Research Paper
Subject Category: Cardiovascular and pulmonary pharmacology
British Journal of Pharmacology (2007) 152, 101–111; doi:10.1038/sj.bjp.0707357; published online 25 June 2007
Role of InsP3 and ryanodine receptors in the activation of capacitative Ca2+ entry by store depletion or hypoxia in canine pulmonary arterial smooth muscle cells
L C Ng1,3, S M Wilson2,3, C E McAllister1 and J R Hume1
- 1Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA
- 2Department of Pharmacology, University of Mississippi School of Pharmacy, University, MS, USA
Correspondence: Professor JR Hume, Department of Pharmacology/318, School of Medicine, University of Nevada, 1664 North Virginia Street, Reno, NV 89557, USA. E-mail: joeh@med.unr.edu
3These authors contributed equally to this work.
Received 20 April 2007; Revised 25 May 2007; Accepted 29 May 2007; Published online 25 June 2007.
Abstract
Background and purpose:
Experiments were performed to determine if capacitative Ca2+ entry (CCE) in canine pulmonary arterial smooth muscle cells (PASMCs) is dependent on InsP3 receptors or ryanodine receptors as induction of CCE is dependent on simultaneous depletion of the functionally separate InsP3- and ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ 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 Ca2+ were made by imaging fura-2 loaded individual myocytes that were perfused with physiological buffered saline solution with or without Ca2+.
Key results:
Treating myocytes with 10 
M cyclopiazonic acid (CPA), removing extracellular Ca2+, and briefly applying 10 mM caffeine and 10 M 5-hydroxytryptamine (5-HT) depleted SR Ca2+ stores. Extracellular Ca2+ reintroduction caused cytosolic [Ca2+] to elevate above baseline signifying CCE. The InsP3 receptor inhibitors 2-aminobiphenylborate (50-75 M; 2-APB) and xestospongin-C (20 M; XeC) abolished CCE. Yet, CCE was unaffected by 10 M or 300 M ryanodine or 10 M dantrolene, which modify ryanodine receptor activity. Higher dantrolene concentrations (50 M), however, can inhibit both ryanodine receptors and InsP3 receptors, did reduce CCE. In contrast, CCE activated by hypoxia was unaffected by XeC (20 M).
Conclusions and implications:
The results provide evidence that CCE activated by depletion of both InsP3 and ryanodine SR Ca2+ stores in canine PASMCs is dependent on functional InsP3 receptors, whereas the activation of CCE by hypoxia appears to be independent of functional InsP3 receptors.
Keywords:
fura-2, sarcoplasmic reticulum, xestospongin-C, 5-hydroxytryptamine, 2-aminobiphenylborate, dantrolene, cyclopiazonic acid, hypoxia
Abbreviations:
2-APB, 2-aminobiphenylborate; [Ca2+]i, intracellular Ca2+ concentration; CCE, capacitative Ca2+ entry; CIF, calcium influx factor; CPA, cyclopiazonic acid; CRAC, calcium release-activated channels; DMSO, dimethyl sulphoxide; HPV, hypoxic pulmonary vasoconstriction; InsP3, inositol 1,4,5-triphosphate; ISOC, store-operated currents; PASMCs, pulmonary arterial smooth muscle cells; PSS, physiological saline solution; SERCA, sarcoplasmic–endoplasmic reticulum Ca2+ ATPase; SR, sarcoplasmic reticulum; TRPC, canonical transient receptor potential; XeC, xestospongin-C
