates calcium signaling in PASMCs likely via upregulation of TRPC expression, leading to enhanced SOCE and basal [[[Ca.sup.2+]].sub.i] in PASMCs, and by this mechanism contributes to pulmonary vascular remodeling during pulmonary arterial hypertension. calcium signaling; intracellular calcium concentration doi: 10.1152/ajpcell.00040.2010.

Hypoxia appears to promote contraction [hypoxic pulmonary vasoconstriction (HPV)] of bovine pulmonary arteries (BPA) through removal of a peroxide-mediated relaxation. This study examines the roles of BPA Nox oxidases and mitochondria in the HPV response. Inhibitors of Nox2 (0.1 mM apocynin and 50 [micro]M gp91-dstat) and mitochondrial electron transport (10 [micro]M antimycin and rotenone) decreased superoxide generation in BPA without affecting contraction to 25 mM KC1 or the HPV response. Transfection of BPA with small inhibitory RNA (siRNA) for Nox2 and Nox4 decreased Nox2 and Nox4 protein expression, respectively, associated with an attenuation of superoxide detection, without affecting 25 mM KC1 contraction. However, Nox4 siRNA, but not Nox2, attenuated HPV in BPA. A Nox4 inhibitor plumbagin (10 [micro]M) increased basal force, decreased superoxide detection and peroxide release, and caused BPA to relax under hypoxia. Although acute removal of peroxide with 0.1 mM ebselen increased 25 mM KC1 contraction and decreased hypoxic contraction, prolonged treatment with ebselen only decreased hypoxic contraction without affecting 25 mM KCI contraction, suggesting basal peroxide levels also maintain a contractile mechanism not removed by acute hypoxia. Organ culture of BPA with transforming growth factor (TGF)-[beta]1 (4 nM) increased Nox4 expression, superoxide, peroxide, and the HPV response. Thus Nox2 and mitochondria are sources for superoxide generation in BPA, which do not appear to influence the HPV response. However, peroxide derived from superoxide generated by Nox4 appears to maintain a basal relaxation in BPA under normoxic conditions, which is removed under hypoxia leading to HPV. Peroxide generated by Nox4 may also function to maintain a contractile mechanism, which is not reversed by acute hypoxia. hydrogen peroxide; hypoxic pulmonary vasoconstriction; mitochondria; Nox oxidase; oxygen sensor doi: 10.1152/ajpheart.01228.2009.

Guignabert C, Alvira CM, Alastalo T-P, Sawada H, Hansmann G, Zhao M, Wang L, El-Bizri N, Rabinovitch M. Tie2-mediated loss of peroxisome proliferator-activated receptor-[gamma] in mice causes PDGF receptor-[beta]-dependent pulmonary arterial muscularization. Am J Physiol Lung Cell Mol Physiol 297: L1082-L1090, 2009. First published October 2, 2009; doi: 10.1152/ajplung.00199.2009.--Peroxisome proliferator-activated receptor (PPAR)-[gamma] is reduced in pulmonary arteries (PAs) of patients with PA hypertension (PAH), and we reported that deletion of PPAR[gamma] in smooth muscle cells (SMCs) of transgenic mice results in PAH. However, the sequelae of loss of PPAR[gamma] in PA endothelial cells (ECs) are unknown. Therefore, we bred Tie2-Cre mice with PPAR[[gamma].sup.flox/flox] mice to induce EC loss of PPAR[gamma] (Tie2 PPAR[[gamma].sup.-/-]), and we assessed PAH by right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and muscularized distal PAs in room air (RA), after chronic hypoxia (CH), and after 4 wk of recovery in RA (Rec-RA). The Tie2 PPAR[[gamma].sup.-/-] mice developed spontaneous PAH in RA with increased RVSP, RVH, and muscularized PAs vs. wild type (WT); both genotypes exhibited a similar degree of PAH following chronic hypoxia, but Tie2 PPAR[[gamma].sup.-/-] mice had more residual PAH compared with WT mice after Rec-RA. The Tie2 PPAR[[gamma].sup.-/-] vs. WT mice in RA had increased platelet-derived growth factor receptor-[beta] (PDGF-R[beta]) expression and signaling, despite an elevation in the PPAR[gamma] target apolipoprotein E, an inhibitor of PDGF signaling. Inhibition of PDGF-R[beta] signaling with imatinib, however, was sufficient to reverse the PAH observed in the Tie2 PPAR[[gamma.sup.-/-] mice. Thus the disruption of PPAR[gamma] signaling in EC is sufficient to cause mild PAH and to impair recovery from CH-induced PAH. Inhibition of heightened PDGF-R[beta] signaling is sufficient to reverse PAH in this genetic model. endothelial cells; platelet-derived growth factor receptor-[beta]; pulmonary remodeling; smooth muscle cell; platelet-derived growth factor doi: 10.1152/ajplung.00199.2009

Endothelium-denuded bovine pulmonary arteries (BPA) contract to hypoxia through a mechanism potentially involving removing a superoxide-derived hydrogen peroxide-mediated relaxation. BPA organ cultured for 24 h with 0.1 mM cobalt chloride (CO[CL.sub.2]) to increase the expression and activity of heme oxygenase-1 (HO-1) is accompanied by a decrease in 5 [micro]M lucigenin-detectable superoxide and an increase in horseradish peroxidase-luminol detectable peroxide levels. Force development to KC1 in BPA was not affected by increases in HO-1, but the hypoxic pulmonary vasoconstriction (HPV) response was decreased. Organ culture with a HO-I inhibitor (10 [micro]M chromium mesoporphyrin) reversed the effects of HO-1 on HPV and peroxide. Treatment of HO-1-induced BPA with extracellular catalase resulted in reversal of the attenuation of HPV without affecting the force development to KC1. Increasing intracellular peroxide scavenging with 0.1 mM ebselen increased force development to KC1 and partially reversed the decrease in HPV seen on induction of HO-1. HO-1 induction increases extracellular (ec) superoxide dismutase (SOD) expression without changing Cu,Zn-SOD and Mn-SOD levels. HO-1-induced BPA rings treated with the copper chelator 10 mM diethyldithiocarbamate to inactivate ecSOD and Cu,Zn-SOD showed increased superoxide and decreased peroxide to levels equal to non-HO-1-induced rings, whereas the addition of SOD to freshly isolated BPA rings attenuated HPV similar to HO-1 induction with CO[Cl.sub.2]. Therefore, HO-1 induction in BPA increases ecSOD expression associated with enhanced generation of peroxide in amounts that may not be adequately removed during hypoxia, leading to an attenuation of HPV. catalase; cobalt; hydrogen peroxide; superoxide; extracellular superoxide dismutase doi: 10.1152/ajpheart.00315.2009

Using adapted retroviruses for gene delivery is a modern and powerful tool in biological research as well as a promising approach for gene therapy. An important limitation for the extensive use of retroviral vectors is the low infection rate in target cells such as pulmonary vascular endothelial cells due to the insufficient infectivity of standard retrovirus supernatants that can only be overcome by complicated methods of vires concentration. This paper describes two easy methods to augment target cell infectivity, first by increasing the retroviral titer in the medium collected from packaging cells by stimulation of viral propagation with dexamethasone, and second, by increasing target cell sensitivity to retroviral infection by the glucocorticoid receptor antagonist, mifepristone. Using this method, we increased the infectivity of pulmonary microvascular endothelial cells from 16% to 85%. We demonstrate that mifepristone increased the susceptibility of target cells to retroviruses without increasing the viral titer of the supernatant. Dexamethasone, but not mifepristone, increased expression of delivered proteins such as GFP that are important for early identification of infected cells. Each, or both step(s), may be included in a standard protocol for retrovirus propagation and infection of target cells. retroviruses; pulmonary artery endothelium; gene delivery

Steady-state wave intensity (WI) analysis indicates that characteristic midsystolic falls in fetal pulmonary trunk (PT) and artery (PA) blood flow are due to an extremely large backward-running compression wave ([BCW.sub.ms]) that 1) originates from the pulmonary microvasculature by a combination of cyclical pulmonary vasoconstriction and vascular reflection of the forward-running compression wave ([FCW.sub.is]) associated with impulsive right ventricular ejection, and 2) is transmitted into the PT. However, no information is available about the dynamic properties of PA [BCW.sub.ms] and its contribution to beat-to-beat regulation of pulmonary hemodynamics. Accordingly, beat-by-beat WI analysis was performed during brief increases in ventricular contractility accompanying an extrasystole (ES) in nine anesthetized late-gestation fetal sheep instrumented with PT and left PA micromanometer catheters to measure pressure (P) and transit-time flow probes to obtain blood velocity (U). WI was calculated as the product of P and U rates of change. At steady state, the magnitude of PA [BCW.sub.ms], and its associated P and U changes ([DELTA]P and [DELTA]U, respectively), were similar to those of [FCW.sub.is]. The PA [FCW.sub.is] and [BCW.sub.ms], and their accompanying [DELTA]P and [DELTA]U, were all transiently potentiated after an ES. Beat-by-beat PA [FCW.sub.is]-[BCW.sub.ms] wave area, [DELTA]P and [DELTA]U relationships were highly linear ([R.sup.2] [greater than or equal to] 0.91) with slopes of 1.36-1.47 (P < 0.001), consistent with the presence of a vasoconstrictor component in PA [BCW.sub.ms]. PA-PT [BCW.sub.ms] area and [DELTA]P and [DELTA]U relationships were also linear ([R.sup.2] [greater than or equal to] 0.77) with slopes of 0.23-0.64 (P < 0.001). These results indicate that the fetal PA [BCW.sub.ms] contributes to beat-to-beat regulation of not only PA but also PT hemodynamics. fetal pulmonary blood flow; fetal pulmonary blood pressure; postextrasystolic potentiation

