Effect of 2,3-butanedione monoxime on force of contraction and protein phosphorylation in bovine smooth muscle

The aim of the study was to investigate the effects of the putative protein phosphatase (PP) activator 2,3-butanedione monoxime (BDM) in vascular smooth muscle. BDM concentration-dependently increased PP activity in homogenates of bovine coronary arteries and led to dephosphorylation of various smooth muscle proteins in P-32-labeled bovine aortic smooth muscle cells. In isolated bovine coronary artery rings (CARs) the effects of 10 mmol/l BDM on force of contraction (FOC) under conditions of depolarization by 75 mmol/l KCl and PP inhibition by 100 mu mol/l cantharidin were investigated. At the end of contraction experiments CARs were freeze-clamped and myosin light chain (MLC20) phosphorylation was determined by two-dimensional gel electrophoresis. Pretreatment of CARs with BDM reduced KCl-induced FOC to 42 +/- 3% vs. 118 +/- 1% (no BDM) and cantharidin-induced FOC to 102 +/- 2% vs. 120 +/- 7%; (no BDM) compared to a former KCI contraction (= 100%;). Moreover, BDM increased the amount of unphosphorylated MLC20, up to 56 +/- 2% vs. 36 +/- 5% (no BDM) and 38 +/- 2% vs. 21 +/- 1% (no BDM), respectively, demonstrating the central role of MLC20 phosphorylation in initiating smooth muscle contraction. In KCl precontracted CARs BDM decreased FOC to 37 +/- 3% vs. 100 +/- 1% (no BDM) but did not affect MLC20 phosphorylation, suggesting an uncoupling of force maintenance and MLC20 phosphorylation. In contrast, BDM neither affected FOC nor MLC20 phosphorylation in CARs precontracted with cantharidin. These results strengthen the hypothesis that PP activation by BDM only occurs on the holoenzyme level, e.g. by affecting regulatory subunits.