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

• Published in: American Journal of Physiology: Cell Physiology Vol. 294; no. 1; p. C313
• Main Authors: Ng, Lih Chyuan, Kyle, Barry D, Lennox, Alison R, Shen, Xiao-Ming, Hatton, William J, Hume, Joseph R
• Format: Journal Article
• Language: English
• Published: United States 01-01-2008
• Subjects: Actins - metabolism; Animals; Caffeine - pharmacology; Calcium - metabolism; Calcium Channel Blockers - pharmacology; Calcium Channels - drug effects; Calcium Channels - metabolism; Calcium Signaling; Calcium-Transporting ATPases - antagonists & inhibitors; Calcium-Transporting ATPases - metabolism; Cell Hypoxia; Cells, Cultured; Dogs; Enzyme Inhibitors - pharmacology; Imidazoles - pharmacology; Indoles - pharmacology; Inositol 1,4,5-Trisphosphate - metabolism; Muscle Contraction; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - enzymology; Muscle, Smooth, Vascular - metabolism; Myocytes, Smooth Muscle - drug effects; Myocytes, Smooth Muscle - enzymology; Myocytes, Smooth Muscle - metabolism; Nifedipine - pharmacology; Oxygen - metabolism; Pulmonary Artery - metabolism; Ryanodine Receptor Calcium Release Channel - metabolism; Serotonin - metabolism; Sodium-Calcium Exchanger - antagonists & inhibitors; Sodium-Calcium Exchanger - metabolism; Thiourea - analogs & derivatives; Thiourea - pharmacology
• ISSN: 0363-6143
• DOI: 10.1152/ajpcell.00258.2007

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