Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia

Recent evidence supports a prominent role for Rho kinase (ROK)-mediated pulmonary vasoconstriction in the development and maintenance of chronic hypoxia (CH)-induced pulmonary hypertension. Endothelin (ET)-1 contributes to the pulmonary hypertensive response to CH, and recent studies by our laboratory and others indicate that pulmonary vascular reactivity following CH is largely independent of changes in vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca(2+)](i)). In addition, CH increases generation of reactive oxygen species (ROS) in pulmonary arteries, which may underlie the shift toward ROK-dependent Ca(2+) sensitization. Therefore, we hypothesized that ROS-dependent RhoA/ROK signaling mediates ET-1-induced Ca(2+) sensitization in pulmonary VSM following CH. To test this hypothesis, we determined the effect of pharmacological inhibitors of ROK, myosin light chain kinase (MLCK), tyrosine kinase (TK), and PKC on ET-1-induced vasoconstriction in endothelium-denuded, Ca(2+)-permeabilized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Further experiments examined ET-1-mediated, ROK-dependent phosphorylation of the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1. Finally, we measured ET-1-induced ROS generation in dihydroethidium-loaded small pulmonary arteries and investigated the role of ROS in mediating ET-1-induced, RhoA/ROK-dependent Ca(2+) sensitization using the superoxide anion scavenger, tiron. We found that CH increases ET-1-induced Ca(2+) sensitization that is sensitive to inhibition of ROK and MLCK, but not PKC or TK, and correlates with ROK-dependent MYPT1(Thr696) phosphorylation. Furthermore, tiron inhibited basal and ET-1-stimulated ROS generation, RhoA activation, and VSM Ca(2+) sensitization following CH. We conclude that CH augments ET-1-induced Ca(2+) sensitization through ROS-dependent activation of RhoA/ROK signaling in pulmonary VSM.     

L-type Ca(2+) channels, resting [Ca(2+)](i), and ET-1-induced responses in chronically hypoxic pulmonary myocytes

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(2+) channels contributes to 1) maintenance of resting intracellular Ca(2+) concentration ([Ca(2+)](i)), 2) increased [Ca(2+)](i) in response to ET-1 (10(-8) M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca(2+)](i) in PASMCs from intrapulmonary arteries of rats exposed to 10% O(2) for 21 days was 293.9 +/- 25.2 nM (vs. 153.6 +/- 28.7 nM in normoxia). Resting [Ca(2+)](i) was decreased after extracellular Ca(2+) removal but not with nifedipine (10(-6) M), an L-type Ca(2+) channel antagonist. After CH, the ET-1-induced increase in [Ca(2+)](i) was reduced and was abolished after extracellular Ca(2+) removal or nifedipine. Removal of extracellular Ca(2+) reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca(2+)](i) in PASMCs from chronically hypoxic rats does not require activation of L-type Ca(2+) channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca(2+)](i).     

Chronic hypoxia augments protein kinase G-mediated Ca2+ desensitization in pulmonary vascular smooth muscle through inhibition of RhoA/Rho kinase signaling

Pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO) is enhanced in pulmonary arteries from rats exposed to chronic hypoxia (CH) compared with controls. Furthermore, in contrast to control arteries, relaxation to NO following CH is not reliant on a decrease in VSM intracellular free calcium ([Ca(2+)](i)). We hypothesized that enhanced NO-dependent pulmonary vasodilation following CH is a function of VSM myofilament Ca(2+) desensitization via inhibition of the RhoA/Rho kinase (ROK) pathway. To test this hypothesis, we compared the ability of the NO donor, spermine NONOate, to reverse VSM tone generated by UTP, the ROK agonist sphingosylphosphorylcholine, or the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate in Ca(2+)-permeabilized, endothelium-denuded pulmonary arteries (150- to 300-microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca(2+)](i). We further examined effects of NO on levels of GTP-bound RhoA and ROK membrane translocation as indexes of enzyme activity in arteries from each group. We found that spermine NONOate reversed Y-27632-sensitive Ca(2+) sensitization and inhibited both RhoA and ROK activity in vessels from CH rats but not control animals. In contrast, spermine NONOate was without effect on PKC-mediated vasoconstriction in either group. We conclude that CH mediates a shift in NO signaling to promote pulmonary VSM Ca(2+) desensitization through inhibition of RhoA/ROK.     

Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries

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(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 microm 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(2+) concentration ([Ca(2+)](i)). Basal VSM [Ca(2+)](i) was not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 microM), 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 microM) 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 microm ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 microM) but not by GF109203X. VSM [Ca(2+)](i) was unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200-300 microm ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament Ca(2+) sensitization.     

