Phosphate transport by rat intestinal basolateral-membrane vesicles.

The characteristics of phosphate transport across intestinal basolateral membranes of the rat were determined by using enriched preparations in which uphill Na+-dependent D-glucose transport could not be demonstrated, but ATP-dependent Ca2+ transport was present. Phosphate transport was saturable, Na+-dependent and exhibited Michaelis-Menten kinetics. Vmax. was 51.1 +/- 4.2 pmol/10 s per mg of protein and Km was 14 +/- 3.9 microM. The transport process was electroneutral. Tracer-exchange experiments and counter-transport studies confirmed the presence of a Na+-Pi carrier at the basolateral membrane. The presence of inside-positive membrane potential did not enhance phosphate uptake, indicating that the Na+ effect is secondary to the presence of the Na+-Pi carrier rather than an induction of positive membrane potential. The stoichiometry of this carrier at pH 7.4 was 2 Na+:1 phosphate, as shown by direct studies utilizing the static-head method. These studies are the first to determine the presence of a phosphate carrier at the basolateral membrane.     

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.     

Intestinal calcium transport in spontaneously hypertensive rats (SHR) and their genetically matched WKY rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.     

Dicarboxylate transport in renal basolateral and brush-border membrane vesicles.

Characteristics of succinate transport were determined in basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated in parallel from rabbit renal cortex. The uptake of succinate was markedly stimulated by the imposition of an inwardly directed Na+ gradient, showing an "overshoot" phenomenon in both membrane preparations. The stimulation of succinate uptake by an inwardly directed Na+ gradient was not significantly affected by pH clamp or inhibition of Na(+)-H+ exchange. The Na(+)-dependent and -independent succinate uptakes were not stimulated by an outwardly directed pH gradient. The Na dependence of succinate uptake exhibited sigmoidal kinetics, with Hill coefficients of 2.17 and 2.38 in BLMV and BBMV, respectively. The Na(+)-dependent succinate uptake by BLMV and BBMV was stimulated by a valinomycin-induced inside-negative potential. The Na(+)-dependent succinate uptake by BLMV and BBMV followed a simple Michaelis-Menten kinetics, with an apparent Km of 22.20 +/- 4.08 and 71.52 +/- 0.14 microM and a Vmax of 39.0 +/- 3.72 and 70.20 +/- 0.96 nmol/(mg.min), respectively. The substrate specificity and the inhibitor sensitivity of the succinate transport system appeared to be very similar in both membranes. These results indicate that both the renal brush-border and basolateral membranes possess the Na(+)-dependent dicarboxylate transport system with very similar properties but with different substrate affinity and transport capacity.     

65Zn2+ Transport by lobster hepatopancreatic lysosomal membrane vesicles

In crustaceans, the hepatopancreas is the major organ system responsible for heavy metal detoxification, and within this structure the lysosomes and the endoplasmic reticulum are two organelles that regulate cytoplasmic metal concentrations by selective sequestration processes. This study characterized the transport processes responsible for zinc uptake into hepatopancreatic lysosomal membrane vesicles (LMV) and the interactions between the transport of this metal and those of calcium, copper, and cadmium in the same preparation. Standard centrifugation methods were used to prepare purified hepatopancreatic LMV and a rapid filtration procedure, to quantify 65Zn2+ transfer across this organellar membrane. LMV were osmotically reactive and exhibited a time course of uptake that was linear for 15-30 sec and approached equilibrium by 300 sec. 65Zn2+ influx was a hyperbolic function of external zinc concentration and followed Michaelis-Menten kinetics for carrier transport (Km = 32.3 +/- 10.8 microM; Jmax = 20.7 +/- 2.6 pmol/mg protein x sec). This carrier transport was stimulated by the addition of 1 mM ATP (Km = 35.89 +/- 10.58 microM; Jmax = 31.94+/-3.72 pmol/mg protein/sec) and replaced by an apparent slow diffusional process by the simultaneous presence of 1 mM ATP+250 microM vanadate. Thapsigargin (10 microM) was also a significant inhibitor of zinc influx (Km = 72.87 +/- 42.75 microM; Jmax =22.86 +/- 4.03 pmol/mg protein/sec), but not as effective in this regard as was vanadate. Using Dixon analysis, cadmium and copper were shown to be competitive inhibitors of lysosomal membrane vesicle 65Zn2+ influx by the ATP-dependent transport process (cadmium Ki = 68.1 +/- 3.2 microM; copper Ki = 32.7 +/- 1.9 microM). In the absence of ATP, an outwardly directed H+ gradient stimulated 65Zn2+ uptake, while a proton gradient in the opposite direction inhibited metal influx. The present investigation showed that 65Zn2+ was transported by hepatopancreatic lysosomal vesicles by ATP-dependent, vanadate-, thapsigargin-, and divalent cation-inhibited, carrier processes that illustrated Michaelis-Menten influx kinetics and was stimulated by an outwardly directed proton gradient. These transport properties as a whole suggest that this transporter may be a lysosomal isoform of the ER Sarco-Endoplasmic Reticulum Calcium ATPase.     

Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast.

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/K(+)-adenosine triphosphate (ATPase), 37-fold in [3H] dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a Km Ca2+ of 0.18 +/- 0.05 microM and Vmax of 0.93 +/- 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration-dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to 10(-6) M nitrendipine nor to 10(-4) M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 +/- 0.02 microM and Vmax 3.4 +/- 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10(-6) M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.     

Pantothenate-sodium cotransport in renal brush-border membranes

The mechanism of pantothenate transport into rabbit renal brush-border membrane vesicles was studied. Under voltage-clamped conditions, an inward NaCl gradient induced the transient accumulation of pantothenate against its concentration gradient, indicating Na+/pantothenate cotransport. K+, Rb+, Li+, NH4+, and choline+ were ineffective in replacing Na+. Pantothenate analogs, D-glucose, and various carboxylic acids did not inhibit Na+-dependent pantothenate transport, suggesting that this system is specific for pantothenate. Kinetic analysis of the Na+-dependent pantothenate uptake revealed a single transport system which obeyed Michaelis-Menten kinetics (Km = 16 microM and Vmax = 6.7 pmol X mg-1 X 10 s-1). Imposition of an inside-negative membrane potential caused net uphill pantothenate accumulation in the presence of Na+ but absence of a Na+ gradient, indicating that Na+/pantothenate cotransport is electrogenic. The relationship between extravesicular Na+ concentration and pantothenate transport measured under voltage-clamped conditions was sigmoidal: a Hill coefficient (napp) of 2 and a [Na+]0.5 of 55 mM were calculated. It is suggested that an anionic pantothenate1- molecule is cotransported with two Na+ to give a net charge of +1. The coupling of pantothenate transport to the Na+ electrochemical gradient may provide an efficient mechanism for reabsorption of pantothenate in the kidney.     

Na+-dependent transport of alpha-aminoisobutyrate in isolated basolateral membrane vesicles from rat parotid glands

Basolateral plasma membranes were prepared from rat parotid gland after centrifugation in a self-orienting Percoll gradient. K+-dependent phosphatase [Na+ + K+)-ATPase), a marker enzyme for basolateral membranes, was enriched 10-fold from tissue homogenates. Using this preparation, the transport of alpha-aminoisobutyrate was studied. The uptake of alpha-aminoisobutyrate was Na+-dependent, osmotically sensitive, and temperature-dependent. In the presence of a Na+ gradient between the extra- and intravesicular solutions, vesicles showed an 'overshoot' accumulation of alpha-aminoisobutyrate. Sodium-dependent alpha-aminoisobutyrate uptake was saturable, exhibiting an apparent Km of 1.28 +/- 0.35 mM and Vmax of 780 +/- 170 pmol/min per mg protein. alpha-Aminoisobutyrate transport was inhibited considerably by monensin, but incubating with ouabain was without effect. These results suggest that basolateral membrane vesicles, which possess an active amino acid transport system (system A), can be prepared from the rat parotid gland.     

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.     

