PubMed Health⌕ Search

Biomedical subjects

K M Giacomini

Publications and source records attributed to K M Giacomini.

At least 19 recordsLinked to original sources
PubMed2005

Functional analysis of genetic variants in the human concentrative nucleoside transporter 3 (CNT3; SLC28A3).

The human concentrative nucleoside transporter, CNT3 (SLC28A3), plays an important role in mediating the cellular entry of a broad array of physiological nucleosides and synthetic anticancer nucleoside analog drugs. As a first step toward understanding the genetic basis for interindividual differences in the disposition and response to antileukemic nucleoside analogs, we examined the genetic and functional diversity of CNT3. In all, 56 variable sites in the exons and flanking intronic region of SLC28A3 were identified in a collection of 270 DNA samples from US populations (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). Of the 16 coding region variants, 12 had not been previously reported. Also, 10 resulted in amino-acid changes and three of these had total allele frequencies of >/=1%. Nucleotide diversity (pi) at nonsynonymous and synonymous sites was estimated to be 1.81 x 10(4) and 18.13 x 10(4), respectively, suggesting that SLC28A3 is under negative selection. All nonsynonymous variants, constructed by site-directed mutagenesis and expressed in Xenopus laevis oocytes, transported purine and pyrimidine model substrates, except for c. 1099G>A (p. Gly367Arg). This rare variant alters an evolutionarily conserved site in the putative substrate recognition domain of CNT3. The presence of three additional evolutionarily conserved glycine residues in the vicinity of p. Gly367Arg that are also conserved in human paralogs suggest that these glycine residues are critical in the function of the concentrative nucleoside transporter family. The genetic analysis and functional characterization of CNT3 variants suggest that this transporter does not tolerate nonsynonymous changes and is important for human fitness.

Adenosine↗
PubMed2001

Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3.

Organic anion transporters (OATs) and organic cation transporters (OCTs) mediate the flux of xenobiotics across the plasma membranes of epithelia. Substrates of OATs generally carry negative charge(s) whereas substrates of OCTs are cations. The goal of this study was to determine the domains and amino acid residues essential for recognition and transport of organic anions by the rat organic anion transporter, rOAT3. An rOAT3/rOCT1 chimera containing transmembrane domains 1-5 of rOAT3 and 6-12 of rOCT1 retained the specificity of rOCT1, suggesting that residues involved in substrate recognition reside within the carboxyl-terminal half of these transporters. Mutagenesis of a conserved basic amino acid residue, arginine 454 to aspartic acid (R454D), revealed that this amino acid is required for organic anion transport. The uptakes of p-aminohippurate (PAH), estrone sulfate, and ochratoxin A were approximately 10-, approximately 48-, and approximately 32-fold enhanced in oocytes expressing rOAT3 and were only approximately 2-, approximately 6-, and approximately 5-fold enhanced for R454D. Similarly, mutagenesis of the conserved lysine 370 to alanine (K370A) suggested that K370 is important for organic anion transport. Interestingly, the charge specificity of the double mutant, R454DK370A, was reversed in comparison to rOAT3-R454DK370A preferentially transported the organic cation, MPP(+), in comparison to PAH (MPP(+) uptake/PAH uptake = 3.21 for the double mutant vs 0.037 for rOAT3). These data indicate that arginine 454 and lysine 370 are essential for the anion specificity of rOAT3. The studies provide the first insights into the molecular determinants that are critical for recognition and translocation of organic anions by a member of the organic anion transporter family.

1-Methyl-4-phenylpyridinium↗
PubMed2001

Transporters involved in the elimination of drugs in the kidney: organic anion transporters and organic cation transporters.

Transporters in the kidney mediate the secretion or reabsorption of many compounds and thereby influence the plasma levels of their substrates. Organic anion transporters and organic cation transporters are two major classes of secretory transporters in the mammalian kidney. During the past decade, significant progress has been made in the cloning, functional expression, and initial characterization of these transporters. To date, five organic cation transporters and nine organic anion transporters have been cloned. In this review, we summarize the available data on organic anion and organic cation transporters, focusing in particular on their molecular characteristics, tissue distribution, and inhibitor and substrate selectivities. Currently we have a good understanding of the inhibitor selectivities for most of these transporters, and with the development of more robust assays, we will soon have a better understanding of their substrate selectivities. Based on the available data, summarized in this review, it appears that many compounds interact with multiple transporters. Furthermore, there appears to be substantial overlap in the selectivities of organic cation transporters, and the same appears true for organic anion transporters. At the present time, it is unclear what the roles of these multiple transporters are in renal drug elimination. With the development of new assays, reagents, and experimental methods, we will soon have a better understanding of the roles of each transporter isoform in the renal elimination of drugs.

