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Myosin phosphatase target subunit 1 (MYPT1) is the regulatory subunit of myosin light chain phosphatase (MLCP). It plays a critical role in vasodilatation induced by cGMP-elevating agents such as nitric oxide (NO). The present study was performed to determine the role of MYPT1 in the development of tolerance of the pulmonary artery to NO. Incubation of isolated porcine pulmonary arteries for 24 or 48 h with DETA NONOate (DETA NO) significantly reduced protein levels of MYPT1 and the leucine zipper-positive (LZ+) isoform of MYPTI but not that of PPlcS. The extent of reduction in total MYPT1 protein level was comparable to that of MYPTI (LZ+). The decrease in MYPT1 protein caused by 48-h DETA NO incubation was prevented by ODQ, an inhibitor of guanylyl cyclase, and by inhibitors of proteasomes (MG-132 and lactacystin) but was not affected by the inhibitor of protein synthesis, cycloheximide. A reduction in MYPT1 protein was also obtained with 8-bromo-cGMP, but this was prevented by Rp-8bromo-PET-cGMP [inhibitor of cGMP-dependent protein kinase (PKG)]. Incubation for 48 h with DETA NO also reduced dephosphorylation of myosin light chain and relaxation of the artery in response to DETA NO, which was prevented by MG-132. These results suggest that the reduction in MYPT1 protein contributes to the development of tolerance of pulmonary arteries to NO. This may result from increased degradation of MYPT1 after prolonged PKG activation. myosin light chain phosphatase; guanosine 3',5'-cyclic monophosphate-dependent protein kinase; vasodilatation doi: 10.1152/ajplung.00340.2009.

We have shown that 20-hydroxy-eicosatetraenoic acid (20-HETE) increases both superoxide and nitric oxide (NO) production in bovine pulmonary artery endothelial cells (BPAECs). The current study was designed to determine mechanisms underlying 20-HETE-stimulated NO release, and particularly the role of NADPH oxidase, reactive oxygen species, and PI3-kinase in stimulated NO release. Intracellular hydrogen peroxide ([H.sub.2][O.sub.2]) and NO production were detected by dichlorofluorescein or dihydrorhodamine and diaminofluorescein fluorescence, respectively. Activation of endothelial nitric oxide synthase (eNOS) (Serl179) and Akt (Ser473) was assessed by comparing the ratio of phosphorylated to total protein expression by Western blotting. Addition of 20-HETE to BPAECs caused an increase in superoxide and hydrogen peroxide, but not peroxynitrite. 20-HETE-evoked activation of Akt and eNOS, as well as enhanced NO release, are dependent on [H.sub.2][O.sub.2] as opposed to superoxide in that these endpoints are blocked by PEG-catalase and not PEG-superoxide dismutase. Similarly, 20-HETE-stimulated NO production in BPAECs is blocked by NADPH oxidase inhibitors apocynin or gp91 blocking peptide, and by PI3-kinase/Akt blockers wortmannin, LY-294002, or Akt inhibitor, implicating NADPH oxidase, PI3-kinase, and Akt signaling pathways, respectively, in this process. Together, these data suggest the following scheme: 20-HETE stimulates NADPH oxidase-dependent formation of superoxide. Superoxide is rapidly dismutated to hydrogen peroxide, which then mediates activation of PI3-kinase/Akt, phosphorylation of eNOS, and enhanced release of NO from eNOS in response to 20-HETE in BPAECs. doi: 10.1152/ajplung.00298.2009 pulmonary endothelium; superoxide; hydrogen peroxide; endothelial nitric oxide synthase

This study investigated the mechanisms underlying epithelium-derived hyperpolarizing factor (EpDHF)type relaxation in rat bronchioles. Immunohistochemistry was performed, and rat bronchioles and pulmonary arteries were mounted in microvascular myographs for functional studies. An opener of small (S[K.sub.ca]) and intermediate (I[K.sub.ca])-conductance calcium-activated potassium channels, NS309 (6,7-dichloro-IH-indole-2,3-dione 3-oxime) was used to induce EpDHF-type relaxation. I[K.sub.ca] and S[K.sub.ca]3 positive immunoreactions were observed mainly in the epithelium and endothelium of bronchioles and arteries, respectively. In 5-hydroxytryptaanine (1 [micro]M)-contracted bronchioles (828 [+ or -] 20 [micro]m, n = 84) and U46619 (0.03 [micro]M)-contracted arteries (720 [+ or -] 24 [micro]m, n = 68), NS309 (0.001-10 [micro]M) induced concentration-dependent relaxations that were reduced by epithelium/endothelium removal and by blocking I[K.sub.ca] channels with charybdotoxin and in bronchioles also by blocking S[K.sub.ca] channels with apamin. Inhibition of cyclooxygenase, nitric oxide synthase, and cytochrome 2C isoenzymes, or blockade of large (B[K.sub.Ca])-conductance calcium-activated potassium channels with iberiotoxin, failed to reduce NS309 relaxation. In contrast to the pulmonary arteries, relaxations to a [[beta].sub.2]-adrenoceptor agonist, salbutamol, were reduced in bronchioles by removing the epithelium or blocking I[K.sub.Ca] and/or S[K.sub.Ca] channels. Extracellular [K.sup.+] (2-20 mM) induced relaxation in both bronchioles and arteries. An inhibitor of [Na.sup.+]-[K.sup.+]-ATPase, ouabain, abolished relaxations to NS309, salbutamol, and [K.sup.+]. These results suggest that I[K.sub.Ca] and S[K.sub.Ca]3 channels are located in the epithelium of bronchioles and endothelium of pulmonary arteries. Analog to the endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in pulmonary arteries, these channels may be involved in EpDHF-type relaxation of bronchioles caused by epithelial [K.sup.+] efflux followed by activation of [Na.sup.+]-[K.sup.+]-ATPase in the underlying smooth muscle layer. doi: 10.1152/ajplung.0020.2009 NS309; salbutamol; potassium; endothelium; GEA 3175

