Substrate specificity of organic cation/H+ exchange in avian renal brush-border membranes

The substrate specificity of the avian renal organic cation exchanger was examined in isolated renal brush-border membrane vesicles. Endobiotic and xenobiotic organic cations (OCs) were tested at a concentration of 100 microM for cis-inhibition of 14C-tetraethylammonium (TEA)/H+ exchange and at 1 mM for trans-stimulation of 14C-TEA efflux. The xenobiotic cations amiloride, cimetidine, mepiperphenidol, procainamide, quinidine, quinine, and ranitidine cis-inhibited TEA uptake >/= 80%; isoproterenol and unlabeled TEA inhibited uptake at least 30%. In contrast, the endogenous cations acetylcholine, choline, and guanidine did not inhibit TEA uptake; however, epinephrine, N1-methylnicotinamide, serotonin, and thiamine inhibited uptake as much as 60%. Each endogenous cation, except thiamine, trans-stimulated TEA efflux, and xenobiotic cations, excluding isoproterenol and TEA, trans-inhibited TEA efflux. The data suggest that the avian renal tubule luminal OC exchanger has greater affinity for xenobiotic cations than for endobiotic cations, but greater transport capacity for endobiotics than for xenobiotics.     

Cloning and characterization of two human polyspecific organic cation transporters

Previously we cloned a polyspecific transporter from rat (rOCT1) that is expressed in renal proximal tubules and hepatocytes and mediates electrogenic uptake of organic cations with different molecular structures. Recently a homologous transporter from rat kidney (rOCT2) was cloned but not characterized in detail. We report cloning and characterization of two homologous transporters from man (hOCT1 and hOCT2) displaying approximately 80% amino acid identity to rOCT1 and rOCT2, respectively. Northern blots showed that hOCT1 is mainly transcribed in liver, while hOCT2 is found in kidney. Using in situ hybridization and immunohistochemistry, expression of hOCT2 was mainly detected in the distal tubule where the transporter is localized at the luminal membrane. After expression in Xenopus laevis oocytes, hOCT1 and hOCT2 mediate tracer influx of N-1-methylnicotinamide (NMN), tetraethylammonium (TEA), and 1-methyl-4-phenylpyridinium (MPP). For cation transport by hOCT2 apparent K(m) and K(i) values were determined in tracer flux measurements. In addition, electrical measurements were performed with voltage-clamped oocytes. Similar to rOCT1, cation transport by hOCT2 was pH independent, electrogenic, and polyspecific; however, the cation specificity was different. In voltage-clamped hOCT2-expressing oocytes, inward currents were induced by superfusion with MPP, TEA, choline, quinine, d-tubocurarine, pancuronium, and cyanine863. Cation transport in distal tubules is indicated for the first time. Here hOCT2 mediates the first step in cation reabsorption. hOCT1 may participate in hepatic excretion of organic cations.     

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.     

Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine

Recently, we cloned the human cation transporter hOCT2, a member of a new family of polyspecific transporters from kidney, and demonstrated electrogenic uptake of tetraethylammonium, choline, N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. Using polymerase chain reaction amplification, cDNA sequencing, in situ hybridization, and immunohistochemistry, we now show that hOCT2 message and protein are expressed in neurons of the cerebral cortex and in various subcortical nuclei. In Xenopus laevis oocytes expressing hOCT2, electrogenic transport of norepinephrine, histamine, dopamine, serotonin, and the antiparkinsonian drugs memantine and amantadine was demonstrated by tracer influx, tracer efflux, electrical measurements, or a combination. Apparent Km values of 1.9 +/- 0.6 mM (norepinephrine), 1.3 +/- 0.3 mM (histamine), 0.39 +/- 0.16 mM (dopamine), 80 +/- 20 microM (serotonin), 34 +/- 5 microM (memantine), and 27 +/- 3 microM (amantadine) were estimated. Measurement of trans-effects in depolarized oocytes and human embryonic kidney cells expressing hOCT2 suggests that there were different rates and specificities for cation influx and efflux. The hypothesis is raised that hOCT2 plays a physiological role in the central nervous system by regulating interstitial concentrations of monoamine neurotransmitters that have evaded high affinity uptake mechanisms. We show that amantadine does not interact with the expressed human Na+/Cl- dopamine cotransporter. However, concentrations of amantadine that are effective for the treatment of Parkinson's disease may increase the interstitial concentrations of dopamine and other aminergic neurotransmitters by competitive inhibition of hOCT2.     

Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1

The previously cloned rat cation transporter rOCT1 detected in renal proximal tubules and hepatocytes (Gründemann, D., Gorboulev, V., Gambaryan, S., Veyhl, M., and Koepsell, H. (1994) Nature 372, 549-552) was expressed in Xenopus oocytes, and transport properties were analyzed using tracer uptake studies and electrophysiological measurements. rOCT1 induced highly active transport of a variety of cations, including the classical substrates for cation transport, such as N-1-methylnicotinamide, 1-methyl-4-phenylpyridinium (MPP), and tetraethylammonium (TEA), but also the physiologically important choline. In oocytes rOCT1 also mediated efflux of MPP, which could be trans-stimulated by MPP and TEA. Cation transport via rOCT1 was electrogenic. In voltage-clamped oocytes, transport of TEA and choline via rOCT1 produced inwardly directed currents, which were independent of extracellular ion composition or pH. The choline- and TEA-induced currents were voltage-dependent at nonsaturating concentrations, and the apparent affinity of these cations was decreased at depolarized voltages. Other substrates transported by rOCT1 were the polyamines spermine and spermidine. Interestingly, the previously described potent inhibitors of rOCT1, cyanine 863, quinine, and D-tubocurarine were substrates themselves. The data indicate that rOCT1 is an effective transport system that is responsible for electrogenic uptake of a wide variety of organic cations into epithelial cells of renal proximal tubules and hepatocytes.     

Peritubular transport of ochratoxin A by single rabbit renal proximal tubules

Epifluorescence microscopy was used to study peritubular transport of the fluorescent mycotoxin ochratoxin A (OTA) into single proximal tubule segments of the rabbit. Initial rates of OTA uptake into S2 segments were saturable and adequately described by Michaelis-Menten kinetics, with an apparent Km of 2.2+/-0.3 microM (SEM). Several lines of evidence indicated that peritubular uptake of OTA in S2 segments was effectively limited to the "classical" organic anion transporter. First, 5 mM p-aminohippurate (PAH) cis-inhibited the uptake of 1 microM OTA into tubules by 96%. Kinetic analysis of the inhibition of OTA uptake by PAH (100 microM to 5 mM) yielded an apparent Ki of 164 microM, similar to the 100 to 200 microM range of Km values previously reported for the peritubular uptake of PAH. Second, efflux of OTA from tubules was trans-stimulated 3.2-fold by the presence of 2.5 mM PAH in the uptake medium. Third, 100 microM alpha-ketoglutarate (alphaKG) trans-stimulated the uptake rate of 1 microM OTA by 1.8-fold. Fourth, besides PAH, other organic anions effectively cis-inhibited the uptake of 1 microM OTA into tubules (inhibitor, % inhibition): 1.5 mM alphaKG, 80%; 1 mM probenecid, 100%; 1 mM piroxicam, 100%; 1 mM octanoate, 100%. In contrast, 1.5 mM tetraethylammonium, an organic cation, blocked uptake of 1 microM OTA by only 7%. The inhibition of OTA uptake into S1 and S3 segments of the proximal tubule was qualitatively similar: 5 mM PAH cis-inhibited the uptake of 1 microM OTA by approximately 95% in both S1 and S3 segments. Thus, peritubular OTA uptake into all segments of the proximal tubule appears to be dominated by its interaction with the classical organic anion transporter. The high-affinity and relatively high capacity of this pathway for OTA suggest that peritubular uptake may be a significant avenue for the entry of this toxin into proximal tubule cells.     

