Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.     

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.     

The kinetics of sulfobromophthalein uptake by rat liver sinusoidal vesicles

The kinetics of bromo[35S]sulfophthalein (35S-BSP) binding by and uptake across the hepatocyte sinusoidal membrane were investigated using isolated rat liver sinusoidal membrane vesicles containing K+ as the principal internal inorganic cation. Uptake of 35S-BSP into vesicles was found to be temperature dependent, with maximum uptake between 35 and 40 degrees C; only binding occurred at or below 15 degrees C. Uptake at 37 degrees C was saturable and resolvable by Eadee-Hofstee analysis into two components: one with high affinity (Km = 53.1 microM) but low capacity, and the second of low affinity (Km = 1150 microM) but high capacity. By pre- or post-incubation, respectively, with unlabelled BSP, trans-stimulation and counter transport of 35S-BSP could also be demonstrated in these vesicles. Uptake was inhibited competitively using 5 microM Rose bengal and 10 microM indocyanine green, and non-competitively using 10 microM DIDS. Taurocholate did not inhibit uptake, and actually enhanced transport at concentrations greater than or equal to 250 microM. Imposition of inwardly directed inorganic ion gradients resulted in the enhancement of 35S-BSP transport when chloride ions were part of this gradient, irrespective of the cation employed whereas there was no apparent cation effect. However, substitution of 10 mM Na+ for 10 mM K+ as the internal cation resulted in a significant increase in uptake in the presence of external K+ as compared to Na+ gradients. This effect was not observed when 10 mM Tris+ was employed as the internal cation. The kinetics of 35S-BSP uptake by isolated sinusoidal membrane vesicles are indicative of facilitated transport. While the observed inorganic ion effects suggest a possible electrogenic component, the driving forces for hepatic BSP uptake remain uncertain. Isolated sinusoidal membrane vesicles provide a useful technique for studying hepatic uptake processes independent of circulatory or subsequent cellular phenomena.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

Chloride transport in control and cystic fibrosis human skin fibroblast membrane vesicles.

Plasma membrane vesicles were isolated from either cystic fibrosis (CF) or non-CF cultured fibroblasts derived from skin biopsies of either foetus, child or adolescent human donors. The total membrane yield was essentially identical for either CF or control membranes. By using a rapid filtration technique, 36Cl uptake by these vesicles was quantitated in the absence and presence of alkali-metal ion-, electrical- and/or pH gradients. In the absence of a pH gradient (pHout = pHin = 7.5), Cl uptake took place downhill in both cases. Either cis K+, cis Na+ or an equimolar mixture of cis Na+ plus K+ caused Cl uptake activation. In the presence of an alkaline-inside pH gradient (pHout/pHin = 5.5/7.5), Cl uptake exhibited an apparent overshoot independently of the presence or absence of any metal-ion gradient. The observed potassium-, sodium- and proton-dependent Cl influx rates were all unaffected by voltage clamping, indicating the existence in these vesicles of electroneutral symport systems of the type Cl-/H+, Cl-/K+ and/or Cl-/Na+; but not 2 Cl-/Na+/K+. In the presence of an inward-directed K+ gradient, valinomycin further increased Cl uptake, both in the presence and in the absence of a pH gradient, indicating the presence of a rheogenic Cl uniport. In absolute quantitative terms, the two different modes (rheogenic and electroneutral) of Cl transport evinced in these vesicles were about 45% lower in CF than in control skin fibroblasts. However, qualitatively, there was no difference between normal and CF cells. The evidence obtained indicates that the CF defect, which is expressed in fibroblast plasma membranes, does not affect specifically either the rheogenic or the electroneutral Cl transport systems. Rather, the CF cells appear to give a smaller yield of closed, functional vesicles, reflected by a significantly smaller apparent intravesicular volume. Because it also affects the transport of D-glucose and L-alanine, this anomaly could be the consequence of a generalized membrane defect characterizing CF fibroblasts.     

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+ 13mM 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 of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). 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.     

A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane.

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.     