Animals↗
PubMed2001

Screening a large reference sample to identify very low frequency sequence variants: comparisons between two genes.

Most human sequence variation is in the form of single-nucleotide polymorphisms (SNPs). It has been proposed that coding-region SNPs (cSNPs) be used for direct association studies to determine the genetic basis of complex traits. The success of such studies depends on the frequency of disease-associated alleles, and their distribution in different ethnic populations. If disease-associated alleles are frequent in most populations, then direct genotyping of candidate variants could show robust associations in manageable study samples. This approach is less feasible if the genetic risk from a given candidate gene is due to many infrequent alleles. Previous studies of several genes demonstrated that most variants are relatively infrequent (<0.05). These surveys genotyped small samples (n<75) and thus had limited ability to identify rare alleles. Here we evaluate the prevalence and distribution of such rare alleles by genotyping an ethnically diverse reference sample that is more than six times larger than those used in previous studies (n=450). We screened for variants in the complete coding sequence and intron-exon junctions of two candidate genes for neuropsychiatric phenotypes: SLC6A4, encoding the serotonin transporter; and SLC18A2, encoding the vesicular monoamine transporter. Both genes have unique roles in neuronal transmission, and variants in either gene might be associated with neurobehavioral phenotypes.

Alleles↗
PubMed2001

A novel proton-dependent nucleoside transporter, CeCNT3, from Caenorhabditis elegans.

In this study, we describe the cloning and characterization of a proton-dependent, broadly selective nucleoside transporter from Caenorhabditis elegans. Recently, we constructed a broadly selective nucleoside transporter which accepts both purine and pyrimidine nucleosides. Based on these studies, we hypothesized that CNTs with novel substrate selectivities exist in nature and that a CNT homolog in the C. elegans genomic database may function as a broadly selective nucleoside transporter. We cloned the cDNA for this transporter, termed CeCNT3 because of its broad selectivity, using polymerase chain reaction-based methods. CeCNT3 is predicted to have 575 amino acid residues (63.4 kDa) with 11 to 14 putative transmembrane domains and exhibits approximately 30% identity to members of the mammalian CNT family. This transporter exhibits a novel substrate selectivity, transporting a wide range of purine and pyrimidine nucleosides (inosine, guanosine, adenosine, uridine, and thymidine) but not cytidine. The apparent Km values for inosine and thymidine are 15.2 +/- 5.3 microM and 11.0 +/- 2.4 microM, respectively. Kinetic studies demonstrate that purine and pyrimidine nucleosides share a common recognition site in the transporter. In contrast to all known members of the mammalian CNT family, CeCNT3-mediated transport of nucleosides is proton-, but not sodium-, dependent. Mutation of tyrosine 332 in CeCNT3 decreased both the maximum uptake rate and apparent Km of thymidine, suggesting that this residue is in the domain of nucleoside recognition and translocation. The broad nucleoside specificity of CeCNT3 may be explained by this and other residues that restrict purine and pyrimidine nucleoside uptake and that discriminate among pyrimidine nucleosides.

Amino Acid Sequence↗
PubMed2001

Localization of GFP-tagged concentrative nucleoside transporters in a renal polarized epithelial cell line.

Many nucleosides undergo active reabsorption within the kidney, probably via nucleoside transporters. To date, two concentrative nucleoside transporters have been cloned, the sodium-dependent purine-selective nucleoside transporter (SPNT) and concentrative nucleoside transporter 1 (CNT1). We report the stable expression of green fluorescence protein (GFP)-tagged SPNT and CNT1 in Madin-Darby canine kidney (MDCK) cells, a polarized renal epithelial line. We demonstrate that the GFP tag does not alter the substrate selectivity and only modestly affects the kinetic activity of the transporters. By using confocal microscopy and functional studies, both SPNT and CNT1 are localized primarily to the apical membrane of MDCK and LLC-PK(1) cells. Apical localization of these transporters suggests a role in renal nucleoside reabsorption and regulation of tubular function via the adenosine pathway.

