Basal-lateral membranes were separated in a self-orienting Percoll (modified colloidal silica) gradient from a heavy microsomal membrane fraction by centrifugation at 48,000g for 0.5 h. The (Na+--K+)-ATPase activity as a marker enzyme for the basal-lateral plasma membrane was 20-fold enriched by this procedure. The adenylate-cyclase activity measured in the basal-lateral membrane fraction was stimulated 6-fold by parathyrin and only up to 1.5-fold by arginine-vasopressin, calcitonin, or isoproterenol. The yield of basal-lateral plasma membranes was 5 to 10 percent of the amount initially present in the homogenate. The method is also applicable to the pig kidney.

Basal-lateral plasma membrane vesicles were isolated from rat duodenum and jejunum by a Percoll gradient centrifugation technique. Ca-uptake into and Ca-release from the vesicles was studied by a rapid filtration method. In the absence of Na (K-medium) at a Ca concentration of 0.05 mmol/liter and pH 7.4, addition of 5 mM MgATP stimulated Ca-uptake up to 10-fold as compared to a control without ATP. Since the Ca-ionophore A23187 (2 microgram/ml) prevented the accumulation of Ca above the equilibrium uptake and rapidly released Ca accumulated by the vesicles in the presence of ATP, it is concluded that the ATP-dependent uptake of Ca involves accumulation of Ca inside the vesicles. The ATP-driven Ca-transport comigrates with the (Na +K)-ATPase and dissociates from the marker enzymes for mitochondrial inner membrane, endoplasmic reticulum and brush border membrane. It is not inhibited by 1 microgram/ml oliomycin or 0.1 mmol/liter ruthenium red. Replacing K by Na inhibits ATP-dependent Ca-uptake by 60%. Efflux of Ca from passively preloaded vesicles is strongly temperature sensitive and enhanced by A23187. An inwardly directed Na-gradient stimulates Ca-efflux as compared to a K-gradient. Addition of gramicidin reduces the Na-stimulation of Ca-efflux, indicating direct coupling of Na and Ca fluxes across basal-lateral membranes. The results suggest that basal lateral membranes possess two distinct mechanisms for Ca-transport: a) ATP-driven Ca-transport and b) Na/Ca-exchange.

Transport mechanisms involved in the small intestinal handling of inorganic phosphate and calcium have been studied by different in vitro methods during the last few years. In concordance with studies on intact epithelial preparations, studies with brush-border and basal-lateral membrane vesicles isolated from the small intestinal epithelial cell revealed that transcellular calcium and inorganic phosphate fluxes are coupled to transcellular sodium flux, i.e., secondary active via coupling to the primary active sodium flux. A sodium-coupled mechanism in the brush-border membrane leads to cellular accumulation of inorganic phosphate. A sodium-coupled mechanism leads to extrusion of calcium from the cell into the serosal interstitium. A primary active transport mediated by the Ca-ATPase and located in the basal-lateral membrane also exists for calcium. Regulation of transcellular phosphate and calcium flux proceeds via altered influx rates at the luminal cell pole.