Myosin light chain phosphorylation and Mn2+-dependent acetylcholine-induced contractions in guinea pig vas deferens

We have reported that noradrenaline but not K+ induced a sustained and dose-dependent contraction without extracellular Ca2+ and Mn2+ in Ca2+-depleted Mn2+loaded vas deferens from the guinea pig. The Mn2+dependent noradrenaline-induced contractions developed without an increase in phosphorylation of 20-kDa myosin light chain. To clarify whether such an unusual Mn2+dependent contraction was induced only by noradrenaline or not, we examined effects of acetylcholine on Ca2+-depleted Mn2+-loaded guinea pig vas deferens. Acetylcholine (1 microM(-1) mM) induced a sustained dose-dependent contraction without extracellular Ca2+ or Mn2+. W-7 (10 microM or 100 microM) and wortmannin (1 microM) both reduced the Mn2+dependent acetylcholine-induced contractions similarly to Ca2+-dependent acetylcholine-induced contractions in isolated vas deferens without either Ca2+ depletion or Mn2+ loading. However, the Mn2+-dependent acetylcholine-induced contractions developed without a significant increase in the phosphorylation of the myosin light chain determined by urea-glycerol polyacrylamide gel electrophoresis and immunoblotting. These results indicate that acetylcholine as well as noradrenaline induces Mn2+-dependent contraction and are consistent with our previous assumption that Mn2+ may preferentially support receptor-mediated contractions in the guinea pig isolated vas deferens. The results also suggest that the activation of myosin light chain kinase is essential for the development of Mn2+-dependent acetylcholine-induced contractions, and that Mn2+ may accelerate formation of non-phosphorylated attached cross-bridges during receptor activation.