Carrier-mediated hepatic uptake of quinolone antibiotics in the rat

The systemic clearance of many quinolone antibiotics is mainly via metabolism and urinary excretion; by contrast, biliary excretion is a major route of elimination for a new quinolone grepafloxacin (GPFX). Accordingly, we studied the hepatic uptake of GPFX because it is the first step in the drug's hepatobiliary transport. The hepatic uptake of GPFX in vivo after i.v. administration was found to approach the hepatic blood flow, suggesting the existence of an effective hepatic uptake mechanism. To clarify this transport mechanism, GPFX uptake by isolated rat hepatocytes was examined and found to consist of a saturable component (Km 173 microM, Vmax 6.96 nmol/min/mg) and a nonspecific diffusion component. The inhibition of GPFX uptake by ATP-depletors and a lack of effect after replacing Na+ with choline demonstrated that the uptake was an Na+-independent carrier-mediated active process. This uptake was inhibited by other quinolones and for lomefloxacin this was competitive in nature. Mutual inhibition studies were undertaken to investigate whether the transporter for GPFX might be the same as other transporters so far identified. GPFX inhibited the uptake of taurocholic acid, pravastatin (organic anion), cimetidine (organic cation) and ouabain (neutral steroid). However, GPFX uptake was not inhibited by these compounds. Confirmation that GPFX uptake is blood flow limited was obtained by extrapolation of the in vitro data based on mathematical modeling. In conclusion, the effective hepatic uptake of quinolone antibiotics are via carrier-mediated active transport, which is distinct from that involved in the transport of bile acids, organic anions, organic cations or neutral steroids.     

Luminal transport system for choline+ in relation to the other organic cation transport systems in the rat proximal tubule. Kinetics, specificity: alkyl/arylamines, alkylamines with OH, O, SH, NH2, ROCO, RSCO and H2PO4-groups, methylaminostyryl, rhodamine, acridine, phenanthrene and cyanine compounds

The efflux of [3H] choline+ from the proximal tubular lumen was measured by using the stop-flow microperfusion method. The 2-s efflux of [3H] choline+ follows kinetics with a Michaelis constant, Km = 0.18 mmol x l-1, maximal flux, Jmax = 0.43 pmol x cm-1 x s-1 and a permeability term = 38.0 micron2 small middle dots-1. Replacement of Na+ by N-methyl-D-glucamine+ or Li+, or a change of luminal pH do not alter choline+ efflux. Replacement of Na+ by Cs+ inhibits 2-s choline+ (0. 01 mmol x l-1) efflux by 22% and replacement by K+ inhibits by 49%, indicating that the electrical potential difference across the brush border membrane acts as driving force for choline+ transport. Comparing the apparent luminal inhibitory constant values for choline (app. Ki,l,choline+) with the chemical structure of inhibiting substrates, it was found that the inhibitory potency of amines with high pKa values, i.e. high basicity, and of quaternary ammonium compounds (tetraethyl to tetrahexylammonium) increases with their hydrophobicity in a similar manner as was observed previously against the contraluminal N1-methylnicotinamide (NMeN+) transporter and the luminal H+/organic cation (N-methyl-4-phenylpyridinium) (MPP+) exchanger. Independently of their hydrophobicity, an increase in the inhibitory potency of the homologous series of aminoquinolines against the choline+ transporter was observed with increasing pKa values, i.e. increasing basicity, as was found previously against the two other organic cation transporters. A third parameter influencing the interaction with the choline+ transporter is the presence of two amino groups with high pKa values or one amino group and a permanent positive charge, as is documented with the two-ring aminostyryl and rhodamine compounds, as well as three-ring aminoacridine, aminophenanthrene and cyanine compounds. Thus with the aminostyryl, pyridinium+, rhodamine, phenanthridium+ and cyanine+ dyes app.Ki,l,choline+ values of between 0.01 and 0.07 mmol x l-1 have been found. A fourth parameter influencing the choline+ transporter is the presence of an OH group on the C atom next to that bearing the N atom (as in choline+) or an ester-OCOR group (acetylcholine+, butyrylcholine+) or a thioester-SCOR-group (acetylthiocholine+, butyrylthiocholine+); or an -OP(OH)2(OR) group (glycerylphosphoryl-choline+), resulting in app.Ki,l,choline+ values of 0.3-1.0 mmol x l-1. Thus the substrates for the luminal choline+ transporter have general features in common with the luminal H+/organic cation exchanger and the contraluminal organic cation transporter, i.e. hydrophobicity and basicity. Additional parameters for interaction are an OH (or similar) group positioned a favourable distance from the N atom or a second amino/ammonium group in multi-ring compounds.     

