Tyrosine phosphorylation-and epidermal growth factor-dependent regulation of the sodium-coupled amino acid transporter B0 in the human placental choriocarcinoma cell line JAR.

We have recently cloned an amino acid transporter from the human placental choriocarcinoma cell line JAR which, when functionally expressed in HeLa cells, induces an amino acid transport activity with characteristics known to be associated with the amino acid transport system B(0) (R. Kekuda, P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, S. Sinha, T.L. Yang-Feng, F.H. Leibach, and V. Ganapathy, J. Biol. Chem. 271, 18657-18661, 1996). The presence of the amino acid transport system B(0) (ATB(0)) has however not been previously described in these cells by functional studies. In the present investigation, we have obtained evidence for the existence of ATB(0) in JAR cells and delineated the functional characteristics of the transporter. The identifying characteristics include Na(+)-dependence and preference for neutral amino acids. In addition, we have used the JAR cells as a model system to investigate the regulatory aspects of ATB(0). Treatment of the cells with the neuroprotective agent aurintricarboxylic acid (ATA) for 16 h leads to a significant increase in ATB(0) activity. This increase is associated with enhanced maximal velocity of the transporter and with increased steady state levels of the transporter mRNA. The effect of ATA is blocked by the tyrosine kinase inhibitor genistein. ATA treatment results in increased tyrosine phosphorylation of two major proteins, 180 kDa and 140 kDa in size. The 180 kDa protein is likely to be the epidermal growth factor (EGF) receptor because exposure of the cells to EGF also leads to enhanced tyrosine phosphorylation of a protein of similar molecular size. Furthermore, the effects of ATA on ATB(0) activity and on ATB(0) mRNA levels can be reproduced by EGF. Treatment of the cells with EGF for 24 h results in a significant increase in ATB(0) activity and this effect is associated with an increase in the maximal velocity of the transporter and with an increase in the steady state levels of the transporter mRNA. These data suggest that ATA influences ATB(0) activity in JAR cells most likely by activating the EGF receptor through tyrosine phosphorylation. It is concluded that the human placental choriocarcinoma cells functionally express the amino acid transport system B(0) and that the expression of the system in these cells is stimulated by EGF.

Cloning and functional characterization of a Na(+)-independent, broad-specific neutral amino acid transporter from mammalian intestine.

We have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with K(t) values in the range of 100-1000 microM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (K(t) values in the range of 10-20 microM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution.

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles.

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(+/-2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T=3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T=3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

Characterization of N5-methyltetrahydrofolate uptake in cultured human retinal pigment epithelial cells.

PURPOSE: To determine the identity of the transport process that is responsible for the uptake of N5-methyltetrahydrofolate, the predominant form of folate in blood, into cultured human retinal pigment epithelial cells. METHODS: Human retinal pigment epithelial cells were cultured on an impermeable plastic support, and the characteristics of the uptake of radiolabeled N5-methyltetrahydrofolate into the cells were investigated. The expression of the folate receptor and the reduced-folate transporter in these cells was evaluated by functional assays and by Northern blot analysis. In addition, the characteristics of N5-methyltetrahydrofolate uptake in these cells were compared with those in folate transport-defective human breast cancer cells that were manipulated to express functionally by transfection the cloned human folate receptor and the human reduced-folate transporter. RESULTS: Transport of N5-methyltetrahydrofolate into these cells occurred by a single saturable process with a Michaelis-Menten constant of 0.13 +/- 0.01 microM. The process was specific for such reduced folates as N5-methyltetrahydrofolate and N5-formyltetrahydrofolate. Nonreduced folate interacted with this transport process only weakly. The transport process was inhibited by anion transport inhibitors. Results of Northern blot analysis indicated the presence of transcripts specific for the reduced-folate transporter in these cells. These cells expressed very small amounts of the folate receptor evidenced from the binding of folate and from the detectable presence of folate receptor-specific transcript, but there was no evidence for participation of the receptor in the observed transport of N5-methyltetrahydrofolate. There was also no evidence in these cells for expression of the folate transporter that prefers nonreduced folate as a substrate. CONCLUSIONS: Transport of N5-methyltetrahydrofolate in human retinal pigment epithelial cells occurs exclusively through the reduced-folate transporter. The folate receptor is expressed at negligible levels in these cells and does not participate in the observed transport. Because the cells were cultured on impermeable supports, making the basolateral membrane inaccessible for transport measurements, it is speculated that the observed findings are related to the transport function of the apical membrane of these polarized cells.

