Luminal transport step of para-aminohippurate (PAH): transport from PAH-loaded proximal tubular cells into the tubular lumen of the rat kidney in vivo.

Proximal tubular cells were loaded for 10 s with [3H]para-aminohippurate ([3H]PAH) by microperfusing the peritubular capillaries with Ringer solution containing 0.05 mmol/l PAH. Immediately thereafter [3H]PAH influx from cells into a column of equilibrium solution injected into the oil-filled tubular lumen was measured by re-aspirating the fluid after 1-10 s of contact time. The rise of luminal PAH concentration within 2 s of contact time was almost linear, reaching a luminal/capillary concentration ratio of 1.6 after 2 s and of 3.2 after 5 s. The 2-s PAH concentration ratio was not changed when different manoeuvres were applied to depolarize proximal tubular cells. Also, the 2-s PAH concentration ratio was not influenced by varying the luminal pH from 6.0 to 8.0 or the luminal Cl- concentration from zero to 134 mmol/l or when either 5 mmol/l urate or 25 mmol/l lactate was in the luminal perfusate. A decrease in the 2-s PAH concentration ratio, i.e. trans-inhibition, was observed when 25 or 50 mmol/l HCO3- (-50%) was in the luminal perfusate. Trans-inhibition was also seen with 5 mmol/l of the following substituted benzoates: 2-hydroxy-benzoate (-58%), 2-methoxy-benzoate (-46%), 2-hydroxy-benzoate-acetyl ester (-36%), 2-hydroxy-3,5-dinitro-benzoate (-48%), 3,5-dichloro-benzoate (-49%), and 2,3,5-trichloro-benzoate (-45%). No effect was seen with benzoate, 3-hydroxy-benzoate, 2-chloro-benzoate, 2-nitro-benzoate, 2,5-dinitro-benzoate, 3-sulfamoyl-benzoate and 4-sulfamoyl-benzoate. However, analogues of the latter two compounds possessing two additional side groups, such as furosemide and piretanide, or a hydrophobic moiety, such as probenecid, were inhibitory (by -62, -41 and -49% respectively). Phenoxyacetate had no effect; however, it inhibited if in addition it had three chloro groups, as in 2,4,5-trichlorophenoxyacetate (-71%) or a hydrophobic carbamoyl side group, as in mersalylic acid (salyrgan, -75%). Benzene-sulfonate trans-inhibited (-33%), as did phenolsulfonphthalein (phenol red, -39%) and sulfofluorescein (-55%). However, the trans-inhibitory effect of the corresponding carboxy-compounds was absent (phenolphthalein) or weaker (fluorescein, -42%). The trans-inhibitory effect of the uricosurics ethacrynic acid (-53%), tienilic acid (-55%) indacrinone (-72%) and benzbromarone (-42%) could be attributed to two chloro or bromo side groups on the benzene ring. Other trans-inhibiting uricosuric substances were indomethacin (-42%), sulfinpyrazone (-38%), losartan (-80%) its metabolite EXP 3174 (-55%), and AA 193 (-65%). These organic acids, with pKa values between 2.8 and 4.9, possess chloro and sulfin groups, as well as heterocyclic 5-ring and hydrophobic ring or chain areas. No significant effect was seen with 5 mmol/l PAH, 2-oxo-glutarate, DIDS, cGMP, prostaglandin E2, cortisol, benzylamiloride, pyrazinoic acid and 25 mmol/l lactate. Our data indicate that in situ the secretory luminal PAH transport proceeds in a non-rheogenic fashion, per exclusionem by anion exchange. The observed trans-inhibition of PAH secretion seems to correlate with the affinity for the luminal PAH transporter and, for uricosuric substances, with their uricosuric potency.

Interaction of Alkyl/Arylphosphonates, phosphonocarboxylates and diphosphonates with different anion transport systems in the proximal renal tubule.

Luminal and contraluminal stop-flow microperfusion was applied, and the apparent Ki values (mmol/l) against the luminal phosphate and the contraluminal p-aminohippurate (PAH), sulfate and dicarboxylate transport systems were evaluated. Luminal phosphate transporter: Among the 20 compounds tested only phosphonoformate (foscarnet), etidronate, and clodronate have a good affinity (app.Ki < 1 mmol/l), whereas the 2-naphthylphosphonates, phosphonoacetate, pamidronate, alendronate and aminomethanediphosphonates have a moderate affinity (app.Ki, 1.6-6.0 mmol/l). The other compounds tested had a low (app. Ki > 6 mmol/l) or no affinity. Contraluminal PAH transporter: The hydrophobic phenyl-, benzyl- or 2-naphthylphosphonates have good to moderate affinity, whereas the less hydrophobic alkylphosphonates, the phosphonocarboxylates (except 4-phosphonobutyrate) and all tested diphosphonates show no interaction. Sulfate transporter: 2-Naphthylmethylphosphonate and 2-naphthylmethyldifluorophosphonate have a good affinity (app.Ki

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.

