Characterization of the defect in the Na(+)-phosphate transporter in vitamin D-resistant hypophosphatemic mice.

Hypophosphatemic vitamin D-resistant rickets is the most common form of vitamin D-resistant rickets in man. The hypophosphatemic mouse model (Hyp) is phenotypically and biochemically similar to the human disease. Biochemically, hypophosphatemia is the hallmark of this disorder. The cause of the hypophosphatemia is thought to be secondary to a defect in the renal and/or intestinal Na(+)-phosphate transporter. The current studies were designed to investigate and characterize the localization of the defect in the Na(+)-phosphate transporter in this disorder. Phosphate uptake by renal brush border membrane vesicles (BBMV) showed a significant decrease in the slope of the initial rate of phosphate uptake in (Hyp) compared with control mice (0.009 versus 0.013, respectively). The slopes representing initial rates of phosphate uptake by jejunal BBMV were similar in (Hyp) and control mice (0.004 and 0.004, respectively). Kinetics of jejunal Na(+)-dependent phosphate uptake showed a Vmax of 0.63 +/- 0.12 and 0.64 +/- 0.12 nmol/mg protein/15 s in (Hyp) and control mice, respectively, whereas Km values were 0.12 +/- 0.08 and 0.2 +/- 0.11 mM, respectively. Similar kinetic analysis in the kidney showed a Vmax of 0.32 +/- 0.06 and 1.6 +/- 0.1 (p less than 0.01) and Km of 0.07 +/- 0.06 and 0.39 +/- 0.05 (p less than 0.02) in (Hyp) and control mice, respectively. Na(+)-dependent D-glucose uptake by BBMVs of intestine and kidney showed typical overshoot phenomena in (Hyp) and control mice. In order to explore these findings further, Na(+)-phosphate transporter expression from intestine and kidney was accomplished by microinjection of 50 ng of poly(A)+ RNA into Xenopus laevis oocytes. Na(+)-dependent phosphate uptake was expressed 6 days after the microinjection of intestinal and kidney poly(A)+ RNA from control mice. However, expression of the transporter from (Hyp) mice occurred only from the intestine, and not from the kidney. The decrease in the expression of the Na(+)-dependent phosphate transporter was not secondary to accelerated efflux of phosphate or decreased metabolism in oocytes injected with poly(A)+ RNA from (Hyp) mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of human intestinal brush border membrane vesicle Na+-dependent phosphate uptake by phosphophloretin derivatives

Hyperphosphatemia and II(o) hyperparathyroidism are common and severe complications of chronic renal failure. Reduced dietary phosphorus has been shown to be an effective treatment in reducing serum phosphate and serum PTH. 2(')-Phosphophloretin inhibited small intestine apical membrane Na(+)/phosphate cotransport and reduced serum phosphate in adult rats. 2(')-PP and phosphoesters of phloretin were tested for inhibition of human small intestine brush border membrane alkaline phosphatase activity and for inhibition of Na(+)-dependent phosphate uptake. The IC(50)'s for inhibition of alkaline phosphatase suggested an order of inhibitory potency of 4-PP > phloretin > 4(')-PP > 2(')-PP. Inhibition of Na(+)-dependent phosphate uptake followed the sequence 2(')-PPz.Gt;4(')-PP > 4-PP > phloretin. These results are consistent with 2(')-PP being a specific inhibitor of human intestinal brush border membrane Na(+)/phosphate cotransport.     

Divalent metal is required for both phosphate transport and phosphate binding to phosphorin, a proteolipid isolated from brush-border membrane vesicles

The Na+-dependent phosphate transport system in the brush border of rabbit kidney exhibits a positive requirement for a divalent metal ion. Treatment of the brush-border membrane vesicles (BBMV) with a divalent metal chelator in combination with the divalent metal ionophore A23187 dramatically and selectively decreased the Na+-dependent uptake of phosphate; Na+-independent uptake of phosphate was not affected. The combination of chelator plus A23187 also inhibited uptake of phosphate in the presence of Na+ but in the absence of a gradient for sodium across the BBMV. This indicates that the inhibitor is not a result of an alteration in the Na+ gradient by chelator plus ionophore. The inhibited Na+ gradient-dependent transport of phosphate was restored by removing the chelator and adding Mn2+ to the BBMV. The phosphate-binding proteolipid (phosphorin) isolated from rabbit kidney BBMV binds inorganic phosphate with high affinity and specificity. Binding of phosphate to phosphorin is also inhibited by divalent metal chelators and can be restored by addition of a divalent metal. We conclude that a divalent metal ion is required both for the Na+-dependent phosphate transport in BBMV and for the binding of phosphate to the proteolipid phosphorin. These findings are consistent with our suggestion that phosphorin is a component of the Na+-dependent phosphate transport system in renal brush-border membranes.     

