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1.
Many organic cations are transported across the apical membrane of the proximal tubule by specific saturable mechanisms. The goal of this study was to determine if the transporter for tetraethylammonium (TEA) in the brush border membrane of an established opossum kidney (OK) cell line is glycosylated and to elucidate the function of this glycosylation. The uptake of TEA was determined in OK cell monolayers treated with tunicamycin (TM), a compound that prevents synthesis of the core oligosaccharide precursor molecules. TM exposure significantly decreased the incorporation of [3H]mannose in OK cell proteins and significantly reduced TEA uptake in a time and a concentration dependent manner. No effect of TM exposure on cellular protein synthesis, DNA content, cell viability, or on [3H]proline uptake was observed. The transport of TEA in control cells was characterized by a Km of 26.9 +/- 16.4 microM and a Vmax of 378 +/- 39 pmol/mg of protein/min. TM treatment (1 microgram/ml for 21 h) significantly increased the Km by over 4-fold to 111.5 +/- 18.4 microM while not affecting the Vmax. The apparent KI values of other organic cations known to interact with this transport system were also significantly increased by TM exposure. Estimated KI values of N1-methylnicotinamide, cimetidine, and mepiperphenidol increased by 6-fold, 4-fold, and 2-fold, respectively, after exposure of OK cells to TM. An increased KI for protons was also observed. Additional inhibitors of the N-linked glycosylation pathway, castanospermine, deoxynojirimycin, and deoxymannojirimycin significantly decreased TEA transport, whereas swainsonine had no effect. Our results suggest that the organic cation transporter is glycosylated. The N-linked oligosaccharide side chain appears to be of the hybrid type, and it either directly or indirectly affects the binding site of the transporter for both organic cations and protons. This is the first report describing the importance of glycosylation in the function of the organic cation transporter in the apical membrane of OK cells. 相似文献
2.
Rheault MR Debicki DM O'Donnell MJ 《American journal of physiology. Regulatory, integrative and comparative physiology》2005,289(2):R495-R504
Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules. 相似文献
3.
The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane. 相似文献
4.
We investigated the contribution of the Na(+)/L-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of L-carnitine was observed in the presence of a Na(+) gradient. The uptake of L-carnitine was of high affinity (K(m)=21 microM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for L-carnitine uptake. Therefore, we confirmed the Na(+)/L-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and L-carnitine uptake. The presence of an outward H(+) gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of L-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [14C]TEA uptake, but unlabeled L-carnitine did not stimulate [14C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na(+)/L-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H(+)/organic cation antiporter. 相似文献
5.
Shuichi Ohnishi 《生物化学与生物物理学报:生物膜》2003,1609(2):218-224
We investigated the contribution of the Na+/l-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of l-carnitine was observed in the presence of a Na+ gradient. The uptake of l-carnitine was of high affinity (Km=21 μM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for l-carnitine uptake. Therefore, we confirmed the Na+/l-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and l-carnitine uptake. The presence of an outward H+ gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of l-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [14C]TEA uptake, but unlabeled l-carnitine did not stimulate [14C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na+/l-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H+/organic cation antiporter. 相似文献
6.
Uptake by the liver of the organic cation and essential nutrient choline is required for the hepatic synthesis of phosphatidylcholine. Uptake of other organic cations is also important for the metabolism and secretion of numerous endobiotics and drugs. Although a high affinity mammalian hepatic choline transporter has been kinetically defined, it has not been previously identified. We have developed stable transfectants of BALB/3T3 cells, using a murine member of the organic cation transporter gene family (mOct1/Slc22a1), and used these cells to characterize the transport of the organic cation choline and model organic cation tetraethylammonium (TEA). Functional expression of mOct1/Slc22a1 in BALB/3T3 cells confers the saturable, temperature-dependent uptake of choline with a K(m) of 42 micrometer, and uptake of TEA with a K(m) of 43 micrometer. We subsequently used our cell culture uptake system to kinetically define in HepG2 cells a high affinity choline uptake process, which transports choline with a K(m) similar to that of mOct1/Slc22a1 protein. We also demonstrated that organic cation transport by mOct1/Slc22a1 is inhibited by several organic cations, and that the gene is expressed in the perinatal period, at a time when phosphatidylcholine synthesis increases.We conclude that mOct1/Slc22a1 encodes a high affinity mammalian hepatic choline/organic cation transporter. This transporter may be important for hepatic phosphatidylcholine synthesis, and for the metabolism and secretion of many organic cationic drugs. 相似文献
7.