Stereospecificity in contraluminal and luminal transporters of organic cations in the rat renal proximal tubule.

The effect of chirality on the interaction of substrates with the organic cation transporters in the proximal tubule of rat kidney was investigated. The apparent Ki values of the enantiomers/diastereomers of ephedrine and norephedrine and the stereoisomers of deprenyl, tranylcypromine, disopyramide, verapamil, pindolol and quinine/quinidine were determined against the contraluminal organic cation transporter, the luminal H+/organic cation exchanger and the luminal choline+ transporter, using the stop-flow luminal or contraluminal capillary microperfusion method. The ephedrine/norephedrine enantiomers/diastereomers had apparent Ki values against the contraluminal organic cation transporter in the range of 0.8 to 2.4 mM, and only norpseudoephedrine showed significant enantioselectivity. The same substrates had apparent Ki values against the luminal H+/organic cation exchanger between 3.0 and 15.0 mM, and ephedrine, norephedrine and norpseudoephedrine showed enantioselectivity. The Ki values against the luminal choline+ transporter were even higher (7.2-19.1 mM) and demonstrated no enantioselectivity. The verapamil and deprenyl enantiomers showed selectivity against the luminal choline+ transporter, as did quinine and quinidine against the contraluminal organic cation transporter. In all other instances enantioselectivity was not observed. In no case was the difference in the Ki values of the enantiomers/isomers greater than a factor of 3. The data confirm the high degree of nonspecificity of the renal organic cation transporters. Evaluation of three-dimensional molecular models of the ephedrine enantiomers/diastereomers suggests that the spatial orientations of the amino group and, to a lesser extent, the OH group and possibly the terminal CH3 group are of importance for different interactions with the transporters.

Luminal transport system for H+/organic cations in the rat proximal tubule. Kinetics, dependence on pH; specificity as compared with the contraluminal organic cation-transport system.

The efflux of radiolabelled organic cations from the tubular lumen into proximal tubular cells was investigated by using the stop-flow microperfusion method. The efflux rate increased in the sequence: N1-methylnicotinamide (NMeN+) < cimetidine < tetraethylammonium (TEA+) < N-methyl-4-phenylpyridinium (MPP+). Preloading the animals by i.v. infusion or pre perfusion of the peritubular capillaries with NMeN+ increased the efflux rate of MPP+. Luminal efflux was also augmented when the tubular solution was made alkaline with HCO3- or phosphate, whereby HCO3- is more effective than phosphate. Replacement of Na+ by Cs+ showed no effect. With i.v. preloading the animals with NMeN+ and with 25 mM HCO3- in the luminal perfusate the 2-s efflux follows kinetics with a Michaelis constant Km = 0.21 mmol/l and maximal flux Jmax = 0.42 pmol.cm-1.s-1 and a permeability term with P = 37.7 microns2.s-1. Comparing the apparent luminal inhibitory constant values for MPP+ (Kil,MPP+) with the apparent contraluminal Kicl,NMeN+ values of substrates of homologous series, it was found that (1) limitation by molecular size occurs at the contraluminal cell side earlier than at the luminal cell side; (2) affinity increases with hydrophobicity of the substrates at the luminal cell side, with a steeper or equal slope than at the contraluminal cell side; (3) affinity increases with basicity (i.e. pKa values) at the luminal cell side with a steeper slope than at the contraluminal cell side. Taken together, substrates with low hydrophobicity and low basicity interact at the luminal cell side more weakly than at the contraluminal cell side. On the other hand large, hydrophobic substrates have, at the luminal cell side, a higher affinity than at the contraluminal cell side. Many substrates, however, have equal affinity at the luminal and contraluminal cell sides.

Polysubstrates: substances that interact with renal contraluminal PAH, sulfate, and NMeN transport: sulfamoyl-, sulfonylurea-, thiazide- and benzeneamino-carboxylate (nicotinate) compounds.

