The effect of extracellular Ca2+ on responses to purinoceptor agonists in cultured swine tracheal smooth muscle cells.

Abstract

The effects of extracellular Ca2+ ions on purinergic responses were examined in swine tracheal smooth muscle cells (TSMCs) in primary culture. ATP (1 microM to 1 mM) and alpha, beta-methylene adenosine 5' triphosphate (alpha, beta-Me ATP) (100 microM and 1 mM) concentration-dependently increased [Ca2+]i in the presence and the absence of extracellular Ca2+. Responses to ATP (10 microM to 1 mM) in the presence of extracellular Ca2+ were significantly larger than those in its absence (n=8), whereas those to alpha, beta-Me ATP were not significantly different between the presence (n=7) and the absence (n=8) of extracellular Ca2+. Responses to ATP (1 mM) at extracellular Ca2+ concentration ([Ca2+]o) of 10 and 5 mM were significantly larger than that at extracellular EGTA concentration ([EGTA]o) of 1 mM (p< 0.01, n=5), whereas the responses to alpha, beta-Me ATP (1 mM) at 10 and 5 mM [Ca2+]o were significantly smaller than that at [EGTA]o of 1 mM (p<0.05, n=5). Increasing [Ca2+]o to 5 mM after the application of either 1 mM ATP (n=4) or 1 mM alpha, beta-Me ATP (n=4) in the absence of extracellular Ca2+ (1 mM [EGTA]o) further increased [Ca2+]i, though the increases in [Ca2+]i by agonists in 1 mM [EGTA]o had been already maximal. Incubating cells for 300 s in 5 mM [Ca2+]o before the application of ATP (1 mM) significantly increased the response to the drug than that obtained by incubating cells for 6 sec in 5 mM [Ca2+]o before the drug application (p< 0.01, n=4). However, alpha, beta-Me ATP (1 mM) induced similar responses by incubating cells for 30 or 300 s in 5 mM [Ca2+]o to that by incubating them for 6 s. These results suggest that the effect of alpha, beta-Me ATP in swine TSMCs in primary culture is mainly through Ca2+ release and that its effect on Ca2+ entry is smaller than other nucleotides.