Abstract 979: Capacitative Ca2+ Entry (cce) And Expression Of Canonical Transient Receptor Potential Channel (trpc) Genes Is Greater In Distal Than In Proximal Pulmonary Arterial Smooth Muscle Cells (pasmcs)
TRPC proteins are mammalian homologs of “transient receptor potential” proteins in Drosophila, and are thought to compose store-operated Ca2+ channels that provide pathways for CCE in many cells. In lung, distal pulmonary arteries (DPA) are thought to be more important than proximal pulmonary arteries (PPA) as sites of vasomotor responses, such as hypoxic pulmonary vasoconstriction (HPV). We recently reported that CCE played an important role in HPV, and that 3 subtypes of TRPC proteins (1, 4 and 6) were expressed in rat DPA, as derermined by standard PCR. In the present study, we used quantitative real-time PCR to compare TRPC gene expression in DPA and PPA, as well as PASMCs isolated from these vessels. In both preparations, the order of TRPC mRNA abundance normalized to β-actin mRNA was TRPC1 > TRPC6 > TRPC4 ≫ TRPC5 ≈TRPC3. TRPC2 and TRPC7 were not detected. Expression of TRPC1, TRPC4 and TRPC6 was 1.6, 2.1 and 2.3-fold greater, respectively, in DPA than in PPA, and 2.4, 1.9 and 2.0-fold greater in distal than in proximal PASMCs. We also used fluorescent microscopy and the Ca2+-sensitive dye, Fura-2, to measure CCE in primary cultures of PASMCs perfused with Ca2+-free Kreb’s Ringer Bicarbonate solution containing cyclopiazonic acid and nifedipine to deplete SR calcium stores and prevent Ca2+ entry through voltage dependent Ca2+ channels. Under these conditions, restoration of extracellular Ca2+ caused greater increases in intracellular Ca2+ concentration (Δ [Ca2+]i) and Mn 2+ caused greater quenching of Fura-2 fluorescence (ΔF/F0) in distal than in proximal PASMCs (Δ [Ca2+]i = 493 ± 77 vs. 239 ± 36 nM; ΔF/F0 = −45 ± 10 vs. −27 ± 11% at 10 min). These results suggest that vasoreactivity may be greater in DPA than in PPA because smooth muscle TRPC gene expression and CCE are greater.