Permeabilized Fibers

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,... 

D.D. Kindler, C. Thiffault, N.J. Solenski, J. Dennis, V. Kostecki, R. Jenkins, P.M. Keeney and J.P.Rr. Bennett, Neurotoxic nitric oxide rapidly depolarizes and permeabilizes mitochondria by dynamically opening the mitochondrial transition pore, Mol. Cell. Neurosci., 23 (2003) 559-573. N.R. Ferreira, A. Ledo, J.G. Frade, G.A. Gerhardt, J. Laranjinha and R.M. Barbosa, Electrochemical measurement of endogenously produced nitric oxide in brain slices using Nafion/o-phenylenediamine modified carbon fiber microelectrodes, Anal. Chim. Acta, 535 (2005) 1-7. 

Lynch, G.S., Rafael, J.A., Chamberlain, J.S., and Faulkner, J.A., 2000, Contraction-induced injury to single permeabilized muscle fibers from mdx, transgenic mdx, and control mice, Am J Physiol Cell Physiol, 279, pp C1290-1294. 

Tsaturyan, A. K., Bershitsky, S. Y., Burns, R., and Ferenczi, M. A. (1999a). Structural changes in the actin-myosin crossbridges associated with force generation induced by temperature jump in permeabilized frog muscle fibers. Biophys.J. 77, 354—372. 

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.,... 

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). 

Using freeze-dried fibers, Riiegg and coworkers (1984) estimated the fraction of calmodulin available for activation of MLCK to be in the range of 0.3 to 4 yJVl, which is about 10% of the total cellular calmodulin. This pool is readily exchangeable and therefore rapidly equilibrates with the incubation medium (Riiegg et al., 1984). Similar results were obtained in triton skinned tracheal smooth muscle (Tansey et al., 1994). In contrast, calmodulin appears to be retained in P-escin-permeabilized tracheal smooth muscle (Tansey et al., 1994). 

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). 

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.,... 

Boucek RJ Jr, Buck SH, Scott F, Oquist NL, Fleischer S, Olson RD (1993) Anthracycline-induced tension in permeabilized cardiac fibers evidence for the activation of the calcium release channel of sarcoplasmic reticulum. J Mol Cell Cardiol 25 249-259... 

Norman, J. C., Jones, D., Barry, S. T., Holt, M. R., Cockcroft, S., and Critchley, D. R. (1998). ARFI mediates paxiUin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts. J. Cell Biol. 143, 1981-1995. 

Sharma S, Si giner DA. Permeabil ity measurement methods in porous media of fiber reinforced composites. Applied Mechanics Reviews, 2010 63(2) 020802-020819. DOI 10.1115/1.4001047. 

---