Inhibitory effect of forskolin on myosin phosphorylation-dependent and independent contractions in bovine tracheal smooth muscle.
In bovine tracheal smooth muscle, carbachol (CCh, 1 microM) and high K+ (72.7 mM) induced sustained increases in cytosolic Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction. Forskolin (FK, 1-10 microM) inhibited the CCh-induced increase in [Ca2+]i, MLC phosphorylation and force in parallel. In contrast, FK inhibited the high K(+)-induced contraction and MLC phosphorylation without changing [Ca2+]i. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), CCh (10 microM) and caffeine (20 mM) induced transient increase in [Ca2+]i and contractile force by releasing Ca2+ from cellular store. FK strongly inhibited the CCh-induced Ca2+ transient, but failed to inhibit the caffeine-induced Ca2+ transient. In the absence of external Ca2+, 12-deoxyphorbol 13-isobutylate (DPB, 1 microM) induced sustained contraction without increase in [Ca2+]i and MLC phosphorylation. FK inhibited this contraction without changing [Ca2+]i. In permeabilized muscle, Ca2+ induced contraction in a concentration-dependent manner. FK (10 microM) and cAMP (1-100 microM) shifted the Ca(2+)-force curve to the higher Ca2+ levels. CCh with GTP, GTP gamma S or DPB enhanced contraction in the presence of constant level of Ca2+. Forskolin and cAMP also inhibited the enhanced contractions in the permeabilized muscle. In the permeabilized, thiophosphorylated muscle, ATP induced contraction in the absence of Ca2+. cAMP (300 microM) had no effect on this contraction. These results suggest that forskolin inhibits agonist-induced contraction in tracheal smooth muscle by multiple mechanisms of action; 1) inhibition of MLC phosphorylation by reducing Ca2+ influx and Ca2+ release, 2) inhibition of MLC phosphorylation by changing the MLC kinase/phosphatase balance, and 3) inhibition of regulatory mechanism which is not dependent on MLC phosphorylation.
Tajimi
M
M
Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Japan.
Hori
M
M
Mitsui
M
M
Ozaki
H
H
Karaki
H
H
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D003864:Depression, Chemical; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000187:drug effects*; D009130:Muscle, Smooth / Q000187:drug effects*; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D014132:Trachea / Q000187:drug effects
D018994:Myosin Light Chains; D005576:Colforsin; D000242:Cyclic AMP; D002118:Calcium