Knockdown of canonical transient receptor potential proteins 1, 4, and 6 attenuates store‐operated calcium entry and calcium responses to acute hypoxia in pulmonary arterial smooth muscle cells

Abstract

Acute hypoxia increases intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ entry through store-operated Ca2+ channels (SOCC) in myocytes from distal pulmonary arteries, which are thought to be the major locus of hypoxic pulmonary vasoconstriction. SOCC may be composed of canonical transient receptor potential (TRPC) proteins, and TRPC1, 4, and 6 are expressed in rat distal pulmonary arterial smooth muscle cells (PASMC). To determine if these TRPC contribute to store-operated Ca2+ entry (SOCE) and [Ca2+]i responses to hypoxia in these cells, we used fluorescence microscopy and the Ca2+-sensitive dye, Fura-2, to measure [Ca2+]i and SOCE in rat distal PASMC 24 h after treatment with siRNA targeted to TRPC1, 4, or 6, followed by exposureto normal basal medium for 24 h. As measured by real-time PCR and Western blotting, RNA interference decreased expression of targeted TRPC mRNA and protein by 75–95%, but did not decrease non-targeted TRPC expression. Compared to nontargeted siRNA, treatment with siRNA targeted to TRPC1, 4, or 6 caused 1) 54, 31, and 39% decreases in SOCE, as measured by quenching of Fura-2 fluorescence by Mn2+ at 10 min in cells perfused with Ca2+-free Kreb's Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid and nifedipine; and 2) 84, 44, and 41% decreases in the elevation of [Ca2+]i caused by perfusion with KRBS equilibrated with 4% O2-5% CO2 for 20 min; and 3) no decreases in elevation of [Ca2+]i in cells perfused with KRBS containing 60 mM KCl (equimolar substitution for NaCl) for 10 min. These results suggest that TRPC1, TRPC4, and TRPC6 all contribute to SOCE and [Ca2+]i responses to hypoxia in rat distal PASMC, but that TRPC1 is quantitatively more important than TRPC4 or TRPC6. (Supported by NIH grants: HL-75113 (J T Sylvester), HL-079981 (J Wang), HL-093020 (J Wang)).