(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

No abstract

Recent advances have identified an important role of bone morphogenetic protein 4 (BMP4) in pulmonary vascular remodeling, yet the underlying mechanisms remain largely unexplored. We have previously found that Ca(2+) influx through store-operated calcium channels (SOCC), which are mainly thought to be composed of canonical transient receptor potential (TRPC) proteins, likely contribute to the pathogenic development of chronic hypoxic pulmonary hypertension. In this study, we investigated the effect of BMP4 on expression of TRPC and store-operated Ca(2+) entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs). Real-time quantitative PCR and Western blotting revealed that treatment with BMP4 (50 ng/ml, 60 h) increased TRPC1, TRPC4, and TRPC6 mRNA and protein expression in growth-arrested rat distal PASMCs. Moreover, in comparison to vehicle control, cells treated with BMP4 also exhibited enhanced SOCE, and elevated basal intracellular calcium concentration ([Ca(2+)](i)) as determined by fluorescent microscopy using the Ca(2+) indicator Fura-2 AM. Perfusing cells with Ca(2+)-free Krebs-Ringer bicarbonate solution (KRBS) or KRBS containing SOCC antagonists SKF-96365 or NiCl(2) attenuated the increases in basal [Ca(2+)](i) caused by BMP4. Specific knockdown of BMP4 by small interference RNA significantly decreased the mRNA and protein expression of TRPC1, TRPC4, and TRPC6 and reduced SOCE and basal [Ca(2+)](i) in serum-stimulated PASMCs. We conclude that BMP4 regulates calcium signaling in PASMCs likely via upregulation of TRPC expression, leading to enhanced SOCE and basal [Ca(2+)](i) in PASMCs, and by this mechanism contributes to pulmonary vascular remodeling during pulmonary arterial hypertension.