Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors

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Abstract
1 The regulation of cytosolic Ca2+ concentrations ([Ca2+]i) during exposure to carbachol was measured directly in canine cultured tracheal smooth muscle cells (TSMCs) loaded with fura-2. Stimulation of muscarinic cholinoceptors (muscarinic AChRs) by carbachol produced a dose-dependent rise in [Ca2+]i which was followed by a stable plateau phase. The EC50 values of carbachol for the peak and sustained plateau responses were 0.34 and 0.33 μm, respectively. 2 Atropine (10 μm) prevented all the responses to carbachol, and when added during a response to carbachol, significantly, but not completely decreased [Ca2+]i within 5 s. Therefore, the changes in [Ca2+]i by carbachol were mediated through the muscarinic AChRs. 3 AF-DX 116 (a selective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a selective M3 antagonist) inhibited the carbachol-stimulated increase in [Ca2+]i with pKB values of 6.4 and 9.4, respectively, corresponding to low affinity for AF-DX 116 and high affinity for 4-DAMP in antagonizing this response. 4 The plateau elevation of [Ca2+]i was dependent on the presence of external Ca2+. Removal of Ca2+ by the addition of 2 mm EGTA caused the [Ca2+]i to decline rapidly to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by the addition of Ca2+ (1.8 mm) in the continued presence of carbachol. 5 Ca2+ influx was required for the changes of [Ca2+]i, since the Ca2+-channel blockers, diltiazem (10 μm), nifedipine (10 μm), verapamil (10 μm) and Ni2+ (5 mm), decreased both the initial and sustained elevation of [Ca2+]i in response to carbachol. These Ca2+-channel blockers also decreased the sustained elevation of [Ca2+]i when applied during the plateau phase. 6 In conclusion, we have demonstrated that the initial detectable increase in carbachol-stimulated [Ca2+]i is due to the release of Ca2+ from internal stores, followed by the flux of external Ca2+ into the cells. This influx of extracellular Ca2+ partially involves an L-type Ca2+-channel. M3 muscarinic receptors appear to mediate the Ca2+ mobilization in canine TSMCs.