Following pulmonary artery banding (PAB), the contractile function of right ventricle diminishes over time. Subsequently, the right atrium (RA) has to contract against a higher afterload, but it is unknown to what extent ventricular dysfunction has an effect on the atrial contractility. We hypothesized that right ventricular pressure overload may have an affect on atrial contractility and [Ca.sup.2+] transport protein expression. Therefore, we induced pressure overload of the right ventricle by PAB for 10 wk in rabbits and examined the changes in the expression of [Ca.sup.2+] transport proteins in the atrium. We demonstrate that PAB significantly decreased the expression of sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (Serca) 2a while expression of [Na.sup.+]/[Ca.sup.2+] exchanger-1 was significantly upregulated in the RA but not in the left atria of rabbit hearts, indicating that pressure is the major trigger. A decrease in Serca2a expression was concomitant with a significant decrease in sarcolipin (SLN), possibly indicating a compensatory role of SLN. The decreased expression of SLN was unable to completely restore sarcoplasmic reticulum [Ca.sup.2+] uptake function of Serca2a. Functional contractile assessments in isolated trabeculae showed no difference between PAB- and sham-operated rabbits at 1 Hz but displayed an enhanced force development at higher frequencies and in the presence of isoproterenol, while twitch timing was unaffected. Our results indicate that right ventricular mechanical overload due to PAB affects the expression of the [Ca.sup.2+]-handling proteins in the RA in rabbits. calcium ion transport proteins; pressure overload; sarcoplasmic reticulum

Human cytochrome P-450 epoxygenase enzymes metabolize eicosapentaenoic acid (EPA), an [omega]-3-polyunsaturated fatty acid (PUFA), and leads to the production of 17(18)-epoxyeicosatetraenoic acid, or 17(18)-EpETE. The aim of the present study was to delineate the mode of action of 17(18)-EpETE on human pulmonary artery (HPA) and distal bronchi. Isometric tension measurements demonstrated that 17(18)-EpETE induced concentration-dependent relaxing effects in pulmonary artery and airway smooth muscles. Iberiotoxin (IbTx) and glyburide (Glyb), known [BK.sub.Ca] and [K.sub.ATP] channel inhibitors, respectively, reversed the relaxation induced by 17(18)-EpETE on both tissues types. Microelectrode measurements showed that exogenous addition of 17(18)-EpETE hyperpolarized the membrane potential of HPA and bronchial smooth muscle cells. These induced electrophysiological effects were reversed by the addition of 10 nM IbTx and 10 [micro]M Glyb. Complementary experiments performed on human bronchi, using the planar lipid bilayer reconstitution technique, demonstrated that 17(18)-EpETE activated reconstituted BKca channels at low free [Ca.sup.2+] concentration. Moreover, in bronchi, the relaxing responses induced by 17(18)-EpETE were also related to reduced [Ca.sup.2+] sensitivity of the myofilaments, since free [Ca.sup.2+] concentration-response curves, performed on [beta]-escinpermeabilized cultured explants, were shifted toward higher [Ca.sup.2+]. Together, these results provide new insight into the mode of action of 17(18)-EpETE in lung tissues and highlight this eicosanoid as a potent modulator of tone on both HPA and distal bronchi in vitro, which may be of clinical relevance in the pathophysiology of pulmonary hypertension and airway diseases. 17(18)-eicosatetraenoic acid; membrane potential; potassium channels; isometric tension; relaxation

A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response to acutely applied NO, cGMP analog, or atrial natriuretic peptide (ANP). Twenty-four-hour treatment with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-l,2-diolate (DETA-NO) resulted in > 10-fold decrease in the response to acutely applied DETA-NO. In parallel with this, the relaxant response to acutely applied cGMP analog, [beta]-phenyl-l,[N.sup.2]-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Sp isomer (Sp-8-Br-PET-cGMPS), and ANP decreased. The reduction in ANP responsiveness in PA was not associated with a reduction in cGMP levels evoked by [l0.sup.-6] M ANP. Twenty-four hours in culture and treatment with DETA-NO decreased total cGMP-dependent protein kinase (cGKI) mRNA level compared with that in freshly prepared PA (1.05 [+ or -] 0.12, 0.42 [+ or -] 0.08, and 0.11 [+ or -] 0.01 amol/[micro]g, respectively). Total cGKI protein levels were decreased to a lesser extent by 24 h in culture and further decreased by 24-h DETA-NO treatment compared with that in freshly prepared PA (361 [+ or -] 33, 272 [+ or -] 20, and 238 [+ or -] 25 ng/mg total protein, respectively). Maximal cGMP-stimulated phosphotransferase activity was reduced in 24-h cultured and DETA-NO-treated PA (986 [+ or -] 84, 815 [+ or -] 81, and 549 [+ or -] 78 pmol [P.sub.i] x [min.sup.-1] x mg soluble [protein.sup.-l]), but the cGMP concentration resulting in 50% of maximal phosphotransferase activity was not. cGKI specific activity (maximal cGMP-activated phosphotransferase activity/ng cGKI) was significantly reduced in PA treated with DETA-NO for 24 h compared with freshly prepared and 24-h cultured PA 0.95 [+ or -] 0.22, 2.64 [+ or -] 0.25, and 2.85 [+ or -] 0.28 pmol [P.sub.i] x [min.sup.-1] x ng [cGKI.sup.-1], respectively). We conclude that prolonged NO treatment induces decreased acute NO responsiveness in PA in part by decreasing cGMP sensitivity. It does so by decreasing both cGKI expression and cGKI specific activity. guanosine 3',5'-cyclic monophosphate; guanosine 3'5'-cyclic monophosphate-dependent protein kinase

Hypoxia causes abnormal neonatal pulmonary artery remodeling (PAR) and inhibition of alveolar development (IAD). Transforming growth factor (TGF)-[beta] is an important regulator of lung development and repair from injury. We tested the hypothesis that inhibition of TGF-[beta] signaling attenuates hypoxia-induced PAR and IAD. Mice with an inducible dominant-negative mutation of the TGF-[beta] type II receptor (DNTGF[beta]RII) and nontransgenic wild-type (WT) mice were exposed to hypoxia (12% [O.sub.2]) or air from birth to 14 days of age. Expression of DNTGF[beta]RII was induced by 20 [micro]g/g ZnS[O.sub.4] given intraperitoneally daily from birth. PAR, IAD, cell proliferation, and expression of extracellular matrix (ECM) proteins were assessed. In WT mice, hypoxia led to thicker, more muscularized resistance pulmonary arteries and impaired alveolarization, accompanied by increases in active TGF-[beta] and phosphorylated Smad2. Hypoxia-induced PAR and IAD were greatly attenuated in DNTGF[beta]RII mice given ZnS[O.sub.4] compared with WT control mice and DNTGF[beta]RII mice not given ZnS[O.sub.4]. The stimulatory effects of hypoxic exposure on pulmonary arterial cell proliferation and lung ECM proteins were abrogated in DNTGF[beta]RII mice given ZnS[O.sub.4]. These data support the conclusion that TGF-[beta] plays an important role in hypoxia-induced pulmonaryvascular adaptation and IAD in the newborn animal model. lung development; infant; persistent pulmonary hypertension of the newborn

Myogenic tone in the pulmonary vasculature of normoxic adult animals is minimal or nonexistent. Whereas chronic hypoxia (CH) increases basal tone in pulmonary arteries, it is unclear if a portion of this elevated tone is due to development of myogenicity. Since basal arterial RhoA activity and Rho kinase (ROK) expression are augmented by CH, we hypothesized that CH elicits myogenic reactivity in pulmonary arteries through ROK-dependent vascular smooth muscle (VSM) [Ca.sup.2+] sensitization. To test this hypothesis, we assessed the contribution of ROK to basal tone and pressure-induced vasoconstriction in endothelium-disrupted pulmonary arteries [50-300 [micro]m inner diameter (ID)] from control and CH [4 wk at 0.5 atmosphere (atm)] rats. Arteries were loaded with fura-2 AM to continuously monitor VSM intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). Basal VSM [[[Ca.sup.2+]].sub.i] was not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 [micro]M), caused a concentration-dependent reduction of basal tone in CH arteries but had no effect in control vessels. In contrast, PKC inhibition with GF109203X (1 [micro]M) did not alter basal tone. Furthermore, significant vasoconstriction in response to stepwise increases in intraluminal pressure (5-45 mmHg) was observed at 12, 15, 25, and 35 mmHg in arteries (50-200 [micro]m ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 [micro]M) but not by GF109203X. VSM [[[Ca.sup.2+]].sub.i] was unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200-300 [micro]m ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament [Ca.sup.2+] sensitization. pulmonary hypertension; protein kinase C; vascular smooth muscle; [Ca.sup.2+] sensitization; HA-1077

Extracellular nucleotides, such as ATP, UDP, and UTP, regulate pulmonary vascular tone through P2X and P2Y receptors. Recently, uridine adenosine tetraphosphate ([Up.sub.4]A) was reported as a novel endothelium-derived vasoconstrictive factor. [Up.sub.4]A contains both pufine and pyrimidine moieties, which potentially activate P2X and P2Y receptors. The present study examined the effect of [Up.sub.4]A on contractility of isolated rat pulmonary artery. [Up.sub.4]A at 1-100 [micro]M stimulated contraction in a concentration-dependent manner. [Up.sub.4]A was equipotent as UTP and UDP in the endothelium-denuded artery while much more effective than UTP and UDP in endothelium-intact preparations. The vasoconstrictor effect of [Up.sub.4]A was inhibited by suramin but not IPsI or desensitization of P2X receptors with [alpha],[beta]-methylene-ATP ([alpha],[beta]-Me-ATP). [Up.sub.4]A-induced contraction was also inhibited by pretreatment with thapsigargin, nitrendipine, or EGTA but unaffected by Hl152. Furthermore, unlike ATP and UTP, [Up.sub.4]A did not induce relaxation of endothelium-intact preparations precontracted with phenylephrine. These results suggest that UpaA is a potent vasoconstrictor, but not a vasodilator, of the rat pulmonary artery. UpnA likely acts through a suramin-sensitive P2Y receptor. The contractile effect of [Up.sub.4]A involves the entry of extracellular [Ca.sup.2+] and release of [Ca.sup.2+] from intracellular stores but not [Ca.sup.2+] sensitization via the RhoA/Rho kinase pathway. [Up.sub.4]A, therefore, potentially plays an important role in the regulation of pulmonary vascular tone. extracellular nucleotides; P2 receptors; smooth muscle contraction

