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 μ M cyclopiazonic acid (CPA), removing extracellular Ca 2+ , and briefly applying 10 mM caffeine and 10 μ M 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 μ M; 2‐APB) and xestospongin‐C (20 μ M; XeC) abolished CCE. Yet, CCE was unaffected by 10 μ M or 300 μ M ryanodine or 10 μ M dantrolene, which modify ryanodine receptor activity. Higher dantrolene concentrations (50 μ M), however, can inhibit both ryanodine receptors and InsP 3 receptors, did reduce CCE. In contrast, CCE activated by hypoxia was unaffected by XeC (20 μ M). 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. British Journal of Pharmacology (2007) 152 , 101–111; doi: 10.1038/sj.bjp.0707357