Animals↗
PubMed2001

Functional characteristics and steroid hormone-mediated regulation of an organic cation transporter in Madin-Darby canine kidney cells.

Organic cation transporters (OCT1-3) play an important role in renal elimination of many drugs. The goals of this study were to 1) identify a cell culture model which constitutively expressed OCT2 that could be used to study the characteristics and regulation of this transporter, and 2) to study the mechanisms by which xenobiotics and hormones regulate the activity of OCT2. We characterized the endogenous organic cation transporter (OCT) activity in Madin-Darby canine kidney (MDCK) cells. The activity was localized to the basolateral membrane and was pH and membrane potential-dependent. The uptake of the model organic cation, tetraethylammonium, was saturable (Km, 19.5 +/- 4.6 microM; Vmax, 350 +/- 19.4 pmol/mg of protein/10 min) and was inhibited by known OCT inhibitors (e.g., cimetidine and quinidine). A cDNA fragment (711 base pairs) isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) was greater than 83% identical to OCT2 cDNAs from mammalian species; no OCT1 or OCT3 was detected by RT-PCR, suggesting that OCT2 may be the primary basolateral OCT in MDCK. OCT2 mRNA levels were increased significantly following exposure of MDCK to the steroid hormones, dexamethasone (2.0-fold), hydrocortisone (2.4-fold), and testosterone (1.8-fold) with comparable increases in activity. Other compounds tested, including the cytochrome P450 inducers, rifampicin, phenobarbital, and phenytoin, and the OCT substrates, verapamil and metformin, had no inducing effects. Collectively, these data indicate that MDCK can serve as a useful and convenient tool in screening candidate drugs for interaction with OCT2 and for studying the regulation of this transporter. Furthermore, our data demonstrate that steroid hormones induce the transcription of OCT2 in the kidney.

Animals↗
PubMed2000

Molecular cloning of a Na+-dependent nucleoside transporter from rabbit intestine.

PURPOSE: Substantial species differences in the transport kinetics of nucleosides and therapeutic nucleoside analogs have been observed in various experimental systems. To explain these differences at a molecular level, it is necessary to clone the relevant transporters and examine their functional characteristics in heterologous expression systems. The goal of the present study was to clone the nucleoside transporters present in rabbit, an important preclinical animal model, and to functionally characterize the clone(s). METHODS: A Polymerase Chain Reaction (PCR)-based homology cloning approach in conjunction with Rapid Amplification of cDNA Ends (RACE) was used to isolate a full-length cDNA. Characterization of this transporter was accomplished through heterologous expression in Xenopus laevis oocytes. RESULTS: A full-length cDNA encoding a purine-selective, Na+-dependent nucleoside transporter, rbSPNT1, was isolated from rabbit small intestine. The encoded protein is 658 amino acid residues in length. Hydropathy analysis suggests that rbSPNT1 has 11 to 14 transmembrane domains. In Xenopus laevis oocytes expressing rbSPNT1, the uptake of uridine and inosine was enhanced significantly; uridine transport was inhibited by purine, but not pyrimidine nucleosides. mRNA transcripts for rbSPNT1 were detected primarily in intestine, lung, and kidney and at lower levels in liver, brain, and heart. CONCLUSIONS: A full-length functional nucleoside transporter was cloned. Sequence analysis and functional characterization suggest that rbSPNT1 is the rabbit homolog of the purine-selective nucleoside transporter, N1. The cloned rbSPNT1 can be used to understand the molecular mechanisms responsible for the observed species differences in the transport of nucleosides and therapeutic nucleoside analogs.

Amino Acid Sequence↗
PubMed2000

Interactions of HIV protease inhibitors with a human organic cation transporter in a mammalian expression system.

