The role of store-operated channels in vascular muscle is unclear. Store depletion-activated, non-selective cation channels were recently identified in aorta (Trepakova et al. 2001) and were shown to mediate Ca²⁺ influx and contraction in mouse anococcygeus muscle (Wayman et al. 1999). Here, we tested the hypothesis that store-operated channels play a similar role in pulmonary artery smooth muscle cells (PASMC). Pulmonary arteries were removed from humanely killed Sprague-Dawley rats and mounted in an organ bath for isometric tension measurement, or used to prepare isolated PASMC. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was measured in PASMC using fura-2 fluorescence and membrane current was recorded using whole-cell voltage-clamp techniques. The expression of Trp channel subunits, which are proposed to underlie store-operated channels, was investigated by reverse transcriptase PCR and immunostaining of PASMC with anti-Trp antibodies. Results are reported as means ± S.E.M. Statistical comparisons employed Student’s t test.

In intact arteries, cyclopiazonic acid (CPA; 5-30 µM) produced a slowly developing, sustained contraction, which was 52 ± 5 % (n = 21) reduced by 1 µM nifedipine and abolished in Ca²⁺-free solution. The nifedipine-resistant contraction was 50 % inhibited by 6 µM Cd²⁺, 10 µM Ni²⁺, 600 µM La³⁺ and 7 µM SKF 96365. In PASMC, CPA produced a sustained, dihydropyridine-resistant increase in [Ca²⁺]_i, which resulted from increased Ca²⁺ influx because it was abolished in Ca²⁺-free medium and CPA accelerated fura-2 quenching by extracellular Mn²⁺ (20 µM). This response was 49 ± 12 % (n = 4) and 69 ± 11 % (n = 3) inhibited by 10 µM Ni²⁺ and 7 µM SKF 96365, respectively. In PASMC voltage clamped at -80 mV using the perforated-patch technique, CPA (30 µM) induced a sustained inward current (0.41 ± 0.04 pA pF^-1; n = 76), which persisted in cells dialysed with 5 mM BAPTA. The current reversed direction close to 0 mV, was increased 2- to 3-fold in Ca²⁺-free solution and was also abolished by 200 µM Cd²⁺, 200 µM Ni²⁺ and 50 µM SKF 96365.

These properties indicate the presence of store-operated, non-selective cation channels in PASMC. The pharmacology of the current further suggests that the store-operated channels mediate the CPA-induced increase in [Ca²⁺]_i and contraction. The channels may be formed by Trp channel subunits, because rat PASMC were found to express Trp1, Trp3, Trp4, Trp5 and Trp6 mRNA, as well as Trp1, Trp3, Trp4 and Trp6 channel proteins.

This work was supported by Tenovus Scotland.

This work was supported by Tenovus Scotland.

Trepakova, E.S., Gericke, M., Hirakawa, Y., Weisbrod, R.M., Cohen, R.A. & Bolotina, V.M. (2001). J. Biol. Chem. 276, 7782-7790.

Wayman, C.P., Wallace, P., Gibson, A. & McFadzean, I. (1999). Eur. J. Pharmacol. 376, 325-329.