Na(+)-dependent D-mannose transport at the apical membrane of rat small intestine and kidney cortex

The presence of a Na(+)/D-mannose cotransport activity in brush-border membrane vesicles (BBMV), isolated from either rat small intestine or rat kidney cortex, is examined. In the presence of an electrochemical Na(+) gradient, but not in its absence, D-mannose was transiently accumulated by the BBMV. D-Mannose uptake into the BBMV was energized by both the electrical membrane potential and the Na(+) chemical gradient. D-Mannose transport vs. external D-mannose concentration can be described by an equation that represents a superposition of a saturable component and another component that cannot be saturated up to 50 microM D-mannose. D-Mannose uptake was inhibited by D-mannose > D-glucose>phlorizin, whereas for alpha-methyl glucopyranoside the order was D-glucose=phlorizin > D-mannose. The initial rate of D-mannose uptake increased as the extravesicular Na(+) concentration increased, with a Hill coefficient of 1, suggesting that the Na(+):D-mannose cotransport stoichiometry is 1:1. It is concluded that both rat intestinal and renal apical membrane have a concentrative, saturable, electrogenic and Na(+)-dependent D-mannose transport mechanism, which is different from SGLT1.     

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.     

Decreased D-glucose transport across renal brush-border membrane vesicles from streptozotocin-induced diabetic rats

The uptake of Na(+)-dependent D-glucose by renal brush-border membrane vesicles (BBMV) isolated from streptozotocin-induced diabetic rats was decreased as compared with controls. Since a Vmax of 4.8 nmol/mg protein per 30 s in diabetic BBMV was significantly decreased as compared with that of controls (Vmax = 7.0 nmol/mg protein per 30 s) without changing an apparent affinity for D-glucose, the decrease in the Na(+)-dependent D-glucose uptake in diabetic rats is likely to be due to the reduction in the number of the transporter. These results are also confirmed by the binding study of [3H]phlorizin to diabetic BBMV. When the blood glucose level is lowered in diabetic rats by both the treatment with insulin and starvation, the decreased Na(+)-dependent D-glucose uptake is returned to control level. These results suggest that Na(+)-dependent D-glucose reabsorption through the apical membrane in proximal tubular kidney cells is dynamically regulated by the change in blood glucose level.     

Quantitation of the Na(+)-Pi cotransporter in renal cortical brush border membranes. [14C]phosphonoformic acid as a useful probe to determine the density and its change in response to parathyroid hormone.

To determine the density of Na(+)-Pi symporters in brush border membranes (BBM) from rat renal cortex, [14C] phosphonoformic acid [( 14C] PFA), a competitive inhibitor of Na(+)-Pi cotransport, was employed as a probe. The [14C]PFA binding was measured in BBM vesicles (BBMV) under equilibrated conditions (extra-vesicular Na+, K+, and H+ = intravesicular Na+, K+, and H+) to avoid modulatory effects of these solutes. BBMV were preincubated in media without or with addition of molar excess of Pi (greater than 20 times) to determine the Pi-protectable PFA-binding sites, and then [14C] PFA binding was determined. Only the [14C]PFA binding in the presence of Na+ displaceable by an excess of Pi was saturated and was independent of intravesicular volume of BBMV. This value denoted as "Pi-protectable Na(+)-[14C]PFA binding," was analyzed by Scatchard plot showing BmaxPFA = 375 +/- 129 pmol of PFA/mg protein, KDPFA = 158 +/- 18 microM; the Hill coefficient was congruent to 1. For Na(+)-dependent binding of [3H]phlorizin, in the same BBMV, Bmax = 310 +/- 37 pmol/mg protein and KD V 2.2 +/- 0.5 microM. BBMV prepared from cortex of thyroparathyroidectomized rats infused with phosphaturic doses of parathyroid hormone (PTH) were compared with vehicle-infused controls. Administration of PTH resulted in decrease of BmaxPFA (-38%) and of Na(+)-gradient-dependent uptake of 32Pi (-35%), but KDPFA was not changed. Neither BmaxPhl and KDPhl for Na(+)-phlorizin binding, nor the Na(+)-gradient-dependent uptake of [3H]D-glucose differed between PTH-treated and control rats. We conclude: (a) measurement of Pi-protectable Na(+)-[14C]PFA binding determines numbers and affinity of Na(+)-Pi symporters in renal BBMV; (b) the affinity of PFA for Na(+)-Pi symporter is similar to apparent affinity for Pi (KmPi), as determined from measurements of Na(+)-gradient-dependent 32Pi uptake by BBMV; (c) both Na(+)-Pi symporter and [Na+]D-glucose symporters are present within renal BBM in a similar range of density; (d) PTH decreases the number of Na(+)-Pi cotransporters in BBMV commensurate with the parallel decrease of Na(+)-gradient-dependent Pi transport, whereas the affinity of Na(+)-Pi symporters for Pi is not changed. These observations support the hypothesis that PTH decreases capacity for Na(+)-dependent Pi reabsorption by internalization of Na(+)-Pi symporters in BBM of renal proximal tubules.     