Some N-containing xenobiotics were recently shown to behave as bisubstrates; that is, they interact with and are transported by both the contraluminal transport system for organic anions (PAH) and the contraluminal transport system for organic cations (NMeN). Thus we determined whether other classes of N-containing substrates, such as sulfamoyl-, sulfonylurea-, thiazide- and benzeneamino-carboxylate (nicotinate) compounds, amongst them diuretics and other drugs, also interact with both transporters. To test this, we applied the stop-flow peritubular capillary perfusion method with initial flux measurements and determined app. Ki values for these substrates on PAH, sulfate and NMeN transport. We found that the following compounds interact with 1) the PAH transporter: benzene carboxylates, benzenesulfonylureas and benzenesulfonamides (as long as their acid pKa value is below 9.5). 2) the sulfate transporter: 2-anilinobenzoates, benzenesulfonylureas, polysubstituted sulfamoylbenzoates and some sulfamoylthiazides with electronegative charge accumulation around an anionic site. 3) the NMeN transporter: anilinobenzoates, sulfamoylbenzoates and benzenesulfonamides, if they bear an N-containing pyridine, pyrrolidine, furylmethylamino or thiazide group. There are, however, exceptions when H-bond formation might be responsible for interaction with that transporter. The data confirm the specificity rule for each transporter and the concept that one and the same substrate can match the requirements for several transporters. Thus the loop diuretics furosemide and piretanide, the thiazide diuretics hydrochlorothiazide, cyclopenthiazide and bendroflumethiazide and the sulfonylureas tolbutamide, chlorpropamide and torasemide interact with all three tested transport systems for PAH, sulfate and NMeN. Therefore, they are able to accomplish complex transport interactions with different transporters.

Bisubstrates: substances that interact with renal contraluminal organic anion and organic cation transport systems. I. Amines, piperidines, piperazines, azepines, pyridines, quinolines, imidazoles, thiazoles, guanidines and hydrazines.

In order to evaluate whether N-containing substrates interact with the organic "anion" (p-aminohippurate, PAH) or only with the organic "cation" (N1-methylnicotinamide, NMeN) transport system or with both, the stop-flow peritubular capillary microperfusion method was applied in the rat kidney in situ and the apparent Ki values of several classes or organic substrate against contraluminal NMeN and PAH transport were determined. Organic "anion" and organic "cation" transport are in inverted commas because neither transporter sees the degree of ionization in bulk solution, and they also accept nonionizable substrates [Ullrich KJ, Rumrich G (1992) Pflügers Arch 421:286-288]. Amines must be sufficiently hydrophobic (phenylethylamine, piperidine, piperazine) in order to interact with NMeN transport. Additional Cl, Br, NO2 or other electronegative groups render them inhibitory towards PAH transport also. Such bisubstrate amines were identified as follows: metoclopramide, bromopride, diphenhydramine, bromodiphenhydramine, verapamil, citalopram, ketamine, mefloquine, ipsapirone, buspirone, trazodone, H7 and trifluoperazine. Imidazole analogues interact with both transporters if they bear sufficiently hydrophobic alkyl or aryl groups or electronegative sidegroups. Bisubstrate imidazole analogues are tinidazole, pilocarpine, clonidine, azidoclonidine and cimetidine. Pyridines and thiazoles interact with the NMeN transporter if they have an additional ring-attached NH2 group. Again with an additional Cl, Br, or NO2 group the aminopyridines and aminothiazoles also become inhibitors for the PAH transporter. Amongst the guanidines only substances with several electronegative side-groups such as guanfacine, amiloride, benzylamiloride and ranitidine, interact with both transporters. Amongst the phenylhydrazines only 4-bromophenylhydrazine interacts with the NMeN transporter and 4-nitrophenylhydrazine with both transporters. Quinoline (isoquinoline) and its amino and hydroxy analogues interact with both transporters, their pKa values correlate directly with the affinity to the NMeN transporter and reciprocally with their affinity to the PAH transporter. In experiments with labelled substrates only the sufficiently hydrophilic cimetidine, amiloride and ranitidine show a saturable transport, which can be inhibited by probenecid (apalcillin) and tetraethylammonium in an additive manner. The highly hydrophobic substrates verapamil, citalopram, imipramine, diltiazem and clonidine enter the cell very fast in an unsaturable and uninhibitable manner, apparently in the undissociated form, since N-methyl-4-phenylpyridinium, which--disregarding its ionization--is similarly hydrophobic, shows a transport behaviour similar to that of tetraethylammonium [Ullrich et al. (1991) Pflügers Arch 419:84-92]. Ethidium bromide and dimidium bromide, which have a permanent cationic quaternary nitrogen and two sufficiently electronegative NH2 groups, also interact with both transporters.(ABSTRACT TRUNCATED AT 400 WORDS)

Bisubstrates: substances that interact with both, renal contraluminal organic anion and organic cation transport systems. II. Zwitterionic substrates: dipeptides, cephalosporins, quinolone-carboxylate gyrase inhibitors and phosphamide thiazine carboxylates; nonionizable substrates: steroid hormones and cyclophosphamides.

