Biomedical subjects

V Ganapathy

Publications and source records attributed to V Ganapathy.

At least 163 records · Page 9Linked to original sources

PubMed1994

Human placenta: a direct target for cocaine action.

Use of cocaine during pregnancy is known to have harmful effects on the mother and her fetus. Currently available models describing the pathogenesis of these effects focus on the involvement of cocaine target systems, primarily the noradrenaline transporter, in the mother and the fetus. The placenta which lies between the mother and the fetus is considered only as a 'silent observer' in the whole process of cocaine-induced complications during pregnancy. Recent studies have, however, shown that the placenta expresses several cocaine target proteins such as the noradrenaline transporter, the serotonin transporter, and the sigma receptor. The functions of these proteins are significantly impaired in the presence of cocaine at concentrations known to exist in the plasma of cocaine users. These studies clearly show that the placenta itself is a direct target for cocaine action and that interaction of cocaine with its target proteins in the placenta plays an important role in the pathogenesis of cocaine-induced complications in the mother and her developing fetus.

Carrier Proteins↗

PubMed1994

Relationship between thyroid hormone transport and neutral amino acid transport in JAR human choriocarcinoma cells.

The relationship between the transport of the thyroid hormone T3 and the transport of neutral amino acids was investigated in JAR human placental choriocarcinoma cells. The uptake of leucine, mediated by the amino acid transport system L, was inhibited by T3 and T4, and the nature of inhibition was competitive. Uptake of T3 into the cells was predominantly Na+ independent, and so was that of leucine. However, although an acidic extracellular pH stimulated leucine uptake, the uptake of T3 remained unaffected. In addition, leucine failed to inhibit T3 uptake. The aromatic amino acids phenylalanine and tryptophan were found to inhibit the uptake of T3, but these two amino acids were transported into the cells predominantly via system L. The amino acid transport system T, which is specific for aromatic amino acids, was not detectable in these cells. Treatment of the cells with the calmodulin antagonist CGS 9343 B stimulated the uptake of leucine and tryptophan, but inhibited the uptake of T3. Kinetic analysis of T3 uptake revealed the presence of a single saturable system for this hormone in these cells, and the Michaelis-Menten constant for this system was 0.77 +/- 0.06 microM. Metabolic poisons that interfere with the cellular generation of ATP had no effect on the uptake of T3. It is concluded that in placental choriocarcinoma cells, 1) T3 and T4 are high affinity competitive inhibitors of the amino acid transport system L, 2) uptake of T3 occurs via a specific Na(+)-independent, energy-independent, and saturable mechanism that is unrelated to the amino acid transport systems L and T, 3) the aromatic amino acids phenylalanine and tryptophan interact, although weakly, with the T3 uptake system, and 4) calmodulin-dependent processes participate in the regulation of the T3 uptake system.

Amino Acids↗

PubMed1994

Molecular identity and calmodulin-mediated regulation of the taurine transporter in a human retinal pigment epithelial cell line.

The molecular identity and calmodulin-mediated regulation of the taurine transporter were investigated in a human retinal pigment epithelial cell line (HRPE). Reverse transcription-polymerase chain reaction amplification of HRPE cell mRNA using primer specific for a taurine transporter cloned from human placenta yielded a product of expected size (approximately 0.9 kb) which hybridized to the placental cDNA probe under high stringency conditions. The nucleotide sequence of the product was identical to the sequence of the portion of the placental taurine transporter cDNA flanked by the specific primers. The taurine transporter expressed in the HRPE cell line thus appears to be identical to the transporter cloned from the placenta. Treatment of the HRPE cells with a selective calmodulin antagonist CGS 9343 B (CGS) led to a marked decrease in taurine transport activity. This effect could be reproduced with W-7, another calmodulin antagonist. The inhibition caused by CGS occurred rapidly (t1/2 approximately 10 min). Treatment of the cells with CGS did not affect the transport of leucine, and amino acid not recognized by the taurine transporter as a substrate. The CGS-induced inhibition of taurine transport was accompanied by a decrease in the maximal velocity of the transporter with no detectable change in the substrate affinity. The steady state levels of the transporter mRNA however remained unaffected by CGS treatment. It is concluded that the HRPE cell line expressed a taurine transporter identical to the transporter describe in the human placenta and that the function of this transporter is regulated by calmodulin-dependent processes.