Expression of renal transport systems for inorganic phosphate and sulfate in Xenopus laevis oocytes.

As a first step within an experimental strategy (expression cloning) leading to the structural identification of the two brush-border membrane transport systems for phosphate and sulfate, we have studied the expression of Na(+)-dependent uptake of phosphate and sulfate in Xenopus laevis oocytes injected with rabbit kidney cortex poly(A)+ RNA (mRNA). Na(+)-dependent uptake of phosphate and sulfate was stimulated in a dose- and time-dependent manner up to 20-fold as compared to water-injected controls. After fractionation of the mRNA on a sucrose gradient (or by preparative gel electrophoresis), two neighboring fractions were identified to stimulate Na(+)-dependent phosphate uptake (average size: 3.4 kilobases) and Na(+)-dependent sulfate uptake (average size: 3.7 kilobases). The two transport systems can be discriminated by their inhibition by thiosulfate, which reduced sulfate uptake, but not phosphate uptake. Kinetic characterization of the expressed Na(+)-dependent transport activities results in properties similar to those described for transport activity in renal brush-border membrane vesicles.     

Enhanced glucose absorption in the rat small intestine following repeated doses of 5-fluorouracil

Many studies demonstrated that 5-fluorouracil (5-FU) treatment of rodents caused the damage of small intestine, resulting in the malabsorption, while we recently found that repeated administration of 5-FU to rats increased Na(+)-dependent glucose absorption in the small intestine. This study investigated the cause of enhanced glucose absorption. 3-O-methyl-d-glucose (3-OMG) absorption was examined using the everted intestine technique. d-Glucose uptake, phlorizin binding, Western blot analysis and membrane fluidity were examined using small intestinal brush-border membrane vesicles (BBMV). Repeated oral administration of 5-FU to rats increased Na(+)-dependent 3-OMG absorption in the small intestine, while alkaline phosphatase activity in the small intestine decreased. Na(+)/K(+)-ATPase activity of 5-FU-treated rats was about three-fold higher than that of control rats. Although the amount of Na(+)-dependent glucose co-transporter (SGLT1) in 5-FU-treated rats decreased, the overshoot magnitude of d-glucose uptake in BBMV was not altered. Maximum binding of phlorizin in 5-FU-treated rats was 1.5-fold larger than that of control rats, but not altered the maximal rate of d-glucose absorption, Michaelis constant of d-glucose and dissociation constant of phlorizin. The membrane fluidity of 5-FU-treated rats increased. The enhanced d-glucose absorption in 5-FU-treated rats seems to occur secondarily due to the activation of Na(+)/K(+)-ATPase activity in basolateral membranes (BLM). Because the amounts of SGLT1 in 5-FU-treated rats decreased, the increase of turnover rate of SGLT1 and/or an expression of unknown Na(+)-dependent glucose co-transporter with high affinity for d-glucose and phlorizin sensitivity would contribute to the enhancement of d-glucose transport in 5-FU-treated rats.     

