Thapsigargin-induced tone and capacitative calcium influx in mouse anococcygeus smooth muscle cells.
The sarcoplasmic reticulum Ca-ATPase inhibitor thapsigargin (Tg; 0.4-100 nM) produced concentration-related, strong and sustained contractions of the mouse-isolated anococcygeus muscle; these contractions were dependent on extracellular calcium but were only partially reduced (by about 50%) in the presence of verapamil (10 and 100 microM). The verapamil-resistant component of the Tg-induced contraction was relaxed by the general calcium entry blockers SKF96365 (0.4-40 microM) and cadmium (50-300 microM), and by the tyrosine kinase inhibitor genistein (10-180 microM). In single smooth muscle cells loaded with Fura-2, addition of Tg (100 nM) to calcium-free medium produced a small, transient increase in fluorescence; subsequent addition of calcium (2.5 mM) produced a larger and sustained increase which was abolished on return to calcium-free conditions, but was only partially reduced by verapamil (10 microM; by about 30%). Manganese quenching of Fura-2 was enhanced in cells treated with Tg. The verapamil-resistant calcium influx was reduced by SKF96365 (20 microM) and to a lesser extent by genistein (40 microM); cadmium (200 microM) produced an initial decrease in fluorescence followed by a marked increase. These results demonstrate that, in the mouse anococcygeus, Tg can cause sustained contractions and elevations of calcium influx in the presence of verapamil; the time-course, calcium dependence and, although to a lesser extent, pharmacology of these effects generally support the proposal that excitation-contraction coupling in this tonic smooth muscle involves sustained capacitative calcium influx.
Wallace
P
P
Messengers and Signalling Research Group, School of Biomedical Sciences, King's College London, UK.
Ayman
S
S
McFadzean
I
I
Gibson
A
A
D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005453:Fluorescence; D016257:Fura-2; D019833:Genistein / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008345:Manganese / Q000494:pharmacology; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects*; D009130:Muscle, Smooth / Q000187:drug effects*; D019284:Thapsigargin / Q000494:pharmacology*; D013997:Time Factors; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D007093:Imidazoles; D002104:Cadmium; D008345:Manganese; D019284:Thapsigargin; D014700:Verapamil; D019833:Genistein; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium; D016257:Fura-2