Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

Abstract

To examine the nature of inositol 1,4,5-trisphosphate (IP3)-sensitive and ryanodine (Ryn)-sensitive Ca2+ stores in isolated canine pulmonary arterial smooth cells (PASMC), agonist-induced changes in global intracellular Ca2+ concentration ([Ca2+]i) were measured using fura 2-AM fluorescence. Properties of elementary local Ca2+ release events were characterized using fluo 3-AM or fluo 4-AM, in combination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca2+ stores with Ryn (300 μM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-induced intracellular Ca2+ transients but had little or no effect on the initial IP3-mediated intracellular Ca2+ transient induced by ANG II (1 μM). Cyclopiazonic acid (CPA; 10 μM) abolished IP3-induced intracellular Ca2+ transients but failed to attenuate the initial Caf-induced intracellular Ca2+ transient. These results suggest that in canine PASMC, IP3-, and Ryn-sensitive Ca2+ stores are organized into spatially distinct compartments while similar experiments in canine renal arterial smooth muscle cells (RASMC) reveal that these Ca2+ stores are spatially conjoined. In PASMC, spontaneous local intracellular Ca2+ transients sensitive to modulation by Caf and Ryn were detected, exhibiting spatial-temporal characteristics similar to those previously described for "Ca2+ sparks" in cardiac and other types of smooth muscle cells. After depletion of Ryn-sensitive Ca2+ stores, ANG II (8 nM) induced slow, sustained [Ca2+]i increases originating at sites near the cell surface, which were abolished by depleting IP3 stores. Discrete quantal-like events expected due to the coordinated opening of IP3 receptor clusters ("Ca2+ puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca2+ stores and elementary Ca2+ release events in isolated PASMC.