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Notes: Summary 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 N 1-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.

Notes: Abstract 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 (N 1-methylnicotinamide, NMeN, or tetraethylammonium, TEA) transporter, the stop-flow peritubular capillary perfusion method was applied and app. K i values were evaluated. Zwitterionic hydrophobic dipeptides not only interact with PAH but also with NMeN transport although with lower inhibitory potency (K i,PAH=0.2–1.4; K i,NMeN 614 mmol/l). Amongst the zwitterionic cephalosporins, which all inhibit PAH transport, the amino cephalosporin analogue cefadroxil was identified to interact also with NMeN transport (K i,PAH = 3.0, K i,NMeN=11.2 mmol/l). All Zwitterionic naphthyridine and oxochinoline gyrase inhibitors tested inhibit NMeN transport with app. K i,NMeN values between 1.2 mmol/l and 4.7 mmol/l; the naphthyridine analogues show a good inhibitory potency against PAH transport (K i,PAH ≈ 0.4 mmol/l), the piperazine-containing quinolone analogues have a moderate inhibitory potency (K i,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. K i,PAH ≈ 3.0; app. K i,NMeN ≈ 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 17α 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. K i 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. K i between 0.84mmol/l and 4.9mmol/l and with the NMeN transporter with app. K i 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.

Notes: Abstract In order to evaluate whether N-containing substrates interact with the organic “anion” (p-aminohippurate, PAH) or only with the organic “cation” (N 1-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 K i 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. The data indicate that a molecule qualifies as a bisubstrate if it carries both the essentials for organic anion (PAH) transport: hydrophobicity, sufficient acidity or electron-attracting O, OH, Cl, Br, NO2 groups, plus the essentials for organic cation transport: hydrophobicity, sufficient basicity or electron-donating N-containing groups. The nitrogen atoms in the N-containing molecules quinoline (pK a 4.9), isoquinoline (pK a 5.4) and benzylpyridine (pK a 5.13) are of such low basicity that they apparently can also interact with the PAH transporter. Apparent hydrophobicity (disregarding ionization) determines interaction with the transporters, while real hydrophobicity [log (octanol distribution values)] determines the diffusion through the lipid bilayer of the cell membrane.

Notes: Abstract Using the stop-flow peritubular capillary microperfusion method the inhibitory potency (apparent K i 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. K i, PAH of 3.4, 0.63 and 0.52 mmol/l. The respective app. K i,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 N 1-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: K m=1.5 mmol/l, J max=0.34 pmol s−1 cm−1, r (extracellular/intracellular amount at steady state)=0.91; for cGMP: K m=0.29 mmol/l, J max=0.31 pmol s−1 cm−1, r=0.55. Comparison of app. K i, cGMP with app. K i, 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, F1α, A1, B1, E2, F2α, D2, A2 and B2 with an app. K i, PAH between 0.08 and 0.18 mmol/l. The app. K i of the prostacyclins 6,15-diketo-13,14-dihydroxy-F1α (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. K i, PAH of the prostacyclins PGI2 (0.55 mmol/l), 6-keto-PGF1α (0.77 mmol/l), and 2,3-dinor-6-keto-PGF1α (0.57 mmol/l) as well as that of thromboxane Bin2 (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. $$K_{i,SO_4^{2--} } $$ 5.4, 11.0, 17.9 mmol/l respectively). Contraluminal influx of labelled PGE2 showed complex transport kinetics with a mixed K m=0.61 mmol/l and J max 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. The hypothesis that prostaglandins as well as 8-bromo and dibutyryl cyclic nucleotides permeate cell membranes by simple diffusion because of their lipid solubility must be considered with reservation.