Expression and characterization of the intestinal Na+/glucose cotransporter in COS-7 cells

Cells derived from the simian kidney, COS-7 cells, were transfected with a eucaryotic expression vector (pEUK-C1) containing the clone for the rabbit intestinal Na+/glucose cotransporter. Expression was monitored after transfection with lipofectin by measuring the initial rate of alpha-methylglucopyranoside (MeGlc) uptake. Cells transfected with vector containing the cDNA for the Na+/glucose cotransporter expressed Na(+)-dependent MeGlc transport. Neither control cells nor cells transfected with vector lacking cloned cDNA expressed the cotransporter. Na(+)-dependent MeGlc uptake into transfected cells was saturable (Km 150 microM), phlorizin-sensitive (Ki 11 microM), and inhibited by sugar analogs (D-glucose greater than MeGlc greater than D-galactose greater than 3-O-methyl-D-glucoside greater than D-allose much greater than L-glucose). Europium was able to mimic Na+ in driving MeGIC uptake. Finally, tunicamycin, an inhibitor of asparagine-linked glycosylation, inhibited the expression of Na(+)-dependent MeGlc transport 80%. We conclude that the rabbit intestinal Na+/glucose cotransporter expressed in COS-7 cell exhibits very similar kinetic properties to that in the native brush border and to that expressed in Xenopus oocytes. In addition, N-linked glycosylation appears to be important for functional expression of this membrane protein.     

Inhibition of renal Na(+)-Pi cotransporter by mercuric chloride: role of sulfhydryl groups.

We studied the role of sulfhydryl groups in Na(+)-Pi cotransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na(+)-dependent 32Pi transport in a dose-dependent manner (IC50 = 54 microM). Na(+)-independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium-exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D-glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (greater than 100 microM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 +/- 0.13 to 0.75 +/- 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected against inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na(+)-Pi cotransporter of renal BBM.     

Intestinal transport of monosaccharides and amino acids during postnatal development of mink

Intestinal development is typically studied using omnivores. For comparative purposes, we examined an altricial carnivore, the mink (Mustela vison). In mink, intestinal dimensions increase up to 8 wk after birth and then remain constant (length) or decrease (mass) into maturity despite continuing gains in body mass. Rates of glucose and fructose transport decline after birth for intact tissues but increase for brush-border membrane vesicles (BBMV). Rates of absorption for five amino acids that are substrates for the acidic (aspartate), basic (lysine), neutral (leucine and methionine), and imino acid (proline) carriers increase between birth and 24 h for intact tissues before declining, but increase after 2 wk for BBMV. The proportion of BBMV amino acid uptake that is Na(+)-dependent increases during development but for aspartate is nearly 100% at all ages. Tracer uptake by BBMV can be inhibited by 100 mmol/l of unlabeled amino acid, except for lysine. BBMV uptake of the dipeptide glycyl-sarcosine does not differ between ages, is not Na(+) dependent, and is only partially inhibited by 100 mmol/l unlabeled dipeptide. Despite the ability to rapidly and efficiently digest high dietary loads of protein, rates of amino acid and peptide absorption are not markedly higher than those of other mammals.     

Inhibition of intestinal glucose uptake by aporphines and secoaporphines

The effects of aporphines and secoaporphines on glucose uptake by isolated intestinal brush-border membrane vesicles (BBMV) or basolateral membrane vesicles (BLMV) and glucose absorption during in situ intestinal perfusion were studied. Of the tested compounds, N-allylsecoboldine was the most potent glucose uptake inhibitor, with IC50 values of 159 microM and 121 microM, respectively, for uptake by BBMV and BLMV. While thaliporphine competitively inhibited glucose uptake by both membrane preparations, inhibition by N-allylsecoboldine was competitive using BBMV and noncompetitive using BLMV. In addition, N-allylsecoboldine significantly reduced both glucose absorption during in situ intestinal perfusion and blood glucose levels in the oral glucose tolerance test. The results demonstrate that levels of both aporphines and secoaporphines achievable by oral administration have an inhibitory effect on intestinal glucose uptake and suggest that the hypoglycemic effects of these compounds merit attention.     

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.     