Oct-4 is a transcription factor considered to be one of the defining pluripotency markers in embryonic stem cells. Its expression has also been demonstrated in adult stem cells, tumorigenic cells, and, most recently and controversially, in somatic cells. Oct-4 pseudogenes also contribute to carcinogenesis. Oct-4 may be involved in the excessive proliferation of pulmonary arterial smooth muscle cells (PASMC) in patients with idiopathic pulmonary arterial hypertension (IPAH), contributing to the pathogenesis of IPAH. In this study, we show that Oct-4 isoforms are upregulated in IPAH-PASMC. Human embryonic stem cells (H9 line) and human PASMC from normotensive subjects were used throughout the investigation as positive and negative controls. In addition to significant upregulation of Oct-4 in a population of IPAH-PASMC, HIF-2[alpha], a hypoxia-inducible transcription factor that has been shown to bind to the Oct-4 promoter and induces its expression and transcriptional activity, was also increased. Interestingly, a substantial upregulation of Oct-4 isoforms and HIF-2[alpha] was also observed in normal PASMC exposed to chronic hypoxia. In conclusion, the data suggest that both Oct-4 isoforms are upregulated and potentially have a significant role in the development of vascular abnormalities associated with the pathogenesis of IPAH and in pulmonary hypertension triggered by chronic hypoxia. doi: 10.1152/ajplung.00314.2009 Oct-4 gene; pulmonary arterial smooth muscle cells; hypoxia

We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a proangiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1-3% [O.sub.2]) resulted in time- and [O.sub.2]-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion. vasa vasorum; hypoxia; angiogenesis; ATP release; phosphatidylinositol 3-kinase; Rho; ROCK doi: 10.1152/ajplung.00038.2009.

A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response of this vessel to acutely applied NO and to the [alpha]-adrenoreceptor agonist phenylephrine. Two-hour treatment with the NO donor (Z)-l-[N-(2-amino-ethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETANO) decreased both NO and phenylephrine responsiveness. Twenty-four-hour treatment with DETA-NO resulted in a further reduction in NO responsiveness but no further reduction in phenylephrine responsiveness. Acute addition of soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) had no effect on phenylephrine responsiveness in PA not treated with DETA-NO. ODQ treatment fully restored phenylephrine responsiveness in PA treated with DETA-NO. sGC[[beta].sub.1] subunit protein levels in PA tissue homogenate were 48.6 [+ or -] 6.9, 51.6 [+ or -] 3.5, and 41.3 [+ or -] 2.8 ng/mg total protein for freshly prepared and 2-h and 24-h NO-treated PA, respectively. Steady-state tissue cGMP was not significantly different in control versus NO-treated PA. sGC specific activity in the absence of added NO was measured in PA homogenate and was 0.29 [+ or -] 0.02, 1.38 [+ or -] 0.12, and 0.53 [+ or -] 0.08 [micro]mol cGMP x [min.sup.-1] x mg [sGC.sup.-1], in freshly prepared and 2-h and 24-h NO treated PA, respectively. Ten-minute Hb treatment completely normalized sGC basal activity in homogenates prepared from DETA-NO-treated PA, which was 0.23 [+ or -] 0.02, 0.18 [+ or -] 0.03, and 0.25 [+ or -] 0.04 [micro]mol cGMP x [min.sup.-1] x mg [sGC.sup.-1], in freshly prepared and 2-h and 24-h NO-treated PA, respectively. The kinetics of the Hb reversal of NO-mediated sGC persistent activation do not support sGC covalent modification as the activation mechanism. We conclude that prolonged NO exposure results in a persistently increased sGC specific activity, which accounts for the observed [alpha]-adrenoreceptor agonist hyporesponsiveness. nitric oxide; guanosine 3',5'-cyclic monophosphate; [alpha]-adrenoreceptor agonist

20-Hydroxyeicosatetraenoic acid (20-HETE) is an endogenous cytochrome P-450 product present in vascular smooth muscle and uniquely located in the vascular endothelium of pulmonary arteries (PAs). 20-HETE enhances reactive oxygen species (ROS) production of bovine PA endothelial cells (BPAECs) in an NADPH oxidase-dependent manner and is postulated to promote angiogenesis via activation of this pathway in systemic vascular beds. We tested the capacity of 20-HETE or a stable analog of this compound, 20-hydroxy-eicosa-5(Z),14(Z)-dienoic acid, to enhance survival and protect against apoptosis in BPAECs stressed with serum starvation. 20-HETE produced a concentration-dependent increase in numbers of starved BPAECs and increased 5-bromo-2'-deoxyuridine incorporation. Caspase-3 activity, nuclear fragmentation studies, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays supported protection from apoptosis and enhanced survival of starved BPAECs treated with a single application of 20-HETE. Protection from apoptosis depended on intact NADPH oxidase, phosphatidylinositol 3 (PI3)-kinase, and ROS production. 20-HETE-stimulated ROS generation by BPAECs was blocked by inhibition of PI3-kinase or Akt activity. These data suggest 20-HETE-associated protection from apoptosis in BPAECs required activation of PI3-kinase and Akt and generation of ROS. 20-HETE also protected against apoptosis in BPAECs stressed by lipopolysaccharide, and in mouse PAs exposed to hypoxia reoxygenation ex vivo. In summary, 20-HETE may afford a survival advantage to BPAECs through activation of prosurvival PI3-kinase and Akt pathways, NADPH oxidase activation, and NADPH oxidase-derived superoxide. reduced nicotinamide adenine dinucleotide phosphatase oxidase; reactive oxygen species; phosphatidylinositol 3-kinase; Akt; hypoxia