Recently, we cloned a human organic cation transporter, hOCT1, which is expressed primarily in the liver. hOCT1 plays an important role in the cellular uptake and elimination of various xenobiotics including therapeutically important drugs. HIV protease inhibitors are a new class of therapeutic agents. The purpose of this study was to elucidate the interactions of HIV protease inhibitors with hOCT1 and to determine whether hOCT1 is involved in the elimination of these compounds. We studied the interactions of HIV protease inhibitors with hOCT1 in a transiently transfected human cell line, HeLa. Uptake studies were carried out 40 h post-transfection using the radiolabeled model organic cation, [(14)C]tetraethylammonium (TEA), under different experimental conditions. In cis-inhibition studies, all of the HIV protease inhibitors tested, i.e., indinavir (IC(50) of 62 microM), nelfinavir (IC(50) of 22 microM), ritonavir (IC(50) of 5.2 microM), and saquinavir (IC(50) of 8.3 microM) inhibited TEA uptake in HeLa cells expressing hOCT1. However, none of the HIV protease inhibitors trans-stimulated [(14)C]TEA uptake, suggesting that they are poorly translocated by hOCT1. Nelfinavir, ritonavir, and saquinavir demonstrated an apparent "trans-inhibition" effect. No enhanced uptake of [(14)C]saquinavir was observed in hOCT1 DNA-transfected cells versus empty vector-transfected cells. These data suggest that HIV protease inhibitors are potent inhibitors, but poor substrates, of hOCT1. Some HIV protease inhibitors may potently inhibit the uptake and elimination of cationic drugs that are substrates for hOCT1, leading to potential drug-drug interactions. Other transporters, e.g., MDR1 and MRP1, in HIV-targeted cells may control the intracellular concentrations of HIV protease inhibitors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Kinetic and selectivity differences between rodent, rabbit, and human organic cation transporters (OCT1).

Organic cation transporters play an important role in the absorption, distribution, and elimination of clinical agents, toxic substances, and endogenous compounds. In kidney preparations, significant differences in functional characteristics of organic cation transport between various species have been reported. However, the underlying molecular mechanisms responsible for these interspecies differences are not known. The goal of this study was to determine the kinetics and substrate selectivities of organic cation transporter (OCT1) homologs from mouse, rat, rabbit, and human that may contribute to interspecies differences in the renal and hepatic handling of organic cations. With a series of n-tetraalkylammonium (nTAA) compounds, a correlation between increasing alkyl chain length and affinity for the four OCT1 homologs was observed. However, the apparent affinity constants (K(i)) differed among the species homologs. For the mouse homolog mOCT1, apparent K(i) values ranged from 7 microM for tetrabutylammonium to 2000 microM for tetramethylammonium. In contrast, the human homolog hOCT1 exhibited weaker interactions with the nTAA compounds. Trans-stimulation studies and current measurements in voltage-clamped oocytes demonstrated that larger nTAA compounds were transported at greater rates in oocytes expressing hOCT1, whereas smaller nTAAs were transported at greater rates in oocytes expressing mOCT1 or rOCT1. The rabbit homolog rbOCT1 exhibited intermediate properties in its interactions with nTAAs compared with its rodent and human counterparts. This report demonstrates that the human OCT1 homolog has functional properties distinct from those of the rodent and rabbit OCT1 homologs. The study underscores potential difficulties in extrapolating data from preclinical studies in animal models to humans.

1-Methyl-4-phenylpyridinium↗
PubMed2000

Electrophysiological analysis of the substrate selectivity of a sodium-coupled nucleoside transporter (rCNT1) expressed in Xenopus laevis oocytes.

Nucleoside transporters that mediate cellular uptake of therapeutic nucleoside analogs are major determinants of the pharmacokinetic properties of these compounds. Understanding the substrate selectivity of these transporters is critical in the development of therapeutic nucleoside analogs with optimal pharmacokinetic properties, including high oral bioavailability and tissue-specific distribution. In general, substrate selectivity of nucleoside transporters has been evaluated indirectly by inhibition studies. The purpose of this study was to directly measure the transport of nucleoside analogs by the sodium-coupled pyrimidine-selective transporter rCNT1 using electrophysiology methods. We used a two-electrode voltage clamp assay to investigate the substrate selectivity of rCNT1; 19 structurally diverse nucleosides and nucleoside analogs were studied. Uridine-induced currents in voltage-clamped oocytes expressing rCNT1 were sodium-, voltage-, and concentration-dependent (K(0.5) = 21 microM), and were blocked by adenosine. Uridine-induced currents increased approximately 5-fold upon hyperpolarization of membrane potential from -10 to -150 mV. Uridine, thymidine, and cytidine induced currents in rCNT1-expressing oocytes, whereas guanosine, inosine, and adenosine did not. Uridine, deoxyuridine, and cytidine analogs with modifications at the 3-, 4-, or 5-position were found to be substrates of rCNT1, whereas uridine and cytidine analogs modified at the 6-position were not. In addition, it was found that the 5'-hydroxyl group of the sugar is not required for transport by rCNT1. These results enhance our understanding of the structural basis for substrate selectivity of nucleoside transporters and should prove useful in the development of therapeutic nucleoside analogs.