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G ([PKG.sub.I]). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate [PKG.sub.I] barrier protection because VASP is a cytoskeletal phosphorylation target of [PKG.sub.I] expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by [PKG.sub.I]-mediated phosphorylation of [Ser.sup.157] and [Ser.sup.239.] To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by [H.sub.2][O.sub.2] in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with [PKG.sub.I] expression introduced by adenoviral infection (Ad.PKG). In the absence of [PKG.sub.I] expression, [H.sub.2][O.sub.2] (100-250 [micro]M) caused a transient increased permeability and [pSer.sup.157]-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP [Ser.sup.157] phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by [H.sub.2][O.sub.2]. With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated [H.sub.2] [O.sub.2]-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated [PKG.sub.I]. Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/[PKG.sub.r] to attenuate [H.sub.2][O.sub.2]-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/[PKG.sub.I] on [H.sub.2][O.sub.2]-induced endothelial barrier dysfunction in HPAEC. protein kinase G; cAMP; protein kinase A; small interfering RNA; [H.sub.2][O.sub.2]

Endogenous endothelin (ET)-1 modulates hypoxic pulmonary vasoconstriction (HPV). Accordingly, intravenously applied ETA receptor antagonists reduce HPV, but this is accompanied by systemic vasodilation. We hypothesized that inhalation of an ETA receptor antagonist might act selectively on the pulmonary vasculature and investigated the effects of aerosolized LU-135252 in an experimental model of HPV. Sixteen piglets (weight: 25 [+ or -] 1 kg) were anesthetized and mechanically ventilated at an inspiratory oxygen fraction ([MATHEMATICAL EXPRESSIONS NOT REPRODUCIBLE IN ASCII.]]) of 0.3. After 1 h of hypoxia at [MATHEMATICAL EXPRESSIONS NOT REPRODUCIBLE IN ASCII/] 0.15, animals were randomly assigned either to receive aerosolized LU-135252 as bolus (0.3 mg/kg for 20 rain; n = 8, LU group), or to receive aerosolized saline (n = 8, controls). In all animals, hypoxia significantly increased mean pulmonary arterial pressure (32 [+ or -] 1 vs. 23 [+ or -] 1 mmHg; P < 0.01; means [+ or -] SE) and increased arterial plasma ET-1 (0.52 [+ or -] 0.04 vs. 0.37 [+ or -] 0.05 fmol/ml; P < 0.01) compared with mild hyperoxia at [MATHEMATICAL EXPRESSIONS NOT REPRODUCIBLE IN ASCI.] 0.3. Inhalation of LU-135252 induced a significant and sustained decrease in mean pulmonary arterial pressure compared with controls (LU group: 27 [+ or -] 1 mmHg; controls: 32 [+ or -] 1 mmHg; values at 4 h of hypoxia; P < 0.01). In parallel, mean systemic arterial pressure and cardiac output remained stable and were not significantly different from control values. Consequently, in our experimental model of HPV, the inhaled ETA receptor antagonist LU-135252 induced selective pulmonary vasodilation without adverse systemic hemodynamic effects. inhalation; selective pulmonary vasodilation; pulmonary arterial hypertension

In recent years, transgenic mouse models have been developed to examine the underlying cellular and molecular mechanisms of lung disease and pulmonary vascular disease, such as asthma, pulmonary thromboembolic disease, and pulmonary hypertension. However, there has not been systematic characterization of the basic physiological pulmonary vascular reactivity in normal and transgenic mice. This represents an intellectual 'gap', since it is important to characterize basic murine pulmonary vascular reactivity in response to various contractile and relaxant factors to which the pulmonary vasculature is exposed under physiological conditions. The present study evaluates excitation- and pharmacomechanical-contraction coupling in pulmonary arteries (PA) isolated from wild-type BALB/c mice. We demonstrate that both pharmaco-and electromechanical coupling mechanisms exist in mice PA. These arteries are also reactive to stimulation by [[alpha].sub.1]-adrenergic agonists, serotonin, endothelin-1, vasopressin, and U-46619 (a thromboxane [A.sub.2] analog). We conclude that the basic vascular responsiveness of mouse PA is similar to those observed in PA of other species, including rat, pig, and human, albeit on a different scale and to varying amplitudes. pharmacology; store depletion; excitation-contraction coupling; G protein-coupled receptors

Congenital cardiac defects associated with increased pulmonary blood flow ([Q.sub.p]) produce pulmonary hypertension. We have previously reported attenuated endothelium-dependent relaxations in pulmonary arteries (PA) isolated from lambs with increased [Q.sub.p] and pulmonary hypertension. To better characterize the vascular alterations in the nitric oxide-superoxide system, 12 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). Twin lambs served as controls. PA were isolated from these lambs at 4-6 wk of age. Electron paramagnetic resonance spectroscopy on fourth-generation PA showed significantly increased superoxide anion generation in shunt PA that were decreased to control levels following inhibition of nitric oxide synthase (NOS) with 2-ethyl-2-thiopseudourea. Preconstricted fifth-generation PA rings were relaxed with a NOS agonist (A-23187), a nitric oxide donor [S-nitrosyl amino penicillamine (SNAP)], polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), or [H.sub.2][O.sub.2]. A-23187-, PEG-SOD-, and [H.sub.2][O.sub.2]-mediated relaxations were impaired in shunt PA compared with controls. Pretreatment with PEG-SOD significantly enhanced the relaxation response to A-23187 and SNAP in shunt but not control PA. Inhibition of NOS with nitro-L-arginine or scavenging superoxide anions with tiron enhanced relaxation to SNAP and inhibited relaxation to PEG-SOD in shunt PA. Pretreatment with catalase inhibited relaxation of shunt PA to A-23187, SOD, and [H.sub.2][O.sub.2]. We conclude that NOS catalyzes the production of superoxide anions in shunt PA. PEG-SOD relaxes shunt PA by converting these anions to [H.sub.2][O.sub.2], a pulmonary vasodilator. The redox environment, influenced by the balance between production and scavenging of ROS, may have important consequences on pulmonary vascular reactivity in the setting of increased [Q.sub.p]. congenital heart disease; hydrogen peroxide; nitric oxide

The objective was to determine the impact of intact normoxic and hyperoxia-exposed (95% [O.sub.2] for 48 h) bovine pulmonary arterial endothelial cells in culture on the redox status of the coenzyme [Q.sub.10] homolog coenzyme Q1 (Co[Q.sub.1]). When Co[Q.sub.l] (50 [micro]M) was incubated with the cells for 30 min, its concentration in the medium decreased over time, reaching a lower level for normoxic than hyperoxia-exposed cells. The decreases in Co[Q.sub.1] concentration were associated with generation of Co[Q.sub.1] hydroquinone (Co[Q.sub.1][H.sub.2]), wherein 3.4 times more Co[Q.sub.1][H.sub.2] was produced in the normoxic than hyperoxia-exposed cell medium (8.2 [+ or -] 0.3 and 2.4 [+ or -] 0.4 [micro]M, means [+ or -] SE, respectively) after 30 min. The maximum Co[Q.sub.1] reduction rate for the hyperoxia-exposed cells, measured using the cell membrane-impermeant redox indicator potassium ferricyanide, was about one-half that of normoxic cells (11.4 and 24.1 nmol*[min.sup.-1]*[mg.sup.-1] cell protein, respectively). The mitochondrial electron transport complex I inhibitor rotenone decreased the Co[Q.sub.1] reduction rate by 85% in the normoxic cells and 44% in the hyperoxia-exposed cells. There was little or no inhibitory effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors on Co[Q.sub.l] reduction. Intact cell oxygen consumption rates and complex I activities in mitochondria-enriched fractions were also lower for hyperoxia-exposed than normoxic cells. The implication is that intact pulmonary endothelial cells influence the redox status of Co[Q.sub.1] via complex I-mediated reduction to Co[Q.sub.1][H.sub.2], which appears in the extracellular medium, and that the hyperoxic exposure decreases the overall Co[Q.sub.1] reduction capacity via a depression in complex I activity. quinone; lung

Mice are useful animal models to study pathogenic mechanisms involved in pulmonary vascular disease. Altered expression and function of voltage-gated [K.sup.+] (Kv) channels in pulmonary artery smooth muscle cells (PASMCs) have been implicated in the development of pulmonary arterial hypertension. Kv currents ([I.sub.K(v)]) in mouse PASMCs have not been comprehensively characterized. The main focus of this study was to determine the biophysical and pharmacological properties of [I.sub.K(v)] in freshly dissociated mouse PASMCs with the patch-clamp technique. Three distinct whole cell [I.sub. K(v)] were identified based on the kinetics of activation and inactivation: rapidly activating and noninactivating currents (in 58% of the cells tested), rapidly activating and slowly inactivating currents (23%), and slowly activating and noninactivating currents (17%). Of the cells that demonstrated the rapidly activating noninactivating current, 69% showed [I.sub. K(v)] inhibition with 4-aminopyridine (4-AP), while 31% were unaffected. Whole cell [I.sub. K(V)] were very sensitive to tetraethylammonium (TEA), as 1 mM TEA decreased the current amplitude by 32% while it took 10 mM 4-AP to decrease [I.sub. K(V)] by a similar amount (37%). Contribution of [Ca.sup.2+]-activated [K.sup.+] ([K.sub.ca]) channels to whole cell [I.sub. K(V)] was minimal, as neither pharmacological inhibition with charybdotoxin or iberiotoxin nor perfusion with [Ca.sup.2+]-free solution had an effect on the whole cell [I.sub. K(v)]. Steady-state activation and inactivation curves revealed a window [K.sup.+] current between -40 and -10 mV with a peak at -31.5 inV. Single-channel recordings revealed large-, intermediate-, and smallamplitude currents, with an averaged slope conductance of 119.4 [+ or -] 2.7, 79.8 [+ or -] 2.8, 46.0 [+ or -] 2.2, and 23.6 [+ or -] 0.6 pS, respectively. These studies provide detailed electrophysiological and pharmacological profiles of the native [K.sub.v] currents in mouse PASMCs. Kv channels

