Calcium mobilization by muscarinic cholinergic stimulation in bovine single airway smooth muscle.
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.
Department of Physiology, University of Massachusetts Medical School, Worcester 01655.
J
J
Kajita
H
H
Yamaguchi
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D002217:Carbachol / Q000494:pharmacology*; D002417:Cattle; D002470:Cell Survival; D004075:Diglycerides / Q000494:pharmacology; D016257:Fura-2; D005711:Gallopamil / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D014132:Trachea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D015220:Calcium Channels; D004075:Diglycerides; C044295:1,2-dioctanoylglycerol; D005711:Gallopamil; D002217:Carbachol; D002118:Calcium; D016257:Fura-2