Permeabilized smooth muscle phosphorylation

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-... 

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... 

Phosphorylation of LC20 in permeabilized gizzard smooth muscle by direct addition of PKC (Sutton and Haeberle, 1990) or its constitutively active proteolytic fragment, PKM (Parente et al., 1992), results in phosphorylation of LC20 mainly on Seri 2 with a small amount on Thr. The only mechanical response observed in those studies in response to direct addition of PKC or PKM was an attenuation of Ca +-induced contraction in permeabilized gizzard smooth muscle fibers (Parente et al., 1992). Phorbol ester-induced attenuation of contractile responses has been observed in ileal and uterine smooth muscle (Baraban et al.,... 

Caldesmon has also been shown to inhibit tension development in chemically permeabilized gizzard smooth muscle (Pfitzer etal., 1993). Furthermore, inhibition of caldesmon/F-actin interaction in permeabilized VSM resulted in contractile force generation independently of changes in [Ca +J, supporting the concept that caldesmon may function as a regulator in situ independently of LC20 phosphorylation (Katsuyama et al., 1992). Both caldesmon (Adam et al.,... 

Triton skinned and glycerinated fibers have been very valuable in demonstrating that phosphorylation and dephosphorylation of MLC is sufficient to induce contraction and relaxation (see Section III.B). These preparations have also been used to study the influence of ionic strength (Arheden et al., 1988 Gag-elmann and Guth, 1985), free Mg + (Arner, 1983 Bar-sotti et al., 1987), pH (Mrwa et al., 1974), inorganic phosphate (Schneider et al., 1981), nucleotides such as ATP and ADP (Arner and Hellstrand, 1985) on isometric force development, shortening velocity, and ATP turnover. Some of these experiments have also been carried out in smooth muscle fiber bundles and single smooth muscle cells permeabilized with saponin, (3-escin, or a-toxin (Saida and Nonomura, 1978 lino, 1981 Warshaw et al., 1987 Crichton et al., 1993). 

Exogenous application of GTP analogues or contractile agonists increased the [Ca +J sensitivity of phosphorylation in smooth muscle permeabilized with staphylococcal oi-toxin (Nishimura et al, 1988 Kitazawaeffl/., 1991a). Exogenous application of either histamine or AIF4 (a nonspecific activator of G proteins) to depolarized intact tissues also increased the [Ca +Ji sensitivity of phosphorylation (Rembold,... 

In this section we review data showing that activation of smooth muscle preparations induces tyrosine phosphorylation of several substrates. As shown in Fig. 7, a similar set of substrates (M, 42,000-205,000) are tyrosine phosphorylated during (1) activation of muscarinic receptors in intact taenia coli, (2) vanadate-induced contraction of the same preparation, (3) activation of aj-adrenergic receptors in VSMC cultured from canine femoral artery, and (4) Ca +-activated contraction of permeabilized ileal longitudinal smooth muscle. It is likely that one or more of these substrates... 

FIGU R E 7 A similar set of substrates is tyrosine phosphorylated during activation of either intact taenia coli, cultured VSMC, or staphylococcal a-toxin-permeabilized deal longitudinal smooth muscle. In these experiments, tyrosine.-phosphorylated substrates were detected by immunoblotting with antiphosphotyrosine antibodies and enhanced chemiluminescence technology rather than the less sensitive I-labeled protein A technology used in Fig. 2. Stimulation of guinea pig taenia coli with either 10 jcM carbachol (Carb) or 1.5 mM vanadate (Van) resulted in pronounced tyrosine phosphorylation of at least nine substrates with apparent masses of 42-45, 50, 70, 80-85, 95,100, 110, 116, and 205 kDa. In like fashion, stimulation of canine femoral VSMC with 100 jjlM phenylephrine (PE) resulted in enhanced tyrosine phosphorylation of a similar set of substrates (however, note that qualitative differences were evident with respect to some substrates, such as the one of 205 kDa). Similarly, the same substrates appeared to be tyrosine phosphorylated when permeabilized ileal smooth muscle was contracted with Ca + (pCa 4.5). From Di Salvo et al. (1994), Fig. 5, p. 1438. 

The sufficiency of myosin phosphorylation for contraction in smooth muscle is indisputably demonstrated in experiments where the addition of pro-teolyzed CaM-independent MLCK to permeabilized fibers brings about contraction at very low [Ca +] (Walsh et al, 1982). Furthermore, injection of CaM-independent MLCK into single smooth muscle cells results in contraction under conditions where [Ca +]j remains at resting values (Itoh et al, 1989). These results tend to rule out a model in which thin filaments are fully inhibited in resting muscle, and require Ca2+-dependent disinhibition to support contraction. However, these experiments do not rule out the possibility that thin filament-binding proteins modulate the contractile response. Alterations in the RLC phosphorylation-force relation in intact muscle indicate that collateral regulation can occur. Moreover, experiments with permeabilized fibers demonstrate that thin filament-binding proteins can inhibit force independent of RLC phosphorylation (Pfitzer et al,... 

---