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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Cell culture alters Ca²⁺ entry pathways activated by store-depletion or hypoxia in canine pulmonary arterial smooth muscle cells

¹Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada; and ²Smooth Muscle Research Centre, Regional Development Centre, Dundalk Institute of Technology, Dundalk, Ireland

Submitted 15 June 2007 ; accepted in final form 30 October 2007

Previous studies have shown that, in acutely dispersed canine pulmonary artery smooth muscle cells (PASMCs), depletion of both functionally independent inositol 1,4,5-trisphosphate (IP₃)- and ryanodine-sensitive Ca²⁺ stores activates capacitative Ca²⁺ entry (CCE). The present study aimed to determine if cell culture modifies intracellular Ca²⁺ stores and alters Ca²⁺ entry pathways caused by store depletion and hypoxia in canine PASMCs. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in fura 2-loaded cells. Mn²⁺ quench of fura 2 signal was performed to study divalent cation entry, and the effects of hypoxia were examined under oxygen tension of 15–18 mmHg. In acutely isolated PASMCs, depletion of IP₃-sensitive Ca²⁺ stores with cyclopiazonic acid (CPA) did not affect initial caffeine-induced intracellular Ca²⁺ transients but abolished 5-HT-induced Ca²⁺ transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced Ca²⁺ transients in cultured PASMCs. In cultured PASMCs, store depletion or hypoxia caused a transient followed by a sustained rise in [Ca²⁺]_i. The transient rise in [Ca²⁺]_i was partially inhibited by nifedipine, whereas the nifedipine-insensitive transient rise in [Ca²⁺]_i was inhibited by KB-R7943, a selective inhibitor of reverse mode Na⁺/Ca²⁺ exchanger (NCX). The nifedipine-insensitive sustained rise in [Ca²⁺]_i was inhibited by SKF-96365, Ni²⁺, La³⁺, and Gd³⁺. In addition, store depletion or hypoxia increased the rate of Mn²⁺ quench of fura 2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. We conclude that cell culture of canine PASMCs reorganizes IP₃ and ryanodine receptors into a common intracellular Ca²⁺ compartment, and depletion of this store or hypoxia activates voltage-operated Ca²⁺ entry, reverse mode NCX, and CCE.

capacitative calcium entry; hypoxia; cultured pulmonary artery smooth muscle cells

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