Chronic hypoxia during the course of pregnancy is a common insult to the fetus. However, its long-term effect on the pulmonary vasculature in adulthood has not been described. In this study, the vasorelaxation responses of conduit pulmonary arteries in adult female sheep that were chronically hypoxic as fetuses and raised postnatally at sea level were investigated. Vessel tension studies revealed that endothelium-dependent relaxation responses were attenuated in pulmonary arteries from adult sheep that experienced prenatal hypoxia. Endothelial nitric oxide synthase (eNOS) protein expression was unchanged, but eNOS activity was significantly decreased in pulmonary arteries from prenatally hypoxic sheep. Protein expression of eNOS partners, caveolin-1, calmodulin, and heat shock protein 90 (Hsp90) did not change following prenatal hypoxia. However, the association between eNOS and caveolin-1, its inhibitory binding partner, was significantly increased, whereas association between eNOS and its stimulatory partners calmodulin and Hsp90 was greatly decreased. Furthermore, phosphorylation of [Ser.sup.1177] in eNOS decreased, whereas phosphorylation of [Thr.sup.495] increased, in the prenatally hypoxic pulmonary arteries, events that are related to eNOS activity. These data demonstrate that prenatal hypoxia results in persistent abnormalities in endothelium-dependent relaxation responses of pulmonary arteries in adult sheep due to decreased eNOS activity resulting from altered posttranslational regulation. endothelial nitric oxide synthase; caveolin-1; calmodulin; heat shock protein 90

Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-[beta]1 (TGF-[beta]1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-[beta]1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-[beta]1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-[beta]1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-[beta]1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.

Mitochondria are proposed to be a major oxygen sensor in hypoxic pulmonary vasoconstriction (HPV), a unique response of the pulmonary circulation to low oxygen tension. Mitochondrial factors including reactive oxygen species, cytochrome c, ATP, and magnesium are potent modulators of voltage-gated [K.sup.+] ([K.sub.v]) channels in the plasmalemmal membrane of pulmonary arterial (PA) smooth muscle cells (PASMCs). Mitochondria have also been found close to the plasmalemmal membrane in rabbit main PA smooth muscle sections. Therefore, we hypothesized that differences in mitochondria localization in rat PASMCs and systemic mesenteric arterial smooth muscle cells (MASMCs) may contribute to the divergent oxygen sensitivity in the two different circulations. Cellular localization of mitochondria was compared with immunofluorescent labeling, and differences in functional coupling between mitochondria and [K.sub.v] channels was evaluated with the patch-clamp technique and specific mitochondrial inhibitors antimycin A (acting at complex III of the mitochondrial electron transport chain) and oligomycin A (which inhibits the ATP synthase). It was found that mitochondria were located significantly closer to the plasmalemmal membrane in PASMCs compared with MASMCs. Consistent with these findings, the effects of the mitochondrial inhibitors on [K.sub.v] current ([I.sub.Kv]) were significantly more potent in PASMCs than in MASMCs. The cytoskeletal disruptor cytochalasin B (10 [micro]M) also altered mitochondrial distribution in PASMCs and significantly attenuated the effect of antimycin A on the voltage-dependent parameters of [I.sub.Kv]. These findings suggest a greater structural and functional coupling between mitochondria and [K.sub.v] channels specifically in PASMCs, which could contribute to the regulation of PA excitability in HPV. pulmonary artery; vascular smooth muscle cells; mesenteric artery; [K.sup.+] channel activation; [K.sup.+] channel inactivation; confocal imaging; patch-clamp technique

Both endogenous and exogenous estrogen decrease pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-[alpha] or ER-[beta], and whether the contribution of ERs is stimulus-dependent, remains unknown. We hypothesized that administration of the selective ER-[alpha] agonist propylpyrazole triol (PPT) and/or the selective ER-[beta] agonist diarylpropiolnitrile (DPN) rapidly decreases PA vasoconstriction induced by pharmacologic and hypoxic stimuli via a nitric oxide (NO)-dependent mechanism. PA rings (n = 3-10/group) from adult male Sprague-Dawley rats were suspended in physiologic organ baths. Force displacement was measured. Vasoconstrictor responses to phenylephrine ([10.sup.-8]M - [10.sup.-5]M) and hypoxia ([Po.sub.2] 35-45 mmHg) were determined. Endothelium-dependent and -independent vasorelaxation were measured by generating dose-response curves to acetylcholine ([10.sup.-8]M - [10.sup.-4]M) and sodium nitroprusside ([10.sup.-9]M - [10.sup.-5]M). PPT or DPN ([10.sup.-9]M - 5 x [10.sup.-5]M) were added to the organ bath in the presence and absence of the NO-synthase inhibitor [N.sup.[omega]]-nitro-t.-arginine methyl ester (L-NAME) ([10.sup.-4]M). Selective ER-[alpha] activation (PPT, 5 x [10.sup.-5]M) rapidly ( propylpyrazole triol; diarylpropiolnitrile; phenylephrine; hypoxic pulmonary vasoconstriction; nongenomic effects

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.sup.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-argininearginase pathways. Incubation of PAEC with different concentrations of UA (2.5-15 mg/dl) for 24 h did not affect L-[[sup.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)(2boronoethyl)-L-cysteine prevented UA-induced reduction of A23 187stimulated 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. nitric oxide; endothelial dysfunction; pulmonary hypertension

A member of the TNF receptor family, the p75 neurotrophin receptor ([p75.sup.NTR]) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of [p75.sup.NTR] in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of [p75.sup.NTR] in mouse pulmonary arteries and the putative role of [p75.sup.NTR] in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and [p75.sup.NTR] knockout ([p75.sup.-/-]) mice. Our data indicated that [p75.sup.NTR] is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery tings from [p75.sup.-/-] mice exhibited significantly elevated active tension due to endothelin-1-mediated [Ca.sup.2+] influx. Furthermore, the contraction due to capacitative [Ca.sup.2+] entry (CCE) in response to phenylephrine-mediated active depletion of intracellular [Ca.sup.2+] stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and [p75.sup.-/-] rings. Active tension induced by serotonin, U-46619 (a thromboxane [A.sub.2] analog), thrombin, 4-aminopyridine (a [K.sup.+] channel blocker), and high extracellular [K.sup.+] in [p75.sup.-/-] rings was similar to that in WT rings. Deletion of [p75.sup.NTR] did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact [p75.sup.NTR] signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion. mouse; pulmonary artery; pharmacology; vasoconstriction; store depletion

