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Partial HIF-1[Alpha] deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia. Am J Physiol Lung Cell Mol Physiol 281: L202-L208, 2001.--Chronic hypoxia depolarizes and reduces [K.sup.+] current in pulmonary arterial smooth muscle cells (PASMCs). Our laboratory previously demonstrated that hypoxia-inducible factor-1 (HIF-1) contributed to the development of hypoxic pulmonary hypertension. In this study, electrophysiological parameters were measured in PASMCs isolated from intrapulmonary arteries of mice with one null allele at the Hif1a locus encoding HIF-1[Alpha] [Hif1a(+/-)] and from their wild-type [Hif1a(+/+)] littermates after 3 wk in air or 10% [O.sub.2]. Hematocrit and right ventricular wall and left ventricle plus septum weights were measured. Capacitance, [K.sup.+] current, and membrane potential were measured with whole cell patch clamp. Similar to our laboratory's previous results, hypoxia-induced right ventricular hypertrophy and polycythemia were blunted in Hif1a(+/-) mice. Hypoxia increased PASMC capacitance in Hif1a(+/+) mice but not in Hif1a(+/-) mice. Chronic hypoxia depolarized and reduced K+ current density in PASMCs from Hif1a(+/+) mice. In PASMCs from hypoxic Hif1a(+/-) mice, no reduction in [K.sup.+] current density was observed, and depolarization was significantly blunted. Thus partial deficiency of HIF-1[Alpha] is sufficient to impair hypoxia-induced depolarization, reduction of [K.sup.+] current density, and PASMC hypertrophy. pulmonary hypertension; voltage-gated potassium current; membrane potential; hypoxia-inducible factor-1; pulmonary arterial smooth muscle cell

Hypoxic constriction of porcine distal pulmonary arteries: endothelium and endothelin dependence. Am J Physiol Lung Cell Mol Physiol 280: L856-L865, 2001.--To determine the role of endothelium in hypoxic pulmonary vasoconstriction (HPV), we measured vasomotor responses to hypoxia in isolated seventh-generation porcine pulmonary arteries [is less than] 300 [micro]m in diameter with (E+) and without endothelium. In E+ pulmonary arteries, hypoxia decreased the vascular intraluminal diameter measured at a constant transmural pressure. These constrictions were complete in 30-40 min; maximum at [PO.sub.2] of 2 mmHg; half-maximal at [PO.sub.2] of 40 mmHg; blocked by exposure to [Ca.sup.2+]-free conditions, nifedipine, or ryanodine; and absent in E+ bronchial arteries of similar size. Hypoxic constrictions were unaltered by indomethacin, enhanced by indomethacin plus [N.sup.G]-nitro-L-arginine methyl ester, abolished by BQ-123 or endothelial denudation, and restored in endothelium-denuded pulmonary arteries pretreated with [10.sup.-10] M endothelin-1 (ET-1). Given previous demonstrations that hypoxia caused contractions in isolated pulmonary arterial myocytes and that ET-1 receptor antagonists inhibited HPV in intact animals, our results suggest that full in vivo expression of HPV requires basal release of ET-1 from the endothelium to facilitate mechanisms of hypoxic reactivity in pulmonary arterial smooth muscle. vascular smooth muscle; internal diameter; calcium; acetyl-choline; U-46619; potassium chloride

Shimoda, Larissa A., James S. K. Sham, Tenille H. Shimoda, and J. T. Sylvester. L-type[Ca.sup.2+] channels, resting [[Ca.sup.2+]][sub.i], and ET-1-induced responses in chronically hypoxic pulmonary myocytes. Am J Physiol Lung Cell Mol Physiol 279: L884-L894, 2000.--In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type [Ca.sup.2+] channels contributes to 1) maintenance of resting intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]][sub.i]), 2) increased [[Ca.sup.2+]][sub.i] in response to ET-1 ([10.sup.-8] M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [[Ca.sup.2+]][sub.i] in PASMCs from intrapulmonary arteries of rats exposed to 10% [O.sub.2] for 21 days was 293.9 [+ or -] 25.2 nM (vs. 153.6 [+ or -] 28.7 nM in normoxia). Resting [[Ca.sup.2+]][sub.i] was decreased after extracellular [Ca.sup.2+] removal but not with nifedipine ([10.sup.-6] M), an L-type [Ca.sup.2+] channel antagonist. After CH, the ET-l-induced increase in [[Ca.sup.2+]][sub.i] was reduced and was abolished after extracellular [Ca.sup.2+] removal or nifedipine. Removal of extracellular [Ca.sup.2+] reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [[Ca.sup.2+]][sub.i] in PASMCs from chronically hypoxic rats does not require activation of L-type [Ca.sup.2+] channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [[Ca.sup.2+]][sub.i]. intracellular calcium concentration; endothelin-1; chronic hypoxia; pulmonary hypertension; contraction; voltage-gated calcium channels; smooth muscle; pulmonary artery smooth muscle cells

