Ca(2+)-insensitive sustained contraction of skinned smooth muscle after acidic ADP treatment

After an acidic treatment in the presence of ADP, Triton X-100-skinned rabbit aortic smooth muscle strips were found to develop a large sustained, Ca(2+)-insensitive tension when returned to a relaxing solution with neutral pH. The presence of ADP during treatment was essential for the manifestation of the Ca(2+)-insensitive contraction. This contraction was reversibly eliminated by withdrawal of MgATP or addition of vanadate and was found to be accompanied by an extraordinarily high level of 20-kDa myosin light-chain (MLC20) phosphorylation. The rate constant for dephosphorylation of MLC20 in treated strips was about one-twenty-fifth that in untreated control, when determined after removal of Ca2+, Mg2+, and ATP. Two-dimensional phosphopeptide mapping of tryptic digests of MLC20 showed that most incorporated phosphate was in the peptides which would be phosphorylated by myosin light-chain kinase. These results provide strong evidence that ADP inactivates myosin light-chain phosphatase under acidic conditions.