An improved procedure for the isolation of plasma membranes from rat mesenteric arteries

A plasma membrane enriched fraction has been prepared from rat mesenteric arteries by an improved procedure which eliminated the contamination by cells other than smooth muscle; i.e. those derived from mesenteries and fat tissues. Morphological and enzymatic characterization of the plasma fraction revealed only minimal contamination by other intracellular membrane fragments in the smooth muscle cell. The plasma membrane was concentrated approximately 8-fold from the post nuclear supernatant fraction as suggested by the enrichment of 5′-nucleotidase, alkaline phosphatase and ATP-dependent calcium accumulation activities. Calcium accumulation by various membrane fractions in the presence of ATP was closely associated with plasma membrane marker enzyme activities, and was not affected by azide and oxalate. The ATP-dependent calcium accumulation by plasma membrane enriched fraction was not affected by the sodium ionophore, monensin, but was substantially inhibited by calcium ionophores X537A and A23187. This suggests that the functional properties of the plasma membrane of vascular smooth muscle include calcium extrusion across the cellular membrane via an energy-dependent transport process and that the plasma membrane plays the major role in regulating the intracellular calcium in vascular smooth muscle.