Ovariectomy and estrogen-induced alterations in myocardial contractility in female rabbits: role of the L-type calcium channel

Carnitine is an essential component for mitochondrial beta-oxidation of fatty acid. Using the degenerate primers designed for organic anion transporters and an organic cation transporter, we isolated a novel cDNA encoding a carnitine transporter (CT1) from rat intestine. CT1 encodes a 557-amino-acid protein with 12 putative membrane-spanning domains. When expressed in Xenopus oocytes, CT1 mediated a high-affinity transport of L-carnitine (Km = 25 microM). The replacement of extracellular sodium with Li reduced CT1-mediated L-carnitine uptake to 19.8%. CT1 did not transport typical substrates for either organic anion or organic cation transporters, such as p-aminohippurate and tetraethylammonium. Octanoylcarnitine, acetylcarnitine, and gamma-butyrobetaine showed potent inhibitory effects on CT1-mediated L-carnitine uptake; betaine and d-carnitine showed moderate inhibition. CT1 mRNA was strongly expressed in the testis, colon, kidney, and liver and weakly in the skeletal muscle, placenta, small intestine, and brain. No CT1 expression was detected in the heart, spleen, or lung. The present study provides the molecular basis of carnitine transport in the body.     

Molecular and cell biological analyses for intestinal absorption and renal excretion of drugs

Intestinal absorption and renal tubular secretion are transport processes determining the availability and the disposition of drugs in the body. In this review, our studies on the molecular and cell biological analyses of intestinal absorption and renal secretion of drugs are described. We evaluated the transepithelial transport and the cellular accumulation of peptide-like drugs such as beta-lactam antibiotics and bestatin (a dipeptide-like antineoplastic agent) in the human adenocarcinoma colon cell line, Caco-2, as an in vitro model for studying absorption mechanisms of these drugs. We have found that the transcellular transport of these peptide-like drugs is mediated by both the apically- and basolaterally-localized peptide transporters. To characterize molecular aspects of absorption of the peptide-like drugs, we studied cDNA cloning of H+/peptide cotransporters, PEPT1 and PEPT2, expressed in rats. The rat PEPT1 has been shown to mediate the H- coupled uphill transport of beta-lactam antibiotics across the brush-border membranes of the intestinal and renal epithelia. The rat PEPT2 is expressed predominantly in the kidney, but not in the intestine, mediating tubular reabsorption of the peptide-like drugs. We examined the transcellular transport of organic cations across monolayers of the kidney epithelial cell line, LLC-PK1. We have found that LLC-PK1 cells possess the H+/organic cation antiporter and the membrane potential-sensitive organic cation transporter in the apical and basolateral membranes, respectively, thereby tetraethylammonium (TEA) being transported unidirectionally from the basolateral to the apical side of the monolayers. We have isolated a cDNA encoding a rat kidney-specific organic cation transporter, OCT 2, which transports TEA in a H(+)-gradient independent manner, suggesting that OCT2 is localized to the basolateral membranes of renal tubular cells. In addition, a cDNA encoding a novel rat organic anion transporter, OAT-K1, has been cloned. OAT-K1 is expressed exclusively in the renal proximal tubules, and mediates the transport of methotrexate. Analyses of the molecular and cell biological mechanisms for drug absorption and secretion will provide information for the understanding of organ specific drug transport systems and for the development of drug design and/or drug delivery system.     

