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Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle

Division of Pulmonary & Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224

Submitted 10 September 2003 ; accepted in final form 9 December 2003

Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca²⁺ entry triggered by depletion of Ca²⁺ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca²⁺ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca²⁺ signaling in distal PASMCs, we measured [Ca²⁺]_i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca²⁺-free media containing cyclopiazonic acid (10 µM) and nifedipine (5 µM) to deplete ER Ca²⁺ stores and block voltage-dependent Ca²⁺ channels, restoration of extracellular Ca²⁺ (2.5 mM) caused marked increases in [Ca²⁺]_i whereas MnCl₂ (200 µM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl₃, and NiCl₂, blocked CCE at concentrations that did not alter Ca²⁺ responses to 60 mM KCl (IC₅₀ 6.3, 40.4, and 191 µM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca²⁺ channels could contribute to Ca²⁺ signaling in myocytes from distal intrapulmonary arteries.

cation channel transient receptor potential; intracellular calcium concentration; fura-2; SKF-96365; LaCl₃; NiCl₂

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