Extracellular matrix remodeling has been proposed as one mechanism by which proximal pulmonary arteries stiffen during pulmonary arterial hypertension (PAH). Although some attention has been paid to the role of collagen and metallomatrix proteins in affecting vascular stiffness, much less work has been performed on changes in elastin structure-function relationships in PAH. Such work is warranted, given the importance of elastin as the structural protein primarily responsible for the passive elastic behavior of these conduit arteries. Here, we study structure-function relationships of fresh arterial tissue and purified arterial elastin from the main, left, and right pulmonary artery branches of normotensive and hypoxia-induced pulmonary hypertensive neonatal calves. PAH resulted in an average 81 and 72% increase in stiffness of fresh and digested tissue, respectively. Increase in stiffness appears most attributable to elevated elastic modulus, which increased 46 and 65%, respectively, for fresh and digested tissue. Comparison between fresh and digested tissues shows that, at 35% strain, a minimum of 48% of the arterial load is carried by elastin, and a minimum of 43% of the change in stiffness of arterial tissue is due to the change in elastin stiffness. Analysis of the stress-strain behavior revealed that PAH causes an increase in the strains associated with the physiological pressure range but had no effect on the strain of transition from elastin-dominant to collagen-dominant behavior. These results indicate that mechanobiological adaptations of the continuum and geometric properties of elastin, in response to PAH, significantly elevate the circumferential stiffness of proximal pulmonary arterial tissue. elastic modulus; stiffness; vascular remodeling

Pulmonary vascular remodeling, a major cause for the elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension (PAH), is partially due to increased proliferation of pulmonary arterial smooth muscle cells (PASMC) in the media, resulting in vascular wall thickening. Platelet-derived growth factor (PDGF) is a potent mitogen that may be involved in the progression of PAH. Blockade of PDGF receptors has been demonstrated to have therapeutic potential for patients with severe pulmonary hypertension. Prednisolone is an immunosuppressant shown to have anti-inflammatory and antiproliferative effects on PASMC. This study was designed to investigate whether PDGF and prednisolone affect human PASMC proliferation by regulating the nuclear translocation of NF-[kappa]B (a transcription factor composed of 2 subunits, p50 and p65). Treatment of human PASMC with PDGF (10 ng/ml) significantly increased nuclear translocation of p50 and p65 subunits. Inhibition of NF-[kappa]B activation or nuclear translocation of p50/p65 significantly attenuated PDGF-induced PASMC proliferation (determined by [[sup.3]H]thymidine incorporation). In the presence of prednisolone (200 [micro]M), the PDGF-induced nuclear translocation of p50 and p65 subunits was markedly inhibited (P < 0.05 vs. the cells treated with PDGF alone). These results indicate that PDGF-induced nuclear translocation of NF-[kappa]B may play an important role in stimulating PASMC proliferation (and/or enhancing PASMC survival), whereas prednisolone may exert anti-inflammatory and antiproliferative effects on PASMC by inhibiting NF-[kappa]B nuclear translocation. proliferation; pulmonary arterial hypertension; immunosuppressant; therapy

We have previously reported the participation of mitogen-activated protein, Rho, and phosphoinositide-3 (PI3) kinases in separate pathways in serotonin (5-HT)-induced proliferation of pulmonary artery smooth muscle cells (SMCs). In this study, we investigated the possible participation of phospholipase D (PLD) and phosphatidic acid (PA) in this growth process. 5-HT stimulated a time-dependent increase in [[sup.3]H]phosphatidylbutanol and PA generation. Exposure of SMCs to 1-butanol or overexpression of an inactive mutant of human PLD1R898R blocked 5-HT-induced proliferation. Furthermore, 1-butanol inhibited 5-HT activation of S6K1 and S6 protein, downstream effectors of mammalian target of rapamycin (mTOR), by 80 and 72%, respectively, and partially blocked activation of extracellular signal-regulated kinase (ERK) by 30% but had no effect on other associated signaling pathways. Exogenous PA caused cellular proliferation and revitalized cyclin D1 expression by 5-HT of the 1-butanol-treated cells. PA also reproduced activations by 5-HT of mTOR, S6K1, and ERK. Transfection with inactive human PLD1 reduced 5-HT-induced activation of S6K1 by ~50%. Inhibition of 5-HT receptor 2A (R 2A) with ketaserin blocked PLD activation by 5-HT. Inhibition with PI3-kinase inhibitor failed to block either activation of PLD by 5-HT or PA-dependent S6K1 phosphorylation. Taken together, these results indicate that ligation of the 5-HTR 2A by 5-HT initiates PLD activation in SMCs, and that its product, PA, is an early signaling molecule in 5-HT-induced pulmonary artery SMC proliferation. Signaling by PA produces its downstream effects primarily through the mTOR/S6K1 pathway and to a lesser extent through the ERK pathway. Hydrolysis of cell membrane lipid may be important in vascular effects of 5-HT. pulmonary artery smooth muscle cell proliferation; serotonin; phospholipase D; phosphatidic acid; cell signal transduction