Kinetic analysis of the primary active transport of conjugated metabolites across the bile canalicular membrane: comparative study of S-(2,4-dinitrophenyl)-glutathione and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole glucuronide

Eisai hyperbilirubinemic rat (EHBR) is a mutant strain with a hereditary defect in canalicular multispecific organic anion transporter (cMOAT). We examined the uptake and mutual inhibition of S-(2,4-dinitrophenyl)-glutathione (DNP-SG), which is a typical substrate for cMOAT, and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) glucuronide (E-glu) with canalicular membrane vesicles (CMV) prepared from Sprague-Dawley (SD) and EHBR rats to investigate the multiplicity of the organic anion transporter. The ATP-dependent uptake by CMV from SD rats had an apparent Km of 17.6 microM for DNP-SG and 5.7 microM for E-glu, whereas the corresponding uptake by CMV from EHBR had an apparent Km of 44.6 microM for E-glu. The effects of E-glu, 4-methylumbelliferone glucuronide (4 MUG), E3040 sulfate (E-sul) and 4-methylumbelliferone sulfate (4 MUS) on the uptake of [3H]DNP-SG were also examined. The uptake of [3H]DNP-SG was inhibited by glucuronides (E-glu and 4 MUG) in a concentration-dependent manner, although it was enhanced by the sulfate conjugates (E-sul and 4 MUS). This enhancement was shown to be caused by an increased DNP-SG affinity for the transporter. In CMV from SD rats, although ATP-dependent uptake of [3H]DNP-SG was almost completely inhibited by E-glu, that of [14C]E-glu was only reduced to about 30% of controls by DNP-SG. On the other hand, in CMV from EHBR, the ATP-dependent uptake of [14C]E-glu was not inhibited at all by DNP-SG. Kinetic analysis indicated that E-glu inhibited DNP-SG uptake competitively. In conclusion: 1) cMOAT recognizes both DNP-SG and E-glu, and another transporter present in SD rats is also involved in E-glu transport along with cMOAT; 2) the latter transporter is kinetically similar to the E-glu transporter present in EHBR; 3) E-sul enhances the uptake of DNP-SG by increasing the affinity of glucuronide for the transporter.     

Characterization of the catecholamine extraneuronal uptake2 carrier in human glioma cell lines SK-MG-1 and SKI-1 in relation to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) selective cytotoxicity

Transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) and (-)-norepinephrine was investigated in SarCNU-sensitive SK-MG-1 and -resistant SKI-1 human glioma cell lines. [3H]SarCNU influx was inhibited by SarCNU, sarcosinamide, and (+/-)-epinephrine in SK-MG-1 cells with competitive inhibition observed by (+/-)-epinephrine (Ki = 140 +/- 12 microM) and (+/-)-norepinephrine (Ki = 255 +/- 41 microM). No effect on influx was detected in SKI-1 cells. [3H](-)-Norepinephrine influx was linear to 15 sec in both cell lines and temperature dependent only in SK-MG-1 cells. Influx of [3H](-)-norepinephrine was found to be saturable in SK-MG-1 (K(m) = 148 +/- 28 microM, Vmax = 1.23 +/- 0.18 pmol/microL intracellular water/sec) but not in SKI-1 cells. In SK-MG-1 cells, [3H](-)-norepinephrine influx was found to be inhibited competitively by (-)-epinephrine (Ki = 111 +/- 7 microM) and SarCNU (Ki = 1.48 +/- 0.22 mM). Ouabain and KCl were able to inhibit the [3H](-)-norepinephrine influx in SK-MG-1 cells, consistent with influx being driven by membrane potential. Several catecholamine uptake2 inhibitors were able to reduce significantly the influx of [3H](-)-norepinephrine and [3H]SarCNU with no inhibition by a catecholamine uptake1 inhibitor. These findings suggest that increased sensitivity of SK-MG-1 to SarCNU is secondary to enhanced accumulation of SarCNU mediated via the catecholamine extraneuronal uptake2 transporter, which is not detectable in SKI-1 cells. The introduction of SarCNU into clinical trials will confirm if increased uptake via the catecholamine extraneuronal uptake2 transporter will result in increased antitumor activity.     

