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

Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland Submitted 3 February 2008 ; accepted in final form 16 April 2008 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 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 . hypoxic pulmonary vasoconstriction; calcium signaling; vascular smooth muscle; potassium chloride; stromal interacting molecule 1; canonical transient receptor potential; calcium ion Address for reprint requests and other correspondence: J. T. Sylvester, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore