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

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.