Ca2+ signaling in hypoxic pulmonary vasoconstriction: effects of myosin light chain and Rho kinase antagonists

Antagonists of myosin light chain (MLC) kinase (MLCK) and Rho kinase (ROK) are thought to inhibit hypoxic pulmonary vasoconstriction (HPV) by decreasing the concentration of phosphorylated MLC at any intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMC); however, these antagonists can also decrease [Ca(2+)](i). To determine whether MLCK and ROK antagonists alter Ca(2+) signaling in HPV, we measured the effects of ML-9, ML-7, Y-27632, and HA-1077 on [Ca(2+)](i), Ca(2+) entry, and Ca(2+) release in rat distal PASMC exposed to hypoxia or depolarizing concentrations of KCl. We performed parallel experiments in isolated rat lungs to confirm the inhibitory effects of these agents on pulmonary vasoconstriction. Our results demonstrate that MLCK and ROK antagonists caused concentration-dependent inhibition of hypoxia-induced increases in [Ca(2+)](i) in PASMC and HPV in isolated lungs and suggest that this inhibition was due to blockade of Ca(2+) release from the sarcoplasmic reticulum and Ca(2+) entry through store- and voltage-operated Ca(2+) channels in PASMC. Thus MLCK and ROK antagonists might block HPV by inhibiting Ca(2+) signaling, as well as the actin-myosin interaction, in PASMC. If effects on Ca(2+) signaling were due to decreased phosphorylated myosin light chain concentration, their diversity suggests that MLCK and ROK antagonists may have acted by inhibiting myosin motors and/or altering the cytoskeleton in a manner that prevented achievement of required spatial relationships among the cellular components of the response.     

Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+ responses to hypoxia

Hypoxic pulmonary vasoconstriction (HPV) requires Ca(2+) influx through store-operated Ca(2+) channels (SOCC) in pulmonary arterial smooth muscle cells (PASMC) and is greater in distal than proximal pulmonary arteries (PA). SOCC may be composed of canonical transient receptor potential (TRPC) proteins and activated by stromal interacting molecule 1 (STIM1). To assess the possibility that HPV is greater in distal PA because store-operated Ca(2+) entry (SOCE) is greater in distal PASMC, we measured intracellular Ca(2+) concentration ([Ca(2+)](i)) and SOCE in primary cultures of PASMC using fluorescent microscopy and the Ca(2+)-sensitive dye fura 2. Both hypoxia (4% O(2)) and KCl (60 mM) increased [Ca(2+)](i). Responses to hypoxia, but not KCl, were greater in distal cells. We measured SOCE in PASMC perfused with Ca(2+)-free solutions containing cyclopiazonic acid to deplete Ca(2+) stores in sarcoplasmic reticulum and nifedipine to prevent Ca(2+) entry through L-type voltage-operated Ca(2+) channels. Under these conditions, the increase in [Ca(2+)](i) caused by restoration of extracellular Ca(2+) and the decrease in fura 2 fluorescence caused by Mn(2+) were greater in distal PASMC, indicating greater SOCE. Moreover, the increase in SOCE caused by hypoxia was also greater in distal cells. Real-time quantitative polymerase chain reaction analysis of PASMC and freshly isolated deendothelialized PA tissue demonstrated expression of STIM1 and five of seven known TRPC isoforms (TRPC1 > TRPC6 > TRPC4 > TRPC3 approximately TRPC5). For both protein, as measured by Western blotting, and mRNA, expression of STIM1, TRPC1, TRPC6, and TRPC4 was greater in distal than proximal PASMC and PA. These results provide further support for the importance of SOCE in HPV and suggest that HPV is greater in distal than proximal PA because greater numbers and activation of SOCC in distal PASMC generate bigger increases in [Ca(2+)](i).     

Kinase-dependent activation of voltage-gated Ca2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

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[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) 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[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-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[Ca(2+)](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[Ca(2+)](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[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.     

ROK contribution to endothelin-mediated contraction in aorta and mesenteric arteries following intermittent hypoxia/hypercapnia in rats