Leukotriene C4 inhibits ATP-dependent transport of glutathione S-conjugate across rat heart sarcolemma. Implication for leukotriene C4 translocation mediated by glutathione S-conjugate carrier

Sarcolemmal vesicles prepared from rat heart exhibited ATP-dependent uptake of S-(2,4-dinitrophenyl)glutathione (DNP-SG), which obeyed Michaelis-Menten kinetics with an apparent Km of 21 microM for DNP-SG and a Vmax of 0.27 nmol.10 min-1.mg protein-1. Several model glutathione S-conjugates inhibited DNP-SG uptake, but leukotriene C4 inhibited uptake much more significantly even at lower concentrations (competitive inhibition, Ki = 1.5 microM). However, leukotrienes D4 and E4, which lack the gamma-glutamyl moiety, were less effective. The results suggest that the ATP-dependent transport system has a high affinity for leukotriene C4, and may be responsible for the translocation of this compound.     

Ca2+ transport through the brush border membrane of human placenta syncytiotrophoblasts.

The calcium (Ca2+) uptake by brush border membrane vesicles isolated from fresh human placentas has been characterized. This process was saturable and time- and concentration-dependent. It exhibited a double Michaelis-Menten kinetics, with apparent Km values of 0.17 +/- 0.03 and 2.98 +/- 0.17 mM Ca2+, and Vmax values of 0.9 +/- 0.13 and 2.51 +/- 0.45 pmol.micrograms-1.5 s-1. It was not influenced by the presence of Na+ or Mg2+ in the incubation medium. It was not increased by K+ or anion diffusion potentials, inside negative. At a steady state of 1 mM Ca2+ uptake, a large proportion (approximately 94%) of the Ca2+ was bound to the internal surface of the membranes. Preincubation of these membrane vesicles with voltage-dependent Ca2+ channel blockers (nifedipine and verapamil) had no influence on Ca2+ uptake. However, this uptake was very sensitive to pH. In the absence of a pH gradient, the Ca2+ uptake increased with alkalinity. When the intravesicular pH was kept constant while the pH of the incubation medium was increased, Ca2+ uptake was also stimulated by alkaline pH. In contrast, when the pH of the incubation medium was kept constant and the intravesicular pH was progressively increased, Ca2+ uptake was diminished with alkaline pH. Therefore, H+ gradient (H+ in trans-position greater than H+ in cis-position) favored Ca2+ transport, suggesting a H+/Ca2+ exchange mechanism. Finally, in contrast to the basal plasma membrane, the brush border membrane did not show any ATP-dependent Ca2+ transport activity.     

Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands.

Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein.     

Expression of the hepatocyte Na+/bile acid cotransporter in Xenopus laevis oocytes

The expression of the basolateral Na+/bile acid (taurocholate) cotransport system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of rat liver poly(A)+ RNA into the oocytes resulted in the functional expression of Na+ gradient stimulated taurocholate uptake within 3-5 days. This Na(+)-dependent portion of taurocholate uptake exhibited saturation kinetics (apparent Km approximately 91 microM) and could be inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene. Furthermore, the expressed taurocholate transport activity demonstrated similar substrate inhibition and stimulation by low concentrations of bovine serum albumin as the basolateral Na+/bile acid cotransport system previously characterized in intact liver, isolated hepatocytes, and isolated plasma membrane vesicles. Finally, a 1.5- to 3.0-kilobase size-class of mRNA could be identified that was sufficient to express the basolateral Na+/taurocholate uptake system in oocytes. These results demonstrate that "expression cloning" represents a promising approach to ultimately clone the gene and to further characterize the molecular properties of this important hepatocellular membrane transport system.     

