title: Cell culture alters Ca2+ entry pathways activated by store-depletion or hypoxia in canine pulmonary arterial smooth muscle cells.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557, USA.
authors.forename: Lih Chyuan
authors.initials: LC
authors.lastname: Ng
authors.forename: Barry D
authors.initials: BD
authors.lastname: Kyle
authors.forename: Alison R
authors.initials: AR
authors.lastname: Lennox
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Shen
authors.forename: William J
authors.initials: WJ
authors.lastname: Hatton
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Hume
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D009119:Muscle Contraction; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D011651:Pulmonary Artery / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D000199:Actins; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007211:Indoles; D019837:Ryanodine Receptor Calcium Release Channel; D019831:Sodium-Calcium Exchanger; D012701:Serotonin; D002110:Caffeine; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D013890:Thiourea; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D009543:Nifedipine; D010100:Oxygen; D002118:Calcium; C000543:cyclopiazonic acid