The antidepressant-sensitive dopamine transporter in Drosophila melanogaster: a primordial carrier for catecholamines.
Extracellular concentrations of monoamine neurotransmitters are regulated by a family of high-affinity transporters that are the molecular targets for such psychoactive drugs as cocaine, amphetamines, and therapeutic antidepressants. In Drosophila melanogaster, cocaine-induced behaviors show striking similarities to those induced in vertebrate animal models. Although a cocaine-sensitive serotonin carrier exists in flies, there has been no pharmacological or molecular evidence to support the presence of distinct carrier subtypes for other bioactive monoamines. Here we report the cloning and characterization of a cocaine-sensitive fly dopamine transporter (dDAT). In situ hybridization demonstrates that dDAT mRNA expression is restricted to dopaminergic cells in the fly nervous system. The substrate selectivity of dDAT parallels that of the mammalian DATs in that dopamine and tyramine are the preferred substrates, whereas octopamine is transported less efficiently, and serotonin not at all. In contrast, dDAT inhibitors display a rank order of potency most closely resembling that of mammalian norepinephrine transporters. Cocaine has a moderately high affinity to the cloned dDAT (IC50 = 2.6 microM). Voltage-clamp analysis of dDAT expressed in Xenopus laevis oocytes indicates that dDAT-mediated uptake is electrogenic; however, dDAT seems to lack the constitutive leak conductance that is characteristic of the mammalian catecholamine transporters. The combination of a DAT-like substrate selectivity and norepinephrine transporter-like inhibitor pharmacology within a single carrier, and results from phylogenetic analyses, suggest that dDAT represents an ancestral catecholamine transporter gene. The identification of a cocaine-sensitive target linked to dopaminergic neurotransmission in D. melanogaster will serve as a basis for further dissection of the genetic components of psychostimulant-mediated behavior. (+info)
Norsalsolinol uptake into secretory vesicles via vesicular monoamine transporter and its secretion by membrane depolarization or purinoceptor stimulation in PC12 cells.
The intracellular dynamics of norsalsolinol, a neurotoxin candidate causing parkinsonism-like symptoms, in PC12 cells was studied. We found that dopamine and norsalsolinol are co-localized to secretory granule layer by sucrose density gradient in norsalsolinol-treated PC12 cells. The norsalsolinol was actively taken up into isolated secretory vesicle fraction from PC12 cells with a Km value of 41.5+/-6.8 microM. The uptake of 10 microM of norsalsolinol was sensitive to reserpine (1 microM), an inhibitor of vesicular dopamine transporter, and dopamine, an endogenous substrate, but insensitive to GBR-12909, an inhibitor of dopamine transporter on plasma membrane. In norsalsolinol-treated PC12 cells, exposure to high K+ or ATP resulted in simultaneous release of norsalsolinol and dopamine. Time course of a release of dopamine and that of norsalsolinol evoked by 50 mM KCl or 100 microM ATP corresponded to each other. These releases were dependent on the concentrations of secretagogues. These data suggest that norsalsolinol is taken up with dopamine into secretory vesicle via vesicular catecholamine transporter. (+info)
A Na(+)/Cl(-)-dependent transporter for catecholamines, identified as a norepinephrine transporter, is expressed in the brain of the teleost fish medaka (Oryzias latipes).
We report the isolation, functional characterization, and localization of a Na(+)/Cl(-)-dependent catecholamine transporter (meNET) present in the brain of the teleost fish medaka. This carrier is very similar to the human neuronal norepinephrine transporter (NET) and the human neuronal dopamine transporter (DAT), showing 70 and 64% amino acid identity, respectively. When expressed in COS-7 cells, this transporter mediates the high-affinity uptake of dopamine (K(M) = 290 nM) and norepinephrine (K(M) = 640 nM). Its pharmacological profile reveals more similarities with NET, including a high affinity for the tricyclic antidepressants desipramine (IC(50) = 0.92 nM) and nortriptyline (IC(50) = 16 nM). In situ hybridization on the medaka brain shows that meNET mRNA is present only in a subset of tyrosine hydroxylase-positive neurons found in the noradrenergic areas of the hindbrain, such as the locus ceruleus and area postrema. None of the dopaminergic areas anterior to the isthmus contains any labeled neurons. Neither reverse transcriptase-polymerase chain reaction with degenerate primers specific for gamma-aminobutyric acid transporter/NET nor autoradiographic experiments with [(125)I]3b-(4-iodophenyl)-tropane-2b-carboxylic acid methyl ester revealed an additional catecholamine transporter in the medaka brain. Uptake experiments with medaka brain synaptosomes show an endogenous transport with a pharmacological profile identical to that of the recombinant meNET. Thus, meNET is probably the predominant--if not the only--catecholamine transporter in the medaka fish brain. In view of the highly conserved primary structures and pharmacological properties of meNET, it is tempting to speculate that a specific dopamine transport developed later in vertebrate evolution and probably accompanied the tremendous enlargement of the meso-telencephalic dopaminergic pathways in amniotes. (+info)
Tissue distribution and renal developmental changes in rat organic cation transporter mRNA levels.
