Triton Skinning

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). 

The use of saponin, a plant glycoside, for permeabilization was introduced by Endo and coworkers (1977). Saponin removes the surface membrane without impairment of the functions of SR. The plasma membrane, unlike that of triton skinned smooth muscle (Spedding, 1983), appears fairly intact in electron microscopic pictures of cross sections of permeabilized cells. However, when patches of isolated membranes were viewed face on in homogenates of permeabilized cells, numerous 70- to 80-A holes were visible (Kargacin and Lay, 1987). 

Fibers treated in the described way respond rapidly to increasing Ca + concentrations. Threshold concentrations are around 2 x 10 7 M Ca + and maximum contraction was observed at 10 (jlM Ca +. Maximal force is comparable to or even larger than that observed before saponin treatment. This was taken as a criterion for complete skinning (Endo et ah, 1982). As in triton skinned fibers, tension development depends on the presence of ATP (Endo et ah, 1982). 

III. PROBING THE PHOSPHORYLATION THEORY IN TRITON SKINNED SMOOTH MUSCLE ... 

The fact that calmodulin diffuses out of triton skinned fibers could be used to demonstrate in recon-... 

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). 

Phosphorylation of MLC is generally accepted to be the primary event involved in the initiation of smooth muscle contraction (Kamm and Stull, 1985 Somlyo and Somlyo, 1994). In triton skinned taenia coli, phosphorylation and dephosphorylation of MLC preceded contraction and relaxation, respectively (Kiihn et al.,... 

In intact smooth muscle, phosphorylation often declines while force is maintained, the so-called "latch" state (Dillon etal., 1981). Regulation of the latch state is still poorly understood. It has been very difficult to induce a latch state in skinned smooth muscle. The closest may be triton skinned chicken gizzard, which contracts independent of MLC phosphorylation (Wagner and Ruegg, 1986). In skinned chicken gizzard, but also in other types of smooth muscle (Bialojan et al., 1987 Siegman et al., 1989 Kenney et al., 1990 Schmidt et al, 1995), a steep and nonlinear relation between force and MLC phosphorylation was observed, which was postulated by the "latch model" of Hai and Mur-... 

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). 

As pointed out by Butler et al. (1994), the a-toxin-permeabilized preparation used by Kitazawa et al. might contain endogenous phosphatase inhibitors that have been lost in the Triton-skinned preparation. This could at least partially explain the observed difference in phosphatase rate, and is an intriguing possibility relating to the demonstration that receptor agonists are able to increase Ca2+ sensitivity in neuroeffector-coupled permeabilized preparations by a mechanism involving inhibited myosin light chain phosphatase (MLCP) activity (Kitazawa et al., 1991 Kubota et al.,... 

Independent evidence for an affect of activation on cross-bridge cycling rate comes from measurements showing increased unloaded shortening velocity with increasing activation by in Triton-skinned... 

In Triton skinned smooth muscle, cAMP or the catalytic subunit of cAMP dependent protein kinase inhibits tension development or induces relaxation at submaximal [Ca +] thereby decreasing the Ca -sensitivity of contraction (Kerrick and Hoar 1981, Riiegg et al. 1981, Riiegg and Paul 1982, Sparrow et al. 1984). Inhibition of force was associated with a decrease in LC20 phosphorylation (Riiegg and Pfitzer 1985). The inhibitory... 

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