Notes: Abstract 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 N 1-[3H]methylnicotinamide (NMeN+) and [3H]tetraethylammonium (TEA+) at different concentrations and contact times was measured and the following parameters evaluated: K m,NMeN = 0.54 mmol/l, J max,NMeN = 0.4 pmol s−1 cm−1; K m,TEA = 0.16 mmol/l, J max,TEA = 0.8 pmol s−1 cm−1. TEA+ inhibited NMeN+ transport and NMeN+ the uptake of TEA+. Thereby, the K i values for inhibition correspond closely to the K m 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 HCO 3 − , 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. K i values) of homologous series of primary, secondary, tertiary and hydroxy amines as well as of mono- and bisquarternary ammonium compounds against NMeN+ transport was tested. The inhibitory potency increased in the sequence methyl 〈 ethyl 〈 propyl 〈 butyl and primary 〈 secondary 〈 tertiary amines 〈 quarternary ammonium compounds. With the amines a reversed correlation between K i,NMeN and the octanol/water partition coefficient (log octanol) is seen. With quarternary ammonium compounds the inhibitory potency decreases with increasing molecular size: tetrabutyl- 〉 tetrapentyl- 〉 tetrahexyl- 〉 tetraheptyl 〉 tetraoctylammonium. Introducing two OH groups into triethylamine reduces the inhibitory potency while introduction of two OH groups into diethylamine or three OH groups into triethylamine abolishes the inhibitory potency as a result of reduced hydrophobicity. With choline (trimethylethanolamine) and its analogues the reversed correlation between K i,NMeN and log octanol was also seen. Molecules with a similar hydrophobic moiety to those of the monoammonium compounds, but with two ammonium groups, showed only a small or no inhibitory potency against NMeN+ transport. The data indicate that (a) hydrophobic moieties are important for the interaction with the contraluminal organic cation transporter, and (b) the size of the molecule can be a limiting factor. The reduced or missing interaction of the bisquarternary compound might be caused either by the second charge and/or reduced hydrophobicity and/or too large size of a molecule.

Notes: Abstract A previous observation of decreased serum carnitine concentrations in phenylketonuria (PKU) was investigated in 169 patients either on a strict diet (n=107; median: 8.1 years) or off diet (n=62; median: 15.0 years). Fifty-seven metabolically healthy children (median: 8.5 years) served as controls. PKU patients on a strict diet and older than 2 years had significantly lower serum carnitine concentrations (19.4±5.4 μmol/l) than those off diet (29.6±6.7 μmol/l). PKU patients on diet also had significantly lower concentrations of haemoglobin and serum ferritin than those off diet. A linear correlation existed between total serum carnitine and ferritin concentrations up to 40 μg/l (r=0.52;P〈0.01). As iron is an essential cofactor of carnitine synthesis we conclude that reduced endogenous carnitine synthesis due to an inadequate availability of iron may be a major cause of low serum carnitine concentrations. The low carnitine content of the strict and highly protein-reduced diet additionally contributes to a decrease in the serum carnitine concentration. Our results show that a further optimization of the PKU diet increasing either iron availability or carnitine intake should be considered.

Notes: Abstract In a retrospective study, 34 early treated, normally intelligent adolescents with phenylketonuria (PKU) and their parents were tested with several psychometric personality inventories and self-developed questionaires concerning their psychosocial situation and their disease-and diet-specific knowledge. Results show that the patients are characterized by less autonomy, a more negative evaluation of their scholastic ability, less achievement motivation, less extraversion and impulsiveness, a feeling of not being quite healthy, more grave and a higher level of dependency from their families. The patients saw their whole social situation as being destinctly restricted. Their knowledge concerning disease and diet was alarmingly poor and the majority had great difficulties in managing the diet satisfactorily without parental help. Up to the age of 15 years the serum phenylalanine levels were persistently above the desired range.

Notes: Abstract The daily Phe intakes of normally growing 1- to 6-year-old treated PKU patients were evaluated. The children received protein in amounts that varied from 2.26±0.47 g/kg body weight per day (mean±SD) at the age of 6 to 1.81±0.35 at the age of 72 months. Mean Phe intakes declining from 34±7 at the age of 6 months to 15±5 mg/kg body weight per day at the age of 72 months were required to maintain mean median plasma Phe levels around 6.0 mg/dl.