Vanadate oxidation activates contraction in permeabilized guinea
pig taenia coli without myosin light chain phosphorylation.
Lalli, M. Jane, K. Obara, Richard J. Paul.
Department of Molecular and Cellular Physiology, University of
Cincinnati, College of Medicine
APStracts 3:0227C, 1996.
Phosphorylation of the myosin regulatory light chain (LC20-Pi) is
generally accepted as the major route for activation of smooth
muscle. However, after prior exposure to vanadate (Vi), permeabilized
guinea pig taenia coli smooth muscle fibers contract in the absence
of LC20-Pi. We characterized the Vi-induced contraction and
investigated the mechanism of this novel activation pathway. As
previously reported, addition of Vi to a control contracture (6.6 109
\f M Ca2+) inhibits force (ED50 = 100 (M). In contrast, pre
-incubation with high concentrations of Vi (threshold at 1-2 mM)
elicited a contraction upon subsequent transfer of the fiber to a Vi
-free, Ca2+-free solution. Maximum force of 60% of control was
obtained in fibers pre-incubated in 4 mM Vi for 10 min. This Vi
-induced force was complementary to that elicited by Ca2+, in that
addition of Ca2+ increased force but the total force never exceeded
the initial control. Moreover, after maximal thiophosphorylation of
LC20 with ATP 103 \f S, treatment with Vi did not increase force.
Thus the force elicited by Vi was also complementary to that elicited
by LC20-Pi. Whereas force is an index of the number of activated
crossbridges, shortening velocity (Vmax) reflects crossbridge cycling
rate. Vmax was similar in either Ca2+ and Vi contractures. Addition
of Ca2+ to a Vi-treated fiber increased the velocity to 2-times that
of either alone. After thiophosphorylation, pre-incubation in Vi had
no effect on the Vmax suggesting that Vi affected the number of
activated bridges and not cycle rate. The mechanism of Vi
contractures likely involves oxidation as pre-incubation with 4 mM Vi
and 25 mM DTT did not produce force. Furthermore, after exposure to
Vi, DTT could reverse a Vi-induced contracture in 30 to 60 min.
Subsequently, fibers demonstrated control contraction/relaxation
cycles. Thus, Vi treatment did not cause irreversible damage, such as
the extraction of contractile or regulatory proteins. Potential sites
for this oxidation are proteins at 17 kDa and between 30-40 kDa,
which were not alkylated by N-ethylmaleimide if they were treated in
the presence of Vi or in the rigor state. Vi-induced contractures are
likely mediated by a reversible oxidation that activates crossbridges
similarly to that of LC20-Pi. Activation of smooth muscle by
oxidation may play an important role in oxidant injury.
Received 27 November 1995; accepted in final form 28 June 1996.
APS Manuscript Number C710-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 25 July 1996