F. H. Leibach

Functional expression of the plasma membrane serotonin transporter but not the vesicular monoamine transporter in human placental trophoblasts and choriocarcinoma cells

We investigated the functional expression of the plasma membrane serotonin transporter and the vesicular monoamine transporter in choriocarcinoma cells and normal trophoblasts. The RBL 2H3 cells, a rat basophilic leukaemia cell line, which express both transporters were used for comparison. The choriocarcinoma cells JAr and BeWo were found to possess the plasma membrane serotonin transporter as assessed by the presence of serotonin transport activity in intact cells that was Na(+)-dependent and was sensitive to inhibition by tricyclic and non-tricyclic antidepressants. The activity of the vesicular monoamine transporter in these cells was determined by measuring serotonin transport in digitonin-permeabilized cells. The transport in permeabilized cells was very slow, was not stimulated by ATP and was insensitive to inhibition by reserpine. Under similar conditions, the vesicular monoamine transporter activity was demonstrable in RBL cells, which was stimulated by ATP and was inhibitable by reserpine, bafilomycin A1 (an inhibitor of the V-type H(+)-pump) and carbonyl cyanide p-trifluoromethoxy phenylhydrazone (a protonophore which dissipates transmembrane H+ gradients). In corroboration with these findings, mRNA transcripts hybridizable to the vesicular monoamine transporter cDNA probe were detectable in RBL cells but not in JAr choriocarcinoma cells. Similarly, there was no evidence for the expression of the vesicular monoamine transporter as assessed by Northern blot analysis in normal trophoblasts which were maintained in culture to differentiate to form multinucleated syncytial cells. It is concluded that the trophoblasts and choriocarcinoma cells express the plasma membrane serotonin transporter but not the vesicular monoamine transporter.

Identification of a renal cell line that constitutively expresses the kidney-specific high-affinity H+/peptide cotransporter.

In this study we describe for the first time the identification of a renal cell line that; ex‐presses the kidney‐specific high‐affinity H+/peptide cotransport system. The kidney cell line SKPT‐C193 C1.2 was obtained by SV40 transformation of rat proximal tubular cells. The transport of the dipeptide glycylsarcosine (Gly‐Sar) was studied in this cell line grown as a confluent monolayer on impermeable plastic supports. Uptake of the dipeptide was rapid and was stimulated sixfold by an inwardly directed H+ gradient, with optimal uptake occurring at an extracellular pH of 6.0. The uptake was markedly reduced by the protonophore carbonyl cyanide 4‐ (trifluoromethoxy) phenylhydrazone whether measured at pH 7.5 or 6.0. Intracellular acidification of the cells by NH4CI prepulse also reduced the uptake of glycylsarcosine. The dipeptide uptake was found to be mediated by a high‐affinity transport system with a Michaelis‐Menten constant (Kt) of 67 ± 2 μM and a maximal transport velocity of 1.20 ± 0.02 nmol · 10 min−1 · mg protein−1. Studied over a concentration range of 5 μM to 5 mM, there was no evidence for a second saturable transport component. Di‐ and tripeptides, but not glycine, were strong inhibitors of glycylsarcosine uptake, indicating that these peptides also interact with the transport system with high affinity. Northern blot analysis of poly(A)+RNA from these cells using cDNA probes specific for the human intestinal peptide transporter (PEPT1) or the human kidney‐specific peptide transporter (PEPT2) revealed that the transport system expressed in these cells is PEPT 2. It is concluded that the SKPT‐0193 C1.2 cell line constitutively expresses the kidney‐specific high‐affinity H+/peptide cotransporter described in the proximal tubular epithelial cells of the normal kidney.—Brandsch, M., Brandsch, C., Prasad, P. D., Ganapathy, V., Hopfer, U., Leibach, F. H. Identification of a renal cell line that constitutively expresses the kidney‐specific high‐affinity H+/pep‐ tide cotransporter. FASEBJ. 9, 1489‐1496 (1995)

Characterization of a sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate in human placental choriocarcinoma cells.

