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 32P-labelled 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 micromol/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+/-4% vs. 118+/-1% (no BDM) and cantharidin-induced FOC to 102+/-2% vs. 120+/-7% (no BDM) compared to a former KCl contraction (= 100%). Moreover, BDM increased the amount of unphosphorylated MLC20 up to 56+/-2% vs. 36+/-5% (no BDM) and 28+/-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 47+/-4% 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.
Waurick
R
R
Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität Münster, Germany.
Knapp
J
J
Van Aken
H
H
Bokník
P
P
Neumann
J
J
Schmitz
W
W
D000818:Animals; D001345:Autoradiography; D002193:Cantharidin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000187:drug effects; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation
D009124:Muscle Proteins; D018994:Myosin Light Chains; C004717:diacetylmonoxime; D002193:Cantharidin; D003931:Diacetyl