In order to test what chemical structure is required for a substrate to interact not only with the contraluminal organic anion (p-aminohippurate, PAH) transporter, but also with the organic cation (N1-methylnicotinamide, NMeN, or tetraethylammonium, TEA) transporter, the stop-flow peritubular capillary perfusion method was applied and app. Ki values were evaluated. Zwitterionic hydrophobic dipeptides not only interact with PAH but also with NMeN transport although with lower inhibitory potency (Ki,PAH = 0.2-1.4; Ki,NMeN 6-14 mmol/l). Amongst the zwitterionic cephalosporins, which all inhibit PAH transport, the amino cephalosporin analogue cefadroxil was identified to interact also with NMeN transport (Ki,PAH = 3.0, Ki,NMeN = 11.2 mmol/l). All zwitterionic naphthyridine and oxochinoline gyrase inhibitors tested inhibit NMeN transport with app. Ki,NMeN values between 1.2 mmol/l and 4.7 mmol/l; the naphthyridine analogues show a good inhibitory potency against PAH transport (Ki,PAH approximately 0.4 mmol/l), the piperazine-containing quinolone analogues have a moderate inhibitory potency (Ki,PAH = 1.1-2.5 mmol/l) and the piperazine-containing pipemidic acid did not inhibit PAH transport at all. Zwitterionic thiazolidine carboxylate phosphamides also interact with both transporters (app. Ki,PAH approximately 3.0; app. Ki,NMeN approximately 18.0 mmol/l). The nonionizable oxo- and hydroxy-group-containing corticosteroid hormones also interact with the two transporters. (a) An OH group in position 21 is necessary for interaction with the PAH transporter, but not for interaction with the TEA transporter. (b) Introduction of an OH group in position 17 alpha abolishes interaction with the TEA transporter, but has different effects with the PAH transporter. (c) Introduction of an OH group in position 6 abolishes interaction with both, the PAH and the TEA transporter. (d) A change of the side-group in position 11 of corticosterone from -OH to -H to = O enhances interaction with the PAH transporter but has no effect on the interaction with the TEA transporter. Nonionizable 4- or 5-androstene analogues inhibit both transporters with app. Ki between 0.16 mmol/l and 0.64 mmol/l, if the steroids are soluble in a concentration greater than 1 mmol/l. Nonionizable oxazaphosphorins with more than one chloroethyl group interact with the PAH transporter with app. Ki between 0.84 mmol/l and 4.9 mmol/l and with the NMeN transporter with app. Ki between 3.2 mmol/l and 18.7 mmol/l. Thus a substrate interacts with both transporters if it is sufficiently hydrophobic, possesses acidic and/or electron-attracting plus basic and/or electron-donating groups, or possesses several electron-attracting nonionizable groups (O, OH, Cl). A certain spatial arrangement of the interacting groups seems to be necessary.

Renal transport mechanisms for xenobiotics: chemicals and drugs.

Using the stopped flow tubular lumen or peritubular capillary microperfusion method, the apparent Ki values of a large number of organic anions and cations against the respective transport systems were evaluated. Thereby the luminal transport system for monocarboxylates (lactate), the contraluminal and luminal transport systems for dicarboxylates (succinate), sulfate, and hydrophobic organic cations (tetraethylammonium or N1-methyl-nicotinamide), as well as contraluminal transport system for hydrophobic organic anions (para-aminohippurate, PAH) were characterized and their specificity determined. There is a partially overlapping substrate specificity between the PAH, dicarboxylate, and sulfate transport systems but also between the PAH and organic cation transport system. Xenobiotics and their metabolites are transported mainly by the organic anion (PAH) and organic cation transport systems. To test the complicated interactions possible a shot injection/urinary excretion method with simultaneous measurement of the intracellular concentration was developed. With this approach it is possible to evaluate (a) whether a substrate is net secreted or net reabsorbed, (b) whether interference with other substrates occurs, (c) whether interference takes place at the luminal or contraluminal cell side, and (d) whether cis-inhibition or trans-stimulation is the predominant mode of interaction. Finally, it will be discussed which ability a substrate must have to penetrate the cell membrane via a transporter, through the lipid bilayer, or both.

Cisplatin nephrotoxicity: site of functional disturbance and correlation to loss of body weight.