Expression of folate receptor alpha in the mammalian retinol pigmented epithelium and retina.

PURPOSE: Folic acid is essential for DNA, RNA, and protein synthesis, and deficiencies in folate can lead to nutritional amblyopia and optic neuropathy. The transport of folate from the choroidal blood supply to the retina is only now beginning to be understood. The reduced-folate transporter was reported recently to be present in cultured human retinal pigment epithelial (RPE) cells and is thought to be localized to the apical region of these cells. The authors hypothesize that the RPE plays a role in the vectorial transport of folate from the choroidal blood to the neural retina and uses not only the reduced-folate transporter but also the folate receptor alpha in mediating this transport. The purpose of the present study was to determine whether the folate receptor alpha was present in the RPE and, if so, whether it was distributed along the basolateral membrane of the RPE, supporting a role for the protein in the initial steps of folate transport into the RPE. METHODS: The expression of the folate receptor alpha in mouse RPE was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), functional assays, in situ hybridization, immunohistochemistry, and laser scanning confocal microscopy. RESULTS: RT-PCR analysis, cloning of the RT-PCR product, and subsequent sequencing established that folate receptor alpha mRNA transcripts are expressed in the mouse RPE/choroid and are expressed also in the neural retina. A heterologous functional expression assay using MTX(R)-ZR-75-1 cells showed that the folate receptor alpha cDNA obtained by RT-PCR from the RPE/choroid complex and the neural retina was functional as assessed by the binding of folic acid and by the uptake of N5-methyltetrahydrofolate. In situ hybridization localized the folate receptor alpha mRNA to the mouse RPE cells and to cells of the neural retina. The folate receptor alpha was detected immunohistochemically in the mouse and rat RPE and in several layers of the neural retina. Laser scanning confocal microscopy revealed the distribution of the folate receptor alpha along the basolateral region of the RPE and not the apical region. CONCLUSIONS: The present work represents the first analysis of the folate receptor alpha expression in intact mammalian retina. The receptor is present and functional in mouse RPE. It is distributed specifically along the basolateral surface of the RPE and is proposed to work in a coordinated manner with the reduced-folate transporter in the vectorial transport of folate from the choroidal blood to the neural retina.

Structure, function, and regulation of human cystine/glutamate transporter in retinal pigment epithelial cells.

PURPOSE: The purpose of this investigation was to provide evidence for the expression of the cystine/glutamate transporter (x(c)(-)) in the human retinal pigment epithelial cell line ARPE-19, clone the light chain of the transporter from an ARPE-19 cell cDNA library and study its function, and investigate the regulation of this transporter by nitric oxide (NO) in ARPE-19 cells. METHODS: Uptake of radiolabeled cystine and glutamate was measured in ARPE-19 cells. The functional identity of x(c)(-) in these cells was established by substrate specificity and Na(+)-independence of the uptake process. The human x(c)(-) light chain (human xCT) was cloned from an ARPE-19 cell cDNA library. The functional identity of the cloned human xCT was investigated by heterologous coexpression of the light chain with the heavy chain (human 4F2hc) in HeLa cells. ARPE-19 cells were treated with or without the NO donor 3-nitroso-N:-acetylpenicillamine (SNAP) and the expression of x(c)(-) was studied at the functional and molecular levels. RESULTS: ARPE-19 cells take up cystine as well as glutamate in the absence of Na(+). Substrate specificity studies indicate that although the uptake of cystine in the absence of Na(+) is mediated by multiple amino acid transport systems including x(c)(-), the uptake of glutamate in the absence of Na(+) occurs exclusively via x(c)(-). The human xCT cloned from ARPE-19 cells is a protein of 501 amino acids. These cells express the heavy chain 4F2hc as evidenced from RT-PCR analysis. Coexpression of human xCT with 4F2hc in HeLa cells leads to the induction of cystine and glutamate uptake with characteristics similar to that of x(c)(-). The activity of x(c)(-) in ARPE-19 cells is upregulated by SNAP, and the process is associated with an increase in the expression of xCT with no detectable change in the expression of 4F2hc. CONCLUSIONS: ARPE-19 cells express the cystine/glutamate transporter x(c)(-) (the light chain xCT and the heavy chain 4F2hc) as is evident from functional and molecular studies. NO upregulates this transport system and the process is associated with an increase in xCT mRNA but with no change in 4F2hc mRNA.

