Abstract-Chronic hypoxia (CH) causes pulmonary vasoconstriction because of increased pulmonary arterial smooth muscle cell (PASMC) contraction and proliferation. We previously demonstrated that intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) was elevated in PASMCs from chronically hypoxic rats because of Ca 2ϩ influx through pathways other than L-type Ca 2ϩ channels and that development of hypoxic pulmonary hypertension required full expression of the transcription factor hypoxia inducible factor 1 (HIF-1). In this study, we examined the effect of CH on the activity and expression of store-operated Ca 2ϩ channels (SOCCs) and the regulation of these channels by HIF- Key Words: Ca 2ϩ channels Ⅲ hypoxia Ⅲ hypoxia-inducible factor 1 Ⅲ hypoxic pulmonary vasoconstriction Ⅲ vascular smooth muscle P rolonged exposure to alveolar hypoxia is associated with changes in the pulmonary vasculature including structural remodeling 1,2 and active contraction of vascular smooth muscle. 3,4 Recent evidence suggests that the latter may play a more prominent role in the elevation of pulmonary vascular resistance because the thickened smooth muscle cell layer caused by chronic hypoxia (CH) in small pulmonary arteries has been found to have no significant impact on luminal diameter. 5 Moreover, inhibition of Rho kinase, a mediator of myofilament contractility, has been shown to acutely reverse the increase in pulmonary arterial pressure in chronically hypoxic rats. 6 Based on these findings, it appears that contraction of smooth muscle during CH is the major factor contributing to the pathogenesis of hypoxic pulmonary hypertension.Although the cellular mechanisms underlying the development of pulmonary hypertension remain poorly understood, both growth and contraction of pulmonary arterial smooth muscle cells (PASMCs) are associated with alterations in

Hypoxic pulmonary vasoconstriction (HPV) requires influx of extracellular Ca2+ in pulmonary arterial smooth muscle cells (PASMCs). To determine whether capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCCs) contributes to this influx, we used fluorescent microscopy and the Ca2+-sensitive dye fura-2 to measure effects of 4% O2 on intracellular [Ca2+] ([Ca2+]i) and CCE in primary cultures of PASMCs from rat distal pulmonary arteries. In PASMCs perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca2+ stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+ entry through L-type voltage-operated Ca2+ channels (VOCCs), hypoxia markedly enhanced both the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] and the rate at which extracellular Mn2+ quenched fura-2 fluorescence. These effects, as well as the increased [Ca2+]i caused by hypoxia in PASMCs perfused with normal salt solutions, were blocked by the SOCC antagonists SKF-96365, NiCl2, and LaCl3 at concentrations that inhibited CCE >80% but did not alter [Ca2+]i responses to 60 mM KCl. In contrast, the VOCC antagonist nifedipine inhibited [Ca2+]i responses to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca2+]i caused by hypoxia was completely reversed by perfusion with Ca2+-free KRBS. LaCl3 increased basal [Ca2+]i during normoxia, indicating effects other than inhibition of SOCCs. Our results suggest that acute hypoxia enhances CCE through SOCCs in distal PASMCs, leading to depolarization, secondary activation of VOCCs, and increased [Ca2+]i. SOCCs and CCE may play important roles in HPV.

(2,3,7,8,38,41,45,47,58,59,70) and contraction (20,24,26,28,32 ] i responses to hypoxia in isolated PA (43). One possible explanation for these findings is that hypoxia caused release of Ca 2ϩ from SR in PASMC, leading to SR depletion, activation of store-operated Ca 2ϩ channels (SOCC), and capacitative Ca 2ϩ entry (CCE). Recently, we reported that several so-called "canonical transient receptor potential (TRPC)" proteins were expressed in smooth muscle of distal PA (60), which are thought to be the major vascular locus of HPV (49). These proteins are homologs of TRP and TRP-like proteins that make up Ca 2ϩ channels in Drosophila photoreceptors and are thought to compose mammalian SOCC, many forms of which may also be permeable to Na ϩ and other cations and therefore function as nonselective cation channels (NSCC) (34, 37, 51). Consistent with TRPC expression, we and others demonstrated the presence of CCE in distal PA (51, 60). More recently, we found that acute hypoxia increased [Ca 2ϩ ] i , Ca 2ϩ influx, and CCE in distal PASMC and that these effects were completely blocked by removal of extracellular Ca 2ϩ or SOCC/NSCC antagonists, but not nifedipine (61). These findings are consistent with recent observations from other laboratories (21,22,35) and suggest that HPV may require activation of SOCC in PASMC; however, it is well known that cell isolation and culture can alter cell phenotype. Moreover, changes in PASMC [Ca 2ϩ ] i do not necessarily translate to changes in pulmonary vascular resistance. In the present study, therefore, we assessed the contribution of SOCC and CCE to HPV in isolated lungs, where physiologically relevant pulmonary vasomotor responses can be measured directly. METHODS Isolated Lung PreparationOur protocol was approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University. Male Wistar rats (200 -400 g) were anesthetized with pentobarbital sodium (65 mg/kg ip). A tracheostomy was performed, and the animal was ventilated with room air at a tidal volume of 10 ml/kg and a rate of 30 min Ϫ1(Harvard Rodent Ventilator model 883; Harvard Apparatus, Holliston, MA). A thoracotomy was performed, heparin (100 units) was injected into the right ventricular cavity, and the animal was exsanguinated from the femoral artery. The ventilating gas was changed to 16% O 2-5% CO2. Cannulae were inserted into the main PA and left atrium, which drained into a heated reservoir. The lungs were perfused with