Base Sequence↗

PubMed1993

Sodium- and chloride-dependent, cocaine-sensitive, high-affinity binding of nisoxetine to the human placental norepinephrine transporter.

Maternal-facing brush border membranes prepared from normal term human placentas possess the norepinephrine transporter. We investigated the interaction of nisoxetine with the human norepinephrine transporter by examining the binding of this ligand to the placental brush border membranes. Scatchard analysis revealed that nisoxetine bound with high affinity to a single class of binding sites in the membranes (dissociation constant = 13.8 +/- 0.4 nM). This value obtained from equilibrium experiments matched the value (11.2 nM) which was calculated using the association and dissociation rate constants. The maximal binding capacity (Bmax) was 5.1 +/- 0.1 pmol/mg of protein. The binding exhibited an absolute requirement for Na+ as well as Cl-. Presence of these ions enhanced the binding affinity without affecting Bmax. Kinetic analyses revealed that the coupling ratio of Na+/nisoxetine was 2, whereas the coupling ratio of Cl-/nisoxetine was 1. The binding was most potently inhibited by the ligands of the norepinephrine transporter (desipramine and nomifensine). The ligands of the serotonin transporter (imipramine, paroxetine, and fluoxetine) showed intermediate inhibitory potencies, whereas the ligands of the dopamine transporter (bupropion and GBR 12909) were the least potent. Among the monoamines, dopamine was the most potent inhibitor, followed by norepinephrine and serotonin. Though both cocaine and its analog RTI-55 were powerful inhibitors of the binding, RTI-55 was approximately 150 times more effective than cocaine. The inhibition of binding by norepinephrine, cocaine, and RTI-55 was competitive. Uptake of norepinephrine measured in membrane vesicles was found to be inhibited by treatment of the vesicles with phenylarsine oxide, a reagent specific for vicinal dithiol groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1993

Regulation of the human serotonin transporter. Cholera toxin-induced stimulation of serotonin uptake in human placental choriocarcinoma cells is accompanied by increased serotonin transporter mRNA levels and serotonin transporter-specific ligand binding.

Treatment of confluent cultures of JAR human placental choriocarcinoma cells with cholera toxin or forskolin for 16 h markedly stimulated (2.4-fold) serotonin transport activity in these cells. Cycloheximide, an inhibitor of protein synthesis or actinomycin D, an inhibitor of mRNA synthesis effectively blocked this stimulation. Northern blot analysis revealed that treatment with cholera toxin resulted in severalfold increase in the concentrations of the three mRNA species (6.8, 4.9 and 3.0 kilobases in size) which hybridized to the human placental serotonin transporter cDNA. Under similar conditions, the concentrations of the mRNA species which hybridized to the human placental taurine transporter cDNA or to the human beta-actin cDNA were not affected. Analysis of paroxetine-sensitive binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane to the membranes prepared from control and cholera toxin-treated cells indicated that the maximal binding capacity was increased 2.5-fold by cholera toxin, with no significant change in the binding affinity. Thus, stimulation of serotonin transporter activity in the placental choriocarcinoma cells following cholera toxin treatment is likely a result of an increase in cell surface density of the serotonin transporter protein as a consequence of increased steady state serotonin transporter mRNA levels.

Binding Sites↗

PubMed1993

Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization.

A Na(+)- and Cl(-)-coupled serotonin (5-hydroxytryptamine, 5HT) transporter is expressed on human neuronal, platelet, placental, and pulmonary membranes. The brain 5HT transporter appears to be a principal site of action of therapeutic antidepressants and may mediate behavioral and/or toxic effects of cocaine and amphetamines. Oligonucleotides derived from consensus transporter sequences were used to identify human placental cDNAs highly related to the rat brain 5HT carrier. Transfection of one of these cDNAs into HeLa cells yields a high-affinity (Km = 463 nM), Na(+)- and Cl(-)-dependent 5HT transport activity which can be blocked by selective 5HT transport inhibitors, including paroxetine, fluoxetine, and imipramine, and which is antagonized by cocaine and amphetamine. Sequence analysis reveals a 630-amino acid open reading frame bearing 92% identity to the cloned rat brain 5HT transporter, with identical predicted topological features and conserved sites for posttranslational modifications. Unlike the rodent, where a single mRNA appears to encode 5HT transporters, multiple hybridizing RNAs are observed in human placenta and lung. Somatic cell hybrid and in situ hybridization studies are consistent, however, with a single gene encoding the human 5HT transporter, localized to chromosome 17q11.1-17q12.

