Permeabilized smooth muscle

Although in in vivo circumstances an intracellular free calcium increase apparently acts as the primary modulator of contraction, it can be bypassed in highly permeabilized smooth muscle preparations where the active subunit of MLCK can be introduced to phosphorylate myosin and induce contraction. The MLCK catalyzed phosphorylation of serine-19 is seen as the necessary event in the activation of smooth muscle myosin to form crossbridges. Thus, the rising phase of force during an isometric smooth muscle contraction follows an increase in the degree of phosphorylation of myosin, and that in turn follows the transient rise of (a) cytosolic free Ca, (b) Ca-calmodulin complexes, and (c) the active form of MLCK. The regulation of the intracellular calcium is discussed below. The dynam-... 

Kitazawa T, Kobayashi S, Horiuti K, Somlyo AV, Somlyo AP 1989 Receptor coupled, permeabilized smooth muscle role of the phosphatidylinositol cascade, G proteins and modulation of the contractile response to Ca2+. J Biol Chem 264 5339-5342 Lopez-Lopez JR, Shacklock PS, Balke CW, Wier WG 1995 Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science 268 1042-1045 Marks AR, Fleischer S, Tempst P 1990 Surface topography analysis of the ryanodine receptor/ junctional channel complex based on proteolysis sensitivity mapping. J Biol Chem 265 13143-13149... 

Kitazawa T, Kobayashi S, Horiuti K et al. (1989) Receptor coupled, permeabilized smooth muscle Role of the phosphatidylinositol cascade, G-proteins and... 

IV. KINETIC INVESTIGATIONS IN PERMEABILIZED SMOOTH MUSCLE USING CAGED COMPOUNDS... 

By using caged compounds in combination with permeabilized smooth muscle preparations, valuable information regarding the rate-limiting steps between excitation and contraction (see Somlyo and Somlyo, 1990, for review Somlyo et al., 1992), regulation of Ca + release from the SR (Walker et al., 1987 lino and Endo, 1992), and the kinetics of the cross-bridge cycle (Arner et al., 1987 Somlyo et al, 1988 Fuglsang et al,... 

In triton skinned and in a-toxin-permeabilized smooth muscle preparations, the Ca + sensitivity of force production is also decreased by cGMP (Pfitzer et al., 1984,1986 Nishimura efflZ., 1992). This may be due to an up-regulation of MLCP (Pfitzer etal., 1986). Clear evidence that modulation of the activity of MLCP would affect Ca + sensitivity of tension development was in fact obtained in triton skinned smooth muscle when it was shown that inhibition of MLCP by the black sponge toxin, okadaic acid, increased Ca + sensitivity (Takai et al., 1987 Bialojan et al., 1988). On the other hand, incubation of triton skinned chicken gizzard fibers with a purified phosphatase decreased Ca + sensitivity (Bialojan et al., 1987). 

VI. STUDIES ON THE Ca2+ RELEASE FROM INTRACELLULAR STORES IN PERMEABILIZED SMOOTH MUSCLE ... 

In general, the stores are loaded with Ca + buffered with millimolar concentrations of EGTA (Endo et al., 1977 Saida and Nonomura, 1978 Itoh et al., 1982a,b Saida, 1982). The stores are fully loaded within 3 to 5 min at 1 (jlM Ca2+ (Saida, 1982), whereby the Ca2+ uptake depends on the Ca + concentration (Saida, 1982 Yamamoto and van Breemen, 1986). At Ca + concentrations greater than 1 jlM, a Ca +-induced Ca release was observed (Itoh et al., 1981 Saida, 1982). The deposition of Ca2+ in the SR of saponin-permeabilized smooth muscle was demonstrated by electron probe X-ray microanalysis (Kowasaki et al.,... 

A powerful new approach is the direct measurement of released Ca + with fluorescent Ca + indicators such as fura-2 or fluo-3, whereby the permeabilized smooth muscle has to be placed into microcuvettes (lino, 1991). Since force is no longer required as an indicator, Ca + release may be measured in the absence of MgATP, which allowed the study of adenine nucleotides on the IP3-induced Ca + release without the interference by Ca + uptake (lino, 1991 Hirose and lino, 1994). Inclusion of ryanodine allows a separate study of the IP3- and caffeine-sensitive stores (Hirose et al., 1993). Experimental protocols have been designed that allow the use of high concentrations of EGTA, thereby preventing Ca -mediated feedback regulation of the Ca2+ release (Hirose and lino, 1994). Direct measurement of Ca + release with indicators becomes even more powerful in combination with... 

CD is effective in inhibiting isometric force in Triton-permeabilized gizzard fibers (Szpacenko et al., 1985 Pfitzer et al., 1993). The actin-binding fragment of CD desensitizes the force-RLC phosphorylation relation, and the inhibitory effect is reversed by high concentrations of Ca +ZCaM (Pfitzer et al., 1993). In addition, CD peptides that act to displace CD from thin filaments are effective in enhancing force at fixed Ca + in isolated permeabilized smooth muscle cells (Katsuyama et al.,... 

Siegman MJ, Butler TM, Mooers SU (1989) Phosphatase inhibition with okadaic acid does not alter the relationship between force and myosin light chain phosphorylation in permeabilized smooth muscle. Biochem Biophys Res Commun 161 838-842... 

Trinkle-Mulcahy L, Ichikawa K, Hartshome DJ, Siegman MJ, Butler TM (1995) Thio-phosphorylation of the 130-kDa subunit associatesd with a decreased activity of myosin light chain phosphatase in a-toxin permeabilized smooth muscle. J Biol Chem 270 18191-18194... 

The Ca +-sensitizing effect of activation of heptameric serpentine receptors (e.g., muscarinic, i-adrenergic, endothelin, thromboxane A2, etc.) coupled by G-proteins to the phosphatidylinositol cascade is mimicked, in permeabilized smooth muscles, by GTPyS. These findings indicated the role of one or more G-proteins in Ca -sensitization (Fujiwara et al. 1989 Himpens et al. 1990 Kitazawa et al. 1991 Nishimura et al. 1992 Crichton et al. 1993 Yoshida et al. 1994 Fujihara et al. 1997 lizuka et al. 1997 rev. in Somlyo and Somlyo 1994), and raised the question of the identity of the G-proteins involved and of the downstream Ca +-sensitizing mechanism. RhoA and Rho-associated proteins... 

Sugiyama, T. Goldman, W. F. Measurement of SR free Ca and Mg in permeabilized smooth muscle cells with use of Furaptra. Am. J. Physiol. 1995, 269, C698-C705. 

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