In a newly characterized cultured porcine pulmonary artery (PA) preparation, 24-h treatment with the nitric oxide (NO) donor (Z)-I[N-( 2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) decreased the response to acutely applied DETA-NO compared with 24-h control (--log ECso 6.55 [+ or -] 0.12 and 5.02 [+ or -] 0.21, respectively). Treatment of PA with the cell-permeable superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride, did not change NO responsiveness in either freshly prepared or 24-h DETA-NO-treated PA. cGMP and cAMP phosphodiesterase activities were approximately equal in PA. Twenty-four-hour DETA-NO treatment did not change either cGMP or cAMP phosphodiesterase activities. Twenty-four hours in culture had no significant effect on soluble guanylyl cyclase (sGC) subunit mRNA expression, but 24-h DETA-NO treatment significantly decreased the expression of both sGC[[alpha].sub.1] and sGC[[beta].sub.1]. sGC[[beta].sub.1] protein expression was 42 [+ or -] 4 ng/mg soluble protein. Twenty-four hours in culture without and with DETA-NO reduced sGC[[beta].sub.1] protein expression (36 [+ or -] 3 and 31 [+ or -] 3 ng/mg soluble protein, respectively, P < 0.025). Basal tissue cGMP [[(cGMP).sub.i]] was significantly increased, and NO-induced [(cGMP).sub.i] was significantly decreased by 24-h DETA-NO treatment. [(cGMP).sub.i] normalized to the amount of sGC protein expressed in PA was significantly lower in PA treated for 24 h with DETA-NO compared with both freshly isolated and 24-h cultured PA. We conclude that prolonged NO treatment induces decreased acute NO responsiveness in part by decreasing both sGC expression and sGC-specific activity. nitric oxide; guanosine 3',5'-cyclic monophosphate; pulmonary artery doi:10.1152/ajplung.00368.2006

Reactive oxygen species (ROS) generated from NADPH oxidases and mitochondria have been implicated as key messengers for pulmonary vasoconstriction and vascular remodeling induced by agonists and hypoxia. Since [Ca.sup.2+] mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the [Ca.sup.2+] response and to delineate the [Ca.sup.2+] pathways activated by hydrogen peroxide ([H.sub.2][O.sub.2]) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 [micro]M to 1 mM [H.sub.2][O.sub.2] elicited concentration-dependent increase in intracellular [Ca.sup.2+] concentration in PASMCs, with an initial rise followed by a plateau or slow secondary increase. The initial phase was related to intracellular release. It was attenuated by the inositol trisphosphate (I[P.sub.3]) receptor antagonist 2-aminoethyl diphenylborate, ryanodine, or thapsigargin, but was unaffected by the removal of [Ca.sup.2+] in external solution. The secondary phase was dependent on extracellular [Ca.sup.2+] influx. It was unaffected by the voltage-gated [Ca.sup.2+] channel blocker nifedipine or the nonselective cation channel blockers SKF-96365 and [La.sup.3+], but inhibited concentration dependently by millimolar [Ni.sup.2+], and potentiated by the [Na.sup.+]/[Ca.sup.2+] exchange inhibitor KB-R 7943. [H.sub.2][O.sub.2] did not alter the rate of [Mn.sup.2+] quenching of fura 2, suggesting store- and receptor-operated [Ca.sup.2+] channels were not involved. By contrast, [H.sub.2][O.sub.2] elicited a sustained inward current carried by [Na.sup.+] at -70 mV, and the current was inhibited by [Ni.sup.2+]. These results suggest that [H.sub.2][O.sub.2] mobilizes intracellular [Ca.sup.2+] through multiple pathways, including the I[P.sub.3]- and ryanodine receptor-gated [Ca.sup.2+] stores, and [Ni.sup.2+]-sensitive cation channels. Activation of these [Ca.sup.2+] pathways may play important roles in ROS signaling in PASMCs. nonselective cation channels; sodium-calcium exchange; ryanodine receptor; inositol trisphosphate receptor; pulmonary arteries doi: 10.1152/ajplung.00323.2006

BACKGROUND: Epidemiology studies have linked exposure to pollutant particles to increased cardiovascular mortality and morbidity, but the mechanisms remain unknown. OBJECTIVES: We tested the hypothesis that the ultrafine fraction of [...]

Pulmonary hypertension (PH), defined as a mean pulmonary arterial (PA) pressure of >25 mmHg at rest or >30 mmHg during exercise, is characterized by a progressive and sustained increase in pulmonary vascular resistance that eventually leads to right ventricular failure. Clinically, PH may result from a variety of underlying diseases (Table 1 and Refs. 50, 113, 124). Pulmonary arterial hypertension (PAH) may be familial (FPAH) or sporadic (idiopathic, IPAH), formerly known as primary pulmonary hypertension, i.e., for which there is no demonstrable cause. More often, PAH is due to a variety of identifiable diseases including scleroderma and other collagen disorders, liver disease, human immunodeficiency virus, and the intake of appetite-suppressant drugs such as phentermine and fenfluramine (72). Other, more common, causes of PAH include left ventricular failure (perhaps the most common cause), valvular lesions, chronic pulmonary diseases, sleep-disordered breathing, and prolonged residence at high altitude. This classification, now widely accepted, was first proposed at a meeting in Evian, France, in 1998, and modified in Venice, Italy, in 2003 (124). transforming growth factor-[beta]/bone morphogenetic protein receptor; vasoactive compounds; serotonin transporter; growth factors; knockout mice

The cytochrome P-450 metabolite 20-HETE induces calcium-, endothelial-, and nitric oxide (NO)-dependent relaxation of bovine pulmonary arteries (PA). VEGF is an NO-dependent dilator of systemic arteries and plays a key role in maintaining the integrity of the pulmonary vasculature. We tested the effect of VEGF on PA diameter and tone and the contribution of cytochrome P-450 family 4 (CYP4) to vasoactive effects of VEGF. Bovine PA tings (1 mm in diameter) relaxed with VEGF (0.1-10 nM) in an endothelial- and eNOS-dependent manner. This response was blunted by pretreatment with the CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) as well as a mechanistically different CYP4 inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine. PAs also increased in diameter by 6-12% in the presence of VEGF (10 nM), and this increase was attenuated by DDMS. In contrast to that shown in PAs, 20-HETE constricted bovine renal arteries and did not increase intracellular [Ca.sup.2+] in renal artery endothelial cells as observed in bovine pulmonary artery endothelial cells (BPAECs). VEGF-evoked increases in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in BPAECs were blunted by treatment with DDMS. Both VEGF (10 nM) and 20-HETE (1-5 [micro]M) stimulated NO release from cultured BPAECs, and once again VEGF-induced increases were attenuated by pretreating the cells with DDMS. We conclude that CYP4/20-HETE contributes to VEGF-stimulated NO release and vasodilation in bovine PAs. Given the unique expression of 20-HETE-forming CYP4 in BPAECs vs. systemic arterial endothelial cells, CYP4 may be an important mediator of endothelial-dependent vasoreactivity in PAs. 20-HETE; nitric oxide signaling; vasoreactivity; lung doi: 10.1152/ajplung.00265.2004

Phosphodiesterases (PDE) metabolize cyclic nucleotides limiting the effects of vasodilators such as prostacyclin and nitric oxide (NO). In this study, DNA microarray techniques were used to assess the impact of NO on expression of PDE genes in rat pulmonary arterial smooth muscle cells (rPASMC). Incubation of rPASMC with S-nitroso-L-glutathione (GSNO) increased expression of a PDE isoform that specifically metabolizes cAMP (PDE4B) in a dose- and time-dependent manner. GSNO increased PDE4B protein levels, and rolipram-inhibitable PDE activity was 2.3 [+ or -] 1.0-fold greater in GSNO-treated rPASMC than in untreated cells. The soluble guanylate cyclase (sGC) inhibitor, 1H[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one, and the cAMP-dependent protein kinase inhibitor, H89, prevented induction of PDE4B gene expression by GSNO, but the protein kinase G (PKG) inhibitors, Rp-8-pCPT-cGMPs and KT-5823, did not. Incubation of rPASMC with IL-1[beta] and tumor necrosis factor-[alpha] induced PDE4B gene expression, an effect that was inhibited by L-[N.sup.6]-(1-iminoethyl)lysine, an antagonist of NO synthase 2 (NOS2). The GSNO-induced increase in PDE4B mRNA levels was blocked by actinomycin D but augmented by cycloheximide. Infection of rPASMC with an adenovirus specifying a dominant negative cAMP response element binding protein (CREB) mutant inhibited the GSNO-induced increase of PDE4B gene expression. These results suggest that exposure of rPASMC to NO induces expression of PDE4B via a mechanism that requires cGMP synthesis by sGC but not PKG. The GSNO-induced increase of PDE4B gene expression is CREB dependent. These findings demonstrate that NO increases expression of a cAMP-specific PDE and provide evidence for a novel 'cross talk' mechanism between cGMP and cAMP signaling pathways. cross talk; S-nitroso-L-glutathione; adenosine 3',5'-cyclic monophosphate; guanosine 3',5'-cyclic monophosphate