Examination of sodium/glucose cotransport by using a visible glucose analogue

The glucose derivative, 2,2,6,6-tetramethylpiperidine-1-oxylglucose (TEMPO-glucose) was synthesized and examined for its ability to substitute for glucose as a substrate for the intestinal brush border membrane Na+/glucose cotransporter. TEMPO-glucose inhibited Na(+)-dependent phlorizin binding with an apparent KI of 18 microM and Na(+)-dependent glucose uptake with an apparent KI of 70 microM. The transport competence of TEMPO-glucose was examined by using two measures of transport. The first involved comparing the reversal of trans Na+ inhibition by D-glucose and TEMPO-glucose. The second directly examined Na(+)-dependent TEMPO-glucose uptake by using TEMPO-glucose quenching of intravesicular fluorescein sulfonate fluorescence. Tryptophan fluorescence was sensitive to TEMPO-glucose in a Na(+)-dependent, glucose-inhibitable manner. The bulk of these tryptophans appeared to be located in hydrophobic environments based on Cs(+)-insensitivity. With the reconstituted cotransporter, TEMPO-glucose, and tryptophan quench reagents, the cotransporter was compared in three transport modes: zero trans uptake, zero trans uptake in the presence of a shunt of membrane potential, and substrate exchange. The results suggest that the cotransporter conformation varies depending on its mode of operation and that TEMPO-glucose may be a useful probe for localizing amino acid residues involved in glucose transport.     

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.     

Involvement of rBAT in Na(+)-dependent and -independent transport of the neurotransmitter candidate L-DOPA in Xenopus laevis oocytes injected with rabbit small intestinal epithelium poly A(+) RNA

Although L-3,4-dihydroxyphenylalanine (L-DOPA) is claimed to be a neurotransmitter in the central nervous system (CNS), receptor or transporter molecules for L-DOPA have not been determined. In an attempt to identify a transporter for L-DOPA, we examined whether or not an active and high affinity L-DOPA transport system is expressed in Xenopus laevis oocytes injected with poly A(+) RNA prepared from several tissues. Among the poly A(+) RNAs tested, rabbit intestinal epithelium poly A(+) RNA gave the highest transport activity for L-[(14)C]DOPA in the oocytes. The uptake was approximately five times higher than that of water-injected oocytes, and was partially Na(+)-dependent. L-Tyrosine, L-phenylalanine, L-leucine and L-lysine inhibited this transport activity, whereas D-DOPA, dopamine, glutamate and L-DOPA cyclohexylester, an L-DOPA antagonist did not affect this transport. Coinjection of an antisense cRNA, as well as oligonucleotide complementary to rabbit rBAT (NBAT) cDNA almost completely inhibited the uptake of L-[(14)C]DOPA in the oocytes. On the other hand, an antisense cRNA of rabbit 4F2hc barely affected this L-[(14)C]DOPA uptake activity. rBAT was thus responsible for the L-[(14)C]DOPA uptake activity expressed in X. laevis oocytes injected with poly A(+) RNA from rabbit intestinal epithelium. As rBAT is localized at the target regions of L-DOPA in the CNS, rBAT might be one of the components involved in L-DOPAergic neurotransmission.     

Evidence for an imipramine-sensitive serotonin transporter in human placental brush-border membranes

Serotonin is actively transported into brush-border membrane vesicles isolated from normal human term placentas and an inward-directed NaCl gradient provides the driving force for this process. Uptake is negligible if Na+ is replaced by Li+, K+, Rb+, Cs+ or choline. The presence of Cl- seems necessary for the maximal activity of this Na+-dependent uptake system. Intravesicular K+ (20-40 mM) stimulates serotonin uptake, the stimulation being considerably greater at pH 7.5 than at pH 6.5. But, in the absence of K+, uptake at pH 6.5 was twice the uptake at pH 7.5. Unlabeled serotonin and dopamine inhibit the uptake of radiolabeled serotonin and the IC50 values are 70 nM and 20 microM, respectively. Histamine and 5-hydroxytryptophan do not significantly interact with the system (IC50 greater than 1 mM). Kinetic analysis reveals that serotonin uptake in these vesicles occurs via a single, saturable, high affinity system (Kt = 51 +/- 2 nM; Vmax = 6.4 +/- 0.1 pmol/mg of protein/15 s). The transporter is highly sensitive to inhibition by imipramine (IC50 = 32 nM) and desipramine (IC50 = 160 nM) but relatively insensitive to reserpine and hydralazine.