Tedisamil blocks single large-conductance Ca2+-activated K+ channels in membrane patches from smooth muscle cells of the guinea-pig portal vein
Procedures for isolation of single smooth muscle cells from taenia coli of guinea pigs have been developed. The preparation was performed with a combination of highly purified collagenase prepared by Amano Pharmaceutical Co. (Japan) and papain obtained from Sigma Chemical Co. (Type III). This combination resulted in very high yield of the single cells (39.2 +/- 4.5 X 10(3) cells/mg tissue wet wt) and less cell debris. In the ordinary procedure, commercially available collagenase preparations contaminated with various peptidases have been used. With these enzyme preparations, however, the yield of single cells was dependent on the batch of the preparations, and a large amount of cell debris was contaminated. Combination of the highly purified collagenase and papain resulted in higher yields constantly. Cells, isolated with these enzymes in a medium consisting of 140 mM KCl, 1.0 mM MgCl2, 4.2 mM Hepes, and 5.6 mM glucose (pH 7.4), were spindle shaped. The length of the cells was 185.9 +/- 5.2 micron (n = 90) and the diameter was approximately 12.6 micron. The diameter was not dependent on the cell length. More than 80% of the single cells were viable when examined by trypan blue exclusion technique. Under the depolarized condition, cells remained viable longer because of lower energy consumption, and these cells were contracted by Ca dose dependently. The dose-response relationship was similar to that obtained with intact tissue. Because the cells are constantly available with higher yield, the preparation might be applicable for biochemical research such as ion flux. Details of cell properties under the physiological conditions are under investigation.