Nicotinic Acetylcholine Receptor Antagonistic Activity of Monoamine Uptake Blockers in Rat Hippocampal Slices

The aim of our study was to investigate the effect of different monoamine uptake blockers on the nicotine-evoked release of [3H]noradrenaline ([3H]NA) from rat hippocampal slices. We found that desipramine (DMI), nisoxetine, cocaine, citalopram, and nomifensine inhibit the nicotine-evoked release of [3H]NA with an IC50 of 0.36, 0.59, 0.81, 0.93, and 1.84 microM, respectively. These IC50 values showed no correlation with the inhibitory effect (Ki) of monoamine uptake blockers on the neuronal NA transporter (r = 0.17, slope = 0.02), indicating that the NA uptake system is not involved in the process. In whole-cell patch clamp experiments neither drug blocked Na+ currents at 1 microM in sympathetic neurons from rat superior cervical ganglia, and only DMI produced a pronounced inhibition (52% decrease) at 10 microM. Comparison of the effect of DMI and tetrodotoxin (TTX) on the electrical stimulation- and nicotine-evoked release of [3H]NA showed that DMI, in contrast to TTX, inhibits only the nicotine-induced response, indicating that the target of DMI is not the Na+ channel. Our data suggest that monoamine uptake blockers with different chemical structure and selectivity are able to inhibit the nicotinic acetylcholine receptors in the CNS. Because these compounds are widely used in the therapy of depressed patients, our findings may have great importance in the evaluation of their clinical effects.     

Functional expression of the norepinephrine transporter in cultured rat astrocytes

We assessed the functional expression of the norepinephrine (NE) transporter (NET) in cultured rat cortical astrocytes. Specific [3H]NE uptake increased in a time-dependent manner, and this uptake involves temperature- and Na+-sensitive mechanisms. The Na+-dependent [3H]NE uptake was saturable, and the Km for the process was 539.3 +/- 55.4 nm and the Vmax was 1.41 +/- 0.03 pmol/mg protein/min. Ouabain, a Na+-K+ ATPase inhibitor, significantly inhibited Na+-dependent [3H]NE uptake. The selective NE uptake inhibitor nisoxetine, the tricyclic antidepressants desipramine and imipramine, and the serotonin and NE reuptake inhibitor (SNRI) milnacipran very potently inhibited Na+-dependent [3H]NE uptake. On the other hand, GBR-12935 (a selective dopamine uptake inhibitor), fluvoxamine (a selective serotonin reuptake inhibitor), venlafaxine (a SNRI) and cocaine had weaker inhibitory activities. RT-PCR demonstrated that astrocytes expressed mRNA for the cloned NET protein, which was characterized as neuronal NET. Western blots indicated that anti-NET polyclonal antibody recognized a major band of 80 kDa in astrocytes. These data indicate that the neuronal NET is functionally expressed in cultured rat astrocytes. Glial cells may exert significant control of noradrenergic activity by inactivating NE that escapes neuronal re-uptake in sites distant from terminals, and are thus cellular targets for antidepressant drugs that inhibit NE uptake.     

The depletion of rat cortical norepinephrine and the inhibition of [3H]norepinephrine uptake by xylamine does not require monoamine oxidase activity

Inhibition of monoamine oxidase A through pretreatment of rats with clorgyline (10 mg/kg ip) or the pro-drug MDL 72,394 (0.5 mg/kg ip) did not block the amine-depleting action of xylamine (25 mg/kg ip). Xylamine treatment resulted in a loss of approximately 60% of the control level of norepinephrine in the cerebral cortex. A 1-hr pretreatment, but not a 24-hr pretreatment, with the monoamine oxidase B inhibitor, L-deprenyl (10 mg/kg ip), prevented the depletion of norepinephrine by xylamine. In addition, pretreatment with MDL 72,974 (1.25 mg/kg ip), a monoamine oxidase B inhibitor without amine-releasing or uptake - inhibiting effects, did not protect cortical norepinephrine levels. Inhibition of monoamine oxidase by either MDL 72,974 or MDL 72,394 did not prevent the inhibition of [3H]norepinephrine uptake into rat cortical synaptosomes by xylamine. These data indicate that monoamine oxidase does not mediate the amine-releasing or uptake inhibiting properties of xylamine. The protection afforded by L-deprenyl following a 1-hr pretreatment most probably was due to accumulation of its metabolite, L-amphetamine, which would inhibit the uptake carrier. A functional carrier is required for depletion since desipramine (20 mg/kg ip) administered 1 hr prior to xylamine, was also able to prevent depletion of norepinephrine.     