Functional link between tyrosine phosphorylation and human serotonin transporter gene expression.

Treatment of the JAR human placental choriocarcinoma cells with herbimycin A, an inhibitor of tyrosine kinases, led to an increase in the activity of the serotonin transporter. This effect was accompanied by an increase in the serotonin transporter density and in the steady-state levels of the serotonin transporter mRNA. A treatment time of > 4 h was necessary for herbimycin A to elicit its effect. Actinomycin D and cycloheximide blocked the effect. There was no increase in the steady-state levels of the serotonin transporter mRNA when cells were treated with herbimycin A in the presence of actinomycin D. The herbimycin A-induced increase in the transporter activity was abolished by genistein, another inhibitor of tyrosine kinases. But the increase in the transporter mRNA levels caused by herbimycin A was not affected by genistein. Treatment of cells with herbimycin A resulted in an increase in the tyrosine phosphorylation of specific cellular proteins, suggesting that herbimycin A directly or indirectly activates specific tyrosine kinases. It is concluded that tyrosine phosphorylation is an essential component in the signaling pathways participating in the regulation of the human serotonin transporter gene expression.

Expression of the extraneuronal monoamine transporter in RPE and neural retina.

PURPOSE: Dopamine has several important functions in the retina including a possible role in controlling photoreceptor disk shedding to the RPE. While some cells express a transporter for dopamine, the RPE cell does not, leading us to ask whether the newly described catecholamine transport system, the extraneuronal monoamine transporter (uptake(2)) (also known as organic cation transporter 3 (OCT3), is present in RPE and might function as a transporter for dopamine. OCT3 also accepts histamine as a transportable substrate and so we investigated the interaction of this retinal neurotransmitter with OCT3. METHODS: OCT3 expression in the mouse eye was analyzed by in situ hybridization, Northern blot analysis and RT-PCR. OCT3 function was analyzed in cultured human ARPE-19 cells by monitoring the uptake of 1-methyl-4-phenyl pyridinium (MPP(+)), a neurotoxin, which is a known substrate for OCT3. RESULTS: In situ hybridization analysis showed that OCT3 is expressed in mouse RPE and in several cell types of the neural retina, including photoreceptor, ganglion, amacrine, and horizontal cells. The expression of OCT3 in RPE was confirmed by Northern blot analysis and RT-PCR. The characteristics of MPP( +) uptake in cultured ARPE-19 cells included the stimulation of transport by alkaline pH, high affinity (K(t) = 28 +/- 4 microM), competition with several cationic drugs and monoamine neurotransmitters and sensitivity to steroids. In addition, the uptake of MPP(+) in RPE cells was inhibited by dopamine and histamine with IC(50) values (concentration needed for 50% inhibition) of 637 +/- 84 microM and 150 +/- 20 microM, respectively. CONCLUSIONS. This study provides the first report on the expression and function of an organic cation transporter, OCT3, in the eye and in particular the RPE. The data have physiological and pharmacological relevance as it is likely that OCT3 participates in the clearance of dopamine and histamine from the subretinal space and may also play a key role in the disposition of the retinal neurotoxin MPP(+).