The effect of acylated polyamine derivatives on polyamine uptake mechanism, cell growth, and polyamine pools in Escherichia coli, and the pursuit of structure/activity relationships

Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [14C]putrescine or [14C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis-Menten kinetics (Kt = 24.58 +/- 2.24 microM). In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (Kt = 3.63 +/- 0.43 microM, Kt' = 0.61 +/- 0.10 microM). From the Ki values, it is inferred that N1-5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N1-acetylspermine, and then N1,N12-diacetylspermine. N1-acetylspermidine and N8-acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows: N1,N12-diacetylspermine approximately N1-5-amino-2-nitro-benzoylspermine > N1-acetylspermine. Both N1-acetylspermidine (Ki = 753 +/- 25 microM, Ki' = 128 +/- 5 microM) and N8-acetylspermidine (Ki = 22.4 +/- 0.4 microM, Ki' = 279 +/- 3 microM) also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers. Neither N4,N9-diacetylspermine, nor N1,N4-bis(beta-alanyl)diaminobutane affect the uptake of any polyamine. Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells. Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.     

Kinetic analysis of tetraethylammonium transport in the kidney epithelial cell line, LLC-PK1

PURPOSE: The aims of this study were to establish a kinetic means of analyzing the membrane transport of organic cations in renal epithelial cells, and to simultaneously evaluate drug interactions in apical and basolateral membranes. METHODS: Tetraethylammonium (TEA) transport was measured using LLC-PK1 cell monolayers grown on microporous membrane filters. After incubating the cells with unlabeled TEA or other drugs, apical or basolateral medium was changed to that containing labeled TEA, and transcellular transport and cellular accumulation were measured. Clearance from apical medium to cells (CL12), cells to apical medium (CL21), cells to basolateral medium (CL23) and basolateral medium to cells (CL32) were calculated based on a three compartment model. RESULTS: TEA was accumulated progressively in the monolayers from the basolateral side and was transported unidirectionally to the apical side. CL32 was greater than CL12 and CL23 was greater than CL21. Therefore, the rate limiting step of TEA transport from the basolateral to the apical medium was the cell-to-apical step. Co-incubation of TEA with procainamide decreased the transport parameters of TEA, CL12, CL21 and CL32, whereas that with levofloxacin decreased only CL12 and CL21, not affecting the parameters in basolateral membranes. CONCLUSIONS: Using a simple model, we analyzed the transport of organic cation in kidney epithelial cell line, LLC-PK1. This method can be useful for the analysis of cation transport and drug interactions in the apical and basolateral membranes of renal tubules.     

Social insurance cost of standard discectomy and percutaneous nucleotomy. A retrospective study of 87 social insurance claim files of male blue collar workers