The characteristics of the uptake of the vitamin pantothenate into JAr human placental choriocarcinoma cells were investigated and these cells were found to accumulate the vitamin against a concentration gradient by a Na(+)-dependent process. Substitution of Na+ with over other monovalent cations abolished the uptake completely. The transport process showed no preference for any particular anion. Kinetic analysis indicated the presence of a single saturable transport system with a Michaelis-Menten constant of 2.1 +/- 0.2 microM and a maximal velocity of 341 +/- 12 pmol/mg of protein per 10 min. The dependence of the uptake rate of pantothenate on Na+ concentration exhibited sigmoidal kinetics, indicating interaction of more than one Na+ ion with the transporter. The Hill coefficient for this process was calculated to be 1.6. The Na+/pantothenate coupling ratio being greater than unity suggests that the transport process is electrogenic, resulting in net transfer of positive charge across the membrane. This was confirmed in plasma membrane vesicles prepared from JAr cells where the uptake of pantothenate was found to be significantly stimulated by valinomycin-induced inside-negative K(+)-diffusion potential. Substrate specificity studies showed that, in addition to pantothenate, the transporter interacts with two other vitamins, namely biotin and lipoate. The characteristics of pantothenate uptake in the placental cell line BeWo was also investigated. These cells were also found to express a pantothenate transport system similar to that expressed in the JAr cells.

Inhibition of protein kinase activity and amino acid and α-methyl-d-glucoside transport by diamide

Dizene dicarboxylic acid bis-(N,N-dimethylamide), commonly called diamide, is known to oxidize stoichiometrically intracellular pools of reduced glutathione and inhibit the accumulation of sugars and amino acids by rat kidney slices. Incubation of rat cortical slices in diamide also leads to a significant decrease in the level of endogenous protein kinase activity. The inhibition of sugar and amino acid transport and protein kinase activity by diamide is partially reversible by the addition of exogenous glutathione or other thiols. A comparison of protein kinase activity with amino acid and sugar transport at various concentrations of diamide indicates that there is a high degree of correlation between these two processes.

Partial purification and characterization of the human placental serotonin transporter

The human placental serotonin transporter was solubilized from purified brush border membranes using digitonin as the solubilizing agent. The solubilizate was subjected to wheat germ agglutinin-Sepharose 6B column chromatography, Centricon-100 ultrafiltration and Sepharose 6B gel filtration to yield a partially purified preparation of the serotonin transporter. Specific binding of the high affinity ligand paroxetine was used to monitor the transporter during the solubilization and the purification steps. The enrichment of paroxetine binding in the final preparation was 51-fold compared to the intact brush border membranes, taking into account the inactivation that occurred during purification. The partially purified transporter exhibited paroxetine binding characteristics which were similar to those of the transporter in intact membranes. The transporter in the partially purified preparation bound paroxetine with a high affinity (dissociation constant, 0.21 nM). The binding was inhibitable by serotonin but not by other monoamines, dopamine and norepinephrine, nor by the serotonin precursor 5-hydroxytryptophan. The antidepressants, imipramine, fluoxetine and desipramine inhibited the binding with a rank order of potency of imipramine = fluoxetine > desipramine. The approximate molecular weight of the transporter was assessed by molecular sieve chromatography on Sepharose 6B and was found to be 300,000. When reconstituted into proteoliposomes, the partially purified transporter was able to catalyse NaCl-dependent serotonin transport in these proteoliposomes. The results of this study show that the human placental serotonin transporter can be solubilized, partially purified and reconstituted in a transport-competent form and, in addition, provide some insight into the protein nature of the transporter.

Human placenta as a target organ for cocaine action: Interaction of cocaine with the placental serotonin transporter