We have investigated the effect of an intraperitoneal cisplatin injection (5 mg/kg body weight) into male Wistar rats on: (1) body weight; (2) proximal tubular transport of D-glucose and sulfate across the luminal membrane; (3) transport of p-aminohippuric acid (PAH) and sulfate across the contraluminal membrane; (4) urinary excretion of inulin; (5) urinary excretion and tissue accumulation of sulfofluorescein, and (6) effect of the 'protecting substances', N-Methyl-D-glucamine-dithiocarbamate (NaG), diethyldithiocarbamate, mercaptosuccinate (MS), probenecid, and glycine on parameters 1, 4 and 5. Five days after intraperitoneal application of cisplatin the following effects were observed: (1) body weight was reduced on average by 11% as compared to a 12% increase in control animals; (2) luminal sulfate and D-glucose transport was inhibited correlating with the degree of weight loss; (3) contraluminal PAH transport was also decreased in correlation to the loss of body weight, while contraluminal sulfate transport was not inhibited by cisplatin; (4) inulin excretion was reduced by 45%, the pattern for protection was the same as for the prevention of weight loss; (5) sulfofluorescein (SF) excretion in the urine was reduced by 43%, and (6) accumulation of SF into cortical tissue was augmented. Protecting substances prevented or mitigated weight loss, fall of urinary inulin and SF excretion as well as SF accumulation in cortical tissue with a similar pattern: N-glucamine-dithiocarbamate > mercaptosuccinate approximately probenecid approximately glycine. The data indicate: (1) that luminal (glucose and sulfate) and contraluminal (PAH) transport processes are affected by cisplatin; (2) that contraluminal transport (sulfate) can be unaffected or less affected than luminal transport processes (SF); (3) our method (SF) gives the possibility to monitor the balance between luminal and contraluminal transport steps in vivo; and (4) the correlation of body weight loss with decay of certain renal transport functions and their prevention with similar protecting patterns indicates that a simple index might be useful to monitor the cytotoxic status of an individual.

Renal contraluminal transport systems for organic anions (paraaminohippurate, PAH) and organic cations (N1-methyl-nicotinamide, NMeN) do not see the degree of substrate ionization.

Using the stop-flow peritubular capillary microperfusion method pH dependence of the interaction of different substrates with the contraluminal PAH- and NMeN transporter was investigated. Substrates for both transport systems with pKa values around 7.0 were chosen and the pH of the perfusates was varied between 6.0 and 8.0. The inhibitory potencies (app. Ki values) were determined and the influx into the proximal tubular cells was measured. The app. Ki(NMeN) values of imidazole (pKa 7.03), a substrate for the NMeN-transporter, the app. KiPAH values of the dipeptide tryptophyl-tryptophan (pKa 7.36), a substrate for the PAH-transporter, and the app. Ki,NMeN and Ki,PAH of cimetidine (pKa 6.98) and buspirone (pKa 7.2) which interact with both transport systems, did not vary between perfusate pH 6.0 and 8.0. The same holds for the influx of 3H-cimetidine into proximal tubular cells. The data indicate that both transporters have no preference for the ionized form of their substrates and that the name organic anion and organic cation transporter resides rather on history than on molecular interaction.

Contraluminal transport of organic cations in the proximal tubule of the rat kidney. II. Specificity: anilines, phenylalkylamines (catecholamines), heterocyclic compounds (pyridines, quinolines, acridines).

In order to study the quantitative structure/activity relationship of organic cation transport across the contraluminal side of the proximal renal tubule cell, the stopped-flow capillary microperfusion method was applied and the inhibitory potency (apparent Ki values) of different homologous series of substrates against N1-[3H]methylnicotinamide (NMeN+) transport was evaluated. Aniline and its ring- or N-substituted analogues as well as the aminonaphthalines do not interact with the contraluminal NMeN+ transporter except for the quaternary trimethylphenylammonium and pararosaniline, which bear a permanent positive charge, and for 1,8-bis-(dimethylamino)naphthaline, which forms an intramolecular hydrogen bond. If, however, one or more than one methylene group is interposed between the benzene ring and the amino group, the compounds interact with the contraluminal NMeN+ transporter in proportion to their hydrophobicity parameter, i.e. the octanol/water partition coefficient (log octanol). The catecholamines and other hydroxyl-substituted phenylethyl analogues also follow this rule. In addition, the N-heterocyclic pyridine, quinoline, isoquinoline and acridine analogues also interact with the contraluminal NMeN+ transporter, when their pKa values are higher than 5.0, and, an inverse correlation between pKa and log Ki,NMeN was observed. An exception to this rule are those hydroxy compounds of pyridine, quinoline and isoquinoline that show tautomerism. These compounds slightly inhibit NMeN+ transport despite low pKa values. The quaternary nitrogen compounds of aniline and the N-heterocyclic analogues, as far as tested, all interact with the contraluminal NMeN+ transporter in relation to their hydrophobicity. The data indicate that the contraluminal NMeN+ transporter interacts with N-compounds according to their hydrophobicity and/or according to their basicity (affinity to protons). The reason for deviation of the aniline analogues and the OH-tautomeric heterocyclic N-compounds from this behaviour is discussed.

Contraluminal transport of organic cations in the proximal tubule of the rat kidney. I. Kinetics of N1-methylnicotinamide and tetraethylammonium, influence of K+, HCO3-, pH; inhibition by aliphatic primary, secondary and tertiary amines and mono- and bisquaternary compounds.