Amino Acid Sequence↗

PubMed1993

Solubilization and functional reconstitution of the human placental taurine transporter.

The taurine transporter from purified human placental brush-border membranes was solubilized and reconstituted into proteoliposomes in a functional form. Solubilization was done with 2.5% cholate in the presence of 4 M urea. The proteins in the solubilizate were precipitated with 6% poly(ethylene glycol) and the precipitated proteins were reconstituted into proteoliposomes with an asolectin/protein ratio of 10:1. Under these experimental conditions, the taurine transport activity in the proteoliposomes was maximal. SDS-PAGE analysis of proteins, however, revealed that the proteoliposomes still contained a majority of the proteins originally present in the brush-border membranes. Uptake of taurine in the reconstituted proteoliposomes was obligatorily dependent on the presence of Na+ as well as Cl-. Substitution of Na+ with other monovalent cations such as K+ and Li+ reduced the taurine transport activity drastically. Similarly, substitution of Cl- with other monovalent anions such as SCN-, F-, I- and NO3- could support the transport activity only to a maximum of 30% of the control activity. In the presence of Cl-, the uptake rate was sigmoidally related to Na+ concentration, resulting in a Na+/taurine coupling ratio of 2:1. The apparent dissociation constant for Na+ was about 195 mM. In the presence of Na+, the uptake rate was hyperbolically related to Cl- concentration, indicating a Cl-/taurine coupling ratio of 1:1. The apparent dissociation constant for Cl- was about 205 mM. The NaCl-dependent taurine uptake was stimulated by an inside-negative membrane potential, showing that the uptake process was electrogenic. The uptake system was specific for beta-amino acids. The affinity of the system for taurine was high with an apparent dissociation constant of 2.7 +/- 0.1 microM. It is concluded that the taurine transporter can be dislodged from the placental brush-border membranes and reconstituted in a catalytically active form in proteoliposomes with no significant change in its characteristics.

Anions↗

PubMed1993

Expression of a cocaine-sensitive norepinephrine transporter in the human placental syncytiotrophoblast.

Maternal-facing brush border membrane vesicles isolated from normal term human placentas were found to accumulate norepinephrine in a concentrative manner in the presence of an inwardly directed NaCl gradient. Both Na+ and Cl- were obligatory for maximal uptake. The NaCl-dependent norepinephrine uptake was further stimulated by the presence of K+ or an acidic pH in the intravesicular medium. The uptake process was electrogenic, being stimulated by an inside-negative membrane potential, and this characteristic was observed in the absence as well as in the presence of K+ inside the vesicles. Kinetic analyses revealed that one Na+ and one Cl- were involved per transport of one norepinephrine molecule. The apparent Michaelis-Menten constant for norepinephrine was 104 +/- 5nM. The uptake process exhibited higher affinity for dopamine than for norepinephrine but had low affinity for serotonin and histamine. The uptake of norepinephrine was inhibited very effectively by nomifensine, desipramine, imipramine, and cocaine, but much less effectively by bupropion and GBR 12909. Northern blot analysis with the cDNA of the human (SK-N-SH cell) norepinephrine transporter as the probe revealed that the human placenta contained two mRNAs, 5.8 and 3.6 kb in size, which hybridized to the probe. The JAR human placental choriocarcinoma cells were found unable to accumulate norepinephrine in a NaCl-dependent manner. These cells were also found not to contain mRNAs which hybridized to the norepinephrine cDNA probe in northern blot. It is concluded that the human placental syncytiotrophoblast expresses a cocaine-sensitive norepinephrine transporter and that these findings may be directly relevant and important to the clinical complications of maternal cocaine abuse during pregnancy.

Biological Transport↗

PubMed1993

Regulation of taurine transport in human colon carcinoma cell lines (HT-29 and Caco-2) by protein kinase C.

