A cDNA clone highly related to the rat brain taurine transporter has been isolated from a human placental cDNA library. Transfection of this cDNA into HeLa cells results in a marked elevation of taurine transport activity. The activity of the cDNA-induced transporter is dependent on the presence of Na+ as well as Cl-. The Na+/Cl-/taurine stoichiometry for the cloned transporter is 2:1:1. The transporter is specific for taurine and other beta-amino acids, including beta-alanine, and exhibits high affinity for taurine (Michaelis-Menten constant approximately 6 microM). The clone consists of a coding region 1863 bp long (including the termination codon), flanked by a 376 bp-long 5′ non-coding region and a 625 bp-long 3′ non-coding region. The nucleotide sequence of the coding region predicts a 620-amino acid protein with a calculated M(r) of 69,853. Northern-blot analysis of poly(A)+ RNA from several human tissues indicates a complex expression pattern differing across tissues. The principal transcript, 6.9 kb in size, is expressed abundantly in placenta and skeletal muscle, at intermediate levels in heart, brain, lung, kidney and pancreas and at low levels in liver. Cultured human cell lines derived from placenta (JAR and BeWo), intestine (HT-29), cervix (HeLa) and retinal pigment epithelium (HRPE), which are known to possess Na(+)- and Cl(-)-coupled taurine transport activity, also contain the 6.9 kb transcript. Somatic cell hybrid and in situ hybridization studies indicate that the cloned taurine transporter is localized to human chromosome 3 p24-->p26.

The 5-hydroxytryptamine (5-HT; serotonin) transporter was solubilized from purified human placental brush border membranes by cholate in the presence of urea, and the solubilized transporter was reconstituted into proteoliposomes in a functionally active form. Solubilization of the membranes with cholate in the absence of urea inactivated the transporter. The reconstitution procedure involved precipitation of the solubilized proteins and simultaneous removal of cholate and urea by poly(ethylene glycol), and incorporation of the precipitated proteins into proteoliposomes in the presence of asolectin by a freeze-thaw/sonication technique. Optimal conditions included the use of 6% poly(ethylene glycol) during the precipitation step and an asolectin/protein ratio of 10:1 during the reconstitution step. K+ was present in the reconstitution medium. The reconstituted proteoliposomes showed the ability to transport 5-HT against a concentration gradient when an inwardly directed NaCl gradient was imposed. The 5-HT transport system in the proteoliposomes had an absolute requirement for Na+ and Cl-. The system was specific for 5-HT and was inhibited by imipramine, paroxetine and fluoxetine. The Na+/Cl-/5-HT stoichiometry was found to be 1:1:1. The transport process was electrically silent, indicating that one K+ ion was countertransported for each 5-HT molecule. The reconstituted 5-HT transporter showed high affinity for 5-HT, with an apparent Michaelis-Menten constant of 0.34 +/- 0.01 microM. It is concluded that the human placental 5-HT transporter can be solubilized and reconstituted into proteoliposomes in a transport-competent form and that the characteristics of the reconstituted transporter are similar to those of the native transporter.

We investigated whether highly purified preparations of basal (fetal-facing) membrane isolated from normal term human placentas possess Na(+)-H+ exchanger activity. Uptake of Na+ into basal membrane vesicles was stimulated many-fold by an outwardly directed H+ gradient. This H(+)-gradient-dependent uptake was inhibitable by amiloride and its analogues. Na+ uptake in these vesicles did not occur via a Na+ channel, as it was not influenced by changes in membrane potential and, in addition, was inhibited by benzamil only at high micromolar concentrations. The results indicate that the human placental basal membrane possesses Na(+)-H+ exchanger activity. We then studied whether this exchanger is similar to or distinct from the Na(+)-H+ exchanger described in brush border (maternal-facing) membrane preparations. For this purpose, we compared the pharmacological characteristics of the basal membrane Na(+)-H+ exchanger with those of the brush border membrane Na(+)-H+ exchanger. The basal membrane exchanger was about 20-fold less sensitive to inhibition by amiloride and about 70-fold less sensitive to inhibition by dimethylamiloride than was the brush border membrane exchanger. The exchanger activity in both membrane preparations was inhibitable by clonidine and cimetidine, but the inhibition patterns with these compounds were markedly different between basal and brush border membrane preparations. These data demonstrate that the basal membrane Na(+)-H+ exchanger is distinct from the brush border membrane Na(+)-H+ exchanger. The pharmacological profiles of these exchangers indicate that the human placental brush border membrane possesses the housekeeping or non-epithelial type Na(+)-H+ exchanger (NHE-1), whereas the basal membrane possesses the epithelial or apical type Na(+)-H+ exchanger (NHE-2).