chi-Conopeptide MrIA partially overlaps desipramine and cocaine binding sites on the human norepinephrine transporter

The interactions of chi-conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [3H]norepinephrine (Ki 1.89 microM) than [3H]dopamine (Ki 4.33 microM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA (to 7 microM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [3H]norepinephrine uptake for three mutations (in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrIA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine.     

High affinity stereospecific binding of [3H] cocaine in striatum and its relationship to the dopamine transporter

A high affinity (KD 35 nM) binding site for [3H]cocaine is detected in rat brain striatum present at 2-3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [3H]dopamine ([3H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [3H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [3H]cocaine binding stereospecifically, but with lower potency (IC50 approximately equal to 1 microM) than does cocaine. It is suggested that the DA transporter in striatum is the putative "cocaine receptor." Binding of [3H]cocaine, measured in 10 mM Na2HPO4-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na+ and K+ and by biogenic amines. DA and Na+ reduce the affinity of the putative "cocaine receptor" for [3H]cocaine without changing the Bmax, suggesting that inhibition may be competitive. However, TRIS reduces [3H]cocaine binding noncompetitively while Na+ potentiates it in TRIS buffer. Binding of [3H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [3H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na+ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

Pharmacological characterization of dopamine transport in cultured rat astrocytes.

The effects of GBR-12909 (selective DA uptake inhibitor), zimelidine (selective 5-HT uptake inhibitor) and nisoxetine (selective NE uptake inhibitor) on the uptake of 30 nM [3H]DA into cultured rat astrocytes were examined. [3H]DA uptake was inhibited by approximately 50% by GBR-12909 or zimelidine in a concentration-dependent manner (100 nM to approximately 10 microM). Furthermore, the inhibition curves of GBR-12909 were biphasic, and uptake was completely inhibited by a high concentration of GBR-12909 (100 microM). [3H]DA uptake was also inhibited by approximately 50% by nisoxetine in a concentration-dependent manner (0.1 to approximately 100 nM), and nisoxetine was more potent than GBR-12909 or zimelidine. The inhibitory potencies were in the order nisoxetine > GBR-12909 > zimelidine. The uptake of [3H]DA under Na+-free conditions was approximately 50% of that under normal conditions. Thus, DA was taken up by both Na+-dependent and Na+-independent mechanisms. Nisoxetine (100 nM), zimelidine (100 microM) and GBR-12909 (10 microM) inhibited [3H]DA uptake into astrocytes only in the presence of Na+. On the other hand, this uptake was completely inhibited by a high concentration of GBR-12909 (100 microM) in the absence of Na+. The present data suggest that the Na+-dependent uptake of [3H]DA in cultured rat astrocytes may occur in the NE uptake system. Furthermore, astrocytes express the extraneuronal monoamine transporter (uptake2), which is an Na+-independent system, and this transporter is involved in the inactivation of centrally released DA.     

GBR-12909 and fluspirilene potently inhibited binding of [3H] (+)3-PPP to sigma receptors in rat brain

Fluspirilene and GBR-12909, two compounds structurally similar to BMY-14802 and haloperidol, were assessed for their ability to interact with sigma receptors. Fluspirilene, an antipsychotic agent that interacts potently with dopamine receptors, inhibited the binding of [3H]-(+) 3-PPP (IC50 = 380 nM) more potently than rimcazole, a putative sigma antagonist that was tested clinically for antipsychotic activity. GBR-12909, a potent dopamine uptake blocker, also inhibited the binding of [3H]-(+) 3-PPP with an IC50 of 48 nM. However, other compounds that block the re-uptake of catecholamines, such as nomifensine, desipramine, imipramine, xylamine, benztropine and cocaine, were much weaker than GBR-12909 as sigma ligands. Thus, GBR-12909 and fluspirilene, compounds structurally similar to BMY-14802, are potent sigma ligands.     

