The isolation and characterization of Ca++-accumulating subcellular membrane fractions from cerebral arteries

Abstract

✓ A study was undertaken using differential centrifugation methods to isolate from rabbit cerebral arteries the subcellular microsomal protein fractions capable of actively sequestering Ca++. One isolated protein fraction displayed a relatively large adenosine triphosphate (ATP)-dependent Ca++-accumulating capacity that was completely inhibited by NaN3, and was therefore designated the “mitochondrial fraction.” Electron microscopy confirmed that this fraction consisted of numerous mitochondrial elements. Another isolated membrane fraction possessed a Ca++-accumulating capacity dependent on ATP and oxalate and only partially sensitive to NaN3. In the presence of mersalyl acid or the Ca++ ionophore, A23187, Ca++ uptake by this fraction was inhibited 98.0% and 87.4%, respectively. Electron microscopy revealed that this fraction consisted of numerous membrane vesicles, and measurements of Na+-K+-ATPase (adenosine triphosphatase) activity indicated minimal plasma membrane contamination. It was concluded that this microsomal fraction consisted primarily of sarcoplasmic reticulum. At physiological free [Ca++] levels, Ca++ uptake by this fraction was inhibited by norepinephrine through a process sensitive to tolazoline but not propranolol. The effects on Ca++ uptake of added cyclic adenosine monophosphate (cAMP) alone or with rabbit or bovine protein kinase were inconclusive. The organic Ca++ channel blockers, nifedipine and verapamil, significantly inhibited Ca++ uptake by sarcoplasmic reticulum.