Characterisation of an ATP-dependent Ca2+ transport system in a plasma membrane enriched fraction from rat parotid

A membrane fraction enriched in plasma membrane marker enzymes K+-dependent p-nitrophenyl phosphatase, 5'-nucleotidase and alkaline phosphatase was prepared from rat parotid glands using Percoll self-forming gradient. This fraction contained an ATP-dependent CA2+ transport system which was distinct from those located on the endoplasmic reticulum and mitochondria of parotid glands. The Km for ATP was 0.57 +/- 0.07 mM (n = 3). Nucleotides other than ATP such as ADP, AMP, GTP, CTP, UTP or ITP were unable to support significant Ca2+ uptake. ATP-dependent Ca2+ uptake displayed sigmoidal kinetics with respect to free Ca2+ concentration with a Hill coefficient of 2.02. The K0.5 for Ca2+ was 44 +/- 3.1 nM (n = 3) and the average Vmax was 13.5 +/- 1.1 nmol/min per mg of protein. The pH optimum was 7.2. Trifluorperazine inhibited Ca2+ transport with half maximal inhibition observed at 30.8 microM. Complete inhibition was observed at 70 microM trifluorperazine. Exogenous calmodulin however had no effect on the rate of transport. Na+ and K+ ions activated Ca2+ transport at 20 to 30 mM ion concentrations. Higher concentrations of Na+ or K+ were inhibitory.     

Leucine transport in membrane vesicles from Chironomus riparius larvae displays a mélange of crown-group features

Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane preparation was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of l-leucine, which exhibited saturation kinetics. Parameters were determined both at neutral (Km 33 +/- 5 microM and Vmax 22.6 +/- 6.8 pmol/7s/mg protein) and alkaline (Km 46 +/- 5 microM and Vmax 15.5 +/- 2.5 pmol/7s/mg protein) pH values. At alkaline pH, external sodium increased the affinity for leucine (Km 17 +/- 1 microM) and the maximal uptake rate (Vmax 74.0 +/- 12.5 pmol/7s/mg protein). Stimulation of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments indicated that at alkaline pH, the transport system readily accepts most L-amino acids, including branched, unbranched, and alpha-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, beta-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans.     

Kinetics of D-glucose transport across the intestinal brush-border membrane of the cat

1. D-glucose transport across the intestinal brush-border membrane of the cat, a carnivorous animal, was investigated using isolated brush-border membrane vesicles (BBMV). Kinetic experiments were performed under zero-trans conditions (initial [Na+]in and [Gluc]in = O) with the transmembrane electrical potential difference clamped to zero. 2. D-glucose uptake by the BBMV was strongly stimulated by an inwardly directed Na+-gradient. Uptake under Na+-free conditions seemed to occur by simple diffusion. 3. The apparent kinetic constants (Vmax, Km) of Na+-dependent D-glucose transport were computed by forcing initial uptake rates at 0.002-10.0 mmol/l D-glucose to either a Michaelis-Menten type equation with a single or with two carrier-mediated components. 4. Best fit of the experimental data was obtained with the two-component model indicating the existence of two Na+-dependent carrier-mediated mechanisms. System 1 and system 2 differ with respect to the transport velocity as well as the substrate affinity constants with Vmax being 2.5-fold and Km being 5-fold higher for system 1 compared with system 2.     

Amine uptake into intact mast cell granules in vitro

Histamine, the principal amine of rat peritoneal mast cells, is taken up into isolated granules with intact membranes. Uptake is pH- and concentration-dependent and is not stimulated by the addition of Mg2+-ATP. The saturable uptake has a Km of 91.1 microM and a Vmax of 95.4 pmol (mg of protein)-1 min-1. Uptake is abolished by 5 mM ammonium ion. 5-HT, the other endogenous amine of the granules, and dopamine and tyramine, which do not occur naturally in rat mast cells, each competitively inhibits [3H]-histamine uptake with Ki's close to 1 microM. Reserpine, a putative amine carrier blocker, inhibits uptake at nanomolar concentrations. At high concentrations, uptake of [3H]-5-HT is nonsaturable; at low concentrations, a saturable component is observed with a Km of 1.6 microM. Uptake of [3H]-5-HT is not enhanced by Mg2+-ATP. It is pH-dependent but with a lower apparent pKa than that of histamine. [3H]-5-HT uptake can be completely inhibited by ammonium ions. Amine inhibition of [3H]-5-HT gives nonlinear Dixon plots, and high concentrations of the competing amines or reserpine cannot completely block uptake. We propose a model consistent with these results in which amine uptake occurs by several distinct saturable transport systems. According to the model, histamine is transported by a single system, which also transports 5-HT and dopamine. 5-HT and dopamine are transported by one or more other systems.     

