Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of Rous sarcoma virus. Comparison of transport properties of whole cells and membrane vesicles.

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37 degrees C) had a 2-fold higher rate of 2-deoxy-D-glucose uptake than the same cells cultured at the non-permissive temperature (41 degrees C). However, both the non-transformed and transformed cells had comparable rates of alpha-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41 degrees C or 37 degrees C, displayed carrier-mediated, intravesicular uptake of D-glucose and alpha-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37 degrees C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41 degrees C. The two types of membrane vesicle had similar uptake rates of alpha-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. Km values for stereospecific D-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37 degrees C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37 degrees C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virally-transformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.

Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1.

The LLC-PK1 cells stably transfected with a rat PEPT1 cDNA transported ceftibuten (anion) and cephradine (zwitterion), both oral beta-lactam antibiotics, in a H+-gradient-dependent manner. Diethylpyrocarbonate, a histidine residue modifier, abolished ceftibuten uptake. This inhibition was prevented in the presence of glycylsarcosine or cephradine. When expressed in Xenopus oocytes, replacement of either histidine 57 or histidine 121 of the rat PEPT1 with glutamine by site-directed mutagenesis eliminated ceftibuten and [14C]glycylsarcosine transport activities. Immunostaining of oocyte sections indicated that insertion of the mutant transporters in the plasma membranes was not impaired. These findings suggest that both histidine 57 and histidine 121, which are conserved in the rat, rabbit and human PEPT1, are involved in substrate recognition of this molecule.

Pharmacokinetics and bioavailability of tacrolimus in rats with experimental renal dysfunction.

The effects of renal failure on the pharmacokinetics and bioavailability of tacrolimus were investigated in rats. Experimental renal dysfunction was induced by intraperitoneal injection of cisplatin (5 mg kg(-1)) into rats. The blood concentration of tacrolimus was measured after intravenous and intra-intestinal administration of the drug. The blood concentration of tacrolimus after intravenous administration (1 mg kg(-1)) was slightly increased (up to 1.3 fold) by induction of renal dysfunction. In contrast, the peak tacrolimus concentration after intra-intestinal administration (1 mg kg(-1) or 3 mg kg(-1)) in rats with renal failure was about 2-fold higher than that in normal controls. The bioavailability was increased by about 35% in rats with impaired renal function as compared with normal controls. These results suggested that the bioavailability of tacrolimus, which is mainly metabolized in the liver and intestine after oral administration, is also influenced by renal function.

Effects of fosfomycin and imipenem-cilastatin on the nephrotoxicity of vancomycin and cisplatin in rats.

The nephrotoxicity of vancomycin and cisplatin and the protective effects of fosfomycin and imipenem-cilastatin on renal function have been studied in rats. The renal clearance of vancomycin after the induction of renal dysfunction was also evaluated by calculating the glomerular filtration rate (GFR) and its secretory clearance. Plasma concentrations of creatinine and urea nitrogen increased dose-dependently after vancomycin injection. No such increases were observed after co-treatment with fosfomycin or imipenem-cilastatin. Changes of N-acetyl-beta-D-glucosaminidase activity in the urine of vancomycin-treated rats were not remarkable compared with those in cisplatin-treated animals. The reduced renal clearance of vancomycin in rats with acute renal failure induced by vancomycin was because of a decrease in both GFR and secretory clearance. However, the changes in GFR and secretory clearance were not proportional-the change in GFR was more pronounced than that of secretory clearance in the experimental groups. In addition, the renal clearance of vancomycin was maintained at the control level after co-administration of fosfomycin or imipenem-cilastatin with vancomycin. These results suggest that vancomycin impairs glomerular filtration more markedly than renal tubular function as compared with cisplatin. Co-administration with fosfomycin or imipenem-cilastatin confers significant protection against the nephrotoxic effects of vancomycin.

Black lipid membranes as a model for intestinal absorption of drugs.

