Nonpolar density gradient ultracentrifugation in the direct determination of myocardial subcellular calcium |
| |
Authors: | R C Kolbeck M J Frank |
| |
Affiliation: | Section of Cardiology, Department of Medicine, Medical College of Georgia, Augusta, Georgia 30912, USA |
| |
Abstract: | A new method is described for making direct measurements of compartmentalized subcellular stores of calcium in the isolated perfused dog heart. Cellular calcium was immobilized by freezing the myocardium in diastole with an extremely cold fluorocarbon fluid (-125 degrees C). All subsequent procedures were conducted under conditions which prevented ionic diffusion, either at temperatures well below the freezing point of water or in the absence of water. Sarcolemmal and mitochondrial enriched fractions were segregated from dessicated, homogenized, myocardial tissue by ultracentrifugation utilizing density-gradients composed of blends of silicone and halocarbon on fluids within which physiological salts are insoluble. The total calcium content of these isolated fractions were then determined by atomic absorption spectrophotometry. Ouabain and epinephrine were subsequently used to alter the contractility of the perfused hearts and such contractile alterations were then related to changes noted in the calcium activity of the isolated subcellular fractions. In this study the calcium levels of the enriched mitochondrial fractions were elevated by both ouabain and epinephrine, while the calcium levels of the enriched sarcolemmal fractions were elevated only by ouabain. The advantage of this segregative procedure is that it prevents artifactual intercompartmental calcium rearrangement and preserves calcium levels to those initially fixed in situ at the time of freezing. |
| |
Keywords: | Myocardial calcium metabolism Nonpolar density gradient Ultracentrifugation Sarcolemma Mitochondria Ouabain Epinephrine |
本文献已被 ScienceDirect 等数据库收录! |
|