Exercise elevates shear stress in the supplying conduit artery. Although this is the most relevant physiological stimulus for flow-mediated dilation (FMD), the fluctuating pattern of shear that occurs may influence the shear stress-FMD stimulus response relationship. This study tested the hypothesis that the brachial artery FMD response to a step increase in shear is influenced by the fluctuating characteristics of the stimulus, as evoked by forearm exercise. In 16 healthy subjects, we examined FMD responses to step increases in shear rate in three conditions: stable shear upstream of heat-induced forearm vasodilation ([FH.sub.Stable]); fluctuating shear upstream of heat-induced forearm vasodilation and rhythmic forearm cuff inflation/deflation ([FH.sub.Fluctuating]); and fluctuating shear upstream of exercise-induced forearm vasodilation ([FE.sub.step Increase]). The mean increase in shear rate ([+ or -]SD) was the same in all trials ([FH.sub.Fluctuating]: 51.69 [+ or -] [15.70 s -1]; [FH.sub.Stable]: 52.16 [+ or -] 14.10 [s.sup.-1]; [FE.sub.Step Increase]: 50.14 [+ or -] [13.03.sup.s-1] P = 0.13l). However, the [FH.sub.Fluctuating] and [FE.sub.Step Increase] trials resulted in a fluctuating shear stress stimulus with rhythmic high and low shear periods that were 96.18 [+ or -] 24.54 and 11.80 [+ or -] [7.30s.sup.-1], respectively. The initial phase of FMD (phase I) was followed by a second, delayed-onset FMD and was not different between conditions (phase I: [FH.sub.Fluctuating]: 5.63 [+ or -] 2.15%; [FH.sub.stable]: 5.33 [+ or -] 1.85%; [FE.sub.Step Increase]: 5.30 [+ or -] 2.03%; end-trial: [FH.sub.Fluctuating]: 7.76 [+ or -] 3.40%; [FH.sub.Stable]: 7.00 [+ or -] 3.03%; [FE.sub.Step Increase]: 6.68 [+ or -] 3.04%; P = 0.196). Phase I speed also did not differ (P = 0.685). In conclusion, the endothelium transduced the mean shear when exposed to shear fluctuations created by a typical handgrip protocol. Muscle activation did not alter the FMD response. Forearm exercise may provide a viable technique to investigate brachial artery FMD in humans. Doppler ultrasound; mechanotransduction; forearm exercise; blood flow

Small pulmonary arteries (SPA), 800 [micro]m diameter, show little or no response. It is unknown why different contractile responses occur within the same vascular bed, but activator or repressor proteins within the smooth muscle cell (SMC) can modify myosin phosphatase and myosin light chain kinase (MLCK), thereby influencing the phosphorylation state of myosin light chain (MLC) and ultimately, contraction. Telokin, a protein with a sequence identical to the COOH-terminal domain of MLCK, is expressed in smooth muscle where in its phosphorylated state it inhibits myosin phosphatase, binds to unphosphorylated myosin, and helps maintain smooth muscle relaxation. We measured telokin mRNA and telokin protein in smooth muscle from different diameter feline pulmonary arteries and sought to determine whether changes in the phosphorylation status of telokin and MLC occurred during hypoxia. In pulmonary arteries, telokin expression varied inversely with artery diameter, but cerebral arteries showed neither telokin protein nor telokin mRNA. Although telokin and MLC were distributed uniformly throughout the SPA muscle cell cytoplasm, they were not colocalized. During hypoxia, telokin dephosphorylated, and MLC became increasingly phosphorylated in SPA SMC, whereas in LPA SMC there was no change in either telokin or MLC phosphorylation. When LPA SMC were exposed to phenylephrine, MLC phosphorylation increased with no change in telokin phosphorylation. These results suggest that in SPA, phosphorylated telokin may help maintain relaxation under unstimulated conditions, whereas in LPA, telokin's function remains undetermined. kinase-related protein; cerebral artery

Monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) in rats is preceded by an inflammatory response, progressive endothelial cell membrane disruption, reduction in the expression of caveolin-1, and reciprocal activation of STAT3 (PY-STAT3). Superoxide and NF-KB have been implicated in PAH. To evaluate the role of caveolin-1, PY-STAT3 activation, and superoxide in PAH, MCT-injected rats were treated daily with pyrrolidine dithiocarbamate (PDTC; starting on days 1, 3, and 14 x 2 wk), an inhibitor of inflammation and NF-[kappa]B activation. Hemodynamic data, the expression of inhibitory (I)-[kappa]B[alpha], caveolin-1, and Tie2 (a membrane protein), activation of PY-STAT3 and NF-[kappa]B, and superoxide chemiluminescence were examined. Rats developed progressive PAH at 2 wk post-MCT. There was progressive reduction in the expression of caveolin-1, Tie2, and activation of PY-STAT3 in the lungs. Reduction in I-[kappa]B[alpha] expression was present at 2 and 4 wk post-MCT. Superoxide chemiluminescence and NF-[kappa]B activation were observed only at 2 wk post-MCT and both decreased by 4 wk post-MCT despite progressive PAH. PDTC (starting on days 1 and 3) rescued caveolin-1 and Tie2, reversed MCT-induced PYSTAT3 activation, and attenuated PAH. In addition, PDTC restored I-[kappa]B[alpha] expression and reduced superoxide chemiluminescence at 2 wk but did not inhibit NF-[kappa]B activation despite attenuation of PAH. PDTC had no effect on established PAH. Increased superoxide chemiluminescence and NF-[kappa]B activation appear to be a transient phenomenon in the MCT model. Thus the disruption of endothelial cell membrane integrity resulting in caveolin-I loss and reciprocal activation of PY-STAT3 plays a key role in the MCT-induced PAH. caveolin-l; inhibitory [kappa]B[alpha]; inflammation; PY-STAT3; Tie2

Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states. peroxynitrite; oxidative stress; mitochondria; protein-protein interactions