Black lipid membranes were generated in isotonic buffer (pH 4-5 and pH 6-5) from egg phosphatidylcholine and intestinal lipid, and the permeability to salicylamide, salicylic acid, p-aminobenzoic acid and tryptophan of these membranes was studied. Electrical resistance of intestinal lipid membranes was higher than that of phosphatidylcholine membranes. The presence of cholesterol produced an increase in the electrical resistance of black lipid membranes and a small decrease in the permeability of membranes to drugs. The permeability coefficient of salicylamide, an uncharged drug, was much larger than the coefficients of the charged drugs examined. The values for salicylic acid and p-aminobenzoic acid were much larger than comparable values predicted from their partition coefficients. Intestinal lipid membranes were more permeable to acidic drugs than phosphatidylcholine membranes. It is suggested that phospholipids and other lipid components of the small intestine may play an important role in the membrane permeability to acidic drugs. This method may be of interest in studying the complex processes of drug absorption from intestine.

Pharmacokinetics and pharmacological effect of recombinant human granulocyte colony-stimulating factor conjugated to poly(styrene-co-maleic acid) in rats.

A new derivative of recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been synthesized by conjugating rhG-CSF to poly(styrene-co-maleic acid) (poly(styrene-co-maleic acid)-rhG-CSF) to try to avoid glomerular filtration and thus potentiate the neutrophil-proliferating activity of rhG-CSF. Poly(styrene-co-maleic acid)-rhG-CSF was highly bound to bovine serum albumin (BSA) and the molecular weight of the poly(styrene-co-maleic acid)-rhG-CSF-BSA complex was estimated to be about 90000 by gel filtration. Intravenous administration of poly(styrene-co-maleic acid)-rhG-CSF to normal rats resulted in a dose-dependent increase in neutrophil count. The neutrophil-proliferating activity of poly(styrene-co-maleic acid)-rhG-CSF was about 10 times greater than that of rhG-CSF. After intravenous injection at a dose of 5 microg protein kg(-1) the total clearance of rhG-CSF fell from 71.0 to 32.1 mLh(-1) kg(-1) following poly(styrene-co-maleic acid) modification. An isolated perfusion study in rat kidney showed that the filtered fraction of rhG-CSF was reduced by conjugation with poly(styrene-co-maleic acid). These results suggest that poly(styrene-co-maleic acid)-conjugation can potentiate the neutrophil-proliferating activity of rhG-CSF by reducing, at least in part, its renal clearance.

Relationship between acetazolamide blood concentration and its side effects in glaucomatous patients.

Although acetazolamide, a carbonic anhydrase inhibitor, has an effect of lowering intraocular pressure, a number of side effects have been reported. Therefore, we investigated the relationship between the concentration of acetazolamide and its side effects, including plasma electrolyte imbalance. This study was conducted on 23 glaucomatous patients who received repeated doses of oral acetazolamide for one week or more. The concentrations of total and unbound plasma acetazolamide, as well as in the whole blood from the patients, were measured by high-performance liquid chromatography. The serum creatinine concentration, electrolyte concentrations, and adverse reactions were monitored. We found that plasma concentrations of chloride ion after repeated doses became higher than the normal range. This chloride ion concentration significantly correlated with the acetazolamide concentration in the erythrocytes, but not with the plasma concentration. The patients with erythrocyte acetazolamide concentration more than 20 microg/ml had higher incidences of the side effects. Periodical monitoring of erythrocyte acetazolamide concentration and plasma chloride ion can be easily and safely applied to elderly glaucomatous patients treated with acetazolamide for long periods to prevent overdosage and side effects.

Transport of quinolone antibacterial drugs by human P-glycoprotein expressed in a kidney epithelial cell line, LLC-PK1.

The purpose of this study was to characterize the transport mechanisms involved in the renal tubular secretion of quinolones. The contribution of P-glycoprotein to the transport of quinolones was elucidated using a kidney epithelial cell line, LLC-PK1, and its transfectant derivative cell line, LLC-GA5-COL150, which expresses human P-glycoprotein on the apical membrane. The transcellular transport of levofloxacin, a quinolone antibacterial drug, from the basolateral to apical side was increased in LLC-GA5-COL150 compared with that in LLC-PK1 monolayers. The apparent Michaelis constant and maximum velocity values for the saturable transcellular transport of levofloxacin from the basolateral to apical side in LLC-GA5-COL150 monolayers were 3.0 mM and 45 nmol/mg protein per 15 min, respectively. The increased basolateral-to-apical transport in LLC-GA5-COL150 monolayers was completely inhibited by cyclosporin A and quinidine to the level observed in LLC-PK1 monolayers. In addition, 3 mM levofloxacin inhibited the basolateral-to-apical transport of daunorubicin in LLC-GA5-COL150 monolayers. The basolateral-to-apical transport of another quinolone antibacterial drug, DU-6859a, in LLC-GA5-COL150 monolayers greatly exceeded than that in LLC-PK1 monolayers, and was inhibited by levofloxacin. These findings suggest that quinolone antibacterial drugs are transported by P-glycoprotein, and that P-glycoprotein may contribute at least in part to the renal tubular secretion of quinolones.