S. K. Hong S. Ashwell-Erickson P. Gigliotti R. Elsner 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1982,149(1):19-24
Summary The present study was undertaken to examine the effects of anoxia and/or low pH on active organic ion transport and tissue water and electrolyte distribution in freshly prepared cortical slices from harbor seal and rat kidneys.p-Aminohippurate (PAH) and tetraethylammonium (TEA) were used as a representative organic anion and cation, respectively. The slice uptake of PAH was not influenced by the incubation medium pH over a range of 5.0–7.5 in the harbor seal while it was very sensitive to the medium pH in the rat, decreasing significantly with reductions in pH (Fig. 1). Although the TEA uptake decreased in anoxia in both species, it decreased considerably less in the harbor seal than in the rat (40% vs 74%) (Fig. 2). However, in contrast to the PAH transport system, the TEA uptake decreased significantly at low pH in both species. The magnitudes of increase in intracellular Na and of decrease in intracellular K in anoxia were consistently less in the harbor seal than in the rat, especially at low pH (Fig. 3). However, the pH dependence of Na–K-ATPase activity of the crude renal cortical homogenate was not different between the two species (Fig. 4). These results, together with the previous finding on the anoxic tolerance of the renal organic anion transport system, indicate that (1) the overall anoxic tolerance of active renal transport systems for both organic anions and cations is greater in the harbor seal than in the rat, and (2) the renal organic anion (but not cation) transport system is resistant to low pH in the harbor seal.Abbreviations
2,4-D
2,4-dichlorophenoxyacetate
-
KRP
Krebs-Ringer phosphate buffer
-
PAH
para-aminohippurate
-
S/M
slice to medium concentration ratio
-
TEA
tetraethylammonium 相似文献
8.
Properties and regulation of organic cation transport in freshly isolated human proximal tubules. 总被引:4,自引:0,他引:4
G Pietig T Mehrens J R Hirsch I Cetinkaya H Piechota E Schlatter 《The Journal of biological chemistry》2001,276(36):33741-33746
The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases. 相似文献
9.
Endo T 《Comparative biochemistry and physiology. Toxicology & pharmacology : CBP》2002,131(3):223-229
Cadmium (Cd) uptake and secretion across the apical membrane of epithelial cells was studied using LLC-PK1 cells cultured on Petri dishes and permeable membranes, respectively. Cd accumulation in cells from the apical medium was decreased by low temperature and metabolic inhibitors. A saturable tendency was observed between initial Cd accumulation and increased concentrations of Cd in the apical medium at 37 degrees C, but not at 4 degrees C. Co-incubation with ZnCl2 or CuCl2 competitively decreased Cd accumulation at 37 degrees C. A decrease in the pH of the apical medium markedly decreased Cd accumulation. Pretreatment of cells with an inorganic anion-exchange inhibitor significantly decreased Cd uptake at pH 7.4 in the presence of bicarbonate, but only marginally in its absence. A decrease in the pH of the apical medium increased the secretory (basolateral-to-apical) transport of Cd, with a concomitant decrease in the cellular accumulation of Cd. Co-incubation with Cd and tetraethylammonium, a typical substrate of the organic cation transporter, decreased Cd transport, with a concomitant increase in cellular Cd accumulation. The uptake and secretion of Cd across the apical membrane appear to be partly mediated via an inorganic anion exchanger and a H+ antiport of the organic cation transport system, respectively. Therefore, a decrease in pH of the apical medium markedly decreases Cd accumulation, possibly as a result of not only the decrease in Cd uptake via an inorganic anion exchanger, but also the increase in Cd secretion via the Cd2+/H+ antiport. Further evidence of the antiport was obtained from experiments using brush border membrane vesicles isolated from rat kidney and small intestine. In addition, passive diffusion of Cd appears to be decreased by low temperature and a decrease in pH. 相似文献
10.