Involvement of noradrenaline transporters in S-nitrosocysteine-stimulated noradrenaline release from rat brain slices: existence of functional Na(+)-independent transporter activity

Noradrenaline (NA) can be released by both exocytosis and by the membrane transporter responsible for transmitter uptake. Previously, we reported that S-nitrosocysteine (SNC), an S-nitrosothiol, stimulated [3H]NA release from the rat hippocampus. In this study, we investigated the involvement of the NA transport system in SNC-stimulated NA release from rat brain (cerebral cortex and hippocampus) slices. [3H]NA release by SNC in normal Na(+) (148 mM)-containing buffer from both slices was slightly, but significantly, inhibited by 1 microM desipramine, an NA transporter inhibitor. [3H]NA release in low Na(+) (under 14 mM)-containing buffer was inhibited by over 50% by desipramine. [3H]NA release by tyramine from both slices in normal and low Na(+) buffer was almost completely inhibited by desipramine. [3H]NA uptake into cerebral cortical slices was observed in low Na(+) buffer at 20-30% of normal Na(+) buffer levels. [3H]NA uptake in both normal and low Na(+) buffers was inhibited by desipramine and by SNC. Although [3H]NA uptake in normal Na(+) buffer was almost completely inhibited by 500 microM ouabain, the uptake in low Na(+) buffer was resistant to ouabain. These findings suggest the existence of a functional Na(+)-independent NA transport system and that SNC stimulates NA release at least partially via this system in brain slices.     

PC12 Variants Deficient in Catecholamine Transport

We have isolated PC12 cell variants deficient in transporter-mediated uptake of 3,4-dihydroxyphenylethylamine (dopamine). The variants either were obtained nonselectively, or they were selected by resistance to guanethidine or N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Dopamine uptake into guanethidine-resistant cells occurred with a decreased Vmax; the Km for dopamine and inhibition by guanethidine were normal. MPTP-resistant cells lacked the capacity to take up dopamine. Most of the variants resembled wild-type PC12 in their response to nerve growth factor and the storage and secretion of dopamine. MPTP-resistant cells exhibited several deficiencies in addition to dopamine transport, i.e., no measurable storage of dopamine or acetylcholine and no observable response to nerve growth factor. Wild-type and variant cells were compared with respect to the labeling of cell proteins with [3H]xylamine, which binds covalently to certain proteins apparently only after entering PC12 via the catecholamine transporter. When intact variant cells were used, there was markedly reduced labeling of the proteins by [3H]xylamine. Almost all of these proteins were readily labeled when cell homogenates were exposed to [3H]xylamine. However, MPTP-resistant cells were missing three of these proteins. Northern blot analysis with cDNA clones revealed that the MPTP-resistant cells had markedly reduced levels of several specific mRNA species.     

Regional Differences in 5-Hydroxytryptamine and Catecholamine Uptake in Primary Astrocyte Cultures

The uptake of 3H-labelled 5-hydroxytryptamine (5-HT, serotonin) norepinephrine ([3H]NE), and 3,4-dihydroxyphenylethylamine ([ 3H]dopamine, [3H]DA) was studied in primary astrocyte cultures prepared from the cerebral cortex, corpus striatum, and hippocampal regions of neonatal rat brain. Na+-dependent uptake showed marked regional differences. For [3H]5-HT the magnitude of uptake was corpus striatum greater than or equal to cerebral cortex greater than hippocampus, whereas for [3H]NE the order was hippocampus greater than corpus striatum greater than cerebral cortex. For [3H]DA, only the hippocampal cultures showed significant Na+-dependent uptake. [3H]5-HT uptake was specifically inhibited by 10(-7) M fluoxetine whereas [3H]NE uptake was preferentially inhibited by 10(-7) M desipramine. These results may reflect regional brain specialization and/or different developmental patterns of high affinity uptake of serotonin and catecholamines by astrocytes in situ.     

