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LLC-PK₁ cells stably expressing the human norepinephrine transporter:: A functional model of carrier-mediated norepinephrine release in protracted myocardial ischemia

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 1999年第2期291卷 456-463页

作者： Smith, NCE Levi, R Cornell Univ Weill Med Coll Dept Pharmacol New York NY 10021 USA

In myocardial ischemia, adrenergic terminals undergo ATP depletion, hypoxia, and intracellular pH reduction, causing the accumulation of axoplasmic norepinephrine (NE) and intracellular Na+ [via the Na+-H+ exchanger (NHE)]. This forces the reversal of the Na+- and Cl--dependent NE transporter (NET), triggering massive carrier-mediated NE release and, thus, arrhythmias. We have now developed a cellular model of carrier-mediated NE release using an LLC-PK1 cell line stably transfected with human NET cDNA (LLC-NET). LLC-NET cells transported [H-3]NE and [H-3]N-methyl-4-phenylpyridinium ([H-3]MPP+) in an inward direction. This uptake was abolished by the NET inhibitors desipramine (100 nM) and mazindol (300 nM) and by extracellular Na+ removal. Na+-gradient reversal induced an efflux of H-3-substrate from preloaded LLC-NET cells. Desipramine and mazindol blocked this efflux. Because of its greater intracellular stability and higher sensitivity to Na+ gradient reversal, [H-3]MPP+ proved preferable to [H-3]NE as an NET substrate;therefore, only [H-3]MPP+ was used for subsequent studies. The K+/H+ ionophore nigericin (10 mu M) evoked a large efflux of [H-3]MPP+. This efflux was potentiated by the Na+,K+-ATPase inhibitor ouabain (100 mu M), was sensitive to desipramine, and was blocked by the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA;10 mu M). In contrast, EIPA failed to inhibit the [H-3]MPP+ efflux elicited by the Na+ ionophore gramicidin (10 mu M). Furthermore, [H-3]MPP+ efflux induced by the NHE-stimulant proprionate (25 mM) was negatively modulated by imidazoline receptor activation. Our findings suggest that LLC- NET cells are a sensitive model for studying transductional processes of carrier-mediated NE release associated with myocardial ischemia.

关键词： 1-甲基-4-苯基吡啶/代谢阿米洛利/类似物和衍生物阿米洛利/药理学生物转运主动地昔帕明/药理学药物相互作用短杆菌肽/药理学 LLC-PK1细胞马吲哚/药理学模型生物学心肌缺血/代谢尼日利亚菌素/药理学去甲肾上腺素/分泌受体药物/代谢钠/生理学猪时间因素动物人类

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Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1 (hOCT1/SLC22A1)

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ANNALS OF HUMAN GENETICS 1999年第6期63卷 473-482页

作者： Hayer, M Bönisch, H Brüss, M Univ Bonn Inst Pharmacol & Toxicol D-53113 Bonn Germany

In this study we report the cloning of four human OCT1 (hOCT1/SLC22A1) isoforms: a long form, hOCT1G/L554, and three shorter forms (hOCT1G/L506, hOCT1G483 and hOCT1G353). All four variants could be identified in the human glioma cell line SK-MG-1, whereas only two isoforms (hOCT1G/L554 and hOCT1G/L506) were found in human liver cDNA. The llOCT1G/L554 represents the full length hOCT1 since the sequence of this clone is more than 99% identical to previously cloned hOCT1 cDNAs. Elucidation of the gene structure of human OCT1 demonstrated that the other isolated isoforms are alternatively spliced variants. The hOCT1 gene consists of 7 exons and 6 introns. When stably expressed in human embryonic kidney (HEK293) cells, only the full length hOCT1 cDNA mediated decynium-22 (D22)-sensitive uptake of tritiated 1-methyl-4-phenylpgridinium ([H-3]-MPP+).

关键词： 1-甲基-4-苯基吡啶/代谢选择性剪接氨基酸序列碱基序列载体蛋白质类/遗传学载体蛋白质类/代谢细胞系克隆分子 DNA引物/遗传学 DNA 互补/遗传学外显子基因表达内含子离子转运膜蛋白质类/遗传学膜蛋白质类/代谢分子序列数据有机阳离子转运子1 蛋白质亚型/遗传学蛋白质亚型/代谢序列同源性氨基酸转染人类

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Transporter-mediated release: A superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 2000年第3期293卷 870-878页

作者： Scholze, P Zwach, J Kattinger, A Pifl, C Singer, EA Sitte, HH Univ Vienna Dept Pharmacol A-1090 Vienna Austria Univ Vienna Brain Res Inst A-1090 Vienna Austria