Transport of procainamide via H(+)/tertiary amine antiport system in rabbit intestinal brush-border membrane.

Transport characteristics of procainamide in the brush-border membrane isolated from rabbit small intestine were studied by a rapid-filtration technique. Procainamide uptake by brush-border membrane vesicles was stimulated by an outward H(+) gradient (pH(in) = 6.0, pH(out) = 7.5) against a concentration gradient (overshoot phenomenon), and this stimulation was reduced when the H(+) gradient was subjected to rapid dissipation by the presence of a protonophore, FCCP. An outward H(+) gradient-dependent procainamide uptake was not caused by H(+) diffusion potential. The initial uptake of procainamide was inhibited by other tertiary amines with N-dimethyl or N-diethyl moieties in their structures, such as triethylamine, dimethylaminoethyl chloride, and diphenhydramine, but not by tetraethylammonium and thiamine. Furthermore, procainamide uptake was stimulated by preloading the vesicles with these tertiary amines (trans-stimulation effect), indicating the existence of a specific transport system for tertiary amines. These findings indicate that procainamide transport in the intestinal brush-border membrane is mediated by the H(+)/tertiary amine antiport system that recognizes N-dimethyl or N-diethyl moieties in the structures of tertiary amines.

Independent organic cation transport activity of Na(+)-L-carnitine cotransport system in LLC-PK(1) cells.

We investigated expression of the Na(+)-L-carnitine cotransport system and its role in transport of tetraethylammonium in a kidney epithelial cell line, LLC-PK(1). L-Carnitine uptake in the LLC-PK(1) cells was markedly stimulated in the presence of Na(+). The uptake was saturable, with Michaelis constant and maximal uptake velocity values of 7.8 microM and 153.7 pmol x mg protein(-1) x 15 min(-1), respectively. Cationic drugs such as tetraethylammonium, cimetidine, and quinidine inhibited L-carnitine uptake. The basolateral-to-apical transport of [(14)C]tetraethylammonium was enhanced markedly in the presence of an H(+) gradient on the apical side at a pH of 5.9. Under the conditions in which Na(+)/L-carnitine cotransport activity was saturable by the addition of 100 microM L-carnitine to the apical-side medium, the basolateral-to-apical transcellular transport of [(14)C]tetraethylammonium was unaffected. These results suggested that the Na(+)-L-carnitine cotransporter is expressed in the apical membranes of LLC-PK(1) cells, and is not responsible for efflux of tetraethylammonium from the cells. Transport of tetraethylammonium appeared to be mediated predominantly by an H(+)/organic cation antiporter in the apical membranes.

Interactions of nonsteroidal anti-inflammatory drugs with rat renal organic anion transporter, OAT-K1.

We recently cloned and characterized the rat kidney-specific organic anion transporter, OAT-K1, which was suggested to mediate renal tubular transport of methotrexate. In this study, we investigated the interactions of nonsteroidal anti-inflammatory drugs (NSAIDs) with OAT-K1 by evaluating the effects of these drugs on renal distribution of methotrexate in vivo, and on methotrexate accumulation in the stably transfected LLC-PK1 cells expressing OAT-K1 (LLC-OAT-K1). NSAIDs such as indomethacin and ketoprofen had significant inhibitory effects on renal accumulation of methotrexate in rats after coadministration. Indomethacin and ketoprofen inhibited methotrexate accumulation by LLC-OAT-K1 cells in a competitive manner with the apparent inhibition constant values of 1. 0 mM and 1.9 mM, respectively. Other NSAIDs including ibuprofen, flufenamate and phenylbutazone also showed potent inhibitory effects on methotrexate accumulation. However, indomethacin was not transported via OAT-K1. These results indicate that NSAIDs have potent inhibitory effects against the OAT-K1-mediated methotrexate transport, which suggests that the OAT-K1 may be one of interaction sites for methotrexate and NSAIDs in the kidney.