Sham, James S. K., Benjamin R. Crenshaw, Jr., Li-Hua Deng, Larissa A. Shimoda, and J. T. Sylvester. Effects of hypoxia in porcine pulmonary arterial myocytes: roles of Ky channel and endothelin-1. Am J Physiol Lung Cell Mol Physiol 279: L262-L272, 2000.--Effects of acute hypoxia on intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2 +]].sub.i]) and cell length were recorded simultaneously in proximal and distal pulmonary (PASMCs) and femoral (FASMCs) arterial smooth muscle cells. Reducing [PO.sub.2] from normoxia to severe hypoxia ([PO.sub.2] [is less than] 10 mmHg) caused small but significant decreases in length and a reversible increase in [[[Ca.sup.2+]].sub.i] in distal PASMCs and a small decrease in length in proximal PASMCs but had no effect in FASMCs, even though all three cell types contracted significantly to vasoactive agonists. Inhibition of voltage-dependent [K.sup.+] ([K.sub.v]) channel with 4-aminopyridine produced a greater increase in [[[Ca.sup.2+]].sub.i] in distal than in proximal PASMCs. In distal PASMCs, severe hypoxia caused a slight inhibition of Ky currents; however, it elicited further contraction in the presence of 4-aminopyridine. Endothelin-1 ([10.sup.-10] M), which itself did not alter cell length or [[[Ca.sup.2+]].sub.i], significantly potentiated the hypoxic contraction. These results suggest that hypoxia only has small direct effects on porcine PASMCs. These effects cannot be fully explained by inhibition of [K.sub.v] channels and were greatly enhanced via synergistic interactions with the endothelium-derived factor endothelin-1. vasoconstriction; calcium; voltage-dependent potassium channel; pulmonary arterial smooth muscle cell

Leach, R. M., D. W. Sheehan, V. P. Chacko, and J. T. Sylvester. Energy state, pH, and vasomotor tone during hypoxia in precontracted pulmonary and femoral arteries. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L294-L304, 2000.--To assess effects of smooth muscle energy state and intracellular pH (pHi) on pulmonary arterial tone during hypoxia, we measured ATP, phosphocreatine, [P.sub.i], and [pH.sub.i] by [sup.31] P-NMR spectroscopy and isometric tension in phenylephrinecontracted rings of porcine proximal intrapulmonary arteries. Hypoxia caused early transient contraction followed by relaxation and late sustained contraction. Energy state and [pH.sub.i] decreased during relaxation and recovered toward control values during late contraction. Femoral arterial rings had higher energy state and lower [pH.sub.i] under baseline conditions and did not exhibit late contraction or recovery of energy state and [pH.sub.i] during hypoxia. In pulmonary arteries, glucose-free conditions abolished late hypoxic contraction and recovery of energy state and [pH.sub.i], but endothelial denudation abolished only late hypoxic contraction. NaCN had little effect at 0.1 and 1.0 mM but caused marked vasorelaxation and decreases in energy state and [pH.sub.i] at 10 mM. These results suggest that 1) regulation of tone, energy state, and [pH.sub.i] differed markedly in pulmonary and femoral arterial smooth muscle, 2) hypoxic relaxation was mediated by decreased energy state or [pH.sub.i] due to hypoxic inhibition of oxidative phosphorylation, 3) recovery of energy state and phi in hypoxic pulmonary arteries was due to accelerated glycolysis mediated by mechanisms intrinsic to smooth muscle, and 4) late hypoxic contraction in pulmonary arteries was mediated by endothelial factors that required hypoxic recovery of energy state and [pH.sub.i] for transduction in smooth muscle or extracellular glucose for production and release by endothelium. vascular smooth muscle; endothelium; cyanide; glucose; hypoxic pulmonary vasoconstriction; phosphocreatine; adenosine 5'-triphosphate; phosphate; swine; phosphorus-31 nuclear magnetic resonance

Shimoda, Larissa A., J. T. Sylvester, and James S. K. Sham. Mobilization of intracellular [Ca.sup.2+] by endothelin-1 in rat intrapulmonary arterial smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L157-L164, 200C,.--Endothelin-1 (ET-1) increases intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for [Ca.sup.2+] mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced [Ca.sup.2+] response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 ([10.sup.-10] to [10.sup.-8] M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [[[Ca.sup.2+]].sub.i]. At [10.sup.-8] M, ET-1 caused a large, transient increase in [[[Ca.sup.2+]].sub.i] ([is greater than] 1 [micro]M) followed by a sustained elevation in [[[Ca.sup.2+]].sub.i] ([is less than] 200 [micro]M). The ET-1-induced increase in [[[Ca.sup.2+]].sub.i] was attenuated ([is less than] 80%) by extracellular [Ca.sup.2+] removal; by verapamil, a voltage-gated [Ca.sup.2+]-channel antagonist; and by ryanodine, an inhibitor of [Ca.sup.2+] release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [[[Ca.sup.2+]].sub.i], but the sustained phase was unaffected. Simultaneously measuring membrane potential and [[[Ca.sup.2+]].sub.i] indicated that depolarization preceded the rise in [[[Ca.sup.2+]].sub.i]. These results suggest that ET-1 initiates depolarization in PASMCs, leading to [Ca.sup.2+] influx through voltage-gated [Ca.sup.2+] channels and [Ca.sup.2+] release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores. calcium-induced calcium release; vascular smooth muscle; potassium chloride; L-type calcium channels