Characterization of poly- and monoclonal antibodies to physalis mottle (tymo) virus.

Polyclonal antibodies were raised against the Physalis mottle virus (PhMV) and its denatured coat protein (PhMV-P). Analysis of the reactivity of the polyclonal antibodies with tryptic peptides of PhMV-P in dot-blot assays revealed that many of the epitopes were common to intact virus and denatured coat protein. Five monoclonal antibodies to the intact virus were obtained using hybridoma technology. These monoclonal antibodies reacted well with the denatured coat protein. Epitope analysis suggested that probably these monoclonal antibodies recognize overlapping epitopes. This was substantiated by epitope mapping using the CNBr digest of PhMV-P in western blots. All the five monoclonals recognized the N-terminal 15 K fragment. Attempts to further delineate the specific region recognized by the monoclonals by various enzymatic cleavages resulted in the loss of reactivity in all the cases. The results indicate that these monoclonals probably recognize epitopes within the N-terminal 15 K fragment of the coat protein.

Characterization of two scleroderma autoimmune antigens that copurify with human ribonuclease P.

Human RNase P has been purified more than 2000-fold from HeLa cells. In addition to the RNA component, H1 RNA, polypeptides of molecular masses 14, 20, 25, 30, 38, and 40 kDa copurify with the enzyme activity. Sera from two different patients with the autoimmune disease scleroderma were used to immunodeplete human RNase P activity. These same sera cross-reacted on immunoblots with two of the copurifying polypeptides, p30 and p38, whereas an autoimmune serum that does not immunodeplete RNase P activity did not react with these proteins. Peptide fragments derived from purified p30 and p38 facilitated the molecular cloning and sequencing of cDNAs coding for these two polypeptides, which are now designated as Rpp30 and Rpp38, respectively. RPP38 cDNA encodes a polypeptide that may be identical to a previously identified antigen of approximately 40 kDa, which is immunoprecipitated by Th and To autoimmune antisera, and that has been implicated as a protein subunit of human RNase P by virtue of its ability to bind to H1 RNA in vitro. The second autoimmune antigen, Rpp30, as such, has not been described previously.

Polarized distribution of interleukin-1 receptors and their role in regulation of serotonin transporter in placenta.

We investigated the expression of interleukin-1 (IL-1) receptors and their involvement in the regulation of the serotonin transporter gene expression in human placenta. IL-1beta is an activator of the serotonin transporter gene expression in JAR human placental choriocarcinoma cells as demonstrated by an increase in the steady-state levels of the transporter mRNA and in serotonin transport activity. This activation is blocked by IL-1 receptor antagonist. Genistein also blocks the effect of IL-1beta, indicating involvement of tyrosine phosphorylation in the process. Treatment of JAR cells with IL-1beta activates mitogen-activated protein kinases and nuclear factor-kappaB. The nuclear factor-kappaB that is responsive to IL-1beta in these cells is the p65 homodimer. Northern blot analysis and reverse transcription-polymerase chain reaction revealed that JAR cells and human placenta express type I and type II IL-1 receptors. The binding sites for (125)I-IL-1beta are localized predominantly in the maternal-facing brush border membrane of the syncytiotrophoblast. These results show that IL-1 in the maternal circulation is likely to play a critical role in the regulation of the serotonin transporter gene expression in the placenta.

Human serotonin transporter: regulation by the neuroprotective agent aurintricarboxylic acid and by epidermal growth factor.