Na+-H+ exchange in cardiac sarcolemmal vesicles

The transport of Na+ by a purified sarcolemmal vesicular preparation from canine ventricular tissue was studied as a function of both internal and external pH. The uptake of Na+ into sarcolemmal vesicles increased upon raising the extravesicular pH of the reaction medium. Half-maximal uptake of Na+ was observed at a pHo of about 8.1 and maximal uptake occurred at pH 8.6. The uptake of Na+ by sarcolemma was also dependent upon the intravesicular pH. Na+ uptake into sarcolemmal vesicles was greatly attenuated in the absence of a H+ gradient across the membrane. Transport of Na+ was potently inhibited by amiloride, a known blocker of Na+-H+ exchange. LiCl was also an effective inhibitor of Na+ transport. In the presence of optimal H+ gradients, Na+ uptake was linear for the first 5 seconds of the reaction and exhibited a Vmax of 290 nmol Na+/mg per min and a KNa of 3.5 mM. These experiments strongly indicate the presence of a Na+-H+ exchange system in cardiac sarcolemma. This activity appeared to be relatively specific for this membrane fraction. The identification of Na+-H+ exchange activity in a sarcolemmal vesicular fraction from the heart will permit extensive characterization of the regulation and kinetics of this antiporter in future investigations.     

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.     

Na+-Dependent Transport of Taurine by Membrane Vesicles of Neuroblastoma ± Glioma Hybrid Cells

The transport of taurine into membrane vesicles prepared from neuroblastoma x glioma hybrid cells 108CC5 was studied. A great part of the taurine uptake by the membrane preparation is due to the transport into an osmotically sensitive space of membrane vesicles. Taurine uptake by membrane vesicles is an active transport driven by the concentration gradient of Na+ across the membrane (outside concentration greater than inside). The Km value of 36 microM for Na+-dependent taurine uptake indicates a high-affinity transport system. The rate of taurine transport by the membrane vesicles is enhanced by the K+ gradient (inside concentration greater than outside) and the K+ ionophore valinomycin. Taurine transport is inhibited by several structural analogs of taurine: hypotaurine, beta-alanine, and taurocyamine. All these results indicate that the taurine transport system of the membrane vesicles displays properties almost identical to those of intact neuroblastoma X glioma hybrid cells.     

Sodium-dependent amino acid transport in reconstituted membrane vesicles from Ehrlich ascites cell plasma membranes

Plasma membranes, isolated from Ehrlich ascites tumor cells, were dissolved in 2% cholate, 4 M urea and then reformed into liposomes upon dialysis at 4 degrees with exogenous phospholipids. Reconstituted vesicles regain the ability to transport amino acids. Na+ was shown to accelerate the uptake of alpha-aminoisobutyrate, phenylalanine, and methionine, but not leucine or epsilon-aminohexanoic acid. With the reconstituted vesicles, methionine, but not leucine, inhibited the uptake of alpha-aminoisobutyrate. An apparent Km value for alpha-aminoisobutyrate uptake of 3.0 mM was obtained. This value is close to that observed with the intact cells and the native membrane vesicles. A Na+ gradient (high Na+ outside) increased alpha-aminoisobutyrate uptake, whereas a reversed gradient (high Na+ inside) increased alpha-aminoisobutyrate efflux. The latter flux was increased by valinomycin, suggesting electrogenic transport. A modest extent of coupling between a Na+ gradient and uphill flow of alpha-aminoisobutyrate was observed.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Taurine transport across hepatocyte plasma membranes: analysis in isolated rat liver sinusoidal plasma membrane vesicles

To elucidate the mechanism of taurine transport across the hepatic plasma membranes, rat liver sinusoidal plasma membrane vesicles were isolated and the transport process was analyzed. In the presence of a sodium gradient across the membranes (vesicle inside less than vesicle outside), an overshooting uptake of taurine occurred. In the presence of other ion gradients (K+, Li+, and choline+), taurine uptake was very small and no such overshoot was observed. Sodium-dependent uptake of taurine occurred into an osmotically active intravesicular space. Taurine uptake was stimulated by preloading vesicles with unlabeled taurine (transstimulation) in the presence of NaCl, but not in the presence of KCl. Sodium-dependent transport followed saturation kinetics with respect to taurine concentration; double-reciprocal plots of uptake versus taurine concentration gave a straight line from which an apparent Km value of 0.38 mM and Vmax of 0.27 nmol/20 s x mg of protein were obtained. Valinomycin-induced K+-diffusion potential failed to enhance the rate of taurine uptake, suggesting that taurine transport does not depend on membrane potential. Taurine transport was inhibited by structurally related omega-amino acids, such as beta-alanine and gamma-aminobutyric acid, but not by glycine, epsilon-aminocaproic acid, or other alpha-amino acids, such as L-alanine. These results suggest that Na+-dependent uptake of taurine might occur across the hepatic sinusoidal plasma membranes via a transport system that is specific for omega-amino acids having 2-3 carbon chain length.     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