The effect of phorbol 12-myristate 13-acetate (PMA), a phorbol ester known to stimulate protein kinase C, on taurine transport was studied in the human colon carcinoma cell line HT-29. PMA (1 microM) was found to inhibit taurine uptake in confluent monolayers of this cell line by approximately 70% after pretreatment of the cells with the compound for 1 h (IC50 = 42.7 +/- 2.6 nM). The inhibitory effect of PMA on the taurine transporter could be confirmed by using beta-alanine, another substrate for the transporter. Kinetic analysis of taurine uptake indicated that the PMA effect was associated with a decrease in the maximal velocity (954 +/- 26 vs. 676 +/- 28 pmol.10 min-1.mg of protein-1) and an increase in the Michaelis-Menten constant (9.8 +/- 0.5 vs. 13.3 +/- 1.0 microM). The inhibition of taurine uptake could be blocked by cotreatment of the cultures with staurosporine, an inhibitor of protein kinase C. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate had no effect. Treatment of the cells with PMA did not alter the uptake of leucine and lysine, stimulated the uptake of aspartic acid, and inhibited the uptake of proline. The PMA effect on taurine uptake was not prevented by cycloheximide, actinomycin D, colchicine, or cytochalasin D. Comparison of the taurine transport activity in HT-29 cells with that in Caco-2, another human colon carcinoma cell line, revealed that the latter cell line also expressed the taurine transporter but at a much reduced level (about one-fifth compared with HT-29).(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗

PubMed1993

Genetic evidence for role of DPP IV in intestinal hydrolysis and assimilation of prolyl peptides.

The functional role of dipeptidyl peptidase IV (DPP IV) in the intestinal hydrolysis and assimilation of prolyl peptides was investigated using Japan F344 rats, which genetically lack this enzyme. USA F344 rats possess normal activity of this enzyme and served as matched controls. Intestinal brush-border membranes from the control rats were able to hydrolyze several proline-containing peptides. The hydrolytic ability of the brush-border membranes from the Japan rats against these peptides was markedly low. The difference in the hydrolytic activities between the two groups of rats was solely due to the absence of DPP IV in the Japan rats. There was no difference in the growth rate between the two groups of rats fed a reference diet whose protein constituents were not rich in proline. When the protein source was changed to gliadin, a proline-rich protein, USA F344 rats maintained their body weight for a 4-wk period on this diet, whereas the Japan rats experienced a significant weight loss under similar conditions. In situ perfusion experiments in intact animals revealed that the ability of morphiceptin (a peptide primarily hydrolyzable by DPP IV), when administered into the intestinal lumen, to block the cholera toxin-induced water secretion was significantly greater in Japan F344 rats than in USA F344 rats, indicating the resistance of morphiceptin to hydrolytic breakdown in the intestinal lumen of the Japan rats. It is concluded that the intestinal DPP IV plays a significant role in the hydrolysis of prolyl peptides and assimilation of proline-rich proteins.

Animals↗

PubMed1993

Expression of IGF-II/Man-6-P receptors on rat, rabbit, and human colon epithelial cells.

Previous experiments from this laboratory have established the presence of receptors for insulin and insulin-like growth factor I (IGF-I) on apical membranes prepared from rabbit colon epithelial cells; however, no receptors for multiplication-stimulating activity (MSA), the rat peptide hormone equivalent of human IGF-II, were found in this tissue. In the current studies, radioligand binding assays, covalent cross-linking experiments, and immunoblot analyses using a polyclonal rabbit antiserum that recognizes the IGF-II/mannose 6-phosphate (Man-6-P) receptor, all confirmed the presence of IGF-II/Man-6-P receptors on membranes prepared from rat and human colon epithelial cells. Exposure of rat colon epithelial cell membrane fractions to 5 mM Man-6-P before incubation with 125I-labeled IGF-II increased radioligand binding. Immunoblot analysis indicated that IGF-II/Man-6-P receptors were present in both unfractionated rat colon membranes and fractions enriched with apical membranes. Rabbit and human colon epithelial cells displayed a different pattern of receptor distribution than rat colon epithelial cells, with more insulin receptors but relatively few IGF-II/Man-6-P receptors. Immunohistochemical studies using a rabbit polyclonal antiserum confirmed that IGF-II/Man-6-P receptors were present on both the apical and the basolateral surfaces of colon epithelial cells.

Animals↗

PubMed1993

Properties of taurine transport in a human retinal pigment epithelial cell line.