To evaluate the effect of cadmium intoxication on renal transport systems for organic anions and cations, transport of p-aminohippurate (PAH) and tetraethylammonium (TEA) were studied in renal cortical plasma membrane vesicles isolated from cadmium-intoxicated rats. Cadmium intoxication was induced by daily injections of CdCl2 (2 mg Cd/kg.day sc) for 2-3 weeks. Renal plasma membrane vesicles were prepared by Percoll gradient centrifugation and magnesium precipitation method. Vesicular uptake of substrate was determined by rapid filtration technique using Millipore filter. The cadmium treatment resulted in a marked attenuation of Na(+)-dependent, alpha-ketoglutarate (alpha KG)-driven PAH uptake in the basolateral membrane vesicle (BLMV), and this was due to a reduction in Vmax and not K(m). The Na(+)-alpha KG symport activity of the BLMV was not affected by 2-week cadmium treatment, but it was significantly inhibited by 3-week cadmium treatment. On the other hand, the alpha KG-PAH antiport activity of the BLMV appeared to be markedly suppressed in 2-week as well as 3-week cadmium-treated animals. The cadmium treatment inhibited the proton gradient-dependent TEA transport in the brush-border membrane vesicle (BBMV), and this was associated with a reduction in Vmax with no change in K(m). These results indicate that cadmium exposures may impair the capacities for organic anion transport in the proximal tubular basolateral membrane and organic cation transport in the luminal membrane. The cadmium effect on organic anion transport is attributed mainly to an inhibition of dicarboxylate-organic anion antiport system.     

Peritubular transport of ochratoxin A in rabbit renal proximal tubules

The transport of the nephrotoxic mycotoxin ochratoxin A across the renal peritubular membrane was examined in suspensions of rabbit renal proximal tubules. Ochratoxin A transport across the peritubular membrane was a high-affinity, low-capacity carrier-mediated process with a Jmax value of 0.12 +/- 0.4 nmol/mg of protein/min and a Km value of 1.4 +/- 0.1 microM. The apparent Michaelis constants for inhibition of [3H]para-aminohippurate (PAH) uptake by ochratoxin A inhibition was 1.5 microM, which is similar to the Km value for ochratoxin A uptake in tubule suspensions and suggests that ochratoxin A could be a substrate for the organic anion pathway. The capacity and affinity for peritubular ochratoxin A transport were 40-fold lower and > 100-fold greater, respectively, than those measured for the peritubular uptake of [3H]PAH in tubule suspensions. A concentration of 2.5 mM PAH, which reduced the uptake of [3H]PAH by 90%, reduced ochratoxin A uptake by only 40% to 50%, whereas probenecid concentrations of 0.6 to 2 mM reduced ochratoxin A accumulation in tubule suspensions up to approximately 80% to 90%. This probenecid-sensitive, PAH-insensitive uptake of ochratoxin A suggested that at least one mediated pathway other than the organic anion transporter was involved in the peritubular uptake of this mycotoxin. A 2 mM concentration of the fatty acid octanoate and 1.5 mM concentration of the nonsteroidal anti-inflammatory agent piroxicam were as effective as probenecid in blocking ochratoxin A uptake. The apparent Ki values for inhibition of ochratoxin A uptake by probenecid, piroxicam and octanoate were 30.5 +/- 7.9, 23.2 +/- 10.4 and 81.5 +/- 8.7 microM, respectively. The ability of octanoic acid to inhibit ochratoxin A transport to the same extent as probenecid and a greater extent than PAH suggests that a separate fatty acid transport pathway may be involved in the accumulation of ochratoxin A by suspensions of rabbit renal proximal tubules.     

Identification of glutathione as a driving force and leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter

oatp1 is an hepatic sinusoidal organic anion transporter that mediates uptake of various structurally unrelated organic compounds from blood. The driving force for uptake on oatp1 has not been identified, although a role for bicarbonate has recently been proposed. The present study examined whether oatp1-mediated uptake is energized by efflux (countertransport) of intracellular reduced glutathione (GSH), and whether hydrophobic glutathione S-conjugates such as leukotriene C4 (LTC4) and S-dinitrophenyl glutathione (DNP-SG) form a novel class of substrates for oatp1. Xenopus laevis oocytes injected with the complementary RNA for oapt1 demonstrated higher uptake of 10 nM [3H]LTC4 and 50 microM [3H]DNP-SG, and higher efflux of [3H]GSH (2.5 mM endogenous intracellular GSH concentration). The oatp1-stimulated LTC4 and DNP-SG uptake was independent of the Na+ gradient, cis-inhibited by known substrates of this transport protein and by 1 mM GSH, and was saturable, with apparent Km values of 0.27 +/- 0.06 and 408 +/- 95 microM, respectively. Uptake of [3H]taurocholate, an endogenous substrate of oatp1, was competitively inhibited by DNP-SG. Of significance, oatp1-mediated taurocholate and LTC4 uptake was cis-inhibited and trans-stimulated by GSH, and [3H]GSH efflux was enhanced in the presence of extracellular taurocholate or sulfobromophthalein, indicating that GSH efflux down its large electrochemical gradient provides the driving force for uptake via oatp1. The stoichiometry of GSH/taurocholate exchange was 1:1. These findings identify a new class of substrates for oatp1 and provide evidence for GSH-dependent oatp1-mediated substrate transport.     