The influence of aurintricarboxylic acid (ATA), a neuroprotective compound, on the serotonin transporter expressed in JAR human placental choriocarcinoma cells was investigated. Treatment of the cells with ATA for 16 h led to a significant stimulation of the serotonin transporter activity. This effect was not observed, however, when the treatment was done for 1-2 h. The stimulatory effect was associated with an increase in the maximal velocity of the transport process with no significant change in the Michaelis-Menten constant. Northern blot hybridization revealed that ATA treatment caused a marked increase in the steady-state levels of serotonin transporter-specific transcripts. Treatment of the cells with ATA was found to increase tyrosine phosphorylation of a 180-kDa protein. The phosphotyrosine content of a protein of a similar molecular size increased dramatically when the cells were exposed to epidermal growth factor (EGF), suggesting that this protein may be the EGF receptor. Treatment of the cells with EGF for 24 h could reproduce the stimulatory effects of ATA on the serotonin transporter activity, the maximal velocity of the transport process, and the steady-state levels of the transporter-specific mRNAs. Genistein, a tyrosine kinase inhibitor, was able to block the stimulatory effect of ATA and EGF. It is concluded that EGF increases the serotonin transporter expression in JAR cells and that the neuroprotective compound ATA produces similar effects on the transporter most likely by activating the EGF receptor through tyrosine phosphorylation.

Cloning and functional expression of the human type 1 sigma receptor (hSigmaR1).

We have isolated a cDNA clone from a human placental choriocarcinoma cell (JAR) cDNA library which codes for a functional type 1 sigma receptor. An RT-PCR product obtained from guinea pig kidney mRNA using primers specific for the guinea pig sigma receptor cDNA was used to screen the cDNA library. The hSigmaR1 cDNA predicts a protein of 223 amino acids with a single putative transmembrane domain. The amino acid sequence exhibits 93% identity with the guinea pig sigma receptor. When functionally expressed in HeLa cells, the hSigmaR1 cDNA enhances the binding of [3H]-haloperidol, a sigma receptor ligand, to the HeLa cell membranes. The inhibitor specificity of the cloned hSigmaR1 indicates that it is the type 1 sigma receptor. Several human tissues, including placenta, liver, and brain, and several human cell lines express the SigmaR1 mRNA (1.7 kb) to a variable extent.

Architecture of physalis mottle tymovirus as probed by monoclonal antibodies and cross-linking studies.

Physalis mottle tymovirus (previously named belladonna mottle virus, Iowa strain) RNA was cross-linked to its coat protein by exposure of the intact virus to ultraviolet light. The site of cross-linking of the coat protein with the RNA was identified as Lys-10 by sequencing the oligonucleotide-linked tryptic peptide obtained upon HPLC separation subsequent to enzymatic digestion of the cross-linked and dissociated virus. Three monoclonal antibodies PA3B2, PB5G9, and PF12C9, obtained using denatured coat protein as antigen, cross-reacted effectively with the intact virus indicating that the epitopes recognized by these monoclonals are on the surface of the virus. Using the peptides generated by digestion with CNBr, clostripain, V-8 protease, or trypsin and a recombinant protein lacking the N-terminal 21 residues expressed from a cDNA clone, it was shown that PA3B2 recognizes the sequence 22-36 on the coat protein while PB5G9 and PF12C9 recognize region 75-110. These results suggest that Lys-10 is one of the specific sites through which the RNA interacts in the intact virus. The polypeptide segment (region 22-36) following this buried portion as well as the epitope within the region 75-110 are exposed in the intact virus. These observations are consistent with the canonical beta-barrel structure observed in certain other plant viruses.

Identification of the histidyl residue obligatory for the catalytic activity of the human H+/peptide cotransporters PEPT1 and PEPT2.