The characteristics of taurine transport were studied in a human retinal pigment epithelial cell line (HRPE). Uptake of taurine into monolayer cultures of the HRPE cells was markedly stimulated by the presence of NaCl in the uptake medium whereas the uptake was negligible in its absence. This NaCl-dependent uptake was an active process as the cells were able to accumulate taurine against a concentration gradient. The uptake rate of taurine was found to be many-fold greater than that of gamma-aminobutyric acid (GABA). Unlabeled taurine and GABA competed with radiolabeled taurine for the uptake process, the former being more effective than the latter. However, uptake of radiolabeled GABA was not affected by unlabeled taurine and GABA. Substrate specificity studies revealed strong interaction of beta-amino acids with the transport system responsible for taurine uptake. alpha-Amino acids failed to inhibit taurine uptake. A specific anion requirement was observed for optimal activity of the taurine transport system and Cl- was the most supportive among several anions tested. Kinetic analyses showed that multiple Na+ and one Cl- were involved in transfer of one taurine molecule. The transport process consisted of a single saturable system with a Michaelis-Menten constant of 2.0 +/- 0.1 microM. These results show that the HRPE cell line expresses a high-affinity taurine transport system. This is the first demonstration of the presence of the taurine transporter in the human retinal pigment epithelium and the HRPE cell line may provide a useful model system for future studies involving taurine transport in the retinal pigment epithelium.

Adult↗

PubMed1993

Characterization and subtype identification of the Na(+)-H+ exchanger in bovine corneal epithelium.

Amiloride analogues with N5-alkyl substitutions are specific high-affinity ligands for the Na(+)-H+ exchanger in various tissues. As a means to characterize the Na(+)-H+ exchanger in the bovine corneal epithelium, we determined the binding properties of [3H] methylisobutylamiloride (MIA) to a fraction enriched in plasma membrane from this tissue. [3H]MIA bound to these membranes in a time, -a temperature-, and -a pH-dependent manner. The binding was optimal at 4 degrees C and at pH 8.5 and it reached equilibrium at 60 min. Under these conditions, specific binding, which was inhibitable by excess unlabeled MIA, was about 85%. Scatchard analysis of this specific binding revealed a single saturable binding component with a Kd of 61 nM and a Bmax of 271 pmoles/mg protein. Inhibition of [3H]MIA specific binding by amiloride analogues showed the following order of potency: MIA > dimethylamiloride (DMA) > benzamil > amiloride. Na+ did not compete with MIA for binding. The effectiveness of clonidine, an alpha 2 agonist, and cimetidine, an H2 receptor antagonist, as inhibitors of Na(+)-H+ exchange activity was also determined because these compounds are used to distinguish between the exchanger subtypes. At concentrations higher than those needed for receptor interaction, clonidine was more effective than cimetidine in decreasing MIA binding. The activity of Na(+)-H+ exchanger, which was measured as the uptake of 22Na+ in the presence of an outwardly directly H+ gradient, was also inhibited by DMA, benzamil and amiloride with the same order of potency as obtained in the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1992

Specific interaction of 5-(N-methyl-N-isobutyl)amiloride with the organic cation-proton antiporter in human placental brush-border membrane vesicles. Transport and binding.

The interaction of 5-(N-methyl-N-isobutyl)amiloride (MIBA) with brush-border membrane vesicles isolated from normal human term placentas was investigated using two parameters: binding and transport. The binding of MIBA to placental membranes was specific and temperature- and pH-dependent, and the apparent dissociation constant (Kd) for the process was 58 +/- 2 microM. The binding was inhibited by other amiloride analogs and also by clonidine and cimetidine with a rank order potency: MIBA > benzamil > dimethylamiloride > amiloride > clonidine > cimetidine. These compounds also inhibited Na(+)-H+ exchanger activity in these membrane vesicles, but with a different order of potency: dimethylamiloride > MIBA > amiloride > benzamil > cimetidine > clonidine. The membrane vesicles were also able to transport MIBA into the intravesicular space, and the transport was stimulated many-fold by the presence of an outwardly directed H+ gradient across the membrane. The H+ gradient was the driving force for uphill accumulation of MIBA inside the vesicles. The transport process was electrically silent. The transport of MIBA was inhibited by other amiloride analogs and by clonidine and cimetidine, and the order of potency was the same as the order with which these compounds inhibited the binding of MIBA. The Michaelis-Menten constant (Kt) for the transport process was 46 +/- 2 microM. The binding as well as the transport were also inhibited by Na+ and Li+. Interestingly, tetraethylammonium and N1-methylnicotinamide, two of the commonly used substrates in organic cation transport studies, failed to inhibit the binding and transport of MIBA. Furthermore, although the outwardly directed H+ gradient-dependent uphill transport of tetraethylammonium could be demonstrated in renal brush-border membrane vesicles, there was no evidence for the presence of a transport system for this prototypical organic cation in placental brush-border membrane vesicles. It is concluded that the human placental brush-border membranes possess an organic cation-proton antiporter which accepts MIBA as a substrate, the low affinity binding site for MIBA observed in these membranes represents this antiporter, and that the placental organic cation-proton antiporter is distinct from the widely studied renal organic cation-proton antiporter.

