Role of InsP ₃ and ryanodine receptors in the activation of capacitative Ca ²⁺ entry by store depletion or hypoxia in canine pulmonary arterial smooth muscle cells

Background and purpose: Experiments were performed to determine if capacitative Ca 2+ entry (CCE) in canine pulmonary arterial smooth muscle cells (PASMCs) is dependent on InsP 3 receptors or ryanodine receptors as induction of CCE is dependent on simultaneous depletion of the functionally separate InsP 3- and ryanodine-sensitive sarcoplasmic reticulum (SR) Ca 2+ stores in these cells. Experimental approach: Myocytes were isolated from canine pulmonary arteries using enzymatic procedures and were used within 8 h of preparation. Measurements of cytosolic Ca 2+ were made by imaging fura-2 loaded individual myocytes that were perfused with physiological buffered saline solution with or without Ca 2+. Key results: Treating myocytes with 10 μM cyclopiazonic acid (CPA), removing extracellular Ca 2+, and briefly applying 10 mM caffeine and 10 μM 5-hydroxytryptamine (5-HT) depleted SR Ca 2+ stores. Extracellular Ca 2+ reintroduction caused cytosolic [Ca 2+] to elevate above baseline signifying CCE. The InsP 3 receptor inhibitors 2-aminobiphenylborate (50-75 μM; 2-APB) and xestospongin-C (20 μM; XeC) abolished CCE. Yet, CCE was unaffected by 10 μM or 300 μM ryanodine or 10 μM dantrolene, which modify ryanodine receptor activity. Higher dantrolene concentrations (50 μM), however, can inhibit both ryanodine receptors and InsP 3 receptors, did reduce CCE. In contrast, CCE activated by hypoxia was unaffected by XeC (20 μM). Conclusions and implications: The results provide evidence that CCE activated by depletion of both InsP 3 and ryanodine SR Ca 2+ stores in canine PASMCs is dependent on functional InsP 3 receptors, whereas the activation of CCE by hypoxia appears to be independent of functional InsP 3 receptors. © 2007 Nature Publishing Group All rights reserved.