The objective of this study was to examine the impact of chronic hyperoxic exposure (95% [O.sub.2] for 48 h) on intact bovine pulmonary arterial endothelial cell redox metabolism of 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ). DQ or durohydroquinone (DQ[H.sub.2]) was added to normoxic or hyperoxiaexposed cells in air-saturated medium, and the medium DQ concentrations were measured over 30 min. DQ disappeared from the medium when DQ was added and appeared in the medium when DQH2 was added, such that after ~15 min, a steady-state DQ concentration was approached that was ~4.5 times lower for the hyperoxia-exposed than the normoxic cells. The rate of DQ-mediated reduction of the cell membrane-impermeant redox indicator, potassium ferricyanide [Fe[(CN).sup.3-.sub.6]], was also approximately twofold faster for the hyperoxia-exposed cells. Inhibitor studies and mathematical modeling suggested that in both normoxic and hyperoxia-exposed cells, NAD(P)H:quinone oxidoreductase 1 (NQO1) was the dominant DQ reductase and mitochondrial electron transport complex III the dominant DQ[H.sub.2] oxidase involved and that the difference between the net effects of the cells on DQ redox status could be attributed primarily to a twofold increase in the maximum NQO1-mediated DQ reduction rate in the hyperoxia-exposed cells. Accordingly, NQO1 protein and total activity were higher in hyperoxia-exposed than normoxic cell cytosolic fractions. One outcome for hyperoxia-exposed cells was enhanced protection from cell-mediated DQ redox cycling. This study demonstrates that exposure to chronic hyperoxia increases the capacity of pulmonary arterial endothelial cells to reduce DQ to DQ[H.sub.2] via a hyperoxia-induced increase in NQO1 protein and total activity. duroquinone; pulmonary endothelial cells; mathematical modeling

Acute hypoxia causes pulmonary vasoconstriction in part by inhibiting voltage-gated [K.sup.+] (Kv) channel activity in pulmonary artery smooth muscle cells (PASMC). The hypoxia-mediated decrease in Kv currents [[I.sub.K(v)]] is selective to PASMC; hypoxia has little effect on [I.sub.K(v)] in mesenteric artery smooth muscle cells (MASMC). Functional Kv channels are homo- and/or heterotetramers of pore-forming [alpha]-subunits and regulatory [beta]-subunits. KCNA5 is a Kv channel [alpha]-subunit that forms functional Kv channels in PASMC and regulates resting membrane potential. We have shown that acute hypoxia selectively inhibits [I.sub.K(v)] through KCNA5 channels in PASMC. Overexpression of the human KCNA5 gene increased [I.sub.K(v)] and caused membrane hyperpolarization in HEK-293, COS-7, and rat MASMC and PASMC. Acute hypoxia did not affect [I.sub.K(v)] in KCNA5-transfected HEK-293 and COS-7 cells. However, overexpression of KCNA5 in PASMC conferred its sensitivity to hypoxia. Reduction of P[O.sub.2] from 145 to 35 mmHg reduced [I.sub.K(v)] by ~40% in rat PASMC transfected with human KCNA5 but had no effect on [I.sub.K(v)] in KCNA5-transfected rat MASMC (or HEK and COS cells). These results indicate that KCNA5 is an important Kv channel that regulates resting membrane potential and that acute hypoxia selectively reduces KCNA5 channel activity in PASMC relative to MASMC and other cell types. Because Kv channels (including KCNA5) are ubiquitously expressed in PASMC and MASMC, the observation from this study indicates that a hypoxia-sensitive mechanism essential for inhibiting KCNA5 channel activity is exclusively present in PASMC. The divergent effect of hypoxia on [I.sub.K(v)] in PASMC and MASMC also may be due to different expression levels of KCNA5 channels. membrane potential; potassium channels; vascular smooth muscle

At bitch, the transition to gas breathing requires the function of endothelial vasoactive agents. We investigated the function of endothelial nitric oxide synthase (eNOS) in pulmonary artery (PA) vessels and endothelial cells isolated from fetal and young (4-wk) sheep. We found greater relaxations to the NOS activator A-23187 in 4-wk-old compared with fetal vessels and that the NOS inhibitor nitro-Larginine blocked relaxations in both groups. Relaxations in 4-wk vessels were not blocked by an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, but were partially blocked by catalase. We therefore hypothesized that activation of eNOS produced reactive oxygen species in 4-wk but not fetal PA. To address this question, we studied NO and superoxide production by endothelial cells at baseline and following NOS stimulation with A-23187, VEGF, and laminar shear stress. Stimulation of NOS induced phosphorylation at serine 1177, and this event correlated with an increase in NO production in both ages. Upon stimulation of eNOS, fetal PA endothelial cells (PAEC) produced only NO. In contrast 4-wk-old PAEC produced superoxide in addition to NO. Superoxide production was blocked by L-NAME but not by apocynin (an NADPH oxidase inhibitor). L-Arginine increased NO production in both cell types but did not block superoxide production. Heat shock protein 90/eNOS association increased upon stimulation and did not change with developmental age. Cellular levels of total and reduced biopterin were higher in fetal vs. 4-wk cells. Sepiapterin [a tetrahydrobiopterin ([BH.sub.4]) precursor] increased basal and stimulated NO levels and completely blocked superoxide production. We conclude that the normal function of eNOS becomes uncoupled after birth, leading to a developmental adaptation of the pulmonary vascular system to produce oxygen species other than NO. We speculate this may be related to cellular production and/or maintenance of [BH.sub.4] levels. endothelial nitric oxide synthase; nitric oxide; superoxide; pulmonary artery; development

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]), suggesting that ET-1 increased [Ca.sup.2+]sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% [O.sub.2] for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type [Ca.sup.2+] channel blocker, to isolate the component of contraction that occurred without a change in [[[Ca.sup.2+]].sub.i]. The mean change in tension caused by ET-1 ([10.sup.-8] M) expressed as a percent of the maximum response to KC1 was 184.0 [+ or -] 39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [[[Ca.sup.2+]].sub.i] in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC. Y-27632; HA- 1077

Endogenous nitric oxide donor compounds (S-nitrosothiols) contribute to low vascular tone by both cGMP-dependent and -independent pathways. We have reported that S-nitrosoglutathione (GSNO) inhibits 5-hydroxytryptamine (5-HT)-mediated pulmonary vasoconstriction via a cGMP-independent mechanism likely involving S-nitrosylation of its G protein-coupled receptor (GPCR) system. Because catecholamines, like 5-HT, constrict lung vessels via a GPCR coupled to [G.sub.q], we hypothesized that S-nitrosothiols modify the [[alpha].sub.1]-adrenergic GPCR system to inhibit pulmonary vasoconstriction by receptor agonists, e.g., phenylephrine (PE). Rat pulmonary artery rings were pretreated for 30 min with and without an S-nitrosothiol, either GSNO or S-nitrosocysteine (CSNO), and constricted with sequential concentrations of PE ([10.sup.-8][-10.sup.-6] M). Effective cGMP-dependence was tested in rings pretreated with soluble guanylate cyclase inhibitors {either 1H-[l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ) or LY-83583} or G kinase inhibitor (KT-5823), and a thiol reductant [dithiothreitol (DTT)] was used to test reversibility of S-nitrosylation. Both S-nitrosothiols attenuated the PE dose response. The GSNO effect was not prevented by LY-83583, ODQ, or KT-5823, indicating cGMP independence. GSNO inhibition was reversed by DTT, consistent with S-nitrosylation or other GSNO-mediated cysteine modifications. In CSNO-treated lung protein, the [[alpha].sub.1]-adrenergic receptor was shown to undergo S-nitrosylation in vitro using a biotin switch assay. Studies of [[alpha].sub.1]-adrenergic receptor subtype expression and receptor density by saturation binding with [sup.125]I-HEAT showed that GSNO decreased [[alpha].sub.1]-adrenergic receptor density but did not alter affinity for antagonist or agonist. These data demonstrate a novel cGMP-independent mechanism of reversible [[alpha].sub.1]-adrenergic receptor inhibition by S-nitrosothiols. nitric oxide; S-nitrosylation; G protein-coupled receptor; guanosine 3',5'-cyclic monophosphate

We previously reported association of eNOS with actin increases eNOS activity. In the present study, regulation of activity of eNOS by actin cytoskeleton during endothelial growth was studied. We found eNOS activity in PAEC increased when cells grew from preconfluence to confluence, eNOS activity was much greater in PAEC in higher density than those in lower density, suggesting increase in eNOS activity during cell growth is caused by increase in cell density. Although eNOS protein contents were also increased when endothelial cells grew from preconfluence to confluence, magnitude of increase in eNOS activity was much higher than increase in eNOS protein content, suggesting posttranslational mechanisms played an important role in regulation of eNOS activity during endothelial growth. Confocal fluorescence microscopy revealed eNOS was colocalized with G-actin in preconfluent cells in perinuclear region, with both G-actin in perinuclear area and cortical F-actin in plasma membrane in confluent cells. There was more [3-actin coimmunoprecipirated with eNOS in Triton X-100-soluble fraction in confluent cells in later growth phase and in high density. Decrease in eNOS association with [beta]-actin by silencing [beta]-actin expression using [beta]-actin siRNA causes inhibition of eNOS activity, NO production, and endothelial monolayer wound repair in PAEC. Moreover, PAEC incubation with cytochalasin D and jasplakinolide resulted in increases in eNOS/actin association and in eNOS activity without changes in eNOS protein content. Yeast two-hybrid experiments suggested strong association between eNOS oxygenase domain and [beta]-actin. These results indicate increase in eNOS association with actin is responsible for greater eNOS activity in confluent PAEC. angiogenesis; regeneration; proliferation; endothelium; nitric oxide