Organic cation transporters (OCTs) are responsible for excretion of cationic substances into urine. Tissue OCT expression may be important for the disposition and excretion of xenobiotics. Therefore, OCT1, OCT2, OCT3, OCTN1, and OCTN2 mRNA levels were measured in adult rat tissues and rat kidney tissue at various stages of development from day 0 to 45. OCT1 mRNA expression was highest in kidney and spleen, moderate in skin, and low in the gastrointestinal tract, brain, lung, thymus, muscle, and prostate. OCT2 mRNA levels were highest in kidney, with low expression in other tissues, and with renal OCT2 levels being approximately 4 times higher in males than that in females. In gonadectomized males, OCT2 mRNA levels were attenuated to female levels, suggesting a role for testosterone in OCT2 expression. OCT3 was moderately expressed in kidney and was highest in blood vessel, skin, and thymus. OCTN1 was expressed in most of the tissues examined, with relatively higher expression in kidney and ileum and lower levels in thymus. Lastly, OCTN2 was expressed abundantly in kidney and ileum, moderately in large intestine, dorsal prostate, bladder, duodenum, and cerebellum, and minimally in thymus, spleen, and cerebral cortex. Renal OCT1, OCTN1, and OCTN2 mRNA levels increased gradually from postnatal day 0 through day 45 in both genders. Renal OCT2 levels remained the same in males and females through day 25 and then dramatically increased only in male kidney after day 30. In summary, OCT mRNA was detected primarily in kidney, and the high level of renal OCT expression may explain why the kidney is a target organ for xenobiotics with cationic properties. (+info)
Reduced expression of organic cation transporters rOCT1 and rOCT2 in experimental diabetes.
Recent reports have documented a functional deficit of organic cation transport in diabetic rats by an unknown mechanism. This study was designed to test the hypothesis that experimental diabetes decreases expression of organic cation transporters at the basolateral membrane. Streptozotocin-induced diabetic rats were maintained for varying durations after induction of diabetes. A second group of age-matched control rats was maintained in a parallel manner. Kinetic analysis of tetraethylammonium accumulation in freshly isolated proximal tubular cells indicated a significantly lower V(max) value for the diabetics versus controls with no statistical difference in K(m) values between the two groups. Cortex sections were processed by standard procedures for Northern and immunoblot analysis. Protein expression of the organic cation transporters rOCT1 and rOCT2 progressively decreased with increasing duration of diabetes. After 21 days of diabetes, rOCT1 and rOCT2 were maximally reduced by 50 and 70%, respectively. Quantification of mRNA expression revealed that the roct1 transcript remained unchanged, whereas the roct2 transcript was decreased by 50% after 14 days of diabetes. Treatment with insulin prevented the reductions in transporter levels. These results support the hypothesis by demonstrating that experimental diabetes decreased expression of both rOCT1 and rOCT2 protein and also of roct2 mRNA accumulation. On the other hand, roct1 mRNA levels were unaffected by the diabetic state. This suggests that differences in rOCT2 protein may result from transcriptional and/or translational changes, whereas rOCT1 deficits may be due to posttranscriptional alterations. (+info)
Transcriptional regulation of murine Slc22a1 (Oct1) by peroxisome proliferator agonist receptor-alpha and -gamma.