Amiloride↗

PubMed1992

Characterization of an ATP-driven H+ pump in human placental brush-border membrane vesicles.

The presence of an ATP-driven H+ pump as measured by H+ uptake upon addition of ATP was not demonstrable in human placental brush-border membrane vesicles when used in their native form, owing to their right-side-out orientation. However, the presence of the H+ pump in these membranes became evident when the membrane vesicles were transiently exposed to 1% cholate, with subsequent removal of the detergent to re-form the vesicles. Apparently, cholate pretreatment reoriented the H+ pump from an inward-facing configuration to outward-facing. Consequently, H+ uptake in response to externally added ATP was easily demonstrable in these cholate-pretreated vesicles by using the delta pH indicator Acridine Orange. In addition, bafilomycin A1-sensitive ATPase activity was measurable in cholate-pretreated vesicles, but not in native intact vesicles, indicating reorientation of the H+ pump. The reoriented H+ pump was electrogenic because H+ uptake was stimulated by an inside-negative anion-diffusion potential or when the vesicles were voltage-clamped. ATP supported H+ uptake with an apparent Km of 260 microM. ITP and GTP supported the pump activity partially, whereas CTP and UTP did not. Mg2+ and Mn2+ were the most preferred bivalent cations. Co2+ and Zn2+ showed partial activity, whereas Ca2+ and Ba2+ showed little or no activity. The pump was inhibited by nanomolar concentrations of bafilomycin A1 and micromolar concentrations of N-ethylmaleimide, p-chloromercuribenzenesulphonate, NN-dicyclohexylcarbodi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was relatively insensitive to oligomycin, vanadate and NaN3. The inhibition by N-ethylmaleimide was protectable by ATP. It is concluded that human placental brush-border membranes possess an ATP-driven H+ pump and that, on the basis of its characteristics, it belongs to the class of vacuolar (V-type) H+ pumps.

Acridine Orange↗

PubMed1992

Reconstitution of the human placental 5-hydroxytryptamine transporter in a catalytically active form after detergent solubilization.

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.

Anions↗

PubMed1992

Modulation of the activity of amino acid transport system L by phorbol esters and calmodulin antagonists in a human placental choriocarcinoma cell line.

We investigated the regulation of the activity of amino acid transport system L in the JAR human placental choriocarcinoma cell line by agents which are known to modulate the activities of three different classes of protein kinases, A-kinase, C-kinase and CaM-kinase. The system L activity was measured by determining the uptake of leucine in these cells, grown as confluent monolayers. Leucine uptake in these cells was predominantly Na(+)-independent, was stimulated by lowering the extracellular pH and was inhibited by hydrophobic neutral amino acids. These characteristics demonstrate that uptake of leucine in this cell line occurs primarily via system L. Treatment of the cells with cholera toxin and forskolin, agents which are known to elevate intracellular cAMP levels, did not have any effect on the activity of system L. 4 beta-Phorbol 12-myristate 13-acetate, an activator of C-kinase, but not the inactive analogue 4 alpha-phorbol 12,13-didecanoate, caused a significant stimulation of system L. The involvement of C-kinase in the phorbol ester-induced stimulation was supported by the finding that staurosporine, an inhibitor of C-kinase, effectively blocked the stimulation. Calmodulin antagonists, calmidazolium, W-7 and CGS 9343 B stimulated system L activity markedly. The potency of these antagonists was in the following order: calmidazolium greater than CGS 9343 B greater than W-7. This stimulatory effect was specific for system L because systems A and ASC were not stimulated by these agents. The stimulation caused by these agents was primarily due to an increase in the maximal velocity, the apparent Km of the system being only minimally affected. It is concluded that the activity of amino acid transport system L in the JAR human placental choriocarcinoma cell line is stimulated by C-kinase, inhibited by CaM-kinase and unaffected by A-kinase.

Alkaloids↗

PubMed1992

Human placental syncytiotrophoblast expresses two pharmacologically distinguishable types of Na(+)-H+ exchangers, NHE-1 in the maternal-facing (brush border) membrane and NHE-2 in the fetal-facing (basal) membrane.

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).

Basement Membrane↗