Effects of fosfomycin and imipenem/cilastatin on nephrotoxicity and renal excretion of vancomycin in rats.

PURPOSE: The effects of fosfomycin and imipenem/cilastatin on the nephrotoxicity of vancomycin were studied in rats, and those on the renal handling of vancomycin were also investigated in perfused kidneys. METHODS: The protective effects of fosfomycin and imipenem/cilastatin on vancomycin nephrotoxicity were evaluated by increases in plasma concentration of creatinine and urea nitrogen in rats. The urinary excretion of vancomycin was measured and analyzed kinetically in the perfused rat kidney. RESULTS: The nephrotoxicity induced by vancomycin (500 mg/kg, i.v.) was inhibited almost completely by co-administration of fosfomycin or imipenem/cilastatin. In the perfused rat kidney, the excretion ratio of vancomycin was less than those of p-aminohippurate and cimetidine, and greater than that of arbekacin, suggesting the secretion and reabsorption of vancomycin in renal tubules. The tissue/perfusate ratios of unbound vancomycin were not significantly changed by co-treatment with fosfomycin or imipenem/cilastatin. Imipenem/cilastatin significantly decreased the excretion ratio of vancomycin. Fosfomycin also decreased vancomycin excretion ratio, although this effect was not significant. CONCLUSIONS: The renal handling of vancomycin was different from those of organic anions and cations and an aminoglycoside antibiotic. The protective effects of fosfomycin and imipenem/cilastatin against the nephrotoxicity of vancomycin might be partly due to the change in renal handling of vancomycin, probably in its tubular secretion/ reabsorption, in rats.

Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney.

We have cloned a cDNA coding for a novel member of organic anion transporter, designated OAT-K1, expressed specifically in the kidney of rats. The rat OAT-K1 cDNA (2788 base pairs) had an open reading frame encoding for a 669-amino acid protein (calculated molecular mass of 74 kDa) which shows 72% identity with the cloned rat liver organic anion transporter, oatp. Northern hybridization and reverse transcription-coupled polymerase chain reaction revealed that the rat OAT-K1 messenger RNA transcript is expressed predominantly in the kidney. By use of stable LLC-PK1 cell monolayers transfected with the rat OAT-K1 cDNA, the transporter was suggested to mediate basolateral uptake of methotrexate, an anionic anticancer drug, but not taurocholate, p-aminohippurate, prostaglandin E2, and leukotriene C4. The methotrexate transport by rat OAT-K1 was unaffected by the presence of Na+ or Cl- gradient. The methotrexate accumulation by the OAT-K1-expressing cells showed saturability with the apparent Km value of 1.0 microM. Folate, sulfobromophthalein, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited the methotrexate accumulation markedly. These findings suggest that the rat OAT-K1 is localized in the basolateral membranes of renal tubules, where it mediates renal clearance of methotrexate from the blood.

Cellular and molecular aspects of drug transport in the kidney.

The kidney plays an important role in the elimination of numerous hydrophilic xenobiotics, including drugs, toxins, and endogenous compounds. It has developed high-capacity transport systems to prevent urinary loss of filtered nutrients, as well as electrolytes, and simultaneously to facilitate tubular secretion of a wide range of organic ions. Transport systems for organic anions and cations are primarily involved in the secretion of drugs in renal tubules. The identification and characterization of organic anion and cation transporters have been progressing at the molecular level. To date, many members of the organic anion transporter (OAT), organic cation transporter (OCT), and organic anion-transporting polypeptide (oatp) gene families have been found to mediate the transport of diverse organic anions and cations. It has also been suggested that ATP-dependent primary active transporters such as MDR1/P-glycoprotein and the multidrug resistance-associated protein (MRP) gene family function as efflux pumps of renal tubular cells for more hydrophobic molecules and anionic conjugates. Tubular reabsorption of peptide-like drugs such as beta-lactam antibiotics across the brush-border membranes appears to be mediated by two distinct H+/peptide cotransporters: PEPT1 and PEPT2. Renal disposition of drugs is the consequence of interaction and/or transport via these diverse secretory and absorptive transporters in renal tubules. Studies of the functional characteristics, such as substrate specificity and transport mechanisms, and of the localization of cloned drug transporters could provide information regarding the cellular network involved in renal handling of drugs. Detailed information concerning molecular and cellular aspects of drug transporters expressed in the kidney has facilitated studies of the mechanisms underlying renal disposition as well as transporter-mediated drug interactions.