Characterization, cDNA cloning, and functional expression of mouse ileal sodium-dependent bile acid transporter

Mouse ileal sodium dependent bile acid transporter (ISBT) was characterized using isolated enterocytes. Only enterocytes from the most distal portion showed Na+-dependent [3H]taurocholate uptake. Northern blot analysis using a probe against mouse ISBT revealed the expression of mouse ISBT mRNA to be restricted to the distal ileum. The Km and Vmax for Na+-dependent [3H]taurocholate transport into isolated ileocytes were calculated as 27 microM and 360 pmol/mg protein/min, respectively. Uptake of [3H]taurocholate was inhibited by N-ethylmaleimide. We have cloned ISBT cDNA from mouse ileum. The cDNA included the entire open reading frame coding 348 amino acid protein with seven hydrophobic segments and two N-glycosylation sites. COS-7 cells transfected with the expression vector containing this cDNA expressed Na+-dependent [3H]taurocholate uptake activity with a Km of 34 microM.     

Evidence for an imipramine-sensitive serotonin transporter in human placental brush-border membranes

Serotonin is actively transported into brush-border membrane vesicles isolated from normal human term placentas and an inward-directed NaCl gradient provides the driving force for this process. Uptake is negligible if Na+ is replaced by Li+, K+, Rb+, Cs+ or choline. The presence of Cl- seems necessary for the maximal activity of this Na+-dependent uptake system. Intravesicular K+ (20-40 mM) stimulates serotonin uptake, the stimulation being considerably greater at pH 7.5 than at pH 6.5. But, in the absence of K+, uptake at pH 6.5 was twice the uptake at pH 7.5. Unlabeled serotonin and dopamine inhibit the uptake of radiolabeled serotonin and the IC50 values are 70 nM and 20 microM, respectively. Histamine and 5-hydroxytryptophan do not significantly interact with the system (IC50 greater than 1 mM). Kinetic analysis reveals that serotonin uptake in these vesicles occurs via a single, saturable, high affinity system (Kt = 51 +/- 2 nM; Vmax = 6.4 +/- 0.1 pmol/mg of protein/15 s). The transporter is highly sensitive to inhibition by imipramine (IC50 = 32 nM) and desipramine (IC50 = 160 nM) but relatively insensitive to reserpine and hydralazine.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.     

Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.     

Expression of a Human Cocaine-Sensitive Dopamine Transporter in Xenopus laevis Oocytes

The human dopamine transporter was expressed in Xenopus laevis oocytes following injection of mRNA isolated from human brain substantia nigra. The specific accumulation of [3H]dopamine into these oocytes was time and Na+ dependent. Furthermore, [3H]dopamine accumulation was prevented by coincubation of oocytes with dopamine (100 microM) or with the dopamine uptake inhibitors GBR 12909 (1 microM) or cocaine (3 microM). In contrast, oocyte injection of mRNA isolated from human globus pallidus, an area devoid of dopamine neuron perikarya, did not elicit expression of the dopamine transporter. Oocyte expression of the human dopamine transporter can be used for the further characterization and cloning of this low-abundance membrane protein.     

Irreversible Binding and Recovery of the Norepinephrine Uptake System Using an Alkylating Derivative of Norepinephrine

The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.     

Comparison of dopamine uptake and release in vitro in sheep and rat striatum.

Cocaine inhibits tritium-labeled dopamine ([3H]DA) uptake in rat (IC50 approximately 400 nM) and sheep (IC50 approximately 1 microM) striatum. GBR 12909, a selective DA uptake inhibitor, potently inhibits [3H]DA uptake in rat (IC50 less than 10 nM), but is less effective (only 60% of the uptake is inhibited at a concentration of 10 microM) and less potent (IC50 approximately 300 nM) in sheep. [3H]DA release from slices of rat or sheep striatum is stimulated by potassium (15-50 mM). In the presence of nomifensine (10 microM), cocaine (10 microM) had no effect on potassium-stimulated [3H]DA release in either species. [3H]DA release is increased by N-methyl-D-aspartate (NMDA) (10-1000 microM) in rat striatum but NMDA did not stimulate [3H]DA release in sheep striatum. These findings suggest that NMDA receptors either are absent from or do not regulate release of preloaded [3H]DA in sheep striatum.     