Modulators of the protein kinase C system influence biliary excretion of cationic drugs

To investigate whether hepatobiliary transport of organic cations is under regulatory control, we studied transport of tri-n-butylmethylammonium in the isolated perfused rat liver and in isolated rat hepatocytes. Transport was investigated in the presence of modulators of the protein kinase C and the cyclic AMP second-messenger system. In the isolated perfused rat liver, it was observed that compounds modulating protein kinase C activity clearly affected the biliary excretion process of the cation tri-n-butylmethylammonium. Phorbol 12-myristate 13-acetate, a compound that directly stimulates protein kinase C, elevated the biliary excretion rate of tri-n-butylmethylammonium in a concentration-dependent manner, reaching a twofold increase at 60 nmol/L of the phorbol ester. The inactive derivative 4 alpha-phorbol 12, 13-didecanoate (60 nmol/L) did not show any effect. Vasopressin (48 nmol/L), a receptor-mediated activator of protein kinase C, stimulated the excretion rate of the cation by about 50%. Staurosporin (1 mumol/L), an inhibitor of protein kinase C, clearly decreased the biliary excretion rate of the cation and also blocked its stimulation by phorbol 12-myristate 13-acetate. Neither phorbol 12-myristate 13-acetate nor vasopressin (at concentrations ranging from 10(-9) to 10(-6) mol/L) affected the initial uptake velocity of tri-n-butylmethylammonium in isolated hepatocytes and isolated perfused livers, whereas staurosporin (1 mumol/L) showed only a modest inhibition of the uptake of the cation. It is inferred that the effect of protein kinase C modulators on hepatobiliary transport of organic cations occurs at the level of carrier-mediated transport in the canalicular membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isoquinoline derivatives isolated from the fruit of Annona muricata as 5-HTergic 5-HT1A receptor agonists in rats: unexploited antidepressive (lead) products

The fruit and the leaves of Annona muricata (Annonaceae) are used in traditional medicine for their tranquillizing and sedative properties. Extracts of the plant have been shown to inhibit binding of [3H]rauwolscine to 5-HTergic 5-HT1A receptors in calf hippocampus, and three alkaloids, annonaine (1), nornuciferine (2) and asimilobine (3), isolated from the fruit have been shown to have IC50 values of 3 microM, 9 microM and 5 microM, respectively, although in ligand-binding studies it was not possible to determine whether interaction of these ligands with the receptor was agonistic or antagonistic. This paper presents the results of functional assays of the alkaloids. The inhibition of cAMP accumulation was tested in NIH-3T3 cells stably transfected with the 5-HT1A receptor from man. None of the alkaloids showed antagonistic properties towards the 5-HT1A receptors because in the antagonistic tests no influence on the forskolin-stimulated increase of cAMP level was detected. Full agonistic properties were measured for all three compounds; the inhibition constants (Ki) for 1, 2 and 3 were < 10 microM. Inhibition of the binding of the radioligand to the 5-HT1A receptor was observed in every ligand-binding assay performed with the alkaloids; the Ki values for 1, 2 and 3 were in the microM range. These results imply that the fruit of Annona muricata possesses anti-depressive effects, possibly induced by compounds 1, 2 and 3, and that in the past potent leads for the development of anti-depressive therapeutics have not been used.