Shimoda, Larissa A., J. T. Sylvester, and James S. K. Sham. Chronic hypoxia alters effects of endothelin and angiotensin on [K.sup.+] currents in pulmonary arterial myocytes. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L431-L439, 1999.--We tested the hypothesis that chronic hypoxia alters the regulation of [K.sup.+] channels in intrapulmonary arterial smooth muscle cells (PASMCs). Charybdotoxin-insensitive, 4-aminopyridine-sensitive voltage-gated [K.sup.+] ([K.sub.V,CI]) and [Ca.sup.2+]-activated [K.sup.+] ([K.sub.Ca]) currents were measured in freshly isolated PASMCs from rats exposed to 21 or 10% [O.sub.2] for 17-21 days. In chronically hypoxic PASMCs, [K.sub.V,CI] current was reduced and [K.sub.Ca] current was enhanced. 4-Aminopyridine (10 mM) depolarized both normoxic and chronically hypoxic PASMCs, whereas charybdotoxin (100 nM) had no effect in either group. The inhibitory effect of endothelin (ET)- 1 ([10.sup.-7] M) on [K.sub.V,CI] current was significantly reduced in PASMCs from chronically hypoxic rats, whereas inhibition by angiotensin (ANG) II ([10.sup.-7] M) was enhanced. Neither ET-1 nor ANG II altered [K.sub.Ca] current in normoxic PASMCs; however, both stimulated [K.sub.Ca] current at positive potentials in chronically hypoxic PASMCs. These results suggest that although modulation of [K.sub.V,CI] and [K.sub.Ca] channels by ET-1 and ANG II is altered by chronic hypoxia, the role of these channels in the regulation of resting membrane potential was not changed. voltage-gated potassium current; calcium-activated potassium current; membrane potential

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca2+ concentration ([Ca2+]i) by inducing Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx through store- and voltage-operated Ca2+ channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca2+ release, we measured [Ca2+]i with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP3R), respectively, or 4% O2 caused rapid transient increases in [Ca2+]i, indicating intracellular Ca2+ release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca2+- ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca2+ release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca2+ release to caffeine and hypoxia but not norepinephrine. The IP3R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca2+ release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca2+]i that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca2+]i induced by hypoxia, as well as the subsequent Ca2+ influx that sustained this increase, required release of Ca2+ from both RyR and IP3R, and 2) the SR Ca2+ stores accessed by RyR, IP3R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca2+-ATPase. [ABSTRACT FROM AUTHOR]

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ [Ca2+]i) occurring through activation of voltage-dependent Ca2+ channels (VDCC) even though ET-1-induced depolarization via inhibition of K+ channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ [Ca2+]i in PASMCs from rats exposed to CH (10% O2, 3 wk) using the Ca2+-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ [Ca2+]i by > 70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ [Ca2+]i to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ [Ca2+]i in PASMCs occurs via Ca2+ influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase. [ABSTRACT FROM AUTHOR]

Lu W, Wang J, Peng G, Shimoda LA, Sylvester JT. Knockdown of stromal interaction molecule I attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol 297: Ll7-L25, 2009. First published April 24, 2009; doi: 10.1152/ajplung.00063.2009. Stromal interaction molecule 1 (STIMI) is a recently discovered membrane-spanning protein thought to sense lumenal Ca2+ in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of Car-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER Ca2+ depletion. To evaluate the role of STIMI and a closely related protein, STIM2, in Ca2+ signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the Ca2+-sensitive dye, fura 2, to measure basal intracellular Ca2+ concentration ([Ca2+]i), store-operated Ca2+ entry (SOCE), and increases in [Ca2+]i caused by acute hypoxia (4% O2) or depolarization (60 mmoI/I KCI) in cells treated with small interfering RNA targeted to STIMI (siSTIM1) or STIM2 (siSTIM2). As determined by real-time quantitative PCR analysis (qPCR), STIMI mRNA was 200-fold more abundant than STIM2 in untreated control PASMC. siSTIM I and siSTIM2 caused specific and sigflificant knockdown of both mRNA measured by qPCR and protein measured by Western blotting. siSTIM 1 markedly inhibited SOCE and abolished the sustained [Ca2+]i response to hypoxia but did not alter the initial transient [Ca2+]i response to hypoxia, the [Ca2+]i response to depolarization, or basal [Ca2+]i. The only effect of siSTIM2 was a smaller inhibition of SOCE. We conclude that STIM1 was quantitatively more important than STIM2 in activation of SOC in rat distal PASMC and that the increase in [Ca2+]i induced by acute hypoxia in these cells required SR Ca2+ release and STIMI-dependent activation of SOC. [ABSTRACT FROM AUTHOR]