Effects of Monovalent Ions on the Transport of Noradrenaline Across the Plasma Membrane of Neuronal Cells (PC-12 Cells)

The dependence on Na+, K+, and Cl- of uptake and accumulation of [3H]noradrenaline was studied in plasma membrane vesicles isolated from PC-12 pheochromocytoma cells. Plasma membrane vesicles accumulated [3H]noradrenaline when an inward-directed gradient for Na+ and an outward-directed gradient for K+ were imposed across the vesicle membrane. Under these conditions, initial rates of uptake of [3H]noradrenaline were saturable (Km = 0.14 microM) and inhibited by a series of substrates and inhibitors of "uptake". The IC50 values were positively correlated with those for inhibition of uptake into intact PC-12 cells. Uptake and accumulation of [3H]noradrenaline in plasma membrane vesicles were absolutely dependent on external Na+ and Cl-; they were dependent on an inwardly directed gradient for Na+ but less dependent on an inwardly directed gradient for Cl-. Internal K+ strongly enhanced uptake and accumulation of [3H]noradrenaline. Rb+, but not Li+, had the capacity to replace internal K+. Two explanations are proposed for this effect of internal K+: (a) creation of a K+ diffusion potential (inside negative) provides a driving force for inward transport, and/or (b) K+ increases the turnover rate by formation of a highly mobile potassium-carrier complex. A hypothetical scheme for the transport of noradrenaline is presented.     

Na(+)- and Cl(-)-dependent [3H]GABA binding to catfish brain particles

The uptake of [3H]GABA by homogenates of catfish brain was previously shown to be temperature-sensitive and sodium-dependent, and to display saturation kinetics. The present study is a continuation of this work and was undertaken to characterize the initial binding of [3H]GABA to its transport system. [3H]GABA binding to catfish brain particles at 4 degrees C displayed saturability and was totally dependent on both Na+ and Cl-, the optimum concentrations of which were 150 mM and 75 mM, respectively. The effects of a number of drugs on binding were established. Unlabelled GABA was the most potent inhibitor (IC50 = 3.2 microM). The structural analogues nipecotic acid and guvacine were also strongly inhibitory. Interestingly, verapamil, a Ca2+ channel blocker, also inhibited [3H]GABA binding (IC50 = 38 microM). Harmaline, known to compete for Na+ binding in other transport systems, did not appear to influence Na+ binding but was effective at displacing [3H]GABA. These results suggest that the interaction of GABA with its carrier is similar to that found in the mammalian nervous system and is further evidence that GABA is involved in neurotransmission in catfish brain.     

Characterization of carrier-mediated efflux in human embryonic kidney 293 cells stably expressing the rat serotonin transporter: a superfusion study

Human embryonic kidney 293 cells stably transfected with the rat plasmalemmal serotonin transporter (rSERT) were incubated with 5-[3H]hydroxytryptamine ([3H]5-HT) and superfused. Substrates of the rSERT, such as p-chloroamphetamine (PCA) or methylenedioxymethamphetamine, concentration-dependently increased basal efflux of [3H]5-HT. 5-HT reuptake blockers (e.g., imipramine, citalopram) also caused an enhancement of [3H]5-HT efflux, reaching about half the maximal effect of the rSERT substrates. In uptake experiments, both groups of substances concentration-dependently inhibited 5-HT uptake. EC50 values obtained in superfusion experiments significantly correlated with IC50 values from uptake studies (r2 = 0.92). Addition of the Na+,K(+)-ATPase inhibitor ouabain (100 microM) to or the omission of K+ from the superfusion buffer accelerated basal efflux. The effect of PCA (10 microM) was markedly enhanced by both measures, whereas the effect of uptake inhibitors remained unchanged. When [3H]MPP+, a substrate with low affinity for the rSERT, was used instead of [3H]5-HT for labeling the cells, uptake inhibitors failed to augment efflux. By contrast, PCA accelerated [3H]MPP+ efflux, and its effect was strongly enhanced in the presence of ouabain. The results suggest that the [3H]5-HT efflux caused by substrates of rSERT is carrier-mediated, whereas efflux induced by uptake inhibitors is a consequence of interrupted high-affinity reuptake that is ongoing even under superfusion conditions.