Available evidence indicates that there are two types of Na(+)-H+ exchangers, type A (housekeeping type) and type B (epithelial or apical type), in mammalian cells. We have recently reported, using isolated membrane vesicles, that these two types can be differentiated by their relative sensitivities to inhibition by clonidine and cimetidine [Kulanthaivel, Leibach, Mahesh, Cragoe & Ganapathy (1990) J. Biol. Chem. 264, 1249-1252]. The present study was undertaken to determine whether this approach is also effective in cultured cells. The JAR human placental choriocarcinoma cell line and the opossum kidney (OK) cell line, when grown as confluent monolayer cultures on an impermeable plastic support, express Na(+)-H+ exchanger activity which is measurable by determining Na+ uptake into the cells from the culture medium. The JAR cell Na(+)-H+ exchanger was found to be about 100 times more sensitive to inhibition by dimethylamiloride than the OK cell Na(+)-H+ exchanger. Inhibition studies with clonidine and cimetidine were able to differentiate between these two exchangers very clearly. Cimetidine was 18 times more potent than clonidine in inhibiting the JAR cell Na(+)-H+ exchanger. In contrast, clonidine was at least 8 times more potent than cimetidine in inhibiting the Na(+)-H+ exchanger of the OK cell. The results show that the JAR cell expresses the type A Na(+)-H+ exchanger, whereas the OK cell expresses the type B Na(+)-H+ exchanger. This approach also proved to be very effective in correctly identifying the type of Na(+)-H+ exchanger in a third cell line (HeLa). It is concluded that the relative susceptibility to inhibition by clonidine and cimetidine offers an easy and efficient means of differentiating between the two types of Na(+)-H+ exchangers in cultured cells.

The JAR human placental choriocarcinoma cell line transports taurine, concentrating it over 1000-fold inside the cell. The transport system is energized by a Na+ gradient and exhibits an absolute requirement for Cl-. Neutral beta-amino acids such as beta-alanine and hypotaurine effectively compete with the system, whereas neutral alpha-amino acids such as alanine, leucine and alpha-aminoisobutyric acid do not. The transport system interacts with gamma-aminobutyric acid to an appreciable extent. Kinetic analysis reveals that the taurine transport system in this cell line is of a high-affinity and low-capacity type (apparent dissociation constant 2.3 +/- 0.3 microM; maximal velocity 88.5 +/- 5.0 pmol/3 min per mg of protein). Pretreatment of the JAR choriocarcinoma cells with phorbol 12-myristate 13-acetate results in the inhibition of the taurine transport system in a dose-dependent manner. The inhibition is blocked by co-treatment of the cells with staurosporine, an inhibitor of protein kinase C. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, has no effect on the transport system. These data show that the choriocarcinoma cells express a taurine transporter with characteristics similar to those of the taurine transporter described in the normal human placenta, and that the activity of the transporter in these cells is under the regulatory control of protein kinase C.

Brush-border membrane vesicles isolated from normal human term placentas were shown to accumulate succinate transiently against a concentration gradient, when an inward-directed Na+ gradient was imposed across the membrane. This uptake was almost totally due to transport into intravesicular space, non-specific binding to the membranes being negligible. The dependence of the initial uptake rate of succinate on Na+ concentration exhibited sigmoidal kinetics, indicating interaction of more than one Na+ ion with the carrier system. The Hill coefficient for this ion was calculated to be 2.7. The Na+-dependent uptake of succinate was electrogenic, resulting in the transfer of positive charge across the membrane. Kinetic analysis showed that succinate uptake in these vesicles occurred via a single transport system, with an apparent affinity constant of 4.8 +/- 0.2 microM and a maximal velocity of 274 +/- 4 pmol/20 s per mg of protein. Uptake of succinate was strongly inhibited by various C4 or C5 dicarboxylic acids, whereas monocarboxylic acids, amino acids and glucose showed little or no effect. Li+ and K+ could not substitute for Na+ in the uptake process. Instead, Li+ was found to have a significant inhibitory effect on the Na+-dependent uptake of succinate.

The Na+-H+ exchanger of the human placental brush-border membrane was inhibited by pretreatment of the membrane vesicles with a histidyl-group-specific reagent, diethyl pyrocarbonate and with a carboxy-group-specific reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. In both cases the inhibition was irreversible and non-competitive in nature. But, if the membrane vesicles were treated with these reagents in the presence of amiloride, cimetidine or clonidine, there was no inhibition. Since amiloride, cimetidine and clonidine all interact with the active site of the exchanger in a mutually exclusive manner, the findings provide evidence for the presence of essential histidyl and carboxy groups at or near the active site of the human placental Na+-H+ exchanger. This conclusion was further substantiated by the findings that Rose Bengal-catalysed photo-oxidation of histidine residues as well as covalent modification of carboxy residues with NN′-dicyclohexylcarbodi-imide irreversibly inhibited the Na+-H+ exchanger and that amiloride protected the exchanger from inhibition caused by NN′-dicyclohexylcarbodi-imide.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.