Calmodulin loss in vascular smooth muscle following Triton X-100 or saponin skinning.
The calmodulin (CaM) content of intact and chemically skinned strips of rat caudal artery was measured using a 125I-CaM radioimmunoassay. The total CaM measured following homogenization of arterial tissue with EGTA and EGTA/Triton X-100 was 2.58 mumol/kg wet tissue. Based on a smooth muscle volume of 40%, this value corresponds to a cellular CaM concentration of 6.5 microM. Approximately 97% of total CaM was soluble and approximately 3% was EGTA-nonextractable. Permeabilization of the plasmalemma with 0.15 mg/ml saponin or 0.5% Triton X-100 caused significant detergent-dependent loss of CaM. At the end of a 1 h skinning period, tissues exposed to saponin lost 30% of total CaM. By comparison, tissues skinned under the same conditions with Triton X-100 lost 50%. During a subsequent 4 h exposure to relaxing solution, total tissue CaM continued to decline. The exponential loss over the 5 h period was described by a first order model having diffusible and nondiffusible CaM components. The diffusible CaM component of saponin skinned tissue (59%) was significantly less than the diffusible component of those skinned with Triton X-100 (88%); however, the rate coefficients for CaM diffusion (0.78 h-1 and 0.91 h-1, respectively) did not statistically differ. The nondiffusible component of CaM was significantly larger in saponin treated strips (42%) than in Triton X-100 permeabilized tissue (12%). Arterial strips skinned with Triton X-100, which were subsequently exposed to relaxing solution for up to 22 h, lost significantly more CaM than those retained in Triton X-100 skinning solution for a comparable duration.(ABSTRACT TRUNCATED AT 250 WORDS)
Gardner
J P
JP
Department of Physiology, UMDNJ-New Jersey Medical School, Newark 07103-2757.
Stout
M A
MA
Harris
S R
SR
D000818:Animals; D001158:Arteries; D002147:Calmodulin / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004058:Diffusion; D004533:Egtazic Acid; D005260:Female; D008297:Male; D009126:Muscle Relaxation; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D017830:Octoxynol; D009994:Osmolar Concentration; D011092:Polyethylene Glycols / Q000494:pharmacology*; D051381:Rats; D011921:Rats, Inbred WKY; D012503:Saponins / Q000494:pharmacology*; D012995:Solubility
D002147:Calmodulin; D012503:Saponins; D011092:Polyethylene Glycols; D004533:Egtazic Acid; D017830:Octoxynol