Saponin, smooth muscle permeabilization

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). 

Smooth muscle preparations permeabilized with saponin, p-escin, or a-toxin have significantly contributed to the understanding of Ca2+ release from intra-... 

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.,... 

---