Alkaline phosphatase (ALP) refers to a group of nonspecific phosphomonoesterases located primarily in cell plasma membrane. It has been described in different cell lines that ecto-ALP is directly or indirectly involved in the modulation of organic cation transport. We aimed to investigate, in Caco-2 cells, a putative modulation of 1-methyl-4-phenylpyridinium (MPP(+)) apical uptake by an ecto-ALP activity. Ecto-ALP activity and (3)H-MPP(+) uptake were evaluated in intact Caco-2 cells (human colon adenocarcinoma cell line), in the absence and presence of a series of drugs. The activity of membrane-bound ecto-ALP expressed on the apical surface of Caco-2 cells was studied at physiological pH using p-nitrophenylphosphate as substrate. The results showed that Caco-2 cells express ALP activity, characterized by an ecto-oriented active site functional at physiological pH. Genistein (250 micro M), 3-isobutyl-1-methylxanthine (1 mM), verapamil (100 micro M), and ascorbic acid (1 mM) significantly increased ecto-ALP activity and decreased (3)H-MPP(+) apical transport in this cell line. Orthovanadate (100 micro M) showed no effect on (3)H-MPP(+) transport and on ecto-ALP activity. On the other hand, okadaic acid (310 nM) and all trans-retinoic acid (1 micro M) significantly increased (3)H-MPP(+) uptake and inhibited ecto-ALP activity. There is a negative correlation between the effect of drugs upon ecto-ALP activity and (3)H-MPP(+) apical transport (r = -0.9; P = 0.0014). We suggest that apical uptake of organic cations in Caco-2 cells is affected by phosphorylation/dephosphorylation mechanisms, and that ecto-ALP activity may be involved in this process. 相似文献
11.
It is becoming increasingly evident that ingested products, such as wine, may have profound effects on the therapeutic efficacy of certain drugs. As various xeno- and endobiotics are organic cations, the purpose of our study was to examine the modulation of organic cations intestinal apical uptake by red (RW) and white wine (WW). For this purpose, we used RW, WW, the same alcohol-free wines, phenolic compounds and ethanol. The uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP+) was evaluated in Caco-2 cells, an intestinal epithelial cell model. RW and alcohol-free RW increased 3H-MPP+ apical uptake, although the effect of alcohol-free RW was less pronounced. On the other hand, WW and alcohol-free WW decreased the organic cation uptake but the effect of alcohol-free WW was more pronounced. Our results show that the total content in phenolic compounds was 7 times higher, and the dialysis index was about 4 times higher in RW compared to WW. Ethanol, in the same concentration found in wine, caused a significant decrease in 3H-MPP+ apical uptake. The solution containing high molecular weight compounds from dialyzed RW increased 3H-MPP+ apical uptake. In conclusion, the results suggest that RW may increase and WW may reduce the intestinal absorption of organic cations present in the diet, such as drugs or vitamins (e.g. thiamine and riboflavin). As ethanol alone decreased the uptake of MPP+, and alcohol-free RW and WW had a lower potency than intact wine upon the transport, the presence of ethanol is probably important for the solubilisation/bioavailability of the components endowed with the transport modulating activity. 相似文献
12.
