Transcellular transport of calcium and inorganic phosphate in the small intestinal epithelium.

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.

Ca++-transport across basal-lateral plasma membranes from rat small intestinal epithelial cells.

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.

A simple isolation method for basal-lateral plasma membranes from rat kidney cortex.

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.

Regulation of Na+-Pi cotransport by 1,25-dihydroxyvitamin D3 in rabbit duodenal brush-border membrane.

Brush-border membrane vesicles were isolated from rabbit duodenum by a Mg2+ precipitation method, and phosphate transport was analyzed by a rapid filtration technique. Uptake of inorganic phosphate (Pi) was stimulated by an inwardly directed sodium gradient, indicating the operation of a Na-Pi cotransport system in brush-border membrane vesicles. Treatment of the animals with ethane-1-hydroxy-1,1-diphosphonate (EHDP), which is known to decrease the circulating levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], reduced within 3 days the sodium-dependent Pi transport in the brush-border vesicles. Injections of 1,25(OH)2D3 into rabbits increased within 9 h the sodium-dependent Pi transport in membranes from EHDP-treated animals as well as in untreated ones. The Na-D-glucose cotransport system appeared to be unaffected by these maneuvers. These results suggest that the Na-Pi cotransport system is an important site of regulation of intestinal transepithelial Pi transport by 1,25(OH)2)D3.

Polar distribution of sodium-dependent and sodium-independent transport system for L-lactate in the plasma membrane of rat enterocytes.

The uptake of L-lactate by rat small intestinal brush-border and basal-lateral plasma membrane vesicles has been studied. L-Lactate uptake by the isolated membrane vesicles is osmotically sensitive and represents predominantly transport into an intravesicular space and not binding to the membranes. The transport of L-lactate across the brush-border membrane is stimulated by sodium, whereas the transport across the basal-lateral plasma membrane is sodium-independent. In both types of membrane vesicles L-lactate is transported faster than D-lactate and L-lactate transport is inhibited by alpha-cyano-cinnamic acid. L-Lactate transport across basal-lateral membranes is inhibited by D-lactate and pyruvate and transstimulated by L-lactate and pyruvate. The polar distribution of transport system for L-lactate in the plasma membrane of rat enterocytes--a Na+/L-lactate cotransport system in the brush-border membrane and a facilitated diffusion system in the basal-lateral membrane--can explain the fact that in the intact epithelium L-lactate produced by cell metabolism is preferentially released on the serosal side and could enable the cell to perform vectorial, secondary active transport of L-lactate from the intestinal lumen to the serosal compartment.

Sodium ion/L-lactate co-transport in rabbit small-intestinal brush-border-membrane vesicles.

Uptake of L-lactate into rabbit jejunal brush-border-membrane vesicles prepared by a Ca2+-precipitation procedure was studied by a rapid filtration technique with L-[14C]-lactate as tracer. Transport of L-lactate into an intravesicular (osmotically reactive) space could be established. An inwardly directed NaCl gradient (outside 21 mM/inside 0mM) stimulated the uptake of L-lactate at 15 s 2-4-fold compared with that observed with an equal KCl gradient. A transient accumulation of L-lactate inside the vesicles (overshoot) was observed in the presence of an NaCl gradient. Gradients of LiCl, RbCl, CsCl or choline chloride were not able to replace NaCl in the stimulation of L-lactate uptake. L-Lactate uptake was saturable only in the presence of Na+. D-Lactate, DL-thiolactate (2-DL-mercaptopropionate), pyruvate and propionate inhibited the Na+-stimulated L-lactate uptake; D-lactate, thiolactate and pyruvate provoked trans-stimulation of L-lactate uptake. Artificially imposed diffusion potentials (inside negative) did not exert any effect on the Na+-dependent L-lactate uptake. The results are consistent with the existence of an electroneutral Na+/L-lactate co-transport system in the brush border of rabbit small intestine.