The role of chloride in taurine transport across the human placental brush-border membrane.

Taurine, a sulfated beta-amino acid, is conditionally essential during development. A maternal supply of taurine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na(+)-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na(+)-dependent, carrier-mediated taurine transport with an apparent Km of 4.00 +/- 0.22 microM and a Vmax of 11.72-0.36 pmol mg-1 protein 20 s-1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential to transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl-. In addition, a Cl(-)-gradient alone (under Na(+)-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 +/- 0.8 pmol mg-1 protein 20 s-1) and an overshoot compared to Na+, Cl- equilibrated conditions (0.60 +/- 0.06 pmol mg-1 protein 20 s-1). A Cl(-)-gradient (Na(+)-equilibrated) also stimulated uptake of [3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl- suggested that 1 Cl- ion is involved in Na+/taurine cotransport. We conclude that Na(+)-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na+/Cl-/taurine symport.     

Presence of 5-hydroxytryptamine (serotonin) transport coupled with vacuolar-type H(+)-ATPase in neurosecretory granules from bovine posterior pituitary

Electrogenic H(+)-ATPase was found in neurosecretory granules from bovine posterior pituitary. This enzyme was sensitive to bafilomycin, a specific inhibitor of vacuolar H(+)-ATPase, and was inactivated completely by cold treatment in the presence of MgATP and NaNO3. Immunoblot analysis showed the presence of immunologically identical polypeptides (72, 57, and 34 kDa) in the ATPases of the neurosecretory granules and chromaffin granules. The granules showed MgATP-dependent activity for 5-hydroxytryptamine (serotonin) uptake. This uptake was temperature-dependent and showed saturation kinetics (apparent Km for 5-hydroxytryptamine, 2 microM) and counter-flow. Reserpine and tetrabenazine at 1 microM inhibited the uptake, whereas imipramine at 2 microM had no effect. Dopamine, epinephrine and norepinephrine were also inhibitory. The uptake was abolished by various treatments that dissipated the electrochemical H+ gradient or inhibited the H(+)-ATPase. These results indicate that a vacuolar type H(+)-ATPase in the neurosecretory granules forms an electrochemical H+ gradient that drives 5-hydroxytryptamine uptake by a specific transport system. A similar granular fraction from the anterior pituitary had no ATP-dependent activity for 5-hydroxytryptamine uptake.     

Characterization of the ascorbic acid transport by 3T6 fibroblasts

Ascorbic acid transport by 3T6 mouse skin fibroblasts has been characterized using radiometric technique with L-[1-14C]ascorbic acid under the conditions in which oxidation of ascorbic acid was prevented by addition of 1 mM thiourea. The ascorbate transport is temperature-dependent with the energy of activation E and Q10 of 13.3 kcal/mol and 2.0, respectively. The transport requires energy and exhibits Michaelis-Menten kinetics with an apparent Km of 112 microM and Vmax of 158 pmol/min per mg protein, when the extracellular Na+ concentration is 150 mM. The ascorbate transport requires presence of extracellular Na+ and can be inhibited by ouabain treatment. At 40 and 200 microM ascorbate concentrations, respectively, 1.4 and 1.0 moles of Na+ bound the transporter molecule per each mole of ascorbate transported. Increased Na+ binding to the transporter at lower ascorbate concentration may signify multiple Na+-binding sites or ascorbate concentration dependent conformational changes in the transporter molecule. Increasing Na+ concentration decreases Km without affecting Vmax, suggesting that Na+ increases affinity of ascorbate for the transporter molecule without affecting translocation process. An increase in ascorbate concentration reduces the number of Na+ bound to the transporter from 1.4 to 1.0. The ascorbate transport is stimulated by Ca2+ and other divalent cations. The mechanism of stimulation by Ca2+ is not clear. Calcium increases both the Km and Vmax. The data presented support the hypothesis that the ascorbate transport by 3T6 fibroblasts is an energy and temperature-dependent active process driven by the Na+ electrochemical gradient. A potent inhibitor of ascorbate transport is also demonstrated in human serum.