Histidyl residues are known to be essential for the catalytic function of the H(+)-coupled peptide transporters expressed in the intestine and the kidney, most likely participating in the binding and translocation of H+. Three histidyl residues are conserved among the intestinal and renal peptide transporters (PEPT1 and PEPT2, respectively) from different animal species. In hPEPT1, these residues are His-57, His-121, and His-260. The corresponding residues in hPEPT2 are His-87, His-142, and His-278. We have individually mutated each of these histidyl residues in hPEPT1 and in hPEPT2 and compared the catalytic function of the mutants with that of their respective wild type transporters by expressing the transporters in Xenopus laevis oocytes and also in HeLa cells. His-57 in hPEPT1 and His-87 in hPEPT2 were found to be absolutely essential for catalytic activity because the corresponding mutants had no detectable peptide transport activity. His-121 in hPEPT1 is not essential since mutation of this residue did not impair transport function. His-142 in hPEPT2 was found to play a significant role in the maintenance of transport function but was not found to be obligatory because the mutant had appreciable transport activity. The obligatory histidyl residue (His-57 in hPEPT1 and His-87 in hPEPT2) is located in an almost identical topological position in both transporters, near the extracellular surface of the second putative transmembrane domain. The second conserved histidyl residue is located in the fourth putative transmembrane domain in hPEPT1 as well as in hPEPT2. The third conserved histidyl residue is present in the cytoplasmic loop between the transmembrane domains 6 and 7 and is unlikely to play any significant role in the binding and translocation of H+ and this was supported by the findings that mutation of this histidyl residue in hPEPT1 did not interfere with transport function. The loss of transport function of hPEPT1 and hPEPT2, when His-57 in hPEPT1 and His-87 in hPEPT2 were mutated, was not due to alterations in protein expression because the expression levels of these mutants were similar to those of the respective wild type transporters in HeLa cells as assessed by immunoblot analysis. Confocal analysis of immunofluorescence in X. laevis oocytes expressing the wild type and the three histidine mutants of hPEPT1 showed that the transporter protein is expressed exclusively in the plasma membrane and that the level of expression is comparable among the wild type and the three mutants. These site-directed mutagenesis studies clearly show that His-57 in hPEPT1 and His-87 in hPEPT2 are the most critical histidyl residues necessary for the catalytic function of these transporters.

Cloning and functional characterization of a potential-sensitive, polyspecific organic cation transporter (OCT3) most abundantly expressed in placenta.

We have isolated a cDNA from rat placenta which, when expressed heterologously, mediates the transport of a wide spectrum of organic cations. The cDNA codes for a protein of 551 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is expressed most abundantly in the placenta and moderately in the intestine, heart, and brain. The expression is comparatively low in the kidney and lung and is undetectable in the liver. This transporter is distinct from the previously cloned organic cation transporters (OCT1, OCT2, NKT, NLT, RST, and OCTN1). When expressed in HeLa cells, the cDNA induces the transport of tetraethylammonium and guanidine. Competition experiments indicate that this transport process recognizes a large number of organic cations, including the neurotoxin 1-methyl-4-phenylpyridinium, as substrates. The cDNA-induced transport is markedly influenced by extracellular pH. However, when expressed in Xenopus laevis oocytes, the cDNA-induced transport is electrogenic, associated with the transfer of positive charge into the oocytes. Under voltage clamp conditions, tetraethylammonium evokes inward currents that are concentration- and potential-dependent. This potential-sensitive organic cation transporter, designated as OCT3, represents a new member of the OCT gene family.

Cannabinoid receptors and their role in the regulation of the serotonin transporter in human placenta.

