Basolateral Plasma Membrane Vesicles Research Articles

Taurocholate transport by basolateral plasma membrane vesicles isolated from developing rat liver

Taurocholate transport was characterized in basolateral plasma membrane vesicles prepared from the livers of 14-day-old Sprague-Dawley rats using a self-generating Percoll gradient method. Liver plasma membrane protein yield, intravesicular volume, and enrichments of various marker enzymes were similar to those obtained for vesicles from adult rat liver. The basolateral marker enzyme Na+-K+-ATPase was enriched 26-fold in the suckling rat basolateral membrane fraction while the bile canalicular marker enzymes alkaline phosphatase and Mg2+-ATPase were enriched only 3- and 5-fold, respectively. The activities of marker enzymes for endoplasmic reticulum, mitochondria, or lysosomes were not enriched compared with homogenate. In the presence of an inwardly directed 100 mM Na+ gradient, vesicle accumulation of taurocholate transiently reached a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot") in suckling and adult membrane vesicles, but the initial rate of taurocholate entry and peak intravesicular accumulation were markedly decreased in suckling compared with adult membrane vesicles. In the presence of an inwardly directed 100 mM K+ gradient, the rate of uptake was slower, and no overshoot occurred in either suckling or adult rat vesicles. The decreased rate of Na+-coupled taurocholate uptake by membrane vesicles from suckling rat liver could not be explained on the basis of more rapid dissipation of the transmembrane Na+ gradient. Kinetic studies demonstrated saturable, Na+-dependent taurocholate uptake for both suckling and adult vesicles. However, the Vmax for taurocholate uptake in suckling rat vesicles was less than half of the adult rate (2.46 +/- 0.13 vs. 5.25 +/- 0.22 nmol X mg prot-1 X min-1, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland.

Ca2+ transport was studied by using basolateral plasma membrane vesicles from rat parotid gland prepared by a Percoll gradient centrifugation method. In these membrane vesicles, there were two Ca2+ transport systems; Na+/Ca2+ exchange and ATP-dependent Ca2+ transport. An outwardly directed Na+ gradient increased Ca2+ uptake. Ca2+ efflux from Ca2+-preloaded vesicles was stimulated by an inwardly directed Na+ gradient. However, Na+/Ca2+ exchange did not show any 'uphill' transport of Ca2+ against its own gradient. ATP-dependent Ca2+ transport exhibited 'uphill' transport. An inwardly directed Na+ gradient also decreased Ca2+ accumulation by ATP-dependent Ca2+ uptake. The inhibition of Ca2+ accumulation was proportional to the external Na+ level. Na+/Ca2+ exchange was inhibited by monensin, tetracaine and chlorpromazine, whereas ATP-dependent Ca2+ transport was inhibited by orthovanadate, tetracaine and chlorpromazine. Oligomycin had no effect on either system. These results suggest that in the parotid gland cellular free Ca2+ is extruded mainly by an ATP-dependent Ca2+ transport system, and Na+/Ca2+ exchange may modify the efficacy of that system.

Active sodium transport in basolateral plasma membrane vesicles from rat kidney proximal tubular cells

Inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can accumulate Na + actively in two ways. Mode 1, which is K +-independent, is ouabain-insensitive and is inhibited by furosemide and mode 2, which is K +-dependent, is inhibited by ouabain and is insensitive to furosemide. The presence of Mg 2+ and ATP in the incubation medium is essential for both modes of Na + uptake to proceed and in both cases, the nucleotide is hydrolyzed during the process. These results are consistent with the idea of the existence, in these membranes, of two Na + pumps: one, which can work in the absence of K + (Na + pump) and another, which needs K + to work (Na + + K + pump).

Evidence for carrier-mediated chloride/bicarbonate exchange in canalicular rat liver plasma membrane vesicles.