The interaction of cocaine and its analog 2 beta-carbomethoxy-3 beta-(4-iodophenyl) tropane (RTI-55) with the human placental serotonin transporter was investigated. The function of the placental serotonin transporter was assayed using three different approaches: serotonin uptake in purified human placental brush border membrane vesicles, paroxetine binding in placental brush border membrane vesicles, and serotonin uptake in cultured human placental choriocarcinoma cells. The interaction of cocaine and RTI-55 with the transporter was studied by determining the effects of these compounds on the transporter function. Cocaine and RTI-55 were found to be potent inhibitors of the transporter in all three approaches. The inhibition was competitive in nature in all cases. The inhibition constant (Ki) for cocaine was not influenced significantly by the duration of time allowed for the compound to interact with the transporter. In contrast, the inhibition constant for RTI-55 was influenced markedly by this parameter. The longer the time allowed for interaction of RTI-55 with the transporter, the smaller was the Ki value. These results suggest that the association kinetics for the interaction of cocaine and RTI-55 with the placental serotonin transporter are significantly different. When equilibrium interaction was allowed, cocaine inhibited the function of the transporter with a Ki of 0.09 microM. It is concluded that cocaine and its analog RTI-55 are potent inhibitors of the function of the serotonin transporter that is expressed in the normal human placenta and in cultured human placental choriocarcinoma cells. Since the reported values for cocaine concentration in the blood of cocaine users are several-fold higher than the inhibition constant for cocaine, the present study strongly suggests that the function of the placental serotonin transporter may be severely impaired by maternal use of cocaine during pregnancy. These findings may be relevant to fetal and placental complications of cocaine abuse during pregnancy.

Inhibition of amino acid accumulation in slices of rat kidney cortex by diamide

Abstract 1. 1.The oxidizing agent diazenedicarboxylic acid bis-( N , N -dimethylamide), common name diamide, has been used to lower the concentration of GSH in rat kidney cortex slices. 2. 2.The ability to accumulate l -amino acids against a concentration gradient was decreased in diamide-treated slices. 3. 3.Studies with six different amino acids have shown that this inhibition of amino acid accumulation is not specific for one amino acid or one group of related amino acids. 4. 4.The addition of exogenous GSH to slices that have been treated with diamide leads to a significant recovery of the ability to concentrate amino acids. 5. 5.Dithiothreitol and mercaptoethanol also reverse the effect of diamide, but are less effective than GSH.

The effect of diamide and glutathione on the uptake of α-methyl-d-glucoside by slices of rat kidney cortex

The uptake of alpha-methyl-D-glucoside was stimulated in slices of rat kidney cortex by pretreatment with reduced glutathione. Diamide, an oxidizind agent with high specificity for GSH, caused an inhibition of alpha-methyl-D-glucoside uptake. These effects appeared to be related specifically to GSH, since dithiothreitol and mercaptoethanol did not increase alpha-methyl-D-glucoside uptake, and were not as effective as GSH in reversing the effects of diamide. GSH and diamide had no effect on the uptake of another sugar analog, 3-O-methylglucose, which is not actively transported. Kinetic studies indicated that GSH increased the apparent V without affecting K-m. The results are discussed in terms of the possible role of GSH in the process of sugar transport.

Specific inhibition of protein kinase by diazenedicarboxylic acid bis-(N,N-dimethylamide)

Abstract Diazenedicarboxylic acid bis (N,N-dimethylamide), commonly called diamide, inhibits purified protein kinase isolated from swine kidney. The extent of inhibition is increased in the presence of cysteine, reduced glutathione and dithiothreitol. The inhibition of protein kinase is reversible and greater than 80% of the activity of an inhibited preparation can be recovered by dialysis. Diamide appears to be specific for the inhibition of protein kinase. The activities of various other kinases acting on low molecular weight substrates were unaffected by diamide. Diamide is the first known low molecular weight inhibitor of protein kinase and may prove to be an extremely useful agent in elucidating the physiological role of this enzyme.

Glutathione in the isolated perfused rabbit kidney

Abstract Perfusion of the isolated rabbit kidney at normothermia leads to a marked depletion in the concentration of reduced glutathione in the kidney tissue. After 90 min of perfusion at 36 ± 1°C, the concentration of GSH found in the renal cortex is more than 80% lower than the levels found in fresh tissue. Comparable perfusion under hypothermic conditions reduced the amount of glutathione depletion considerably; however, after 90 min of perfusion at 15 ± 1°C, renal cortex GSH levels were still nearly 50% lower than the levels found in fresh tissue. It was found that the addition of GSH to the perfusate completely alleviated this glutathione-depletion phenomenon.