Animals↗
PubMed1999

Serine 318 is essential for the pyrimidine selectivity of the N2 Na+-nucleoside transporter.

Molecular cloning has isolated two subtypes of Na+-nucleoside transporters; one is pyrimidine-selective (N2), and the other is purine-selective (N1). Using chimeric rat N2/N1 transporters, we previously demonstrated that transmembrane domains (TM) 8 and 9 are the major sites for substrate binding and discrimination. Interestingly, when TM8 of N2 was replaced by that of N1, the resulting chimera, T8, lost the pyrimidine selectivity of N2 and accepted both purine and pyrimidine nucleosides. Five residues differ between rat N2 and N1 in TM8. To identify the critical residues responsible for transport selectivity, the five residues in N2 were systematically changed to their equivalents in N1. Replacing the serine residue at position 318 to its equivalent N1 residue, glycine, caused N2 to lose its selectivity for pyrimidine nucleosides and accept purine nucleosides as substrates. In contrast, replacing the other four residues did not change the pyrimidine selectivity of N2. Furthermore, when glycine 318 in chimera T8 was changed back to serine, the chimeric transporter regained pyrimidine selectivity. These observations suggest that serine 318 is located in the nucleoside permeation pathway and is responsible for the substrate selectivity of N2. An adjacent residue, glutamine 319, was found to be important in modulating the apparent affinity for nucleosides.

Amino Acid Sequence↗
PubMed1999

Characterization of a bioengineered chimeric Na+-nucleoside transporter.

Na+-dependent nucleoside transporters mediate the intracellular uptake of purine and pyrimidine nucleosides. The N1, N2, and N3 Na+-nucleoside transporters differ in substrate selectivity. N1 is purine-selective, N2 is pyrimidine-selective, and N3 is broadly selective. Recently, we created a chimeric transporter, T8, from the cloned rat N1 and N2 transporters. Whereas most chimeric proteins exhibit the characteristics of one of the two parent proteins, limited studies suggested that T8 possesses either a combined substrate selectivity of N1 and N2 or the selectivity of N3. The purpose of this study was to determine the substrate profile, transport mechanisms, and Na+-coupling stoichiometry of T8 and to compare these measurements with those of wild-type N1, N2, and N3. In Xenopus laevis oocytes expressing T8, Na+-dependent uptake of 3H-labeled purine (adenosine, inosine, and guanosine) and pyrimidine nucleosides (uridine, thymidine, and cytidine) was significantly enhanced (3.5-18.6-fold), which suggests that T8 accepts both purine and pyrimidine nucleosides as permeants. T8-mediated uptake of [3H]thymidine was competitively inhibited by inosine, and T8-mediated uptake of [3H]inosine was competitively inhibited by thymidine, which suggests that purine and pyrimidine nucleosides share a common binding site. Base-modified ribo- and 2'-deoxyribonucleosides were potent inhibitors of T8. In contrast, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and 3'-azidothymidine, which are known inhibitors of N1 or N2, did not inhibit T8-mediated uptake. These data suggest that the substrate profile of T8 is not a combination of those of N1 and N2; rather, it is similar to that of N3. However, the Na+/nucleoside stoichiometric ratio of T8 was determined to be 1, consistent with both N1 and N2 but different from N3.

Animals↗
PubMed1999

The interaction of n-tetraalkylammonium compounds with a human organic cation transporter, hOCT1.

Polyspecific organic cation transporters in epithelia play an important role in the elimination of many endogenous bioactive amines and therapeutically important drugs. Recently, the first human organic cation transporter (hOCT1) was cloned from liver. The purpose of the current study was to determine the effect of molecular size and hydrophobicity on the transport of organic cations by hOCT1. We studied the interaction of a series of n-tetraalkylammonium (n-TAA) compounds (alkyl chain length, N, ranging from 1 to 6 carbons) with hOCT1 in a transiently transfected human cell line, HeLa. [14C]tetraethylammonium (TEA) uptake was measured under different experimental conditions. Both cis-inhibition and trans-stimulation studies were carried out. With the exception of tetramethylammonium, all of the n-TAAs significantly inhibited [14C]TEA uptake. A reversed correlation of IC50 values (range, 3.0-260 microM) with alkyl chain lengths or partition coefficients (LogP) was observed. trans-Stimulation studies revealed that TEA, tetrapropylammonium, tetrabutylammonium, as well as tributylmethylammonium trans-stimulated TEA uptake mediated by hOCT1. In contrast, tetramethylammonium and tetrapentylammonium did not trans-stimulate [14C]TEA uptake, and tetrahexylammonium demonstrated an apparent "trans-inhibition" effect. These data indicate that with increasing alkyl chain lengths (N >/= 2), n-TAA compounds are more poorly translocated by hOCT1 although their potency of inhibition increases. Similar findings were obtained with nonaliphatic hydrocarbons. These data suggest that a balance between hydrophobic and hydrophilic properties is necessary for binding and subsequent translocation by hOCT1.