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular Ca2+ concentration ([Ca2+]i), suggesting that ET-1 increased Ca2+ sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% O2 for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type Ca2+ channel blocker, to isolate the component of contraction that occurred without a change in [Ca2+]i. The mean change in tension caused by ET-1 (10(-8) M) expressed as a percent of the maximum response to KCl was 184.0+/-39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [Ca2+]i in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC.

In pulmonary arterial smooth muscle cells (PASMCs), voltage-gated K+ (Kv) channels play an important role in regulating membrane potential, cytoplasmic free Ca2+ concentration, and pulmonary vasomotor tone. Previous studies demonstrated that exposure of rats to chronic hypoxia decreased Kv channel function in PASMCs from distal pulmonary arteries (dPA). To determine whether this decrease in function was due to decreased expression of Kv channel proteins and which Kv proteins might be involved, we analyzed Kv channel gene expression in intact, endothelium-denuded dPAs obtained from rats exposed to 10% O2 for 3 wk. Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv4.3, and Kv9.3 channel alpha-subunits and Kv1, Kv2, and Kv3 beta-subunits were expressed in rat dPAs. Exposure to chronic hypoxia decreased mRNA and protein levels of Kv1.1, Kv1.5, Kv1.6, Kv2.1, and Kv4.3 alpha-subunits in dPAs but did not alter gene or protein expression of these channels in aorta. Furthermore, chronic hypoxia did not alter the mRNA levels of beta-subunits in dPAs. These results suggest that diminished transcription of Kv alpha-subunits may reduce the number of functional Kv channels in dPAs during prolonged hypoxia, causing the decreased Kv current previously observed in PASMCs and leading to pulmonary artery vasoconstriction.

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.

We addressed the mechanism by which antigen contracts trachea isolated from actively sensitized mice. Trachea were isolated from mice (C57BL/6J) that had been actively sensitized to ovalbumin (OVA). OVA (10 μg ml −1 ) caused histamine release (∼70% total tissue content), and smooth muscle contraction that was rapid in onset and short-lived (t 1/2 < 1 min), reaching approximately 25% of the maximum tissue response. OVA contraction was mimicked by 5-HT, and responses to both OVA and 5-HT were sensitive to 10 μm-ketanserin (5-HT 2 receptor antagonist) and strongly inhibited by atropine (1 μm). Epithelial denudation had no effect on the OVA-induced contraction. Histological assessment revealed about five mast cells/tracheal section the vast majority of which contained 5-HT. There were virtually no mast cells in the mast cell-deficient (sash−/−) mouse trachea. OVA failed to elicit histamine release or contractile responses in trachea isolated from sensitized mast cell-deficient (sash−/−) mice. Intracellular recordings of the membrane potential of parasympathetic neurons in mouse tracheal ganglia revealed a ketanserin-sensitive 5-HT-induced depolarization and similar depolarization in response to OVA challenge. These data support the hypothesis that antigen-induced contraction of mouse trachea is epithelium-independent, and requires mast cell-derived 5-HT to activate 5-HT 2 receptors on parasympathetic cholinergic neurons. This leads to acetylcholine release from nerve terminals, and airway smooth muscle contraction.

Autonomic control of heart rate is mediated by cardioinhibitory parasympathetic cholinergic neurons located in the brainstem and stimulatory sympathetic noradrenergic neurons. During embryonic development the survival and cholinergic phenotype of brainstem autonomic neurons is promoted by brain-derived neurotrophic factor (BDNF). We now provide evidence that BDNF regulates heart rate by a mechanism involving increased brainstem cardioinhibitory parasympathetic activity. Mice with a BDNF haploinsufficiency exhibit elevated resting heart rate, and infusion of BDNF intracerebroventricularly reduces heart rate in both wild-type and BDNF+/− mice. The atropine-induced elevation of heart rate is diminished in BDNF+/− mice and is restored by BDNF infusion, whereas the atenolol-induced decrease in heart rate is unaffected by BDNF levels, suggesting that BDNF signaling enhances parasympathetic tone which is diminished with BDNF haploinsufficiency. Whole-cell recordings from pre-motor cholinergic cardioinhibitory vagal neurons in the nucleus ambiguus indicate that BDNF haploinsufficiency reduces cardioinhibitory vagal neuron activity by increased inhibitory GABAergic and diminished excitatory glutamatergic neurotransmission to these neurons. Our findings reveal a previously unknown role for BDNF in the control of heart rate by a mechanism involving increased activation of brainstem cholinergic parasympathetic neurons.