To determine whether anion exchangers might play a role in hepatic bile formation, we looked for the presence of Cl-:OH- and Cl-:HCO3- exchange in highly purified canalicular (c) and basolateral (bl) rat liver plasma membrane (LPM) vesicles. In cLPM vesicles, a pH gradient (7.7 in/6.0 out) stimulated 36Cl- uptake twofold above values obtained during pH-equilibrated conditions (7.7 in = out). When 50 mM HCO3- was also present inside the vesicles, the same pH gradient (7.7 in/6.0 out) resulted in Cl- uptake to levels fourfold above pH- and HCO3--equilibrated controls and two- to threefold above Cl- equilibrium (overshoot). Initial rates of both pH and HCO3- gradient-stimulated Cl- uptake were completely inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). A valinomycin-induced K+ diffusion potential (inside positive) also stimulated Cl- uptake in cLPM, but this conductive Cl- pathway was insensitive to DIDS. The DIDS-sensitive, pH and HCO3- gradient-stimulated Cl- uptake demonstrated: saturation with Cl- (Km approximately 6.3 mM; Vmax approximately 51 nmol X mg-1 X min-1); partial inhibition by bumetanide (26%), furosemide (33%), probenecid (37%), and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (49%); cis-inhibition by chloride and nitrate but not by sulfate and various organic anions, and independence from the membrane potential. These data demonstrate the presence of an electroneutral Cl-:OH- and Cl-:HCO3- exchanger in rat liver canalicular membranes that favors Cl-:HCO3- exchange. In contrast, no evidence was found for the presence of a Cl-:HCO3- (OH-) exchange system in blLPM vesicles. Furthermore, neither blLPM nor cLPM vesicles exhibited Na+-stimulatable Cl- uptake, indicating the absence of a NaCl co-transport system in either LPM subfraction. These findings are consistent with a functional role for a Cl-:HCO3- (OH-) exchanger in canalicular bile formation.

620 MECHANISMS OF TAURINE TRANSPORT BY PLASMA MEMBRANE FROM RAT LIVER

Taurine (TAU) availability dictates the conjugation pattern of bile acids; this amino acid may also contribute to regulation of hepatocyte osmolarity, ion flux and membrane function. Therefore, we used basolateral (sinusoidal) liver plasma membrane vesicles (LPMV) prepared from suckling (14d) and adult rats (J Biol Chem 259:9295, 1984) to characterize the mechanisms of taurine uptake by the liver. In the presence of an inwardly directed 100 mM Na gradient, TAU (10 μM) was rapidly taken up by LPMV from 14d and from adult rats and transiently accumulated at a concentration two-fold above equilibrium (“overshoot”). In contrast, with a 100 mM K+-gradient, uptake was slower and no “overshoot” was observed. Na+-dependent TAU uptake was significantly increased by a negatively charged intravesicular electrical potential (vesicles preloaded with 50 mM K+-gluconate in the presence of the K+-ionophore, valinomycin). The initial velocity (5 seconds) of Na+-dependent TAU uptake was significantly greater in LPMV from 14d rats compared to adult rats (2.87 ± 0.29+[SE] vs. 0.63 ± 0.14 pmoles·mg−1 protei·5 sec−1, p < 0.001). Na+-stimulated TAU uptake, measured under “voltage-clamped” conditions, was saturable in LPMV from 14d rats (Km 92.1 ± 24 μM; Vmax 351 ± 40 pmoles·mg−1 protein·min−1). Summary: Hepatic uptake of taurine is stimulated by a Na+-gradient, associated with net movement of positive charge (electrogenic) and saturable. In contrast to other solutes, e.g., bile acids, taurine transport by the liver is well developed in early life perhaps reflecting the needs of the immature animal for this essential nutrient.

The effect of ethanol and calcium on fluid state of plasma membranes of rat hepatocytes

Basolateral (blLPM) and canalicular (cLPM) plasma membrane vesicles were isolated from rat liver to compare membrane fluidity, fluidity responses to membrane perturbants, and the relationship between fluidity and a membrane protein function such as carrier-mediated taurocholate transport. Membrane fluidity was measured by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as a probe. Uptake of [ 3H] taurocholate was measured by a rapid Millipore filtration technique. blLPM were more fluid than cLPM. Ethanol produced a concentration-dependent fluidizing effect on both membrane preparations, the change being greater in blLPM. Incubation with calcium for 2 hr at 37°C rendered both membrane preparations more rigid, again the cLPM being more resistant to perturbation. There was a linear correlation between an increase in membrane fluidity and inhibition of taurocholate uptake into blLPM in the presence of increasing concentrations of ethanol. The data support the concept that membrane lipid fluidity is an important regulator of membrane protein functions and hence also of overall cellular activity.

