Cellular mechanisms for Ca2+ mobilization by muscarinic cholinergic stimulation were investigated in dispersed bovine airway smooth muscle cells by the fura-2 method. Because the dissociation constant (Kd) of the intracellular fura-2 was not known, we first determined the Kd as 386 +/- 6 nM, which ranged from 368 to 394 nM. By using the Kd, resting intracellular Ca2+ concentration ([Ca2+]i) was calculated to be 177 +/- 11 nM (n = 22). Carbachol (CCh) at 10(-7) - 10(-5) M caused two distinct increases in [Ca2+]i; an initial transient which reached 1.3-2.7 microM followed by a sustained increase in [Ca2+]i of 250-400 nM. The sustained Ca2+ elevation was not observed in the absence of extracellular Ca2+. D 600 (10(-5) - 3 x 10(-5) M) also eliminated this response. An analogue of diacylglycerol (DAG), 1,2-dioctanoyl-sn-glycerol, caused a gradual increase in [Ca2+]i to 303 +/- 24 nM (n = 13), which was equivalent to the sustained response to 10(-7) and 10(-6) M CCh and was again blocked by D 600. These findings are consistent with the idea that inositol 1,4,5-trisphosphate releases stored Ca2+ as the main cause for the initial transient and that activation of DAG by CCh may result in the sustained increase in [Ca2+]i through voltage-dependent Ca2+ channels.
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