We reported previously that intermittent hypoxia with CO(2) to maintain eucapnia (IH-C) elevates plasma endothelin-1 (ET-1) and arterial pressure. In small mesenteric arteries (sMA; inner diameter = 150 microm), IH-C augments ET-1 constrictor sensitivity but diminishes ET-1-induced increases in intracellular Ca(2+) concentration, suggesting IH-C exposure increases both ET-1 levels and ET-1-stimulated Ca(2+) sensitization. Because Rho-associated kinase (ROK) can mediate Ca(2+) sensitization, we hypothesized that augmented vasoconstrictor sensitivity to ET-1 in arteries from IH-C-exposed rats is dependent on ROK activation. In thoracic aortic rings, ET-1 contraction was not different between groups, but ROK inhibition (Y-27632, 3 and 10 microM) attenuated ET-1 contraction more in IH-C than in sham arteries (50 +/- 11 and 78 +/- 7% vs. 41 +/- 12 and 48 +/- 9% inhibition, respectively). Therefore, ROK appears to contribute more to ET-1 contraction in IH-C than in sham aorta. In sMA, ROK inhibitors did not affect ET-1-mediated constriction in sham arteries and only modestly inhibited it in IH-C arteries. In ionomycin-permeabilized sMA with intracellular Ca(2+) concentration held at basal levels, Y-27632 did not affect ET-1-mediated constriction in either IH-C or sham sMA and ET-1 did not stimulate ROK translocation. In contrast, inhibition of myosin light-chain kinase (ML-9, 100 microM) prevented ET-1-mediated constriction in sMA from both groups. Therefore, IH-C exposure increases ET-1 vasoconstrictor sensitivity in sMA but not in aorta. Furthermore, ET-1 constriction is myosin light-chain kinase dependent and mediated by Ca(2+) sensitization that is independent of ROK activation in sMA but not aorta. Thus ET-1-mediated signaling in aorta and sMA is altered by IH-C but is dependent on different second messenger systems in small vs. large arteries.     

Reduced store-operated Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats

Chronic hypoxia (CH)-induced pulmonary hypertension may influence basal endothelial cell (EC) intracellular Ca(2+) concentration ([Ca(2+)](i)). We hypothesized that CH decreases EC [Ca(2+)](i) associated with membrane depolarization and reduced Ca(2+) entry. To test this hypothesis, we assessed 1) basal endothelial Ca(2+) in pressurized pulmonary arteries and freshly isolated ECs, 2) EC membrane potential (E(m)), 3) store-operated Ca(2+) current (I(SOC)), and 4) store-operated Ca(2+) (SOC) entry in arteries from control and CH rats. We found that basal EC Ca(2+) was significantly lower in pressurized pulmonary arteries and freshly isolated ECs from CH rats compared with controls. Similarly, ECs in intact arteries from CH rats were depolarized compared with controls, although no differences were observed between groups in isolated cells. I(SOC) activation by 1 muM thapsigargin displayed diminished inward current and a reversal potential closer to 0 mV in cells from CH rats compared with controls. In addition, SOC entry determined by fura 2 fluorescence and Mn(2+) quenching revealed a parallel reduction in Ca(2+) entry following CH. We conclude that differences in the magnitude of SOC entry exist between freshly dispersed ECs from CH and control rats and correlates with the decrease in basal EC [Ca(2+)](i). In contrast, basal EC Ca(2+) influx is unaffected and membrane depolarization is limited to intact arteries, suggesting that E(m) may not play a major role in determining basal EC [Ca(2+)](i) following CH.     

Inhibition of hypoxic pulmonary vasoconstriction by antagonists of store-operated Ca2+ and nonselective cation channels

Previous studies indicated that acute hypoxia increased intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) influx, and capacitative Ca(2+) entry (CCE) through store-operated Ca(2+) channels (SOCC) in smooth muscle cells from distal pulmonary arteries (PASMC), which are thought to be a major locus of hypoxic pulmonary vasoconstriction (HPV). Moreover, these effects were blocked by Ca(2+)-free conditions and antagonists of SOCC and nonselective cation channels (NSCC). To test the hypothesis that in vivo HPV requires CCE, we measured the effects of SOCC/NSCC antagonists (SKF-96365, NiCl(2), and LaCl(3)) on pulmonary arterial pressor responses to 2% O(2) and high-KCl concentrations in isolated rat lungs. At concentrations that blocked CCE and [Ca(2+)](i) responses to hypoxia in PASMC, SKF-96365 and NiCl(2) prevented and reversed HPV but did not alter pressor responses to KCl. At 10 microM, LaCl(3) had similar effects, but higher concentrations (30 and 100 microM) caused vasoconstriction during normoxia and potentiated HPV, indicating actions other than SOCC blockade. Ca(2+)-free perfusate and the voltage-operated Ca(2+) channel (VOCC) antagonist nifedipine were potent inhibitors of pressor responses to both hypoxia and KCl. We conclude that HPV required influx of Ca(2+) through both SOCC and VOCC. This dual requirement and virtual abolition of HPV by either SOCC or VOCC antagonists suggests that neither channel provided enough Ca(2+) on its own to trigger PASMC contraction and/or that during hypoxia, SOCC-dependent depolarization caused secondary activation of VOCC.     