The transcription factor nuclear factor of activated T cells (NFAT) resides in the cytoplasm in resting cells and upon stimulation is dephosphorylated, translocates to the nucleus, and becomes transcriptionally active. NFAT is commonly activated by stimulation of receptors coupled to [Ca.sup.2+] mobilization; however, little is known about the regulation of NFAT in pulmonary vascular smooth muscle. The aim of this study was to investigate regulation of NFAT in human and rat intralobar pulmonary artery by two constrictors: phenylephrine (PE) and 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 metabolite formed endogenously in lungs. Immunostaining of smooth muscle cells revealed cytoplasmic localization of NFAT in untreated cells, and PE or 20-HETE induced translocation to the nucleus, with maximal effect at 30 min. Cyclosporin A and FK-506 (both 1 [micro]M) inhibited NFAT translocation, indicating involvement of calcineurin. Moreover, the Rho-kinase blocker Y-27632 prevented translocation. Translocation of NFAT was confirmed by Western blots, with NFAT3 the prominent isoform in pulmonary artery. Constrictors caused calcineurin-sensitive translocation of NFAT to nuclei in intact arteries, demonstrating regulation in native tissue. To investigate a role for [Ca.sup.2+], cells were loaded with fura-2. Whereas PE caused an acute transient rise of [Ca.sup.2+]i, 20-HETE caused a prolonged low amplitude rise of [Ca.sup.2+]i. The involvement of Rho-kinase in PE- and 20-HETE-induced NFAT3 translocation in pulmonary artery suggests a level of control not previously recognized in smooth muscle. Constrictors of the pulmonary vasculature not only cause acute responses but also activate NFAT, which may alter gene expression in pulmonary health and disease. transcription factor; Western blot; Rho kinase; phenylephrine; 20-HETE; nuclear factor of activated T cells

The effects of inhibitors of CaMKII on intracellular [Ca.sup.2+] signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [[[Ca.sup.2+]].sub.i] transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular [Ca.sup.2+] resulted in a dose-dependent increase of [[[Ca.sup.2+]].sub.i] consisting of a rapid and transient [Ca.sup.2+] spike followed by a small sustained plateau phase of elevated [[[Ca.sup.2+]].sub.i]. Exposure to KN-93 in the absence of extracellular [Ca.sup.2+] caused a transient rise of [[[Ca.sup.2+]].sub.i] suggesting that exposure to CaMKII inhibitors directly triggered release of [Ca.sup.2+] from intracellular endoplasmic reticulum (ER) [Ca.sup.2+]stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released [Ca.sup.2+] largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (I[P.sub.3]) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced [Ca.sup.2+] response, suggesting that exposure to KN-93 affects [Ca.sup.2+] release from an I[P.sub.3]-sensitive store. Depletion of [Ca.sup.2+] stores by exposure to ATP or to the ER [Ca.sup.2+] pump inhibitor tapsigargin triggered robust capacitative [Ca.sup.2+] entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates [Ca.sup.2+] handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of [Ca.sup.2+] from intracellular stores and activation of CCE. [Ca.sup.2+]/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry

PTHrP has important roles in lung development and function. Here we determined the vasomotor responses of isolated pulmonary arteries and veins of newborn and adult sheep to PTHrP. In vessels constricted with endothelin-1, PTHrP (PTHrP 1-34) caused greater relaxation of veins than of arteries. In both vessel types, relaxation to the peptide was less in adult than in newborn vessels. In newborn lambs, PTHrP-induced relaxation was not affected by endothelium removal, inhibition of eNOS, or inhibition of adenylyl cyclases by SQ-22536. However, relaxation was attenuated by 4-aminopyridine, inhibitor of voltage-dependent potassium channels, in both arteries and veins, and by charybdotoxin, inhibitor of calcium-activated potassium channels, in veins. When vessels were saturated with 8-BrcAMP (3 x [10.sup.-4] M), to eliminate relaxation mediated by endogenous cAMP, PTHrP-induced relaxation was partially attenuated. In vessels treated with 8-BrcAMP (3 x [10.sup.-4] M), 4-aminopyridine but not charybdotoxin inhibited relaxation induced by PTHrP 1-34 in both arteries and veins. Radioimmunoassay showed that, in the presence of a general phosphodiesterase inhibitor, PTHrP caused a concentration-dependent increase in intracellular cAMP content in arteries and veins, which was largely abolished by SQ-22536. Our results demonstrate that PTHrP is a potent vasodilator of pulmonary vessels, with a greater effect in veins than in arteries. Relaxation induced by the peptide contains both cAMP-dependent and -independent components. In both arteries and veins, voltage-dependent potassium channels mediate the response to PTHrP, at least in part, in a cAMP-independent fashion; and in veins, calcium-activated potassium channels may be stimulated by elevated cAMP levels. vasodilation; cAMP; potassium channel; perinatal lungs

Increased resistance of the small blood vessels within the lungs is associated with pulmonary hypertension and results from a decrease in size induced by the contraction of their smooth muscle cells (SMCs). To study the mechanisms that regulate the contraction of intrapulmonary arteriole SMCs, the contractile and [Ca.sup.2+] responses of the arteriole SMCs to 5-hydroxytrypamine (5-HT) and KC1 were observed with phase-contrast and scanning confocal microscopy in thin lung slices cut from mouse lungs stiffened with agarose and gelatin. 5-HT induced a concentration-dependent contraction of the arterioles. Increasing concentrations of extracellular KC1 induced transient contractions in the SMCs and a reduction in the arteriole luminal size. 5-HT induced oscillations in [[[Ca.sup.2+]].sub.i] within the SMCs, and the frequency of these [Ca.sup.2+] oscillations was dependent on the agonist concentration and correlated with the extent of sustained arteriole contraction. By contrast, KC1 induced [Ca.sup.2+] oscillations that occurred with low frequencies and were preceded by small, localized transient [Ca.sup.2+] events. The 5-HT-induced [Ca.sup.2+] oscillations and contractions occurred in the absence of extracellular [Ca.sup.2+] and were resistant to [Ni.sup.2+] and nifedipine but were abolished by caffeine. KC1-induced [Ca.sup.2+] oscillations and contractions were abolished by the absence of extracellular [Ca.sup.2+] and the presence of [Ni.sup.2+], nifedipine, and caffeine. Arteriole contraction was induced or abolished by a 5-H[T.sub.2]-specific agonist or antagonist, respectively. These results indicate that 5-HT, acting via 5-H[T.sub.2] receptors, induces arteriole contraction by initiating [Ca.sup.2+] oscillations and that KC1 induces contraction via [Ca.sup.2+] transients resulting from the overfilling of internal [Ca.sup.2+] stores. We hypothesize that the magnitude of the sustained intrapulmonary SMC contraction is determined by the frequency of [Ca.sup.2+] oscillations and also by the relaxation rate of the SMC. KEY WORDS: confocal microscopy * pulmonary hypertension * airways * arteriole * serotonin

Hypoxia/reoxygenation-induced changes in endothelial permeability are accompanied by endothelial actin cytoskeletal and adherens junction remodeling, but the mechanisms involved are uncertain. We therefore measured the activities of the Rho GTPases Rac 1, RhoA, and Cdc42 during hypoxia/reoxygenation and correlated them with changes in endothelial permeability, remodeling of the actin cytoskeleton and adherens junctions, and production of ROS. Dominant negative forms of Rho GTPases were introduced into cells by adenoviral gene transfer and transfection, and inhibitors of NADPH oxidase, PI3 kinase, and Rho kinase were used to characterize the signaling pathways involved. In some experiments constitutively activated forms of RhoA and Racl were also used. We show for the first time that hypoxia/reoxygenation-induced changes in endothelial permeability result from coordinated actions of the Rho GTPases Racl and RhoA. Racl and RhoA rapidly respond to changes in oxygen tension, and their activity depends on NADPH oxidase- and PI3 kinase-dependent production of ROS. Racl acts upstream of RhoA, and its transient inhibition by acute hypoxia leads to activation of RhoA followed by stress fiber formation, dispersion of adherens junctions, and increased endothelial permeability. Reoxygenation strongly activates Racl and restores cortical localization of F-actin and VE-cadherin. This effect is a result of Racl-mediated inhibition of RhoA and can be prevented by activators of RhoA, L63RhoA, and lysophosphatidic acid. Cdc42 activation follows the RhoA pattern of activation but has no effect on actin remodeling, junctional integrity, or endothelial permeability. Our results show that Rho GTPases act as mediators coupling cellular redox state to endothelial function. reactive oxygen species; adherens junctions; actin cytoskeleton

Pyrrolizidine alkaloids initiate disease in the lung (pulmonary hypertension), liver (veno-occlusive disease and cirrhosis), and kidneys (afferent arteriolar block and mesangiolysis) by inducing a megalocytotic phenotype in target endothelial and parenchymal cells. A 'hit-and-run' type of exposure to the bioactive pyrrolizidine results, within 2-3 days, in enlarged cells with large nuclei and enlarged Golgi and endoplasmic reticulum, while the cells remain in [G.sub.2]/M block. In the present study, we recapitulated monocrotaline pyrrole (MCTP)-induced megalocytosis in cultures of bovine pulmonary arterial endothelial cells (PAEC), human Hep3B hepatocytes, human type II-like alveolar epithelial cells (A549), and human pulmonary arterial smooth muscle cells (PASMC) and investigated the subcellular mechanism involved. There was an inverse relationship between reduction in caveolin (Cav)-1 levels and stimulation of promitogenic STAT3 and ERK1/2 cell signaling. In megalocytotic PAEC, the Golgi scaffolding protein GM130 was shifted from membranes with heavy density to those with a lighter density. This lighter Golgi fraction was enriched for hypo-oligomeric Cav-1, indicating dysfunctional trafficking of cargo. Immunofluorescence imaging studies confirmed the trapping of Cav-1 in a GM130-positive Golgi compartment. There was an increase in [Ser.sup.25] phosphorylation of GM130 (typically a prelude to Golgi fragmentation and mitosis) and increased association between pGM130, cdc2 kinase, and Cav-l. Nevertheless, megalocytotic MCTP-treated cells showed reduced entry into mitosis upon stimulation with 2-methoxyestradiol (2-ME), reduced 2-ME-induced Golgi fragmentation, and a slowing of Golgi reassembly after nocodazole-induced fragmentation. These data suggest that a disruption of the trafficking and mitosis sensor functions of the Golgi may represent the subcellular mechanism leading to MCTP-induced megalocytosis ('the Golgi blockade hypothesis'). pulmonary hypertension; caveolin-1; pyrrolizidine alkaloids