The molecular mechanisms of organic cation transport by rat OCT2 was examined in the Xenopus oocyte expression system. When extracellular Na+ ions were replaced with K+ ions, uptake of tetraethylammonium (TEA) by OCT2-expressing oocytes was decreased, suggesting that TEA uptake by OCT2 is dependent on membrane potential. Kinetic analysis revealed that the decreased TEA uptake by ion substitution was caused at least in part by decreased substrate affinity. Acidification of extracellular buffer resulted in decreased uptake of TEA, whereas TEA efflux from OCT1- and OCT2-expressing oocytes was not stimulated by inward proton gradient, in consistent with basolateral organic cation transport in the kidney. Inhibition of TEA uptake by various organic cations revealed that apparent substrate spectrum of OCT2 was similar with that of OCT1. However, the affinity of procainamide to OCT1 was higher than that to OCT2. Uptake of 1-methyl-4-phenylpyridinium was stimulated by OCT2 as well as OCT1, but uptake of levofloxacin, a zwitterion, was not stimulated by both OCTs. These results suggest that OCT2 is a multispecific organic cation transporter with the characteristics comparable to those of the basolateral organic cation transporter in the kidney. 相似文献
13.
Functional analysis of rat renal organic anion transporter OAT-K1: bidirectional methotrexate transport in apical membrane. 总被引:1,自引:0,他引:1
Renal organic anion transporter OAT-K1 was stably transfected in MDCK cells and examined for its transport characteristics and membrane localization. OAT-K1 mediated both uptake and efflux of methotrexate in the apical membranes. Immunoblotting showed that the apparent molecular mass of the expressed OAT-K1 was 50 kDa, which was comparable to that found in the rat renal brush-border membranes. The OAT-K1-mediated methotrexate transport was significantly inhibited in the presence of several organic anions such as folate and sulfobromophthalein. These findings suggest that OAT-K1 mediates bidirectional methotrexate transport across the apical membranes, and may be involved in the renal handling of methotrexate. 相似文献
14.
Effect of piperonyl butoxide on organic anion and cation transport in rabbit kidneys 总被引:1,自引:0,他引:1
C H Kuo B M Tune J B Hook 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1983,174(2):165-171
Piperonyl butoxide has been shown to reduce accumulation of cephaloridine in rabbit renal cortex; however, the mechanism responsible for this effect remains unclear. Cephaloridine is a zwitterion and its accumulation in renal cortex has been suggested to be regulated by both organic anion and cation transport systems. Thus, it was of interest to determine the effect of piperonyl butoxide on renal transport of p-aminohippurate (PAH, an organic anion) and tetraethylammonium (TEA, an organic cation). Although pretreatment with piperonyl butoxide markedly inhibited renal cortical uptake of cephaloridine, the same treatment had less inhibitory effect on either PAH or TEA uptake. Efflux of PAH from preloaded renal cortical slices was enhanced by pretreatment with piperonyl butoxide; however, TEA efflux was unaffected. Thus, piperonyl butoxide appears to have effects on renal membrane functions which result in differential effects on PAH, TEA, and cephaloridine transport. 相似文献
15.
E K Park S K Hong J Goldinger G Andres B Noble 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1985,180(1):174-184
Organic ion transport across the basolateral membrane of proximal tubules was measured by means of the tissue slice technique in each of the four different stages of Heymann nephritis. Impairment of both organic anion and cation transport was detected early in Stage 2, and became more severe in Stage 3 of Heymann nephritis. The decreased transport function was associated with extensive damage to proximal tubule cells, including loss of brush border microvilli and basal infoldings. Despite these abnormalities of structure and function, oxygen consumption of proximal tubule cells remained essentially normal. Partial recovery of organic cation transport was noted late in Heymann nephritis (Stage 4). Recovery of the cation transport function was associated with a partial restoration of brush border microvilli and basal infoldings to proximal tubule cells. However, organic anion transport remained depressed throughout the entire course of disease. Impairment of organic ion transport in rats with Heymann nephritis appeared to result from damage to basolateral membrane transport elements rather than general deterioration of the metabolic machinery of proximal tubule cells. Decreased organic cation transport appeared to be the consequence of a reduction in the number of carrier sites, a phenomenon that could have resulted from decreased membrane surface area. However, the depression of organic anion transport was associated with decreased substrate affinity of the anion carrier, indicating that qualitative, rather than quantitative changes, were primarily responsible for that defect. Specific antibody-mediated damage to the anion transport elements in basolateral membranes of proximal tubules is postulated to occur in Heymann nephritis. 相似文献
16.