p-Aminohippurate transport in basal-lateral membrane vesicles from rabbit renal cortex: stimulation by pH and sodium gradients

p-Aminohippuric acid (PAH) uptake was studied in basal-lateral membrane vesicles prepared from rabbit renal cortex. An outwardly directed hydroxyl gradient (pHo = 6.0, pHi = 7.6) stimulated PAH uptake slightly over that when the internal and external pH values were equal at 7.6. A 100 mM sodium gluconate gradient directed into the basal-lateral membrane vesicles increased PAH uptake about 2-fold over that when N-methyl-D-glucamine or potassium gluconate gradients were present. When hydroxyl and sodium gradients were simultaneously imposed (pHo = 6.0, pHi = 7.6 and 100 mM sodium gluconate extravesicularly) PAH uptake was stimulated greater than with the pH or Na+ gradient alone. In fact, an 'overshoot' was observed. Countertransport experiments showed that either intravesicular PAH or intravesicular PAH and Na+ could stimulate 3H-PAH uptake. Probenecid, an inhibitor of organic anion transport, inhibited both the hydroxyl-stimulated and Na+ gradient-stimulated PAH uptake but the greatest inhibition by probenecid was seen when the hydroxyl and sodium gradients were both present. Thus, it is proposed that the driving force for PAH accumulation across the basal-lateral membrane of the proximal tubule is a transport system which moves Na+ and PAH into the cell for an hydroxyl ion leaving the cell, i.e. a sodium-dependent anion-anion exchange system.     

Calcium/sodium exchange in purified secretory vesicles from bovine neurohypophyses

Purified secretory vesicles isolated from bovine neurohypophyses take up Na+ under the same circumstances where an efflux of Ca2+ takes place, suggesting a Na+/Ca2+ exchange. Potassium cannot substitute for Na+ in this process. Also, a Ca2+/Ca2+ exchange can occur. Inhibiting the latter process by Mg2+ allowed to estimate an apparent KM of 0.7 microM free Ca2+ and a maximal uptake of 1.5 nmol X mg protein-1 X min-1 Ca2+ in exchange for Na+. The vesicles did not contain plasma membrane marker (Na+/K+ ATPase) as shown by distribution analyses on the density gradients on which they were purified. Similarly, distribution studies also showed that no other ATPase activity could be detected in the purified vesicle fraction. It is concluded that a Na+/Ca2+ exchange is operating across the secretory vesicle membrane and that it is not directly dependent on ATP hydrolysis.     

A monoclonal antibody against a Na(+)-L-glutamate cotransporter from rat brain.

A monoclonal mouse IgM antibody (Z8E9) was raised against the Na(+)-L-glutamate cotransporter from rat brain. In a preparation of brain plasma membrane vesicles, Z8E9 binds specifically to a polypeptide with an apparent molecular weight of 70,000 and inhibits Na+ gradient-dependent L-glutamate cotransport (up to 50%) in brain membrane vesicles. In the membrane vesicles, the antibody does not alter the membrane permeability for Na+ and K+ nor the Na+ gradient-dependent uptake of gamma-aminobutyric acid. Kinetic experiments showed that Z8E9 does not alter the K0.5 values for L-glutamate and Na+ activation of L-glutamate transport. However, an apparent cooperativity observed for L-glutamate activation was increased, and the Vmax of L-glutamate transport was decreased. Immunostaining of rat cerebellum identified antigenic sites of Z8E9 in Golgi epithelial cells and astrocytes (by light and electron microscopy), whereas no labeling at nerve terminals was detected. The data suggest that a component of a Na(+)-L-glutamate cotransporter subtype has been identified that is specific for glia cells in brain.     

Na+ + Cl- -gradient-driven, high-affinity, uphill transport of taurine in human placental brush-border membrane vesicles

Uptake of taurine in human placental brush-border membrane vesicles was greatly stimulated in the presence of an inwardly-directed Na+ + Cl- -gradient and uphill transport of taurine could be demonstrated under these conditions. Na+ as well as Cl- were obligatory for this uptake and both ion gradients could energize the uphill transport. This Na+ + Cl- -gradient-dependent taurine uptake was stimulated by an inside-negative membrane potential, demonstrating the electrogenicity of the process. The uptake system was highly specific for beta-amino acids and the Km of the system for taurine was 6.5 +/- 0.4 microM.