Because systems controlled by basal NAD(P)H oxidase activity appear to contribute to differences in responses of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to hypoxia, we characterized the Nox oxidases activities present in these vascular segments and how cytosolic NAD(P)H redox systems could be controlling oxidase activity. BPA generated ~60-80% more lucigenin (5 [micro]M) chemiluminescence detectable superoxide than BCA. Apocynin (10 [micro]M), a NAD(P)H oxidase inhibitor, and 6-aminonicotinamide (1 mM), a pentose phosphate inhibitor (PPP), both attenuated (approximately by 50-70%) superoxide detected in BPA and BCA. There was no significant difference in the expression of Nox2 or Nox4 mRNA or protein detected by Western blot analysis. NADPH and NADH increased superoxide in homogenates and isolated microsomal membrane fractions in a manner consistent with BPA and BCA having similar levels of oxidase activity. BPA had 4.2-fold higher levels of NADPH than BCA. The activity and protein levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting PPP enzyme generating cytosolic NADPH, were 1.5-fold higher in BPA than BCA. Thus BPA differ from BCA in that they have higher levels of G6PD activity, NADPH, and superoxide. Because both arteries have similar levels of Nox expression and activity, elevated levels of cytosolic NADPH may contribute to increased superoxide in BPA. glucose-6-phosphate dehydrogenase; lactate; NAD(P)H oxidase; Nox2; Nox4; pentose phosphate pathway.

Vascular NADPH oxidases have been shown to be a major source of reactive oxygen species (ROS). Recent studies have also implicated ROS in the proliferation of vascular smooth muscle cells. However, the components required for activation of the NADPH oxidase complex have not been clearly elucidated. Here we demonstrate that ROS generation in ovine pulmonary arterial smooth muscle cells (PASMCs) requires the activation of Rac1, implicating this protein as an important subunit of the NADPH oxidase complex. Our results, using a geranylgeranyl transferase inhibitor (GGTI-287), demonstrated a dose-dependent inhibition of Racl activity and ROS production. This was associated with an inhibition of PASMC proliferation with an arrest at [G.sub.2]/M. The inhibition of Rac1 by GGTI-287 led us to more specifically target Racl to investigate its role in the generation of ROS and cellular proliferation. To accomplish this, we utilized a dominant negative Rac1 (N17Rac1) and a constitutively active Racl (V12Racl). These two forms of Racl were transiently expressed in PASMCs using adenovirus-mediated gene transfer. N17Rac1 expression resulted in decreased cellular Racl activity, whereas V12Racl infection showed increased activity. Compared with controls, the V12Racl-expressing cells had higher levels of ROS production and increased proliferation, whereas the N17Rac1-expressing cells had decreased ROS generation and proliferation and cell cycle arrest at [G.sub.2]/M. However, the inhibition of cell growth produced by N17Rac1 overexpression could be overcome if cells were co-incubated with the Cu,Zn superoxide dismutase inhibitor DETC. These results indicate the importance of Rac1 in ROS generation and proliferation of vascular smooth muscle cells. vascular reduced nicotinamide adenine dinucleotide phosphate oxidase

An insight into the causes and the ways to prevent pulmonary arterial Hypertension is highlighted. Pulmonary arterial Hypertension comprises a group of clinical and pathological entities with similar features buy a variety of underlying causes.

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% [O.sub.2]) or hypoxic (15% [O.sub.2]) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 [+ or -] 0.5 vs. 26.6 [+ or -] 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane [A.sub.2] mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [p[D.sub.2] (= -log E[C.sub.50]): 6.51 [+ or -] 0.1 vs. 7.05 [+ or -] 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy. pulmonary hypertension; endothelium; cardiac hypertrophy

We previously reported that angiotensin II stimulates an increase in nitric oxide production in pulmonary artery endothelial cells. The aims of this study were to determine which receptor subtype mediates the angiotensin II-dependent increase in nitric oxide production and to investigate the roles of the angiotensin type 1 and type 2 receptors in modulating angiotensin II-dependent vasoconstriction in pulmonary arteries. Pulmonary artery endothelial cells express both angiotensin II type 1 and type 2 receptors as assessed by RT-PCR, Western blot analysis, and flow cytometry. Treatment of the endothelial cells with PD-123319, a type 2 receptor antagonist, prevented the angiotensin II-depcndent increase in nitric oxide synthase mRNA, protein levels, and nitric oxide production. In contrast, the type 1 receptor antagonist losartan enhanced nitric oxide synthase mRNA levels, protein expression, and nitric oxide production. Pretreatment of the endothelial cells with either PD-123319 or an anti-angiotensin II antibody prevented this losartan enhancement of nitric oxide production. Angiotensin II-dependent enhanced hypoxic contractions in pulmonary arteries were blocked by the type 1 receptor antagonist candesartan; however, PD-123319 enhanced hypoxic contractions in angiotensin II-treated endothelium-intact vessels. These data demonstrate that angiotensin II stimulates an increase in nitric oxide synthase mRNA, protein expression, and nitric oxide production via the type 2 receptor, whereas signaling via the type 1 receptor negatively regulates nitric oxide production in the pulmonary endothelium. This endothelial, type 2 receptor-dependent increase in nitric oxide may serve to counterbalance the angiotensin II-dependent vasoconstriction in smooth muscle cells, ultimately regulating pulmonary vascular tone. nitric oxide synthase; angiotensin type 2 receptor; pulmonary endothelium

The reversibility of tissue remodeling is of general interest to medicine. Pulmonary arterial tissue remodeling during hypertension induced by hypoxic breathing is well known, but little has been said about the recovery of the arterial wall when the blood pressure is lowered again. We hypothesize that tissue recovery is a function of the oxygen concentration, blood pressure, location on the vascular tree, and time. We measured the changes of blood pressure, vessel lumen, vessel wall thicknesses, and opening angle of each segment of the blood vessel at its zero-stress state after step changes of the oxygen concentration in the breathing gas. The zero-stress state of each vessel is emphasized because it is important to the analysis of stress and strain and in morphometry. Experimental results are presented as histories of tissue parameters after step changes of the oxygen level. Tissue characteristics are examined under the hypothesis that they are linearly related to changes in the local blood pressure. Under this linearity hypothesis, each aspect of the tissue change can be expressed as a convolution integral of the blood pressure history with a kernel called the indicial response function. It is shown the indicial response function for rising blood pressure is different from that for falling blood pressure. This difference represents a major nonlinearity of the tissue remodeling process of the blood vessels. indicial response function | blood vessel opening angle at zero-stress state | nonlinearity of tissue remodeling

Accumulating evidence suggests that changes in both 5-hydroxytryptamine (5-HT) receptor activity and in the levels of reactive oxygen species (ROS) play an important role in regulating pulmonary artery (PA) vascular responsiveness, particularly in the setting of pulmonary hypertension. Therefore, we hypothesized that increased levels of superoxide enhance 5-HT-induced PA constriction. With the use of a small-vessel bioassay, 5-HT (0.01-10 [micro]M) induced a concentration-dependent vasoconstriction in isolated wild-type murine intrapulmonary arteries (100-150 [micro]m diameter) that was enhanced by both removal of the endothelium and by treatment with either [N.sup.G]-nitro-L-arginine methyl ester (30 [micro]M) or xanthine (10 [micro]M) + xanthine oxidase (0.005 U/ml). PA isolated from extracellular superoxide dismutase (EC-SOD) knockout mice also showed enhanced constriction. On the other hand, PA constriction to 5-HT was attenuated by either the addition of GR-127935 (0.1 [micro]M, a selective inhibitor of 5-[HT.sub.1B1D] receptor) or copper/zinc-containing superoxide dismutase (Cu/Zn SOD, 150 U/ml) and in PA isolated from transgenic mice overexpressing human EC-SOD. With the use of both oxidative fluorescent confocal microscopy and lucigenin-enhanced chemiluminescence, superoxide levels were increased significantly after 5-HT-induced PA vasoconstriction. This increase in superoxide levels could be blocked by the exogenous addition of Cu/Zn SOD (150 U/ml) or by apocynin (30 [micro]M, an inhibitor of NADPH oxidase) but was not affected by [gp91.sup.phox] knockout mice. Overall, our results are consistent with 5-HT increasing vascular smooth muscle superoxide production via an NADPH oxidase pathway that is independent of [gp91.sup.phox], which leads to increases in extracellular superoxide levels, which in turn enhances 5-HT-induced murine pulmonary vasoconstriction. extracellular superoxide; 5-hydroxytryptamine; murine pulmonary artery; reduced nicotinamide adenine dinucleotide

