COMPARISON OF THE EFFECTS OF 2,3-BUTANEDIONE MONOXIME ON FORCE PRODUCTION, MYOSIN LIGHT-CHAIN PHOSPHORYLATION AND CHEMICAL ENERGY USAGE IN INTACT AND PERMEABILIZED SMOOTH AND SKELETAL-MUSCLES

The primary goal of this study was to determine the utility of 2,3-but anedione monoxime as a tool for determining and separating the chemica l energy usage associated with force production from that of force-ind ependent, or'activation' processes in smooth and skeletal muscles. We determined the effects of 2,3-butanedione monoxime on force production , myosin light chain phosphorylation and high energy phosphate usage i n intact and permeabilized smooth (rabbit taenia coli) and skeletal (m ouse extensor digitorum longus) muscles. In the intact taenia coli, 2, 3-butanedione monoxime depressed the tonic phase of the tetanus, contr actures evoked by high potassium (90 mM) and by carbachol (10(-5) M) a nd the small force response evoked by these agonists after treatment w ith D-600 (10(-5) M). In the electrically stimulated intact taenia col i 2,3-butanedione monoxime (0-20 mM) caused a proportional inhibition of tetanic force output, myosin light chain phosphorylation and high e nergy phosphate usage (ED(50) similar to 7 mM for all these parameters ). At 20 mM 2,3-butanedione monoxime, force and energy usage fell to n ear zero and the degree of myosin light chain phosphorylation decrease d to resting values, indicating a shut-down of both force-dependent an d force-independent energy usage at high concentrations of 2,3-butaned ione monoxime. In permeabilized taenia coli, 2,3-butanedione monoxime had little or no depressant effects on force production, ATPase activi ty or calcium sensitivity. 2,3-butanedione monoxime had a very modest inhibitory effect on the in vitro motility of unregulated actin filame nts interacting with thiophosphorylated myosin. In solution, 2,3-butan edione monoxime inhibited myosin light chain kinase, but not the phosp hatase (SMP-IV). These results suggest that the major effect of 2,3-bu tanedione monoxime is not on the contractile proteins themselves, but rather on calcium delivery during excitation, thereby reducing the deg ree of activation of myosin light chain kinase and subsequent activati on of myosin by light chain phosphorylation. Thus, 2,3-butanedione mon oxime is not useful for the determination of the energetics of activat ion processes in smooth muscle because of its inhibition of both force -dependent and force-independent processes. In contrast, in the intact mouse extensor digitorum longus, 2,3-butanedione monoxime inhibits te tanic force production (ED(50) similar to 2 mM) to a much greater exte nt than myosin light chain phosphorylation. When 2,3-butanedione monox ime was used to manipulate force production in muscles at L(0) it was found that similar to 60% of the total energy usage was force-independ ent and the remainder was force-dependent. In the permeabilized extens or digitorum longus treated with 12 mM 2,3-butanedione monoxime, there was a decrease in calcium-activated force production and a decrease i n calcium sensitivity. The effects of 2,3-butanedione monoxime were co nsiderably greater in the intact than in the permeabilized mouse exten sor digitorum longus. At 2,3-butanedione monoxime concentrations that block force production in the intact muscle, the effects on in vitro m otility were small, yet far greater than those on smooth muscle myosin . These results suggest that 2,3-butanedione monoxime has a direct eff ect on the contractile proteins, but what cannot be ignored is the dec rease in myosin light chain phosphorylation in the skeletal muscle, wh ich, like the decreased force output, may result from a reduction in c alcium release from the sarcoplasmic reticulum. For these reasons, the use of 2,3-butanedione monoxime to probe the components of energy usa ge during the contraction of skeletal muscle requires considerable cau tion and a full definition of its actions.