Interaction and transport of thiazide diuretics, loop diuretics, and acetazolamide via rat renal organic anion transporter rOAT1.

The renal tubular secretion of thiazides and loop diuretics via the organic anion transport system in renal tubules is required for them to reach their principal sites of action. Similarly, acetazolamide, a diuretic clinically administered for glaucoma, is excreted from the kidney by glomerular filtration and tubular secretion. In this study, we investigated the interaction and transport of these diuretics via the rat renal organic anion transporter rOAT1 by using Xenopus laevis oocyte expression system. p-[(14)C]Aminohippurate (PAH) uptake by rOAT1-expressing oocytes was inhibited in the presence of a thiazide (chlorothiazide, cyclothiazide, hydrochlorothiazide), a loop diuretic (bumetanide, ethacrynic acid, furosemide), or a carbonic anhydrase inhibitor (acetazolamide, ethoxzolamide, methazolamide). Dixon plot analysis demonstrated that the inhibition constant (K(i)) value was 1.1 mM for acetazolamide, 150 microM for hydrochlorothiazide, 9.5 microM for furosemide, and 5. 5 microM for bumetanide. Kinetic analysis revealed that acetazolamide inhibited rOAT1 competitively and that inhibition style of furosemide was a mixture of competitive and noncompetitive. [(14)C]PAH efflux was significantly enhanced when the rOAT1-expressing oocytes were incubated in the presence of unlabeled PAH, alpha-ketoglutarate, acetazolamide, chlorothiazide, or hydrochlorothiazide. rOAT1 stimulated acetazolamide uptake, which was inhibited by probenecid. Although the loop diuretics had little trans-stimulation effect on [(14)C]PAH efflux via rOAT1, the rOAT1-mediated furosemide uptake was observed. These findings suggest that rOAT1 contributes, at least in part, to the renal tubular secretion of acetazolamide, thiazides, and loop diuretics.

Secretory mechanisms of grepafloxacin and levofloxacin in the human intestinal cell line caco-2.

Grepafloxacin and levofloxacin transport by Caco-2 cell monolayers was examined to characterize the intestinal behavior of these quinolones. The levels of transcellular transport of [(14)C]grepafloxacin and [(14)C]levofloxacin from the basolateral to the apical side were greater than those in the opposite direction. The unidirectional transport was inhibited by the presence of excess unlabeled quinolones, accompanied by increased accumulation. The inhibitory effects of cyclosporin A plus grepafloxacin on basolateral-to-apical transcellular transport and cellular accumulation of [(14)C]grepafloxacin were comparable to those of cyclosporin A alone, indicating that the transport of grepafloxacin across the apical membrane was mainly mediated by P-glycoprotein. On the other hand, basolateral-to-apical transcellular transport of [(14)C]levofloxacin in the presence of cyclosporin A was decreased by unlabeled levofloxacin, grepafloxacin, and enoxacin, accompanied by significantly increased cellular accumulation. The organic cation cimetidine, organic anion p-aminohippurate, and the multidrug resistance-related protein (MRP) modulator probenecid did not affect the transcellular transport of [(14)C]grepafloxacin or [(14)C]levofloxacin in the presence of cyclosporin A. The basolateral-to-apical transcellular transport of levofloxacin in the presence of cyclosporin A showed concentration-dependent saturation with an apparent Michaelis constant of 5.6 mM. In conclusion, these results suggested that basolateral-to-apical flux of quinolones was mediated by P-glycoprotein and a specific transport system distinct from organic cation and anion transporters and MRP.

