Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors.
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 microM, respectively. 2. Atropine (10 microM) 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 119 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 microM), nifedipine (10 microM), verapamil (10 microM) and Ni2+ (5 mM), decreased both the initial and sustained elevation of [Ca2+], in response to carbachol. These Ca2+-channel blockers also decreased the sustained elevation of [Ca2+], when applied during the plateau phase.6. In conclusion, we have demonstrated that the initial detectable increase in carbachol-stimulated[Ca2+]J 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.
Yang
C M
CM
Department of Pharmacology, Chang Gung Medical College, Tao-Yuan, Taiwan.
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Y L
YL
Wang
Y Y
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D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D007424:Intracellular Fluid / Q000378:metabolism; D008297:Male; D018727:Muscarinic Antagonists; D009130:Muscle, Smooth / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D011976:Receptors, Muscarinic / Q000145:classification / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013268:Stimulation, Chemical; D014132:Trachea / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D004533:Egtazic Acid; D002217:Carbachol; D002118:Calcium