Inhibition of smooth-muscle myosin-light-chain phosphatase by Ruthenium Red

Ruthenium Red (RuR) is widely used as an inhibitor of ryanodine receptor Ca 2+ release channels, but has additional effects such as the induction of Ca 2+ sensitization of contraction of permeabilized smooth muscles. To address the mechanism underlying this process, we examined the effects of RuR on contractility in permeabilized guinea-pig ileum and on the activity of myosin-light-chain phosphatase (MP). RuR increased the force at submaximal [Ca 2+ ] (pCa 6.3) approx. 4-fold. This effect was not observed after thiophosphorylation of MP. RuR also seemed capable of preventing the thiophosphorylation of MP, suggesting a direct interaction of RuR with MP. Consistent with this possibility, smooth-muscle MP was inhibited by RuR in a concentration-dependent manner (IC 50 23 µ M). Exogenous calmodulin significantly increased RuR-induced contraction at pCa 6.3 but had little effect on contraction induced by microcystin at this [Ca 2+ ]. Ca 2+ -independent contraction was induced by RuR (EC 50 843 µ M) and by microcystin (EC 50 59nM) but the maximal force induced by RuR was smaller than that induced by microcystin. The addition of 300 µ M RuR enhanced the contraction induced by 30nM microcystin but markedly decreased that induced by 1 µ M microcystin. Such a dual action of RuR on microcystin-induced effects was not observed in experiments using purified MP. We conclude that the RuR-induced Ca 2+ sensitization of smooth-muscle contraction is due to the direct inhibition of MP by RuR.