We assessed the roles of the protein kinase C (PKC) and the tyrosine kinase (TK) signaling pathways in regulating capacitative calcium entry (CCE) in human pulmonary artery smooth muscle cells (PASMCs) and investigated the effects of intravenous anesthetics (midazolam, propofol, thiopental, ketamine, etomidate, morphine, and fentanyl) on CCE in human PASMCs. Fura-2-loaded human PASMCs were placed in a dish (37[degrees]C) on an inverted fluorescence microscope. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) was measured as the 340/380 fluorescence ratio in individual PASMCs. Thapsigargin, a sarcoplasmic reticulum [Ca.sup.2+]-adenosine triphosphatase inhibitor, was used to deplete intracellular [Ca.sup.2+] stores after removing extracellular [Ca.sup.2+]. CCE was then activated by restoring extracellular [Ca.sup.2+] (2.2 mM). The effects of PKC activation and inhibition, TK inhibition, and the intravenous anesthetics on CCE were assessed. Thapsigargin caused a transient increase in [[[Ca.sup.2+]].sub.i]. Restoring extracellular [Ca.sup.2+] caused a rapid peak increase in [[[Ca.sup.2+]].sub.i], followed by a sustained increase in [[[Ca.sup.2+]].sub.i]; i.e., CCE was stimulated in human PASMCs. PKC activation attenuated (P < 0.05), whereas PKC inhibition potentiated (P < 0.05), both peak and sustained CCE. TK inhibition attenuated (P < 0.05) both peak and sustained CCE. Midazolam, propofol, and thiopental each attenuated (P < 0.05) both peak and sustained CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. Our results suggest that CCE in human PASMCs is influenced by both the TK and PKC signaling pathways. Midazolam, propofol, and thiopental each attenuated CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. protein kinase C; tyrosine kinase

Reactive oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Racl, and a peptide-based inhibitor of NADPH oxidase subunit [p47.sup.phox] association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac 1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of [p47.sup.phox] and tyrosine phosphorylation of [p47.sup.phox] in a time-dependent manner as well as increased activated Racl/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid. superoxide; Rac1/2; hydrogen peroxide; CYP4A; reactive oxygen species

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 ([Cavl.sup.-/-]) 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 [Cavl.sup.-/-] mice than wild-type (WT), and increased PVR in [Cavl.sup.-/-] mice was attributed to precapillary sites. Treatment with [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) to inhibit NOS activity raised PVR by 42% in WT but 82% in [Cavl.sup.-/-] mice, indicating greater NO-mediated pulmonary vasodilation in [Cavl.sup.-/-] mice compared with WT. Pulmonary vasculature of [Cavl.sup.-/-] 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 [Cavl.sup.-/-] mice compared with WT suggestive of vascular remodeling. Fluorescent agarose casting also showed markedly decreased density of pulmonary arteries and artery filling defects in [Cavl.sup.-/-] 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 [Cavl.sup.-/-] mice compensates partly for the vascular structural abnormalities by promoting pulmonary vasodilation. nitric oxide; pulmonary hypertension; fluorescent angiography; corrosion casting

The aim of the present study was to provide a mechanistic insight into how 20-hydroxyeicosatetraenoic acid (20-HETE) relaxes distal human pulmonary arteries (HPAs). This compound is produced by to-hydroxylase from free arachidonic acid. Tension measurements, performed on either fresh or 1 day-cultured pulmonary arteries, revealed that the contractile responses to 1 [micro]M 5-hydroxytryptamine were largely relaxed by 20-HETE in a concentration-dependent manner (0.01-10 [micro]M). Iberiotoxin pretreatments (10 nM) partially decreased 20-HETE-induced relaxations. However, 10 [micro]M indomethacin and 3 [micro]M SC-560 pretreatments significantly reduced the relaxations to 20-HETE in these tissues. The relaxing responses induced by the eicosanoid were likely related to a reduced [Ca.sup.2+] sensitivity of the myofilaments since free [Ca.sup.2+] concentration ([[Ca.sup.2+]])-response curves performed on [beta]-escin-permeabilized cultured explants were shifted toward higher [[Ca.sup.2+]]. 20-HETE also abolished the tonic responses induced by phorbol-ester-dibutyrate (a PKC-sensitizing agent). Western blot analyses, using two specific primary antibodies against the PKC-potentiated inhibitory protein CPI-17 and its PKC-dependent phosphorylated isoform pCPI- 17, confirmed that 20-HETE interferes with this intracellular process. We also investigated the effect of 20-HETE on the activation of Rho-kinase pathway-induced [Ca.sup.2+] sensitivity. The data demonstrated that 20-HETE decreased U46619-induced [Ca.sup.2+] sensitivity on arteries. Hence, this observation was correlated with an increased staining of [p116.sup.Rip], a RhoA-binding protein. Together, these results strongly suggest that the 20-hydroxyarachidonic acid derivative is a potent modulator of tone in HPAs in vitro. 20-hydroxyeicosatetraenoic acid; calcium sensitivity; tension measurement

This study examines in endothelium-denuded bovine pulmonary arteries the effects of increasing heme oxygenase-I (HO-1) activity on relaxation and soluble guanylate cyclase (sGC) activation by nitric oxide (NO). A 24-h organ culture with 0.1 mM cobalt chloride (COC12) or 30 [micro]M Co-protoporphyrin IX was developed as a method of increasing HO-1 expression. These treatments increased HO-1 expression and HO activity by approximately two- to fourfold and lowered heme levels by 40-45%. Induction of HO-1 was associated with an attenuation of pulmonary arterial relaxation to the NO-donor spermine-NONOate. The presence of a HO-1 inhibitor 30 [micro]M chromium mesoporphyrin during the 24-h organ culture (but not acute treatment with this agent) reversed the attenuation of relaxation to NO seen in arteries co-cultured with agents that increased HO-1. Relaxation to isoproterenol, which is thought to be mediated through cAMP, was not altered in arteries with increased HO-1. Inducers of HO-1 did not appear to alter basal sGC activity in arterial homogenates or expression of the [[beta].sub.1]-subunit of sGC. However, the increase in activity seen in the presence of 1 [micro]M spermine-NONOate was attenuated in homogenates obtained from arteries with increased HO-1. Since arteries with increased HO-I had decreased levels of superoxide detected by the chemiluminescence of 5 [micro]M lucigenin, superoxide did not appear to be mediating the attenuation of relaxation to NO. These data suggest that increasing HO-1 activity depletes heme, and this is associated with an attenuation of pulmonary artery relaxation and sGC activation responses to NO. cGMP; cobalt protoporphyrin; chromium mesoporphyrin; superoxide

