VANADATE OXIDATION ACTIVATES CONTRACTION IN SKINNED SMOOTH-MUSCLE WITHOUT MYOSIN LIGHT-CHAIN PHOSPHORYLATION

Phosphorylation of the myosin regulatory light chain (LC20-P-i) is the major route of smooth muscle activation. However, after prior exposur e to vanadate, permeabilized guinea pig taenia coli smooth muscle cont racts in the absence of LC20-P-i. We characterized the vanadate-induce d contraction and investigated the mechanism of this novel activation pathway. Addition of vanadate to a control contracture (6.6 mu M Ca2+) inhibits force (effective dose for 50% response was similar to 100 mu M). In contrast, preincubation with high concentrations of vanadate ( threshold at 1-2 mM) elicited a contraction on subsequent transfer of the fiber to a vanadate-free, Ca2+-free solution. Maximum isometric fo rce of similar to 60% of control was obtained in fibers preincubated i n 4 mM vanadate for 10 min. Addition of Ca2+ to a vanadate-induced con tracture increased force, but the total force never exceeded the initi al control. After maximal thiophosphorylation of LC20 with adenosine 5 '-O-(3-thiotriphosphate), treatment with vanadate did not increase for ce. Unloaded shortening velocity (V-max) was similar in Ca2+ and vanad ate contractures and was additive. After thiophosphorylation, preincub ation in vanadate had no effect on V-max suggesting that vanadate affe cted the number of activated bridges and not cycle rate. Vanadate mech anisms likely involve oxidation, since preincubation with 4 mM vanadat e and 25 mM dithiothreitol (DTT) did not produce force. DTT could reve rse a vanadate-induced contracture in 30-60 min. Subsequently, fibers demonstrated control contraction/relaxation cycles. Thus vanadate trea tment did not cause irreversible damage, such as the extraction of pro teins. Potential oxidation sites are proteins at 17 kDa and between 30 and 40 kDa, which were not alkylated by N-ethylmaleimide if they were treated in the presence of vanadate or in the rigor state. Vanadate-i nduced contractures are Likely mediated by a reversible oxidation that activates cross bridges similarly to that of LC20-P-i and may play an important role in oxidant injury.