In order to study the characteristics of contraluminal organic cation transport from the blood site into proximal tubular cells the stopped-flow capillary perfusion method was applied. The disappearance of N1-[3H]methylnicotinamide (NMeN+) and [3H]tetraethylammonium (TEA+) at different concentrations and contact times was measured and the following parameters evaluated: Km,NMeN = 0.54 mmol/l, Jmax,NMeN = 0.4 pmol s-1 cm-1; Km,TEA = 0.16 mmol/l, Jmax,TEA = 0.8 pmol s-1 cm-1. TEA+ inhibited NMeN+ transport and NMeN+ the uptake of TEA+. Thereby, the Ki values for inhibition correspond closely to the Km values for uptake. Similar inhibitory potencies of ten organic cation against TEA+ and NMeN+ transport provide further evidence for a common transport system. Omission of HCO3-, or Na+ and addition of K+ (with or without Ba2+) reduce NMeN+ transport, while omission of K+ (with or without valinomycin) or addition of thiocyanate has no effect. Since the manoeuvres that depolarize contraluminal electrical potential difference reduce NMeN+ transport, cell-negative electrical potential difference is suggested as a driving force for contraluminal organic cation transport from the interstitium into the cell. Furthermore, the inhibitory potency (app. Ki values) of homologous series of primary, secondary, tertiary and hydroxy amines as well as of mono- and bisquaternary ammonium compounds against NMeN+ transport was tested. The inhibitory potency increased in the sequence methyl less than ethyl less than propyl less than butyl and primary less than secondary less than tertiary amines less than quaternary ammonium compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraluminal p-aminohippurate transport in the proximal tubule of the rat kidney. VIII. Transport of corticosteroids.

Using the stop-flow peritubular capillary microperfusion method contraluminal transport of corticosteroids was investigated (a) by determining the inhibitory potency (apparent Ki values) of these compounds against p-aminohippurate (PAH), dicarboxylate (succinate) and sulphate transport and (b) by measuring the transport rate of radiolabelled corticosteroids and its inhibition by probenecid. Progesterone did not inhibit contraluminal PAH influx but its 17 alpha- and 6 beta-hydroxy derivatives inhibited with an app. Ki of 0.36 mmol/l. Introduction of an OH group in position 21 of progesterone, to yield 11-deoxycorticosterone, augments the inhibitory potency considerably (app. Ki, PAH of 0.07 mmol/l). Acetylation of the OH-group in position 21 of 11-deoxycorticosterone, introduction of an additional hydroxy group in position 17 alpha to yield 11-deoxycortisol or in position 11 to yield corticosterone brings the app. Ki, PAH back again into the range of 0.2-0.4 mmol/l. Acetylation of corticosterone or introduction of a third OH group to yield cortisol does not change the inhibitory potency, but, omission of the 21-OH group or addition of an OH group in the 6 beta position reduces or abolishes it. Cortisol and its derivatives prednisolone, dexamethasone and cortisone exert similar inhibitory potencies (app. Ki, PAH 0.12-0.27 mmol/l). But again, omission of the 21-OH group in cortisone or addition of a 6 beta-OH group reduces or even abolishes the inhibitory potency against PAH transport. The interaction of corticosterone was not changed when 11 beta, 18-epoxy ring (aldosterone) was formed. On the other hand, the interaction was considerably augmented if the 11-hydroxy group was changed to an oxo group in 11-dehydrocorticosterone (app. Ki, PAH 0.02 mmol/l). When the A ring of corticosterone is saturated and reduced to 3 alpha, 11 beta-tetrahydrocorticosterone the inhibitory potency is not changed very much. But if more than four OH or oxo groups are on the pregnane skeleton or if the OH in position 21 is missing, the inhibitory potency decreases drastically (app. Ki, PAH 0.7-1.7 mmol/l). Introduction of a 21-ester sulphate into corticosterone, cortisol and cortisone does not change app. Ki, PAH very much. Glucuronidation, however, reduces it (app. Ki, PAH approximately 1.2 mmol/l). None of the tested corticosteroids interacts, in concentrations applicable, with dicarboxylate transport and only the sulphate esters interact with sulphate transport. Radiolabelled cortisol, D-aldosterone, 11-dehydrocorticosterone, and corticosterone are rapidly transported into proximal tubular cells. With the latter three compounds no sign of saturation and no transport inhibition with probenecid could be seen.(ABSTRACT TRUNCATED AT 400 WORDS)

Contraluminal p-aminohippurate transport in the proximal tubule of the rat kidney. VII. Specificity: cyclic nucleotides, eicosanoids.