This study of larval Drosophila melanogaster examined the effects of injecting the prototypical organic cation tetraethylammonium (TEA) into the hemocoel or adding TEA and/or other organic cations to the diet. Mortality, hemolymph TEA levels, and Malpighian tubule TEA secretion rates were measured. The LD50 for dietary TEA was 158.4 mM and mortality increased if competitive inhibitors of organic cation transporters were also included in the diet. Mortality increased from 24% on TEA (100 mM) alone to 83 and 67% when the diet contained both TEA and quinidine (10 mM) or cimetidine (100 mM), respectively. TEA-selective microelectrode measurements indicated that hemolymph TEA concentration was approximately 3% of that in the diet for larvae maintained on TEA-enriched diet for 24 h. Malpighian tubules isolated from larvae exposed to dietary TEA excreted more TEA than did tubules from controls fed a TEA-free diet. However, the rate of decline of hemolymph TEA concentration following ingestion or injection of TEA into the hemocoel was greater than that explicable by rates of active transport by the gut and Malpighian tubules (MTs). We propose that TEA concentrations in the hemolymph are reduced not only by active transport across the MTs and gut, but also by diffusion into the gut. The latter pathway is particularly important when larvae previously maintained upon TEA-enriched diet are transferred to a TEA-free diet. The ingestion of TEA-free food not only clears the gut lumen, but also creates a TEA-free compartment into which TEA may passively diffuse from the hemolymph. 相似文献
17.
J J Mertens J G Weijnen W J van Doorn B Spenkelink J H Temmink P J van Bladeren 《Chemico-biological interactions》1988,65(3):283-293
Monolayers of LLC-PK1 cells, a cell line with features typical of proximal tubular epithelial cells, were treated at the apical and basolateral side with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBD-GSH) and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBD-NAC). Apical treatment with PCBD-GSH (greater than 20 microM) resulted in cytotoxicity, which could be inhibited by acivicin and aminooxyacetic acid (AOAA), inhibitors of gamma-glutamyltranspeptidase (gamma GT) and beta-lyase respectively. In contrast apical treatment with PCBD-NAC was only toxic at high concentrations (greater than 850 microM), and this effect could hardly be inhibited by AOAA. Basolateral treatment of confluent LLC-PK1 monolayers, grown on porous membranes, with PCBD-GSH gave a much smaller response than apical treatment, consistent with the fact that gamma GT is predominantly present at the apical side. Basolateral treatment even with high concentrations of PCBD-NAC (1.1 mM) did not show an increase in cytotoxicity when compared to the effect after apical treatment. These results suggest the absence of an organic anion transporter, by which these conjugates in vivo are transported into the cells from the basolateral side. This supposition was substantiated in a study of transcellular transport of the model ions tetraethyl ammonium (TEA) and para-aminohippurate (PAH), in LLC-PK1 monolayers, grown as indicated above. No active PAH transport could be demonstrated, whereas an active TEA transport was present. The absence of an organic anion transporter limits the usefulness of LLC-PK1 cells for the study of nephrotoxicity of compounds, like PCBD-NAc, needing this transport to enter the cells. However, the finding of an active basolateral organic cation transporter, together with the presence of gamma GT, dipeptidase and beta-lyase, makes this system especially interesting for testing all compounds that use this transporter or these enzymes in order to elicit toxicity. 相似文献
18.
