Abstract-Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca 2ϩ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca 2ϩ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store-and receptor-operated Ca 2ϩ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca 2ϩ stores, caused dramatic increase in cation entry measured by Mn 2ϩ quenching of fura-2 and by Ca 2ϩ transients. OAG-induced responses were Ϸ700-fold more resistant to La 3ϩ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin-and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2-to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca 2ϩ or inhibition of store-operated Ca 2ϩ entry with La 3ϩ and SK&F-96365 reversed the elevated basal [Ca 2ϩ ] i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store-and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension. Key Words: pulmonary hypertension Ⅲ transient receptor potential channels Ⅲ store-operated Ca 2ϩ channels Ⅲ receptor-operated Ca 2ϩ channels P rolonged exposure to alveolar hypoxia causes pulmonary hypertension with profound vascular remodeling and increase in vasomotor tone. The increase in vascular tone is in part attributable to alterations in vasoconstricting and vasorelaxing influences imposed by the endothelially derived and circulating factors. 1 Recent evidence indicates that chronic hypoxia also causes intrinsic changes in ionic balance and Ca 2ϩ homeostasis in pulmonary arterial smooth muscle cells (PASMCs), including membrane depolarization, elevation in resting [Ca 2ϩ ] i , and changes in electrophysiological and Ca 2ϩ responses to vasoconstrictors and vasodilators. [2][3][4][5][6] The mechanism for alteration in Ca 2ϩ homeostasis in hypoxic PASMCs is controversial. Previous studies found significant suppression of voltage-gated K ϩ (K V ) currents and K V channel expression in PASMCs isolated fr...

Transient receptor potential melastatin-(TRPM) and vanilloid-related (TRPV) channels are nonselective cation channels pertinent to diverse physiological functions. Multiple TRPM and TRPV channel subtypes have been identified and cloned in different tissues. However, their information in vascular tissue is scant. In this study, we sought to identify TRPM and TRPV channel subtypes expressed in rat deendothelialized intralobar pulmonary arteries (PAs) and aorta. With RT-PCR, mRNA of TRPM2, TRPM3, TRPM4, TRPM7, and TRPM8 of TRPM family and TRPV1, TRPV2, TRPV3, and TRPV4 of TRPV family were detected in both PAs and aorta. Quantitative real-time RT-PCR showed that TRPM8 and TRPV4 were the most abundantly expressed TRPM and TRPV subtypes, respectively. Moreover, Western blot analysis verified expression of TRPM2, TRPM8, TRPV1, and TRPV4 proteins in both types of vascular tissue. To examine the functional activities of these channels, we monitored intracellular Ca 2ϩ transients ([Ca 2ϩ ]i) in pulmonary arterial smooth muscle cells (PASMCs) and aortic smooth muscle cells (ASMCs). The TRPM8 agonist menthol (300 M) and the TRPV4 agonist 4␣-phorbol 12,13-didecanoate (1 M) evoked significant increases in [Ca 2ϩ ]i in PASMCs and ASMCs. These Ca 2ϩ responses were abolished in the absence of extracellular Ca 2ϩ or the presence of 300 M Ni 2ϩ but were unaffected by 1 M nifedipine, suggesting Ca 2ϩ influx via nonselective cation channels. Hence, for the first time, our results indicate that multiple functional TRPM and TRPV channels are coexpressed in rat intralobar PAs and aorta. These novel Ca 2ϩ entry pathways may play important roles in the regulation of pulmonary and systemic circulation. transient receptor potential channels; calcium signaling; nonselective cation channels THE TRANSIENT RECEPTOR POTENTIAL (TRP) channels belong to a large superfamily of cation channels that have diverse physiological functions in both nonexcitable and excitable cells (26,33). On the basis of sequence homology, the TRP superfamily can be divided into three major subfamilies of canonical (TRPC), melastatin-related (TRPM), and vanilloid-related (TRPV) channels, as well as four more distant subfamilies of polycystin-related (TRPP), mucolipin-related (TRPML), ankyrin-related (TRPA), and no mechanoreceptor potential C, or NOMPC (TRPN), channels and/or proteins.The classic or canonical TRPC subfamily consists of seven members (TRPC1-7), which have attracted enormous attention because of their putative roles as store-operated and receptoroperated cation channels. In vascular smooth muscle, it is generally accepted that TRPC1 channels are related to storeoperated Ca 2ϩ entry, which can be activated by depletion of Ca 2ϩ stores (1,4,17,22,41,48), whereas TRPC6 (and TRPC3) channels are involved in receptor-operated Ca 2ϩ entry, which can be activated directly by diacylglycerol in a PKC-independent manner (13,15,18,22). TRPC channels have been shown to play pivotal roles in vasoconstriction induced by ␣-adrenoceptor agonists, vasopressin, endo...

Ca+ sparks originating from ryanodine receptors (RyRs) are known to cause membrane hyperpolarization and vasorelaxation in systemic arterial myocytes. By contrast, we have found that Ca2+ sparks of pulmonary arterial smooth muscle cells (PASMCs) are associated with membrane depolarization and activated by endothelin-1 (ET-1), a potent vasoconstrictor that mediates/modulates acute and chronic hypoxic pulmonary vasoconstriction. In this study, we characterized the effects of ET-1 on the physical properties of Ca2+ sparks and probed the signal transduction mechanism for spark activation in rat intralobar PASMCs. Application of ET-1 at 0.1-10 nM caused concentration-dependent increases in frequency, duration, and amplitude of Ca2+ sparks. The ET-1-induced increase in spark frequency was inhibited by BQ-123, an ETA-receptor antagonist; by U-73122, a PLC inhibitor; and by xestospongin C and 2-aminoethyl diphenylborate, antagonists of inositol trisphosphate (IP3) receptors (IP3Rs). However, it was unrelated to sarcoplasmic reticulum Ca2+ content, activation of L-type Ca2+ channels, PKC, or cADP ribose. Photorelease of caged-IP3 indicated that Ca2+ release from IP3R could cross-activate RyRs to generate Ca2+ sparks. Immunocytochemistry showed that the distributions of IP3Rs and RyRs were similar in PASMCs. Moreover, inhibition of Ca2+ sparks with ryanodine caused a significant rightward shift in the ET-1 concentration-tension relationship in pulmonary arteries. These results suggest that ET-1 activation of Ca2+ sparks is mediated via the ETA receptor-PLC-IP3 pathway and local Ca2+ cross-signaling between IP3Rs and RyRs; in addition, this novel signaling mechanism contributes significantly to the ET-1-induced vasoconstriction in pulmonary arteries.

Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca 2ϩ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, ϳ15-20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca 2ϩ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca 2ϩ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) ϭ 193.3 Ϯ 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca 2ϩ sparks (FDHM ϭ 44.6 Ϯ 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca 2ϩ