Sodium-stimulated active transport of aminoisobutyric acid by reconstituted vesicles from partially purified plasma membranes of mouse fibroblasts transformed by simian virus 40.

Plasma membrane fractions isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40 were partially purified by treatment with dimethylmaleic anhydride followed by extraction with 2% cholate. The extracted proteins were combined with exogenous phospholipids and eluted through a Sephadex G50 column. Reconstituted vesicles thus obtained were shown to possess the ability of Na+-stimulated transport of alpha-aminoisobutyric acid. The simultaneous addition of NaSCN and alpha-aminoisobutyric acid to these vesicles produced a transient accumulation above the equilibrium level (overshoot, active transport). The Na+-stimulated transport of alpha-aminoisobutyric acid was sensitive to the accompanying anion and to the temperature of incubation. The results demonstrate that partially purified membrane proteins of mouse fibroblast cells can be incorporated into the liposomes that have the characteristics of Na+-stimulated and electrochemically sensitive active transport of alpha-aminoisobutyric acid.

Functional characteristics and membrane localization of rat multispecific organic cation transporters, OCT1 and OCT2, mediating tubular secretion of cationic drugs.

We have isolated a kidney-specific organic cation transporter, rat OCT2, which is distinct from rat OCT1 (Okuda M, Saito H, Urakami Y, Takano M and Inui K (1996) Biochem Biophys Res Commun 224:500-507). In our study, the functional characteristics and membrane localization of OCT1 and OCT2 were investigated by uptake studies using MDCK cells transfected with rat OCT1 or OCT2 cDNA (MDCK-OCT1 or MDCK-OCT2) and immunological studies. Tetraethylammonium (TEA) uptake by both MDCK-OCT1 and MDCK-OCT2 cells was markedly elevated when TEA was added to the basolateral medium, but not to the apical medium. Efflux of TEA from MDCK-OCT1 and MDCK-OCT2 cells was not changed by extracellular pH from 5.4 to 8.4, whereas TEA uptake by both transfectants was decreased by acidification of extracellular medium. Apparent Km values for TEA uptake by MDCK-OCT1 and MDCK-OCT2 cells were 38 and 45 microM, respectively. Although various hydrophilic organic cations such as 1-methyl-4-phenylpyridinium, cimetidine, quinidine, nicotine, N1-methylnicotinamide and guanidine markedly inhibited TEA uptake by both MDCK-OCT1 and MDCK-OCT2 cells, there were no significant differences in the apparent inhibition constants (Ki) against these organic cations between both transfectants. Furthermore, immunological studies using a polyclonal antibody against OCT1 revealed that OCT1 was expressed in the basolateral membranes but not in the brush-border membranes of the rat kidney. These results suggested that both OCT1 and OCT2 are basolateral-type organic cation transporters with broad substrate specificities, mediating tubular secretion of cationic drugs.

Distinct characteristics of transcellular transport between nicotine and tetraethylammonium in LLC-PK1 cells.

To clarify the mechanisms of the renal tubular secretion of nicotine, we studied transport of nicotine in the kidney epithelial cell line LLC-PK1. The transcellular transport of nicotine from the basolateral side to the apical side of the LLC-PK1 monolayers grown on membrane filters was much greater than that of tetraethylammonium. The basolateral-to-apical transport of nicotine was stimulated by lowering the pH of the apical side, accompanied by a decrease in the accumulation of nicotine. The accumulation of nicotine from the basolateral side was inhibited by unlabeled nicotine, cotinine, tetraethylammonium, cimetidine and quinidine. The uptake of nicotine across the apical membrane was inhibited by unlabeled nicotine and quinidine but not by tetraethylammonium or cimetidine. Pretreatment with p-chloromercuribenzene sulfonate caused a decrease in the transcellular transport of tetraethylammonium but not of nicotine. These results suggest that nicotine undergoes vectorial transport from basolateral side to the apical side of LLC-PK1 monolayers in a H+ gradient-dependent manner, corresponding to the secretion in the renal tubules. Nicotine transport in LLC-PK1 cells could be mediated by a transport system that is distinct from the transport system for tetraethylammonium.