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.     

Chronic hypoxia upregulates pulmonary arterial ASIC1: a novel mechanism of enhanced store-operated Ca2+ entry and receptor-dependent vasoconstriction

Acid-sensing ion channel 1 (ASIC1) is a newly characterized contributor to store-operated Ca(2+) entry (SOCE) in pulmonary vascular smooth muscle (VSM). Since SOCE is implicated in elevated basal VSM intracellular Ca(2+) concentration ([Ca(2+)](i)) and augmented vasoconstriction in chronic hypoxia (CH)-induced pulmonary hypertension, we hypothesized that ASIC1 contributes to these responses. To test this hypothesis, we examined effects of the specific pharmacologic ASIC1a inhibitor, psalmotoxin 1 (PcTX1), on vasoconstrictor and vessel wall [Ca(2+)](i) responses to UTP and KCl (depolarizing stimulus) in fura-2-loaded, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) Wistar rats. PcTX1 had no effect on basal vessel wall [Ca(2+)](i), but attenuated vasoconstriction and increases in vessel wall [Ca(2+)](i) to UTP in arteries from control and CH rats; normalizing responses between groups. In contrast, responses to the depolarizing stimulus, KCl, were unaffected by CH exposure or PcTX1. Upon examining potential Ca(2+) influx mechanisms, we found that PcTX1 prevented augmented SOCE following CH. Exposure to CH resulted in a significant increase in pulmonary arterial ASIC1 protein. This study supports a novel role of ASIC1 in elevated receptor-stimulated vasoconstriction following CH which is likely mediated through increased ASIC1 expression and SOCE.     

Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution

Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) through controlling voltage-gated K(+) channel expression and membrane potential (E(m)) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca(2+)](cyt) activates Rho-kinase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced [Ca(2+)](cyt), by either polyamine depletion or exposure to the Ca(2+)-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca(2+)](cyt) also inhibited cell migration after wounding. Elevation of [Ca(2+)](cyt) induced by the Ca(2+) ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via [Ca(2+)](cyt) can interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration.     

Mobilization of intracellular Ca(2+) by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca(2+) mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca(2+) response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca(2+)](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca(2+)](i) (>1 microM) followed by a sustained elevation in [Ca(2+)](i) (<200 nM). The ET-1-induced increase in [Ca(2+)](i) was attenuated (<80%) by extracellular Ca(2+) removal; by verapamil, a voltage-gated Ca(2+)-channel antagonist; and by ryanodine, an inhibitor of Ca(2+) release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca(2+)](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca(2+)](i) indicated that depolarization preceded the rise in [Ca(2+)](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.     

Altered membrane lipid domains limit pulmonary endothelial calcium entry following chronic hypoxia

Agonist-induced Ca(2+) entry into the pulmonary endothelium depends on activation of both store-operated Ca(2+) (SOC) entry and receptor-operated Ca(2+) (ROC) entry. We previously reported that pulmonary endothelial cell SOC entry and ROC entry are reduced in chronic hypoxia (CH)-induced pulmonary hypertension. We hypothesized that diminished endothelial Ca(2+) entry following CH is due to derangement of caveolin-1 (cav-1) containing cholesterol-enriched membrane domains important in agonist-induced Ca(2+) entry. To test this hypothesis, we measured Ca(2+) influx by fura-2 fluorescence following application of ATP (20 μM) in freshly isolated endothelial cells pretreated with the caveolar-disrupting agent methyl-β-cyclodextrin (mβCD; 10 mM). Cholesterol depletion with mβCD attenuated agonist-induced Ca(2+) entry in control endothelial cells to the level of that from CH rats. Interestingly, endothelial membrane cholesterol was lower in cells isolated from CH rats compared with controls although the density of caveolae did not differ between groups. Cholesterol repletion with a cholesterol:mβCD mixture or the introduction of the cav-1 scaffolding peptide (AP-cav; 10 μM) rescued ATP-induced Ca(2+) entry in endothelia from CH arteries. Agonist-induced Ca(2+) entry assessed by Mn(2+) quenching of fura-2 fluorescence was also significantly elevated by luminal AP-cav in pressurized intrapulmonary arteries from CH rats to levels of controls. Similarly, patch-clamp experiments revealed diminished inward current in response to ATP in cells from CH rats compared with controls that was restored by AP-cav. These data suggest that CH-induced pulmonary hypertension leads to reduced membrane cholesterol that limits the activity of ion channels necessary for agonist-activated Ca(2+) entry.     

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-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, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.     

Endothelin-1 increases intracellular Ca(2+) in rabbit pulmonary artery smooth muscle cells through phospholipase C

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.