Mg 2+-ATP-dependent sodium transport in inside-out basolateral plasma membrane vesicles from guinea-pig small intestinal epithelial cells

The transport of sodium into inside-out basolateral plasma membrane vesicles from small intestinal epithelial cells has been examined. It was found, under equilibrium conditions, that binding of 22Na represents approx. 55% of the total uptake during an equilibration period of 30 min; 45% of the total uptake correspond to passive sodium entry in the vesicle space. In addition to binding and to passive Na + entry, two distinct mechanisms capable of accumulating sodium in the intravesicular space can be demonstrated when ATP is added to the incubation medium. One transports sodium actively in the absence of potassium, whereas the other requires the presence of potassium in the interior of the vesicles. The two mechanisms can also be differentiated by their affinities for sodium, their optimal pH and by their behaviour towards different inhibitors. Thus, the mechanism that transports sodium in the absence of potassium is refractory to ouabain, but is inhibited by ethacrynic acid and furosemide, whilst the mechanism that accumulates sodium inside the vesicles in the presence of internal potassium is strongly inhibited by ouabain, is weakly inhibited by ethacrynic acid and is insensitive of furosemide. ATP is a specific stimulator of both processes, and the requirement for magnesium is absolute in both cases.

Ca 2+-stimulated, Mg 2+-independent ATP hydrolysis and the high affinity Ca 2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg 2+-dependency of Ca 2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg 2+- and Ca 2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg 2+ with a K m of 13 μ M in the absence or presence of 1 μ M free Ca 2+. At free Mg 2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg 2+ -independent, but is strongly stimulated by submicromolar Ca 2+ concentrations K m  0.25 μM, V max  24 μmol P i/h per mg protein). The Ca 2+-stimulated ATP hydrolysis strongly decreases at higher Mg 2+ concentrations. The Ca 2+-stimulated Mg 2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg 2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca 2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca 2+-pumping in renal basolaterals and on Ca 2+-ATPase in erythrocyte ghosts suggest that the Ca 2+-pumping enzyme requires Mg 2+. In contrast, a role of the Ca 2+-stimulated Mg 2+-independent ATP hydrolysis in active Ca 2+ transport across basolateral membranes is rather unlikely.

Driving Forces for Taurocholate Transport across Basolateral and Canalicular Rat Liver Plasma Membrane Vesicles

Driving Forces for Taurocholate Transport across Basolateral and Canalicular Rat Liver Plasma Membrane Vesicles

Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. Evidence for an electrogenic canalicular organic anion carrier.

The driving forces for taurocholate transport were determined in highly purified canalicular (cLPM) and basolateral rat liver plasma membrane (LPM) vesicles. Alanine transport was also examined for comparison. Inwardly directed Na+ but not K+ gradients transiently stimulated [3H]taurocholate (1 microM) and [3H]alanine (0.2 mM) uptake into basolateral LPM 3-4- fold above their respective equilibrium values (overshoots). Na+ also stimulated [3H]taurocholate countertransport and tracer exchange in basolateral LPM whereas valinomycin-induced inside negative K+ diffusion potentials stimulated alanine uptake but had no effect on taurocholate uptake. In contrast, in the "right-side out" oriented cLPM vesicles, [3H]taurocholate countertransport and tracer exchange were not dependent on Na+. Efflux of [3H]taurocholate from cLPM was also independent of Na+ and could be trans-stimulated by extra-vesicular taurocholate. Furthermore, an inside negative valinomycin-mediated K+ diffusion potential inhibited taurocholate uptake into and stimulated taurocholate efflux from the cLPM vesicles. These studies provide direct evidence for a "carrier mediated" and potential-sensitive conductive pathway for the canalicular excretion of taurocholate. In addition, they confirm the presence of a possibly electroneutral Na+-taurocholate cotransport system in basolateral membranes of the hepatocyte.

A new method for the rapid isolation of basolateral plasma membrane vesicles from rat liver. Characterization, validation, and bile acid transport studies.