Kwok B Yamauchi A Rajesan R Chan L Dhillon U Gao W Xu H Wang B Takahashi S Semple J Tamai I Nezu J Tsuji A Harper P Ito S 《American journal of physiology. Regulatory, integrative and comparative physiology》2006,290(3):R793-R802
The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of human (h) novel organic cation transporters (OCTN) 1 and hOCTN2 (a Na+-dependent carnitine carrier with Na+-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC OCTs such as hOCT1 and extraneuronal monoamine transporter (EMT)/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of ATB0+ (an amino acid transporter with a Na+/Cl(-)-dependent carnitine transport activity), and Fly-like putative transporter 2/OCT6 (a splice variant of carnitine transporter 2: a testis-specific Na+-dependent carnitine transporter). TEA uptake was pH dependent. Carnitine uptake was dependent on Na+, and partly on Cl-, compatible with hOCTN2 and ATB0+ function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by K(m) of 5.1 microM for carnitine and 1.6 mM for TEA, apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake but required supratherapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotic challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/nutrient transporters in human mammary gland drug transfer. 相似文献
19.
Platinum complex toxicity in cultured renal epithelia. 总被引:1,自引:0,他引:1
BACKGROUND: Cisplatin is a potent antitumor drug but its clinical use is limited by nephrotoxic side effects. We have found recently, that nephrotoxicity of platinum complexes is related to basolateral organic cation transport. In this study, effects of cell culture conditions on platinum complex toxicity and organic cation transport were investigated by an in vitro system that utilizes the high TransEpithelial Electrical Resistance (TEER) of the C7-clone of the MDCK-(Madin-Darby-Canine-Kidney) cells. METHODS: TEER and caspase-3 activity of cells in microfilter membrane cups were measured after exposure of apical or basolateral membranes to 100 microM cis-, oxali-, or carboplatin. Caspase-3 activity after platinum complex exposure and uptake of the cation ASP+ (4-(4-(diethylamino)styryl)-N-methylpyridinium) of cells on filter membranes and impermeable supports (e.g. culture flasks) were compared. Atomic Force Microscopy (AFM) was used to depict morphometric differences between both culture conditions. RESULTS: In cells on filter membranes, cis-, oxali- and carboplatin induced loss of epithelial monolayer integrity by apoptosis via activation of caspase-3 to different extents. Basolateral application of platinum complexes enhanced toxicity dramatically and uptake of ASP+ from the basolateral side was higher than from the apical medium compartment. Intracellular accumulation of ASP+ was less distinct in cells grown on impermeable supports. Only cisplatin, the most lipophilic investigated complex, induced activation of caspase-3 in these cells. AFM disclosed more prominent cell-cell contacts in cells grown on filter membranes. CONCLUSION: We conclude that toxicity of hydrophilic substances can be underestimated in cells grown on solid supports, if basolateral transport mechanisms are involved. We suggest that unhindered access to basolateral transporters is responsible for higher levels of organic cation uptake and apoptosis in cells on filter membranes, even though more prominent cell-cell contacts indicate a better barrier function. 相似文献
20.
Transport of [14C]tetraethylammonium (TEA), an organic cation, was studied in brush-border (BBMV) and basolateral (BLMV) membrane vesicles isolated from rabbit kidney cortex. In BBMV, the presence of an outwardly directed H+ gradient induced a marked stimulation of TEA uptake against its concentration gradient (overshoot phenomenon), whereas a valinomycin-induced inside-negative potential had no effect on TEA uptake. In BLMV, TEA uptake was significantly stimulated by the presence of an outwardly directed H+ gradient and by an inside-negative potential, but the effect of H+ gradient was absent when the vesicles were chemically 'voltage clamped'. In BBMV, internal H+ stimulated TEA uptake in a non-competitive manner by binding at a site with apparent pKa of 6.87. External H+ inhibited TEA uptake through a direct interaction with the putative H+/organic-cation exchanger at a site with apparent pKa of 6.78. Changing external pH while maintaining the pH gradient constant produced a result similar to that obtained by changing external pH alone. Increasing external H+ showed a mixed-type inhibition of TEA uptake. These results suggest that in the rabbit TEA transport across the basolateral membranes is driven by an inside-negative potential and that transport across the brush-border membrane is driven by a H+ gradient via an electroneutral H+/TEA antiport system. 相似文献