Ca²⁺ sensitization of smooth muscle contractility induced by ruthenium red

Abstract

The effects of ruthenium red (RuR) on contractility were examined in skinned fibers of guinea pig smooth muscles, where sarcoplasmic reticulum function was destroyed by treatment with A-23187. Contractions of skinned fibers of the urinary bladder were enhanced by RuR in a concentration-dependent manner (EC50 = 60 μM at pCa 6.0). The magnitude of contraction at pCa 6.0 was increased to 320% of control by 100 μM RuR. Qualitatively, the same results were obtained in skinned fibers prepared from the ileal longitudinal smooth muscle layer and mesenteric artery. The maximal contraction induced by pCa 4.5 was not affected significantly by RuR. The enhanced contraction by RuR was not reversed by the addition of guanosine 5′- O-(2-thiodiphosphate) or a peptide inhibitor of protein kinase C [PKC-(19—31)]. The application of microcystin, a potent protein phosphatase inhibitor, induced a tonic contraction of skinned smooth muscle at low Ca2+ concentration ([Ca2+]; pCa > 8.0). RuR had a dual effect on the microcystin-induced contraction-to- enhancement ratio at low concentrations and suppression at high concentrations. The relaxation following the decrease in [Ca2+] from pCa 5.0 to >8.0 was significantly slowed down by an addition of RuR. Phosphorylation of the myosin light chain at pCa 6.3 was significantly increased by RuR in skinned fibers of the guinea pig ileum. These results indicate that RuR markedly increases the Ca2+ sensitivity of the contractile system, at least in part via inhibition of myosin light chain phosphatase.

Pharmacological Modulation of Sarcoplasmic Reticulum Function in Smooth Muscle

“…Ureter Guinea pig Isolated strips; double sucrose-gap; membrane potential, contractile activity 10 mM Burdyga and Magura, 1986 Urinary bladder Guinea pig Isolated strips; microelectrode, isometric dynamometry; membrane potential, contractile activity 10 mM Fujii et al, 1990 Urinary bladder Guinea pig Isolated tissue; single sucrose-gap, microelectrode; membrane potential, contractile activity 1, 7, and 15 mM Kurihara and Sakai, 1976b Urinary bladder Guinea pig Isolated tissue; single sucrose-gap; membrane potential, contractile activity 15 mM Kurihara, 1975 Vas deferens Rat Isolated tissue; 45 Ca 2ϩ cellular loading, ͓ 3 H͔-myoinositol incubated, isometric dynamometry; Ca 2ϩ fluxes, accumulation of inositol phosphates, contractile activity 10 mM Khoyi et al, 1993 Vas deferens Rat Isolated segments; isometric dynamometry; contractile activity 10 mM Huang, 1995Huang, 484 1999. Conversely, the relaxation of ileal longitudinal smooth muscle induced by decreasing [Ca 2ϩ ] is reduced by ruthenium red (100 M) (Yamada et al, 1999). These effects are mediated by a direct concentration-dependent inhibition of MLCP, as shown with MLCP purified from guinea pig ileum longitudinal smooth muscle (IC 50 ϭ 23 M) (Yamada et al, 2000).…”

Hypoxia enhances a cGMP-independent nitric oxide relaxing mechanism in pulmonary arteries

“…Because the cGMP-independent mechanism of relaxation to NO appeared to be expressed in the presence of multiple contractile agents that are thought to function through a variety of different signaling systems, approaches were developed to probe some of the key control sites known to be influenced by relaxing mechanisms, including processes that lower intracellular Ca 2ϩ through inhibition of intracellular release, enhancement of intracellular reuptake by SERCA, or attenuation of extracellular influx. In addition, the action of an inhibitor of MLC phosphatase (500 nM microcystin-LR) was examined to detect whether activation of this relaxing mechanism (9,16,27) was involved in the cGMP-independent actions of NO. Therefore, the objective of this study is to characterize the novel observation that hypoxia controls the expression of a cGMP-independent relaxation to NO and to investigate the role of signaling mechanisms that potentially contribute to the expression of vascular relaxation.…”

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

“…This preparation was treated with β-escin to permeabilize the plasma membrane and with the Ca# + ionophore A23187 to destroy SR function. The tonic contraction in pCa 6n3 solution was enhanced by the addition of 300 µM RuR, as described previously [28]. The Ca# + sensitizing effect of RuR was reversed on washout.…”

“…We were therefore surprised to find that, in spite of the demonstrated inhibition of MLCK by RuR via blockade of Ca# + binding to CaM [27], the application of RuR to permeabilized strips prepared from several types of smooth muscle markedly increased the Ca# + sensitivity of the contractile system, concomitant with an increase in LC #! phosphorylation [28]. The features of RuR-induced Ca# + sensitization in permeabilized smooth-muscle strips, e.g.…”