OBJECTIVE: We sought to investigate the expression of cannabinoid receptors in human placenta and BeWo choriocarcinoma cells and study their role in the regulation of the serotonin transporter. STUDY DESIGN: Expression of the 2 types of cannabinoid receptors (CB1 and CB2) in human placenta and BeWo cells was investigated by reverse transcriptase-polymerase chain reaction and Northern blot analysis. The involvement of the receptors in the regulation of the serotonin transporter expression was studied by using a cannabinoid receptor agonist (WIN 55212-2). BeWo cells were treated with the agonist in the presence or absence of forskolin, and the serotonin transporter activity was measured by assessing paroxetine-sensitive serotonin transport. Serotonin transporter density in cell membranes was monitored by measuring paroxetine-sensitive binding of RTI-55, a specific high-affinity ligand for the transporter. Agonist-induced changes in intracellular levels of cyclic adenosine monophosphate were also monitored. RESULTS: Reverse transcriptase-polymerase chain reaction and Northern blot analysis demonstrated unequivocally that human placenta and BeWo cells express both types of cannabinoid receptors. Treatment of BeWo cells with the receptor agonist blocked the activity of the constitutive, as well as the forskolin-induced, serotonin transporter without affecting the serotonin transporter density. This effect is not mediated by alterations in intracellular cyclic adenosine monophosphate levels. CONCLUSION: The results show that cannabinoid receptors are expressed in human placenta and BeWo cells and play a role in the regulation of the serotonin transporter activity. Human placenta is therefore a direct target for cannabinoids, and marijuana use during pregnancy is likely to affect the placental clearance of serotonin through the serotonin transporter.

Human LAT1, a subunit of system L amino acid transporter: molecular cloning and transport function.

We report here on the cloning and functional characterization of human LAT1, a subunit of the amino acid transport system L. The hLAT1 cDNA, obtained from a human placental cDNA library, codes for a protein of 507 amino acids. When functionally expressed in mammalian cells together with the heavy chain of the rat 4F2 antigen (r4F2hc), hLAT1 induces the transport of neutral amino acids. When expressed independently, neither hLAT1 nor r4F2hc was capable of amino acid transport to any significant extent. Thus, the hLAT1-r4F2hc heterodimeric complex is responsible for the observed amino acid transport. The transport process induced by the heterodimer is Na+ independent and is not influenced by pH. It recognizes exclusively neutral amino acids with high affinity. LAT1-specific mRNA is expressed in most human tissues with the notable exception of the intestine.

Cloning and functional expression of a cDNA encoding a mammalian sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin, and lipoate.

Previous studies have shown that a Na+-dependent transport system is responsible for the transplacental transfer of the vitamins pantothenate and biotin and the essential metabolite lipoate. We now report the isolation of a rat placental cDNA encoding a transport protein responsible for this function. The cloned cDNA, when expressed in HeLa cells, induces Na+-dependent pantothenate and biotin transport activities. The transporter is specific for pantothenate, biotin, and lipoate. The Michaelis-Menten constant (Kt) for the transport of pantothenate and biotin in cDNA-transfected cells is 4.9 +/- 1.1 and 15.1 +/- 1.2 microM, respectively. The transport of both vitamins in cDNA-transfected cells is inhibited by lipoate with an inhibition constant (Ki) of approximately 5 microM. The nucleotide sequence of the cDNA (sodium-dependent multivitamin transporter (SMVT)) predicts a protein of 68.6 kDa with 634 amino acids and 12 potential transmembrane domains. Protein data base search indicates significant sequence similarity between SMVT and known members of the Na+-dependent glucose transporter family. Northern blot analysis shows that SMVT transcripts are present in all of the tissues that were tested. The size of the principal transcript is 3.2 kilobases. SMVT represents the first Na+-dependent vitamin transporter to be cloned from a mammalian tissue.

Cloning of the sodium-dependent, broad-scope, neutral amino acid transporter Bo from a human placental choriocarcinoma cell line.

We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expressed in HeLa cells, induces a Na+-dependent amino acid transport system with preference for zwitterionic amino acids. Anionic amino acids, cationic amino acids, imino acids, and N-methylated amino acids are excluded by this system. These characteristics are identical to those described for the amino acid transporter Bo. When expressed in Xenopus laevis oocytes that do not have detectable endogenous activity of the amino acid transporter Bo, the cloned transporter increases alanine transport in the oocytes severalfold and induces alanine-evoked inward currents in the presence of Na+. The cDNA codes for a polypeptide containing 541 amino acids with 10 putative transmembrane domains. Amino acid sequence homology predicts this transporter (hATBo) to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located the ATBo gene to human chromosome 19q13.3.