GTP gamma S-dependent regulation of smooth muscle contractile elements

Abstract

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) increases the sensitivity of the contractile response to activation by Ca2+ in permeabilized tracheal smooth muscle. Increased tension was associated with a proportional increase in myosin light chain phosphorylation. The site of phosphorylation was determined to be serine-19, which corresponds to the site rapidly phosphorylated by myosin light chain kinase. GTP gamma S did not affect the contraction induced by the protein phosphatase inhibitor okadaic acid but did enhance contraction produced by Ca(2+)-independent myosin light chain kinase. In tracheal homogenates Ca(2+)-dependent myosin light chain kinase activity was not affected by GTP gamma S; however, dephosphorylation of 32P-labeled heavy meromyosin by phosphatase was inhibited. Thus GTP gamma S may increase the Ca2+ sensitivity of contractile elements in tracheal smooth muscle by inhibition of protein phosphatase activity toward myosin light chain.

Rho-associated Kinase Directly Induces Smooth Muscle Contraction through Myosin Light Chain Phosphorylation

“…Except for Ca 2ϩ -independent MLC kinase (9, 13), we find no documentation that the exogenous addition of kinases to the cytosol of permeabilized smooth muscle directly exerts contractile responses comparable with findings with CAT. Because the inhibition of myosin phosphatase may possibly be the main mechanism of the G-protein-mediating Ca 2ϩ sensitization of smooth muscle contraction (1,9,24,25,27), the CAT-induced contraction of G-protein-uncoupled smooth muscle permeabilized by Triton X-100 (Fig. 1) may be also mediated by the inhibition of myosin phosphatase.…”

“…However, as the use of Ca 2ϩ indicator revealed that the force/ Ca 2ϩ ratio is variable, the Ca 2ϩ -CaM-dependent MLC kinase pathway cannot solely account for the mechanisms of agonistor GTP␥S-induced increases in the force/Ca 2ϩ ratio, so-called Ca 2ϩ sensitization (1,(5)(6)(7)(8)(9). Thus, an additional mechanism that can regulate Ca 2ϩ sensitization of smooth muscle has been proposed.…”

“…Using membrane permeabilization of smooth muscle, the possibility that monomeric Ras family G-proteins, such as Rho, contribute to Ca 2ϩ sensitization of smooth muscle was demonstrated (10 -12). Direct activation of G-proteins by the application of GTP␥S (8,9), agonists (1,(5)(6)(7)(8), and GTP-activated Rho (10 -12) could exert Ca 2ϩ -sensitizing effects on saponin-or ␤-escin-permeabilized smooth muscle. However, the activated Rho failed to induce Ca 2ϩ sensitization of extensively Triton X-100-permeabilized smooth muscle (11).…”

Pertussis toxin‐sensitive Gi‐proteins and intracellular calcium sensitivity of vasoconstriction in the intact rat tail artery

“…Myosin phosphorylation is the ®nal determinant of smooth muscle contraction; [Ca 2+ ] i sensitivity of tension is correlated to [Ca 2+ ] i sensitivity of myosin light chain (MLC 20 ) phosphorylation (Ikebe et al, 1988;Rembold, 1990). Myosin phosphorylation is modulated by MLC 20 -kinase and MLC 20 -phosphatase which are under the control of G-proteins (Kitazawa et al, 1991;Kubota et al, 1992). Pertussis toxin (PTX), an inhibitor of G i/o -proteins, reduces the vasoconstrictor response to aadrenoceptor agonists in the rat tail artery (Li & Triggle, 1993).…”

Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape.

“…Although in tracellular Ca^"^ levels are the main regulator of MLCK activity, other mechanisms can alter the state of Ser^^ phosphorylation levels while Ca^^ levels remain con stant. In particular, it has been suggested that signals deriving from the Rho family of small GTP-binding pro teins lead to inhibition of myosin phosphatase activity, thus altering Ser'^ phosphorylation levels (Fujiwara et al 1989;Kitazawa et al 1989Kitazawa et al , 1991Kubota et al 1992;Noda et al 1995;Gong et al 1996). Kimura et al (1996) have shown recently that a Rho-binding Ser/Thr kinase (Rho-kinase) can phosphorylate and thereby inhibit the activity of a myosin phosphatase in vitro.…”