The transport and metabolism of organic cationic endobiotics, nutrients, and drugs are essential hepatic functions. Slc22A1 is the basolateral liver transporter mediating the uptake of organic cations; however, little is known about the regulation of this transport protein. Peroxisome proliferator agonist receptor (PPAR)-alpha and -gamma agonists are commonly used agents that regulate many hepatocellular transport functions. Thus the purpose of this study was to examine the effects of PPAR agonists on the hepatic regulation and function of Slc22a1. Mice and H35 cells were administered PPAR-alpha and -gamma agonists, and the effect on Slc22a1 gene expression was measured. We subsequently cloned the Slc22a1 promoter and employed chimeric constructs to assay Slc22a1 gene transcription. The effects of PPAR-agonist treatment on organic cation uptake was also assayed. Slc22a1 expression was increased by PPAR-alpha and -gamma agonist treatment in both murine livers and H35 cells. Gene expression in H35 cells was further increased following transfection with expression vectors of PPAR transcription factors and PPAR agonist treatment. We cloned the promoter region of Slc22a1 and identified a PPAR-response element, and transfections with chimeric Slc22a1; promoter-reporter gene constructs demonstrate that the increased gene expression was transcriptionally regulated. Functional assays confirmed that cells treated with PPAR agonists displayed significant increases in organic cation uptake. PPAR-alpha and -gamma agonists transcriptionally increase Slc22a1 gene expression, and the increased Slc22a1 expression results in enhanced cellular organic cation uptake. These studies may have implication for the uptake of organic cationic drugs and for lipid metabolism. (+info)
Subtype-specific affinity for corticosterone of rat organic cation transporters rOCT1 and rOCT2 depends on three amino acids within the substrate binding region.
The affinity of corticosterone to organic cation transporters (OCTs) is subtype- and species-dependent. For example, the IC50 values for corticosterone inhibition of cation uptake by transporters rOCT1 and rOCT2 are approximately 150 and approximately 4 microM, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that the exchange of three amino acids in the presumed 10th transmembrane alpha helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (Ala443, Leu447, and Gln448 in rOCT1 and Ile443, Tyr447, and Glu448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants, the K(m) values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC50 values for the inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E), the IC50 value for the inhibition of [3H]MPP (0.1 microM) uptake by corticosterone (24 +/- 4 microM) was significantly higher compared with the IC50 value for inhibition of [14C]TEA (10 microM) uptake (5.3 +/- 1.7 microM). This finding suggests an allosteric interaction between transported cation and corticosterone. Because this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP versus TEA and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short-range allosteric effect on the corticosterone binding site. (+info)
A species difference in the transport activities of H2 receptor antagonists by rat and human renal organic anion and cation transporters.
A clinical drug-drug interaction between famotidine (a H2 receptor antagonist) and probenecid has not been reproduced in rats. The present study hypothesized that the species-dependent probenecid sensitivity is due to a species difference in the contribution of renal organic anion and cation transporters. The transport activities of the H2 receptor antagonists (cimetidine, famotidine, and ranitidine) by rat and human basolateral organic anion and cation transporters [human organic anion transporter (hOAT) 1, hOAT2, r/hOAT3, rat organic cation transporter (rOct) 1, and r/hOCT2] were compared using their cDNA transfectants. The transport activities (Vmax/Km) of famotidine (Km, 345 microM) by rOat3 were 8- and 15-fold lower than those of cimetidine (Km, 91 microM) and ranitidine (Km, 155 microM), respectively, whereas the activity by hOAT3 (Km, 124 microM) was 3-fold lower than that of cimetidine (Km, 149 microM) but similar to that of ranitidine (Km, 234 microM). Comparison of the relative transport activity with regard to that of cimetidine suggests that famotidine was more efficiently transported by hOAT3 than rOat3, and vice versa, for ranitidine. Only ranitidine was efficiently transported by hOAT2 (Km, 396 microM). rOct1 accepts all of the H2 receptor antagonists with a similar activity, whereas the transport activities of ranitidine and famotidine (Km, 61/56 microM) by r/hOCT2 were markedly lower than that of cimetidine (Km, 69/73 microM). Probenecid was a potent inhibitor of r/OAT3 (Ki, 2.6-5.8 microM), whereas it did not interact with OCTs. These results suggest that, in addition to the absence of OCT1 in human kidney, a species difference in the transport activity by hOAT3 and rOat3 accounts, at least in part, for the species difference in the drug-drug interaction between famotidine and probenecid. (+info)