Prolonged exposure to decreased oxygen tension causes contraction and proliferation of pulmonary arterial smooth muscle cells (PASMCs) and pulmonary hypertension. Hypoxia-induced inhibition of voltage-gated [K.sup.+] ([K.sub.v]) channels may contribute to the development of pulmonary hypertension by increasing intracellular calcium concentration ([[Ca.sup.2+]][sub.i]). The peptide endothelin-1 (ET-1) has been implicated in the development of pulmonary hypertension and acutely decreases [K.sub.v] channel activity. ET-1 also activates several transcription factors, although whether ET-1 alters [K.sub.v] channel expression is unclear. The hypoxic induction of ET-1 is regulated by the transcription factor hypoxia-inducible factor-1 (HIF1), which we demonstrated to regulate hypoxia-induced decreases in [K.sub.v] channel activity. In this study, we tested the hypothesis that HIF-l-dependent increases in ET-1 lead to decreased [K.sub.v] channel expression and subsequent elevation in [[Ca.sup.2+]][sub.i]. Resting [[Ca.sup.2+]][sub.i] and [K.sub.v] channel expression were measured in cells exposed to control (18% 02, 5% C[O.sub.2]) and hypoxic (4% [O.sub.2], 5% C[O.sub.2]) conditions. Hypoxia caused a decrease in expression of [K.sub.v]l.5 and [K.sub.v]2.1 and a significant increase in resting [[Ca.sup.2+]][sub.i]. The increase in [[Ca.sup.2+]][sub.i] was reduced by nifedipine, an inhibitor of voltage-dependent calcium channels, and removal of extracellular calcium. Treatment with BQ123, an ET-1 receptor inhibitor, prevented the hypoxia-induced decrease in [K.sub.v] channel expression and blunted the hypoxia-induced increase in [[Ca.sup.2+]][sub.i] in PASMCs, whereas ET-1 mimicked the effects of hypoxia. Both hypoxia and overexpression of HIF-1 under normoxic conditions increased ET-1 expression. These results suggest that the inhibition of [K.sub.v] channel expression and rise in [[Ca.sup.2+]][sub.i] during chronic hypoxia may be the result of HIF-1-dependent induction of ET-1. hypoxia-inducible factor-l; pulmonary hypertension

S100A4/Mts-overexpressing mice have thick elastic laminae and mild pulmonary arterial hypertension (PAH), and the occasional older mouse develops occlusive neointimal lesions and perivascular inflammation. We hypothesized that a vasculotropic virus could induce neointimal lesions in the SI00A4/Mts1 mouse by facilitating breakdown of elastin and migration and proliferation of smooth muscle cells. To test this hypothesis, we infected SI00A4/Mtsl mice with gammaherpesvirus 68 ([gamma]HV68). We observed, 6 mo after [gamma]HV68 [4 x [l0.sup.3] plaque-forming units (PFU)], perivascular inflammation in 10/15 S 100A4/Mts I mice and occlusive neointimal formation in 3/10 mice, accompanied by striking degradation of elastin. We then compared the early response after high-dose [gamma]HV68 (4 x [10.sup.6] PFU) in C57B1/6 and SI00A4/Mtsl mice. In S100A4/Mtsl mice only, significant PAH, muscularization of distal vessels, and elastase activity were observed 6 wk after [gamma]HV68. These features resolved by 3 mo without neointimal formation. We therefore infected mice with the M1-[gamma]/HV68 strain that reactivates from latency with higher efficiency and observed neointimal lesions at 3 mo in 2/5 C57B1/6 (5-9% of vessels) and in 5/5 S100A4/Mtsl mice (13-40% of vessels) accompanied by mild PAH, heightened lung elastase activity, and intravascular viral expression. This suggested that enhanced generation of elastin peptides in S100A4/Mtsl mice may promote increased viral entry in the vessel wall. Using SI00A4/Mtsl PA organ culture, we showed, in response to elastase activity, heightened production of elastin peptides associated with invasion of inflammatory cells and intravascular viral antigen. We therefore propose that early viral access to the vessel wall may be a critical determinant of the extent of vascular pathology following reactivation. pulmonary hypertension; [gamma] herpes virus; pulmonary vascular occlusive disease

We have previously demonstrated increased fibroblast growth factor-2 (FGF-2) expression in a lamb model of increased pulmonary blood flow secondary to congenital heart disease, which may contribute to the associated increases in pulmonary arterial muscularization. However, the mechanisms underlying these increases in FGF-2 expression remain to be identified. Initially, we found that exogenous FGF-2 increased endogenous FGF-2 promoter activity and protein levels in ovine pulmonary arterial smooth muscle cells (PASMC). Furthermore, we found that these increases in FGF-2 expression were mediated by increases in superoxide levels via NADPH oxidase activation. In addition, FGF-2-mediated increases in FGF-2 expression and PASMC proliferation were attenuated by inhibition of phosphatidylinositol 3-kinase, Akt, and NADPH oxidase. Increases in FGF-2 expression could be stimulated by other factors known to increase reactive oxygen species (ROS) signaling in PASMC (endothelin-1 and transforming growth factor-[beta]1), whereas antioxidants attenuated these increases. Deletion constructs localized the growth factor- and ROS-sensitive region within the proximal 103 bp of the FGF-2 promoter, and sequence analysis identified a putative hypoxia response element (HRE), a DNA binding site for the ROS-sensitive transcription factor hypoxia-inducible factor-1[alpha] (HIF-1[alpha]). Stabilization of HIF-1[alpha] increased FGF-2 promoter activity, whereas mutation of the putative HRE attenuated FGF-2-induced FGF-2 promoter activity. Furthermore, FGF-2 increased HIF-1[alpha] protein levels and consensus HRE promoter activity in PASMC via antioxidant-sensitive mechanisms. Thus we conclude that FGF-2 can stimulate its own expression in PASMC via NADPH oxidase-mediated activation of ROS-sensitive transcription factors, including HIF-1[alpha]. This positive feedback mechanism may contribute to pulmonary vascular remodeling associated with increased pulmonary blood flow. cell signaling; proliferation; pulmonary hypertension