Basolateral plasma membrane vesicles were prepared from rat liver by a new technique using self-generating Percoll gradients. The method is rapid (total spin time of 2.5 h) and protein yields were high (0.64 mg/g of liver). Transmission electron microscopy studies and measurements of marker enzyme activities indicated that the preparation was highly enriched in basolateral membranes and substantially free of contamination by canalicular membranes or subcellular organelles. High total recoveries for protein yield and marker enzyme activities during the fractionation procedure indicated that enzymatic activity was neither lost (inactivation) nor increased (activation). Thus, the pattern of marker enzyme activities found in the membrane preparation truly reflected substantial enrichment in membranes from the basolateral surface. Analysis of freeze-fracture electron micrographs suggested that approximately 75% of the vesicles were oriented "right-side-out." In order to assess the functional properties of the vesicles, the uptake of [3H]taurocholate was studied. In the presence of a Na+ gradient, taurocholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot"). In the absence of a gradient but in the presence of equimolar Na+ inside and outside of the vesicle, taurocholate uptake was faster than in the absence of Na+. These findings support a direct co-transport mechanism for the uptake of taurocholate and Na+. Kinetic studies demonstrated that Na+-dependent taurocholate uptake was saturable with a Km of 36.5 microM and a Vmax of 5.36 nmol mg-1 protein min-1. The high yield, enzymatic profile and retention of transport properties suggest that this membrane preparation is well suited for studies of basolateral transport.

Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver.

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ-28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.

1α,25-dihydroxy-vitamin D-3 regulates ATP-dependent calcium transport in basolateral plasma membranes of rat enterocytes

Basolateral plasma membrane vesicles of rat small intestinal epithelium accumulate calcium through an ATP-dependent pumping system. The activity of this system is highest in duodenum and decreases towards the ileum. This distribution along the intestinal tract is similar as the active calcium absorption capacity of intact intestinal epithelial segments. ATP-dependent calcium uptake in basolateral membrane vesicles from duodenum and ileum increased significantly after repletion of young vitamin D-3-deficient rats with 1α,25-dihydroxy-vitamin D-3. Ca 2+-ATPase activity in duodenal basolateral membranes increased to the same extend as ATP-dependent calcium transport, but (Na + + K +)-ATPase activity remained unaltered.

ATP-dependent calcium transport and its correlation with Ca 2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca 2+-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH 2)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K m of 0.2 μM Ca 2+ and a V max of 5.3 nmol Ca 2+/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca 2+-ATPase are: K m = 0.25 μM Ca 2+ and V max = 19.2 nmol P i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V max of ATP-dependent calcium uptake amounts to 25 nmol Ca 2+/min per mg protein and of the Ca 2+-ATPase to 23 nmol P i/min per mg protein. The close correlation between kinetic parameters of Ca 2+-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca 2+-pump located in duodenal basolateral plasma membranes.

Calmodulin-regulated, ATP-driven calcium transport by basolateral membranes of rat small intestine.

Uptake of calcium by a membrane fraction enriched in basolateral plasma membrane vesicles from rat small intestine has been investigated using a rapid filtration technique. Calcium accumulation is stimulated by ATP and released by the calcium ionophore A23187. Kinetic studies indicate a calcium concentration of 28 nM for half-maximal stimulation of ATP-driven uptake. Transport is minimally inhibited by oligomycin and ouabain but is reduced 40% by lanthanum and abolished by 50 microM vanadate. The ATP-dependent calcium uptake is stimulated by the calcium regulatory protein, calmodulin, in a dose-dependent fashion. Calmodulin increases both the maximal transport rate and the calcium affinity of the transport mechanism. These results are consistent with the existence of an ATP-dependent, calmodulin-regulated calcium transport mechanism in the basolateral plasma membrane of intestinal epithelial cells, and suggests that calmodulin may modulate transepithelial calcium absorption in addition to controlling cytosolic calcium levels.

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of d-[ 3H]glucose into these vesicles was studied by a rapid filtration technique. (Na + + K +)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of d-glucose uptake was almost unaffected by Na + gradient (no overshoot).

Phenylalanine uptake in isolated renal brush border vesicles

The uptake of l-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up l-phenylalanine by an Na +-dependent, saturable transport system. The apparent affinity of the transport system for l-phenylalanine is 6.1 mM at 100 mM Na + and for Na + 13 mM at 1 mM l-phenylalanine. Reduction of the Na + concentration reduces the apparent affinity of the transport system for l-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference for Na + across the membrane ( η Na 0 > η Na 1 ) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K + gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of l-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na + and is dependent on the concentration difference of the amino acid, on the concentration difference of Na + and on the electrical potential difference. The exit of l-phenylalanine across the basolateral plasma membranes is Na +-independent and probably involves facilitated diffusion.

Coupled transport of sodium and organic solutes.

Coupled transport of sodium and organic solutes.