HEK 293 cells stably expressing the human serotonin transporter (hSERT) were grown on coverslips, preincubated with [H-3]5-hydroxytryptamine (5-HT), and superfused. Substrates of the hSERT [e.g., p-chloroamphetamine (PCA)], increased the basal efflux of [H-3]5-HT in a concentration-dependent manner. 5-HT reuptake blockers (e.g., imipramine, paroxetine) also raised [H-3]5-HT efflux, reaching approximately one-third of the maximal effect of the hSERT substrates. In uptake experiments, both groups of substances inhibited [H-3]5-HT uptake. Using the low-affinity substrate [H-3]N-methyl-4-phenylpyridinium (MPP+) to label the cells in superfusion experiments, reuptake inhibitors failed to enhance efflux. Similar results were obtained using human placental choriocarcinoma (JAR) cells that constitutively express the hSERT at a low level. By contrast, PCA raised [H-3]MPP+ efflux in both types of cells, and its effect was inhibited by paroxetine. The addition of the Na+,K+-ATPase inhibitor ouabain (100 mu M) to the superfusion buffer enhanced basal efflux of [H-3]5-HT-loaded hSERT cells by approximately 2-fold;the effect of PCA (10 mu M) was strongly augmented by ouabain, whereas the effect of imipramine was not. The Na+/H+ ionophore monensin (10 mu M) also augmented the effect of PCA on efflux of [H-3]5-HT as well as on efflux of [H-3]MPP+. In [H-3]5-HT-labeled cells, the combination of imipramine and monensin raised [H-3]5-HT efflux to a greater extent than either of the two substances alone. In [H-3]MPP+-labeled cells, imipramine had no effect on its own and fully reversed the effect of monensin. The results suggest that the [H-3]5-HT efflux caused by uptake inhibitors is entirely due to interrupted high-affinity reuptake, which is ongoing even under superfusion conditions.

关键词： 1-甲基-4-苯基吡啶/代谢载体蛋白质类/生理学细胞系剂量效应关系药物芬氟拉明/药理学肾/代谢膜糖蛋白类/生理学膜转运蛋白质类莫能星/药理学单胺氧化酶抑制剂/药理学神经组织蛋白质类血清素/分泌血清素质膜转运蛋白质类血清素摄取抑制药/药理学转染 p-氯苯丙胺/药理学人类

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In vitro studies of striatal vesicles containing the vesicular monoamine transporter (VMAT2): Rat versus mouse differences in sequestration of 1-methyl-4-phenylpyridinium

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 2000年第2期293卷 329-335页

作者： Staal, RGW Hogan, KA Liang, CL German, DC Sonsalla, PK Univ Med & Dent New Jersey Robert Wood Johnson Med Sch Dept Neurol Piscataway NJ 08854 USA Univ Texas SW Med Ctr Dept Psychiat Dallas TX USA

Significant differences exist in the sensitivity of mice and rats to the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that cannot be explained by differences in exposure to or uptake of 1-methyl-4-phenylpyridinium (MPP+) into dopamine (DA) neurons. MPP+ is also a substrate for the brain vesicular monoamine transporter (VMAT2), and sequestration into synaptic vesicles may be one mechanism of protection against MPP+ toxicity. A greater sequestration of MPP+ into vesicles of DA neurons in rats versus mice could explain the lower vulnerability of DA neurons in the rat to MPP+ toxicity. To test this hypothesis, the kinetics of uptake for [H-3]MPP+ and [H-3]DA as well as [H-3]dihydrotetrabenazine binding to VMAT2 were compared in vesicles isolated from the striata of rats and mice. The K-m value of [H-3]MPP+ transport was similar in the two species. In contrast, the maximal transport rate (V-max) was 2-fold greater in vesicles from rats than in those from mice. Likewise, the K-m value for [H-3]DA transport was similar in both preparations, but the V-max value was 2-fold greater in rat than in mouse vesicles. The B-max value for [H-3]dihydrotetrabenazine binding was also 2-fold greater in striatal vesicles from rats than in those from mice. Electron micrographs demonstrated that vesicles isolated from rats and mice were approximately the same size. Based on these observations, we propose that striatal vesicles from rats have more VMAT2 than vesicles from mice and that this species difference in VMAT2 density may help explain the reduced vulnerability of rat DA neurons to MPP+ neurotoxicity.

关键词： 1-甲基-4-苯基吡啶/代谢可卡因/类似物和衍生物可卡因/代谢多巴胺药/代谢多巴胺摄取抑制剂/代谢指示剂和试剂动力学膜糖蛋白类/代谢膜转运蛋白质类显微镜检查电子新纹状体/药物作用新纹状体/代谢新纹状体/超微结构神经肽类神经毒素类/代谢神经递质药/代谢大鼠 Sprague-Dawley 种特异性突触小泡/药物作用突触小泡/代谢突触小泡/超微结构囊泡生物胺转运蛋白质类囊泡单胺转运蛋白质类动物男(雄)性小鼠大鼠

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

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 2000年第3期292卷 1146-1152页

作者： Dresser, MJ Gray, AT Giacomini, KM Univ Calif San Francisco Dept Biopharmaceut Sci San Francisco CA 94143 USA Univ Calif San Francisco Dept Anesthesia San Francisco CA 94143 USA

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 mu M for tetrabutylammonium to 2000 mM 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.

关键词： 1-甲基-4-苯基吡啶/代谢氨基酸序列载体蛋白质类/化学载体蛋白质类/生理学动力学膜蛋白质类/化学膜蛋白质类/生理学分子序列数据有机阳离子转运子1 种特异性底物特异性四乙基铵化合物/药理学爪蟾光滑动物女(雌)性人类小鼠兔大鼠

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