Cell culture alters [Ca.sup.2+] 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 ([IP.sub.3])- and ryanodine-sensitive [Ca.sup.2+] stores activates capacitative [Ca.sup.2+] entry (CCE). The present study aimed to determine if cell culture modifies intracellular [Ca.sup.2+] stores and alters [Ca.sup.2+] entry pathways caused by store depletion and hypoxia in canine PASMCs. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) was measured in fura 2-loaded cells. [Mn.sup.2+] 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 [IP.sub.3]-sensitive [Ca.sup.2+] stores with cyclopiazonic acid (CPA) did not affect initial caffeine-induced intracellular [Ca.sup.2+] transients but abolished 5-HT-induced [Ca.sup.2+] transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced [Ca.sup.2+] transients in cultured PASMCs. In cultured PASMCs, store depletion or hypoxia caused a transient followed by a sustained rise in [[[Ca.sup.2+]].sub.i]. The transient rise in [[[Ca.sup.2+]].sub.i] was partially inhibited by nifedipine, whereas the nifedipine-insensitive transient rise in [[[Ca.sup.2+]].sub.i] was inhibited by KB-R7943, a selective inhibitor of reverse mode [Na.sup.+]/[Ca.sup.2+] exchanger (NCX). The nifedipine-insensitive sustained rise in [[[Ca.sup.2+]].sub.i] was inhibited by SKF-96365, [Ni.sup.2+], [La.sup.3+], and [Gd.sup.3+]. In addition, store depletion or hypoxia increased the rate of [Mn.sup.2+] 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 [IP.sub.3] and ryanodine receptors into a common intracefiular [Ca.sup.2+] compartment, and depletion of this store or hypoxia activates voltage-operated [Ca.sup.2+] entry, reverse mode NCX, and CCE. capacitative calcium entry; hypoxia; cultured pulmonary artery smooth muscle cells