Byline: M. Ruperez (1), J. Lopez-Herce (1), C. Garcia (1), C. Sanchez (1), E. Garcia (1), D. Vigil (2) Keywords: Cardiac output; Swan--Ganz catheter; Femoral arterial thermodilution technique; Pulse contour analysis Abstract: This study compares the correlation between two methods for the determination of cardiac output--the pulmonary arterial thermodilution technique using the Swan--Ganz catheter and the femoral arterial thermodilution technique using a pulse contour analysis computer (PiCCO) catheter. We performed a prospective animal study using 16 immature Maryland pigs weighing 9 to 16 kg. A 5.5- or 7.5-Fr Swan--Ganz catheter was introduced into the femoral or jugular vein, and a 4- or 5-Fr arterial PiCCO catheter was introduced into the femoral artery. In each animal, we made measurements of cardiac output at 30-minute intervals, simultaneously by pulmonary arterial thermodilution and femoral arterial thermodilution, before, during, and after hemodiafiltration carried out via different venous catheters, recording a total of 78 measurements. The mean Swan--Ganz cardiac output was 2.22 +- 0.94 L/min, and mean PiCCO cardiac output was 1.94 +- 0.80 L/min (no significant difference). The mean difference (bias) of differences (limits of agreement) was 0.2812. The differences between the methods increased with higher cardiac output, but the percentage differences in relation to cardiac output remained stable. Good correlation was found between the two methods: single-measure intraclass correlation was 0.8892 (95% confidence interval, 0.54--0.95). There were no differences between the 5.5- and 7.5-FR Swan--Ganz catheters or between the 4- and 5-Fr PiCCO catheters. Femoral arterial thermodilution cardiac output measurements correlated well with pulmonary arterial thermodilution cardiac output measurements in a pediatric animal model. Author Affiliation: (1) Pediatric Intensive Care Unit, Gregorio Maranon University Hospital, Dr. Castelo 49, 28009 Madrid, Spain (2) Preventive and Quality Control Service, Gregorio Maranon University Hospital, Dr. Castelo 49, 28009 Madrid, Spain, Spain Article History: Registration Date: 01/01/2003 Online Date: 23/12/2003

Deten, Alexander, Huntly Millar, and Heinz-Gerd Zimmer. Catheterization of pulmonary artery in rats with an ultraminiature catheter pressure transducer. Am J Physiol Heart Circ Physiol 285: H2212-H2217, 2003. First published July 24, 2003; 10.1152/ajpheart.00315.2003.-Utilizing new materials and miniaturization techniques, an ultraminiature catheter pressure transducer for catheterization of the pulmonary artery (PA) has been developed and applied in intact, spontaneously breathing, anesthetized rats. The catheter arrangement consists of three components: 1) an SPR-671 ultraminiature pressure transducer (measuring catheter), 2) a plastic introducer (sheath) that is slipped over the measuring catheter, and 3) an external wire mounted on the outside of the introducer for bending its tip. The measuring catheter is first inserted through the right jugular vein into the right ventricle. The introducer is then slipped over it. The tip of the introducer is bent so that there is an angle of ~90[degrees] or less to the shaft. The measuring catheter is advanced across the pulmonary valve into the PA. Measurements of pulmonary arterial pressure were made in five male Long Evans (364 [+ or -] 7 g body wt) and five female Sprague-Dawley (244 [+ or -] 7 g body wt) rats under control conditions. The effects of infusion of norepinephrine (0.1 mg * [kg.sup.-1n * [h.sup.-1] iv for 20-rain duration) were tested in Long Evans rats. Pulmonary arterial systolic pressure measurements were 34.0 [+ or -] 0.8 and 29.5 [+ or -] 0.4 mmHg, and diastolic pressure values were 23.6 [+ or -] 0.8 and 18.1 [+ or -]0.6 mmHg in male Long Evans and female Sprague-Dawley rats, respectively. Norepinephrine induced an increase in pulmonary arterial systolic (40.8 [+ or -] 0.1 mmHg) and diastolic (28.6 [+ or -] 0.4 mmHg) pressures and an elevation in pulmonary vascular resistance from a control value of 0.093 [ + or -] 0.003 to 0.103 [+ or -] 0.004 mmHg. kg. rain. [ml.sup.-1] cardiac output; contractility; norepinephrine; right heart

Byline: Michael R. Pinsky (1) Keywords: Bedside measurements Pulmonary hemodynamics Left ventricular performance Critically ill patients Pulmonary artery occlusion pressure Pulmonary vascular status Abstract: Background. Ppao values are routinely used to assess pulmonary vascular status and LV performance. Regrettably, under many common clinically relevant conditions, even when Ppao values are measured accurately, Ppao values at baseline and in response to therapy often reflect an inaccurate measure of cardiovascular status. Results and conclusions. Thus, caution should be used when applying measures of Ppao in determining therapy if changes in RV volume, hyperinflation, or LV diastolic compliance are simultaneously occurring. Author Affiliation: (1) , Division of Coibical Care Medicine, 604 Scaife Hall, 3550 Terrace Street, PA 15261, Pittsburgh Article History: Received Date: 18/11/2002 Accepted Date: 18/11/2002 Article note: Electronic Publication

Byline: D.S. Hogg (), G. McMurray (), R.Z. Kozlowski () Abstract: This study demonstrates for the first time that endothelial cells freshly isolated from small pulmonary arteries of the rat, based on their electrophysiological profile, possess two distinct populations of cells. Immunohistochemical staining revealed the presence of both anti-Kv1.5 and anti-Kir2.1 immunoreactivity in the endothelium of small pulmonary arteries. Patch-clamp studies demonstrated that 90% of cells studied exhibited an electrophysiological profile that was characterized by a delayed rectifier K+ conductance. However, the remaining 10% of cells studied showed the complete absence of a delayed rectifier K+ current and were characterized by an inward rectifier K+ conductance. Together these results indicate that endothelial cells isolated from rat small pulmonary arteries possess a heterogeneous population of cells that may be distinguished by their markedly different electrophysiological profiles. These different populations of cells may differ in their control of the resting membrane potential of endothelial cells, and thereby altering Ca2+ homeostasis and release of vasoactive compounds. These findings may therefore have important implications for understanding the regulation of pulmonary vascular tone. Author Affiliation: () Department of Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 3DG, United Kingdom, GB () University Department of Pharmacology, , Mansfield Road, Oxford OX1 3QT, United Kingdom, GB Article History: Accepted Date: 30/05/2002

Balyakina, Elena V., Daohong Chen, Mayme L. Lawrence, Suzanne Manning, Richard E. Parker, Scott B. Shappell, and Barbara Meyrick. ET-1 receptor gene expression and distribution in L1 and L2 cells from hypertensive sheep pulmonary artery. Am J Physiol Lung Cell Mol Physiol 283: L42-L51, 2002. First published February 8, 2002; 10.1152/ajplung.00337.2001.--We examined gene and surface expression and activity of the endothelin (ET)-1 receptors (E[T.sub.A] and E[T.sub.b]) in subendothelial (L1) and inner medial (L2) cells from the main pulmonary artery of sheep with continuous air embolization (CAE)-induced chronic pulmonary hypertension (CPH). According to quantitative realtime RT-PCR, basal gene expression of both receptors was significantly higher in L2 than L1 cells, and hypertensive L2 cells showed significantly higher gene expression of E[T.sub.B] than controls. Expression of both genes in hypertensive L1 cells was similar to controls. Fluorescence-activated cell sorter analysis confirmed the increased distribution of E[T.sub.B] in hypertensive L2 cells. Although only the ETA receptors in control L2 cells showed significant binding of [[sup.125]I]-labeled ET-1 at i h, both receptors bound ET-1 to hypertensive cells. Exposure to exogenous ET-1 for 18 h revealed that only the L2 cells internalized ET-1, and internalization by hypertensive L2 cells was significantly reduced when compared with controls. Treatment with E[T.sub.A] (BQ-610) and E[T.sub.B] (BQ-788) receptor antagonists demonstrated that both receptors contributed to internalization of ET-1 in control L2 cells, whereas in hypertensive cells only when both receptor antagonists were used in combination was significant suppression of ET-1 internalization found. We conclude that in sheep receiving CAE, alterations in E[T.sub.B] receptors in cells of the L2 layer may contribute to the maintenance of CPH via alterations in their expression, distribution, and activity. E[T.sub.A] receptor; E[T.sub.B] receptor; smooth muscle cells; pulmonary hypertension; endothelin

Byline: A. Vizcaino (), J. Campbell (), S. Litovsky (), R. Van Praagh () Abstract: We report the third known case of origin of the right and left pulmonary artery branches from the ascending aorta via a short common pulmonary artery. A large unbranching main pulmonary artery opened through a patent ductus arteriosus into the descending thoracic aorta. Preductal coarctation of the aorta and multiple congenital anomalies were also present. This rare cardiovascular malformation facilitates a new anatomic and developmental understanding of truncus arteriosus. Author Affiliation: () Department of Cardiology, Hospital Infantil de Mexico, Dr. Marquez No. 162, Mexico DF, Mexico, MX () Departments of Pathology and Cardiology, Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA, US

Byline: R.G. Bennhagen (), G. Holje (), S. Laurin (), E. Pesonen () Abstract: Pulmonary arteriovenous malformation (PAVM), as a part of Osler-Weber-Rendu Syndrome, in the neonate, is a rare hereditary vascular malformation. Large intrapulmonary right-to-left shunting, causing hypoxaemia and cyanosis, can be a life-threatening condition. Repeated transcatheter coil embolization procedures proved to be a favorable strategy to improve systemic arterial oxygen saturation, with a good outcome in a newborn child. While the radiation dose was high, the use of this amount of radiation was felt to be justified and its effects considered tolerable in the treatment of this patient's serious malformation. Author Affiliation: () Division of Paediatric Cardiology, Department of Paediatrics, Lund University Hospital, 221 85 Lund, Sweden, SE () Department of Radiation Physics, Lund University Hospital, 221 85 Lund, Sweden, SE () Department of Radiology, Lund University Hospital, 221 85 Lund, Sweden, SE