V. Ganapathy

Expression of the cystine-glutamate exchanger (xc-) in retinal ganglion cells and regulation by nitric oxide and oxidative stress.

The cystine-glutamate exchanger, system xc−, mediates the Na+-independent exchange of cystine into cells, coupled to the efflux of intracellular glutamate. System xc− plays a critical role in glutathione homeostasis. Early studies of brain suggested that system xc− was present primarily in astrocytes but not neurons. More recent work indicates that certain brain neurons have an active system xc−. In the retina, system xc− has been demonstrated in Müller and retinal pigment epithelial cells. We have recently suggested that two protein components of system xc−, xCT and 4F2hc, are present in ganglion cells of the intact retina. Here, we have used (1) molecular and immunohistochemical assays to determine whether system xc− is present in primary ganglion cells isolated from neonatal mouse retinas and (2) functional assays to determine whether its activity is regulated by oxidative stress in a retinal ganglion cell line (RGC–5). Primary mouse ganglion cells and RGC–5 cells express xCT and 4F2hc. RGC–5 cells take up [3H]glutamate in the absence of Na+, and this uptake is blocked by known substrates of system xc− (glutamate, cysteine, cystine, quisqualic acid). Treatment of RGC–5 cells with NO and reactive oxygen species donors leads to increased activity of system xc− associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. This is the first report of system xc− in primary retinal ganglion cells and RGC–5 cells. Oxidative stress upregulates this transport system in RGC–5 cells, and the process is associated with an increase in xCT mRNA and protein but no change in 4F2hc mRNA or protein.

Biological functions of SLC5A8, a candidate tumour suppressor

SLC5A8 is a candidate tumour suppressor gene that is silenced in colon cancer, gastric cancer and possibly other cancers in humans. This gene codes for a transporter belonging to the Na(+)/glucose co-transporter gene family (SLC5). The cancer-associated silencing of the gene involves hypermethylation of CpG islands present in exon 1 of the gene. SLC5A8 is expressed in colon, ileum, kidney and thyroid gland. The protein coded by the gene mediates the Na(+)-coupled and electrogenic transport of a variety of monocarboxylates, including short-chain fatty acids, lactate and nicotinate. It may also transport iodide. The normal physiological function of this transporter in the intestinal tract and kidney is likely to facilitate the active absorption of short-chain fatty acids, lactate and nicotinate. One of the short-chain fatty acids that serves as a substrate for SLC5A8 is butyrate. This fatty acid is an inhibitor of histone deacetylases and is known to induce apoptosis in a variety of tumours including colonic tumour. Since butyrate is produced in the colonic lumen at high concentrations by bacterial fermentation of dietary fibre, we speculate that the ability of SLC5A8 to mediate the entry of this short-chain fatty acid into colonic epithelial cells underlies the potential tumour suppressor function of this transporter.

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.

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.

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.

Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.

Folate Transport in Retina and Consequences on Retinal Structure and Function of Hyperhomocysteinemia

Folate is a water-soluble vitamin that is essential for the synthesis of DNA, RNA, and some amino acids. It is required for the proper function of every cell, including those of the retina. This chapter summarizes reported studies of the transport mechanisms by which retinal cells take up folate, including folate receptor α, reduced folate carrier, and proton-coupled folate transporter. It is well known that when folate is deficient, homocysteine levels increase. Genetic mutations can also trigger hyperhomocysteinemia. The second portion of the chapter focuses on the consequences on retina structure and function under conditions of hyperhomocysteinemia. It describes studies in several models in which defects in enzymes associated with the remethylation and transsulfuration pathway are associated with varying degrees of retinal neuropathy and vasculopathy.

Sodium-dependent high-affinity binding of carnitine to human placental brush border membranes

The interaction of carnitine with human placental brush-border membrane vesicles was investigated. Carnitine was found to associate with the membrane vesicles in a Na(+)-dependent manner. The time course of this association did not exhibit an overshoot, which is typical of a Na+ gradient-driven transport process. The absolute requirement for Na+ was noticeable whether the association of carnitine with the vesicles was measured with a short time incubation or under equilibrium conditions, indicating Na(+)-dependent binding of carnitine to the human placental brush-border membranes. The binding was saturable and was of a high-affinity type with a dissociation constant of 1.37 +/- 0.03 microM. Anions had little or no influence on the binding process. The binding process was specific for carnitine and its acyl derivatives. Betaine also competed for the binding process, but other structurally related compounds did not. Kinetic analyses revealed that Na+ increased the affinity of the binding process for carnitine and the Na+/carnitine coupling ratio for the binding process was 1. The dissociation constant for the interaction of Na+ with the binding of carnitine was 24 +/- 4 mM. This constitutes the first report on the identification of Na(+)-dependent high-affinity carnitine binding in the plasma membrane of a mammalian cell. Studies with purified rat renal brush-border membrane vesicles demonstrated the presence of Na+ gradient-driven carnitine transport but no Na(+)-dependent carnitine binding in these membrane vesicles. In contrast, purified intestinal brush-border membrane vesicles posses neither Na+ gradient-driven carnitine transport nor Na(+)-dependent carnitine binding.