Previous studies have shown that, in acutely dispersed canine pulmonary artery smooth muscle cells (PASMCs), depletion of both functionally independent inositol 1,4,5-trisphosphate ([IP.sub.3])- and ryanodine-sensitive [Ca.sup.2+] stores activates capacitative [Ca.sup.2+] entry (CCE). The present study aimed to determine if cell culture modifies intracellular [Ca.sup.2+] stores and alters [Ca.sup.2+] entry pathways caused by store depletion and hypoxia in canine PASMCs. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) was measured in fura 2-loaded cells. [Mn.sup.2+] quench of fura 2 signal was performed to study divalent cation entry, and the effects of hypoxia were examined under oxygen tension of 15-18 mmHg. In acutely isolated PASMCs, depletion of [IP.sub.3]-sensitive [Ca.sup.2+] stores with cyclopiazonic acid (CPA) did not affect initial caffeine-induced intracellular [Ca.sup.2+] transients but abolished 5-HT-induced [Ca.sup.2+] transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced [Ca.sup.2+] transients in cultured PASMCs. In cultured PASMCs, store depletion or hypoxia caused a transient followed by a sustained rise in [[[Ca.sup.2+]].sub.i]. The transient rise in [[[Ca.sup.2+]].sub.i] was partially inhibited by nifedipine, whereas the nifedipine-insensitive transient rise in [[[Ca.sup.2+]].sub.i] was inhibited by KB-R7943, a selective inhibitor of reverse mode [Na.sup.+]/[Ca.sup.2+] exchanger (NCX). The nifedipine-insensitive sustained rise in [[[Ca.sup.2+]].sub.i] was inhibited by SKF-96365, [Ni.sup.2+], [La.sup.3+], and [Gd.sup.3+]. In addition, store depletion or hypoxia increased the rate of [Mn.sup.2+] quench of fura 2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. We conclude that cell culture of canine PASMCs reorganizes [IP.sub.3] and ryanodine receptors into a common intracefiular [Ca.sup.2+] compartment, and depletion of this store or hypoxia activates voltage-operated [Ca.sup.2+] entry, reverse mode NCX, and CCE. capacitative calcium entry; hypoxia; cultured pulmonary artery smooth muscle cells

Endothelin-1 (ET) induces increases in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]), [Ca.sup.2+] sensitization, and contraction of both bronchiole and pulmonary arteriole smooth muscle cells (SMCs) and may play an important role in the pathophysiology of asthma and pulmonary hypertension. However, because it remains unclear how changes in [[[Ca.sup.2+]].sup.i] and the [Ca.sup.2+] sensitivity regulate SMC contraction, we have studied mouse lung slices with phase-contrast and confocal microscopy to correlate the ET-induced contraction with the changes in [[[Ca.sup.2+]].sup.i] and [Ca.sup.2+] sensitivity of bronchiole and arteriole SMCs. In comparison with acetylcholine (ACh) or serotonin (5-HT), ET induced a stronger and long-lasting contraction of both bronchioles and arterioles. This ET-induced contraction was associated with prominent asynchronous [Ca.sup.2+] oscillations that were propagated as [Ca.sup.2+] waves along the SMCs. These [Ca.sup.2+] oscillations were mediated by cyclic intracellular [Ca.sup.2+] release and required external [Ca.sup.2+] for their maintenance. Importantly, as the frequency of the [Ca.sup.2+] oscillations increased, the extent of contraction increased. ET-induced contraction was also associated with an increase in [Ca.sup.2+] sensitivity. In 'model' slices in which the [[[Ca.sup.2+]].sub.i] was constantly maintained at an elevated level by pretreatment of slices with caffeine and ryanodine, the addition of ET increased bronchiole and arteriole contraction. These results indicate that ET-induced contraction of bronchiole and arteriole SMCs is regulated by the frequency of [Ca.sup.2+] oscillations and by increasing the sensitivity of the contractile machinery to [Ca.sup.2+]. confocal microscopy; blood vessels; airways; lung slices; acetylcholine; serotonin

It has been proposed that a hypoxia-induced inhibition of the [Na.sup.+]-[Ca.sup.2+] exchanger (NCX) contributes to hypoxic pulmonary vasoconstriction (HPV). By recording isometric tension development in rat intrapulmonary arteries (IPA), we examined the effect on HPV of maneuvers that reduce the ability of NCX to regulate intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]. In some experiments, fura pentakis(acetoxymethyl) ester-3 (fura PE-3) was also used to monitor [[[Ca.sup.2+]].sub.i]. HPV was elicited in IPA that were pretreated with l0 [micro]M diltiazem and slightly preconstricted with [PGF.sub.2[alpha]], which enhances the hypoxic response. Substitution of [Na.sup.+] with [Li.sup.+] increased HPV and the associated rise in [[[Ca.sup.2+]].sub.i]. Pretreatment with ouabain (100 [micro]M) to diminish the [Na.sup.+] gradient or with the reverse-mode NCX inhibitor KB-R7943 (3 or l0 [micro]M) had no significant effect on HPV. Combined treatment with ouabain and low-[[Na.sup.+]] (24 mM) solution enhanced HPV strongly. The role of NCX in [Ca.sup.2+] extrusion was examined by assessing the decrease in [[[Ca.sup.2+]].sub.i] in [Ca.sup.2+]-free physiological saline solution either containing or lacking [Na.sup.+] following a high [K.sup.+]-induced loading of cellular [[Ca.sup.2+]]. Although the large initial rapid fall in [[Ca.sup.2+]] was [Na.sup.+] independent, final recovery of [[Ca.sup.2+]] to its basal level was delayed in the absence of [Na.sup.+]. Therefore, HPV persisted or was increased under conditions in which forward-mode NCX was already attenuated or prevented, demonstrating that inhibition of NCX by hypoxia is unlikely to initiate HPV. Instead, NCX appears to act to inhibit HPV as would be expected if it is functioning to extrude [Ca.sup.2+]. hypoxia; pulmonary artery