Using the stop-flow peritubular capillary microperfusion method the inhibitory potency (apparent Ki values) of cyclic nucleotides and prostanoids against contraluminal p-aminohippurate (PAH), dicarboxylate and sulphate transport was evaluated. Conversely the contraluminal transport rate of labelled cAMP, cGMP, prostaglandin E2, and prostaglandin D2 was measured and the inhibition by different substrates was tested. Cyclic AMP and its 8-bromo and dibutyryl analogues inhibited contraluminal PAH transport with an app. Ki,PAH of 3.4, 0.63 and 0.52 mmol/l. The respective app. Ki,PAH values of cGMP and its analogues are with 0.27, 0.04 and 0.05 mmol/l, considerably lower. None of the cyclic nucleotides tested interacted with contraluminal dicarboxylate, sulphate and N1-methylnicotinamide transport. ATP, ADP, AMP, adenosine and adenine as well as GTP, GDP, GMP, guanosine and guanine did not inhibit PAH transport while most of the phosphodiesterase inhibitors tested did. Time-dependent contraluminal uptake of [3H]cAMP and [3H]cGMP was measured at different starting concentrations and showed facilitated diffusion kinetics with the following parameters for cAMP: Km = 1.5 mmol/l, Jmax = 0.34 pmol S-1 cm-1, r (extracellular/intracellular amount at steady state) = 0.91; for cGMP: Km = 0.29 mmol/l, Jmax = 0.31 pmol S-1 cm-1, r = 0.55. Comparison of app. Ki,cGMP with app. Ki,PAH of ten substrates gave a linear relation with a ratio of 1.83 +/- 0.5. All prostanoids applied inhibited the contraluminal PAH transport; the prostaglandins E1, F1 alpha, A1, B1, E2, F2 alpha, D2, A2 and B2 with an app. Ki,PAH between 0.08 and 0.18 mmol/l. The app. Ki of the prostacyclins 6,15-diketo-13,14-dihydroxy-F1 alpha (0.22 mmol/l) and Iloprost (0.17 mmol/l) as well as that of leukotrienes B4 (0.2 mmol/l) was in the same range, while the app. Ki,PAH of the prostacyclins PGI2 (0.55 mmol/l), 6-keto-PGF1 alpha (0.77 mmol/l) and 2,3-dinor-6-keto-PGF1 alpha (0.57 mmol/l) as well as that of thromboxane B2 (0.36 mmol/l) was somewhat higher. None of these prostanoids inhibited contraluminal dicarboxylate transport and only PGB1, E2 and D2 inhibited contraluminal sulphate transport (app. Ki,SO4(2-) 5.4, 11.0, 17.9 mmol/l respectively). Contraluminal influx of labelled PGE2 showed complex transport kinetics with a mixed Km = 0.61 mmol/l and Jmax of 4.26 pmol S-1 cm-1. It was inhibited by probenecid, sulphate and indomethacin. Contraluminal influx of PGD2, however, was only inhibited by probenecid. The data indicate that cyclic nucleotides as well as prostanoids are transported by the contraluminal PAH transporter. For prostaglandin E2 a significant uptake through the sulphate transporter occurs in addition.(ABSTRACT TRUNCATED AT 400 WORDS)

Contraluminal para-aminohippurate (PAH) transport in the proximal tubule of the rat kidney. VI. Specificity: amino acids, their N-methyl-, N-acetyl- and N-benzoylderivatives; glutathione- and cysteine conjugates, di- and oligopeptides.

In order to evaluate the specificity of the renal contraluminal PAH transport system for amino acids, oligopeptides and their conjugates, the inhibitory potency of these substances against contraluminal [3H] PAH influx has been determined. For this, inhibition of 3H-PAH flux from the interstitium into cortical tubular cells of the rat kidney in situ has been measured. Apparent Ki values were evaluated by a computer program assuming competitive inhibition. Unconjugated amino acids (glycine, cysteine, alanine, leucine, phenylalanine, tyrosine, aspartate, glutamate, arginine, ornithine and lysine) do not inhibit [3H] PAH influx. The very hydrophobic tryptophan, however, does. N-alpha-methylation does not change this behaviour. N-alpha-acetylation does not evoke interaction with the PAH transporter when it occurs with glycine, cysteine (to yield mercapturic acid), arginine, ornithine and lysine. However, it renders alanine, leucine, phenylalanine, tryptophan, L-aspartate moderately, and L-glutamate strongly, inhibitory. The acetylated D-isomers of alanine, leucine and phenylalanine exert a higher inhibitory potency compared with the respective L-isomers. N-alpha-benzoylation of L-lysine is ineffective. N-alpha-benzoylation, however, evokes interaction with the PAH transporter, when it occurs with ornithine less than arginine less than histidine less than glycine = leucine less than alanine = phenylalanine = aspartate = glutamate. Dipeptides interact with the PAH transporter according to their hydrophobicity (Nozaki scale down to 0.9, Fauchère scale up to 1.0). N-acetylation does not change this behaviour. Hydrophobicity also renders oligopeptides, as angiotensin II, inhibitory against PAH transport. Similarly the anionic angiotensin I converting enzyme inhibitors Captopril, Enalapril and Ramipril inhibit contraluminal PAH influx.