Biological Transport↗
PubMed1999

Functional characterization of a human purine-selective, Na+-dependent nucleoside transporter (hSPNT1) in a mammalian expression system.

Nucleosides and nucleoside analogs are actively transported in the human kidney. With the recent cloning of a purine-selective, Na+-dependent, nucleoside transporter (hSPNT1, also termed hCNT2) from human kidney, it is now possible to study the interaction of nucleosides and nucleoside analogs with this transport protein and gain a more detailed knowledge of the underlying mechanisms of nucleoside transport in the human kidney. In this study we examined the substrate selectivity of hSPNT1 for nucleosides and nucleoside analogs. We determined that the naturally occurring nucleosides adenosine, inosine, and uridine are substrates for this carrier, whereas thymidine is not. The nucleoside analogs (0.5 mM) 2', 3'-dideoxyadenosine; 2',3'-dideoxyinosine; and 2-chloro-2'deoxyadenosine (2CdA), significantly inhibited the uptake of [3H]inosine in HeLa cells transiently transfected with hSPNT1. However, there was no significant Na+-dependent uptake of [3H]2', 3'-dideoxyinosine or [3H]2CdA in the transfected cells, suggesting that these nucleoside analogs are not permeants of hSPNT1. Interestingly, 2CdA was considerably less potent in inhibiting [3H]inosine uptake in HeLa cells expressing hSPNT1 than in cells expressing the rat homolog rSPNT (IC50 = 371 microM versus 13.8 microM), suggesting that there may be notable species differences in the kinetic interactions of some nucleoside analogs with purine- selective nucleoside transporters.

Animals↗
PubMed1998

Molecular cloning and functional expression of a rabbit renal organic cation transporter.

A cDNA encoding an organic cation transporter (rbOCT1) was isolated from rabbit kidney. The cDNA encodes a 554 amino acid protein that is highly homologous to other mammalian organic cation transporters. rbOCT1 mediated 3H-1-methyl-4-phenylpyridinium (3H-MPP+) transport in Xenopus laevis oocytes was saturable, sensitive to membrane potential, and inhibited by various organic cations. rbOCT1 mRNA transcripts are expressed in the kidney, liver, and intestine.

Amino Acid Sequence↗
PubMed1998

Nicotine transport in a human choriocarcinoma cell line (JAR).

Smoking is a major health problem in pregnancy resulting in intrauterine growth retardation and birth complications. Nicotine, a toxic component of cigarette smoke, interferes with amino acid transport in the placenta and stimulates catecholamine release resulting in uteroplacental vasoconstriction. Transplacental transport of nicotine may be an important determinant of placental and fetal exposure. Our aim was to determine the mechanism of nicotine transport in the human choriocarcinoma cell line, JAR, as a model for the placenta. JAR cells were subcultured in 12-well plates following trypsinization at a seeding density of 0.5 x 10(6) cells/well (1.3 x 10(5) cells/cm2). Uptake studies of [3H]nicotine were carried out in JAR cell monolayers on day 2 after plating. [3H]Nicotine uptake was saturable (Km 156 microM), sensitive to temperature, and inhibited by unlabeled nicotine and various organic cations including mecamylamine and quinidine, but not by guanidine, tetraethylammonium (TEA), or neurotransmitters. Counterflux of [3H]nicotine uptake was produced by unlabeled nicotine and mecamylamine but not by cotinine or acetylcholine, consistent with a carrier-mediated transport process. The uptake could be driven by an inside-negative membrane potential or by an outwardly directed pH gradient. This is the first demonstration of a carrier-mediated transport mechanism for nicotine in a human cell line. This transport mechanism may have implications to the disposition of nicotine in the human placenta.

Biological Transport↗