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

Abstract

Hypoxic pulmonary vasoconstriction (HPV) requires Ca2+influx through store-operated Ca2+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 Ca2+entry (SOCE) is greater in distal PASMC, we measured intracellular Ca2+concentration ([Ca2+]i) and SOCE in primary cultures of PASMC using fluorescent microscopy and the Ca2+-sensitive dye fura 2. Both hypoxia (4% O2) and KCl (60 mM) increased [Ca2+]i. Responses to hypoxia, but not KCl, were greater in distal cells. We measured SOCE in PASMC perfused with Ca2+-free solutions containing cyclopiazonic acid to deplete Ca2+stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+entry through L-type voltage-operated Ca2+channels. Under these conditions, the increase in [Ca2+]icaused by restoration of extracellular Ca2+and the decrease in fura 2 fluorescence caused by Mn2+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 [Ca2+]i.