Contraluminal organic anion and cation transport in the proximal renal tubule: V. Interaction with sulfamoyl- and phenoxy diuretics, and with beta-lactam antibiotics.

In order to study the interaction of sulfamoyl- and phenoxy diuretics as well as of beta-lactam antibiotics with the contraluminal anion and cation transport systems the inhibitory potency of these substances against the influx of 3H-para-aminohippurate, 14C-succinate, 35S-sulfate and 3H-N1-methylnicotinamide into cortical tubular cells have been determined. 1.) 2-, 3- and 4-sulfamoylbenzoate inhibit contraluminal PAH influx. N-dipropyl substitution to yield probenecid or ring-substitution to yield furosemide and piretanide augment the inhibitory potency. However, hydrochlorothiazide and acetazolamide exert only a moderate inhibitory potency. Succinate transport was inhibited by furosemide only. Sulfate transport was inhibited by furosemide and 3-sulfamoyl-4-phenoxybenzoate as well as by probenecid, piretanide, hydrochlorothiazide and acetazolamide. 2.) Phenoxyacetate, -propionate, and -butyrate exert increasing inhibition against PAH transport. The weed-killers 2,4-dichloro-, and 2,4,5-trichlorophenoxyacetate (2,4 D and 2,4,5 T) had a similar inhibitory potency, while ethacrynic acid showed a lower and the uricosuric tienilic acid a higher inhibitory potency. None of the compounds of this group interact with contraluminal succinate transport, and only the multiring-substituted compounds 2,4 D, 2,4,5 T, ethacrynic and tienilic acid interact slightly with the sulfate transporter. 3.) The monocarboxylic penicillins benzylpenicillin and phenoxymethylpenicillin as well as the dicarboxylic ticarcillin interact with the contraluminal PAH transport. The aminopenicillin ampicillin had a lower, and apalcillin a higher inhibitory potency than monocarboxylic penicillin. Benzylpenicillin showed small inhibition against succinate transport and ticarcillin against sulfate transport. 4.) The monocarboxylic cephalosporine, 6315 S Shionogi, and the aminocephalosporines, cephalexin and cefadroxil, showed an app. Ki.PAH as the comparable penicillins. The zwitterions cephaloridine and cefpirome did not interact with the PAH transporter, but with the organic cation (NMN) transporter. Amongst the amino-thiazol-containing compounds cefotaxime, ceftriaxone, and cefodizime, increasing interaction with the PAH transporter was seen dependent of a second ionizable anionic group. Compounds with two ionizable anionic groups (cefsulodin, ceftriaxone, cefodizime) exert also a small inhibitory potency against sulfate transport. None of the cephalosporins interacted with the dicarboxylate transporter. The interaction pattern of the tested compounds is in accordance with the specificity requirements for the contraluminal transporters depending on electrical charge and hydrophobicity.

Anion transport through the contraluminal cell membrane of renal proximal tubule. The influence of hydrophobicity and molecular charge distribution on the inhibitory activity of organic anions.

Three different mechanisms of anion transport have been identified for the contraluminal membrane in the proximal tubule of the rat kidney. These mechanisms are specific for the transport of sulfate, dicarboxylate and p-aminohippurate anions. Sulfate transport is inhibited by bivalent organic anions with a distance between the charges of less than 7 A. The sulfate system acts in two modes: in a planar mode for anions with flat charged residues such as COO- and a charge separation of 3-4 A or in a bulky mode for groups such as SO3H- and a charge separation of 4-7 A. Monovalent anions can be accepted if there is a hydrophobic core next to the negative charges. Dicarboxylate transport is inhibited exclusively by anions with two charge centers located within 5 to 9 A, one of those possibly being a partial charge of -0.5 elementary charges. p-Aminohippurate transport is inhibited by monovalent anions, if these have a hydrophobic domain with a minimal length of about 4 A. Bivalent anions inhibit, if they have a charge distance of 6-10 A; both charges can be partial charges of about -0.5 elementary charges. Longer bivalent anions can be effective provided they have a sufficiently large hydrophobic domain. For the sulfate and p-aminohippurate systems it is found that anions with high acidity yield good inhibition. The overlapping specificities of the three systems with respect to charge distance and hydrophobicity allow them to accept a large variety of organic anions.