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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 technique of capillary perfusion and simultaneous luminal stop flow microperfusion the reabsorption of bicarbonate and glycodiazine from the papillary collecting duct was evaluated. Starting with equal H14CO 3 − and3H-glycodiazine concentrations in the luminal and peritubular perfusates, the decrease in the luminal concentration at 10 and 45 s contact time was measured. In control rats with 25 mmol/l HCO 3 − in the perfusates the rate of HCO 3 − reabsorption calculated from the 10 s values was 0.34 nmol cm−2s−1. In acute metabolic acidosis, the rate of bicarbonate reabsorption was 2,3 times higher. In metabolic alkalosis, the rate of bicarbonate absorption dropped to 13% of the control values. Also the 45 s values of acidotic and alkalotic animals differed significantly from each other. With 25 mmol/l glycodiazine in both perfusates the rate of biffer reabsorption as calculated from the 10 s values was 0.76 nmol cm−2s−1 in control rats and did not deviate significantly from this value in acidotic and alkalotic animals. In control rats the bicarbonate reabsorption in % was the same, no matter whether both luminal and capillary perfusate contained 25 mmol/l bicarbonate or 10 mmol/l. In acidotic rats the rate of HCO 3 − reabsorption did not change significantly if all Na+ in the perfusates was replaced by choline (0.88 versus 0.79 nmol cm−2s−1 at 25 mmol/l HCO 3 − ). When in acidotic rats 0.1 mmol/l acetazolamide or 1 mmol/l SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid) was added to both perfusates the rate of HCO 3 − reabsorption dropped by 75 and 58%, respectively. A potassium deficient diet for one week and DOCA administration had no influence on the bicarbonate reabsorption of rats which were on standard diet. The data indicate that (1) the buffer reabsorption from the papillary collecting duct is rather due to H+ ion secretion than to buffer anion reabsorption. (2) The adaptation to metabolic acidosis and alkalosis is specific for bicarbonate and not seen with glycodiazine. (3) Within the concentration range tested the HCO 3 − reabsorption rises linearly with the HCO 3 t- concentration. (4) The HCO 3 − reabsorption in the papillary collecting duct is Na+-independent, it can be inhibited by acetazolamide and SITS, but is not influenced by K+-deficient diet plus DOCA.

Notes: Abstract 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: N 1-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 HCO 3 − or phosphate, whereby HCO 3 − 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 HCO 3 − in the luminal perfusate the 2-s efflux follows kinetics with a Michaelis constant K m=0.21 mmol/l and maximal flux J max=0.42 pmol · cm−1 · s−1 and a permeability term with P=37.7 μm2 · s−1. Comparing the apparent luminal inhibitory constant values for MPP+ $$(Ki_{l,MPP^ + } )$$ with the apparent contraluminal $$Ki_{cl,NMeN^ + }$$ 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.

Notes: Abstract Using the shrinking droplet method and simultaneous perfusion of the peritubular capillaries the isotonic reabsorption of Ringer's solution from the papillary collecting ducts was measured. Under control conditions the volume reabsorption from the papillary collecting ducts wasJ v±SE=2.6±0.1 · 10−5 cm3 · cm−2 · s−1. In rats which were on low Na+ diet,J v increased to 127%, and in adrenalectomized animals it decreased to 34% of the control value. Three hours after application of aldosterone in the adrenalectomized animalsJ v was partially restored to 63% of control rats. Amiloride 10−4 M, added to the luminal perfusate, produced a strong inhibition ofJ v (to 32% of control). Acetazolamide, 10−4 M, added to both perfusates, reducedJ v very strongly (to 40% of control), while omission of bicarbonate reduced it only to 77% of control. Acetazolamide, added to bicarbonate-free perfusates, did not result in a significant further reduction ofJ v. The data indicate that the Na+ reabsorption from the papillary collecting duct is controlled by mineralocorticoids. Furthermore, they suggest the existence of two transport mechanisms in the luminal cell membrane: 1. An amiloride-sensitive entry step and 2. an entry step via a Na+−H+-countertransport mechanism, the latter being less important.

Notes: Abstract The transport ofd-lactate across the epithelium of the late proximal convolution was investigated by two methods: 1. by measuring the zero net flux transtubular concentration difference (Δc tt,45s) and the permeability (P) ofd-lactate and calculating from both the transtubular active transport rate (J lac act ). 2. By measuring the 3.5 s efflux ofd-lactate from the tubular lumen, while blood was flowing through the capillaries. The 3.5 s efflux comprises two components, one going through the brush border (J lac bb ) and one going the paracellular pathway (J lac paracell =P lac·c lac lumen). Both,J lac act andJ lac bb ofd-lactate gave the sameK m 1.9 and 1.7 mmol/l and the same maximal transport rate 3.2 and 2.9 pmol cm−1 s−1. TheK i ofl-lactate tested againstJ lac act andJ lac bb ofd-lactate was also the same: 1.1 and 1.0 mmol/l. These data indicate that under our experimental conditions only the flux through the brush border seems to be rate limiting and thatd-lactate uses the same transport system asl-lactate. When Na+ was omitted from the perfusatesJ lac act disappeared completely, whileJ lac bb was reduced by 64%. These data reflect the Na+ dependence of thed-lactate transport through the brush border. Variation of intra-and extracellular pH by raisingpCO2, omitting HCO 3 − from the perfusates or adding acetazolamide had no effect on the transport ofd-lactate when α-ketoglutarate was used as fuel. However, when acetate was used as fuel, intracellular acidosis brought the reducedJ lac act back to the values obtained with α-ketoglutarate as fuel. It is suggested that this is an effect on a contraluminal transport step. Probenecid (5 mmol/l) and phloretin (0.25 mmol/l) inhibitedJ lac act significantly.J lac bb , however, was only inhibited by probenecid when acetate was used as fuel. These data indicate that both compounds act on thed-lactate exit at the contraluminal cell side, but that probenecid acts in addition at the luminal cell side. SITS (1 mmol/l) augmentedJ lac bb when acetate was used as fuel and is similar to the effect of lowering intracellular pH as described above. The SH reagents mersalyl (1.0 mmol/l) and maleolylglycine (1 mmol/l) did not influenceJ lac bb .

Notes: Summary The rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (α-methyl-d-glycoside), H+ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (Δc) of these substances. In the case of calcium the electrochemical potential differenceΔc +zFci Δϕ/RT) was the criterion employed. The rate of isotonic Na+-absorption (JNa) was measured with the shrinking droplet method. The effect of ouabain on the transport of these substances was tested in the golden hamster and the effect of SITS (4-acetamido-4′isothiocyanatostilbene 2,2′-disulfonic acid) was observed in rats. Ouabain (1 mM) applied peritubularly incompletely inhibited JNa (80%), but in combination with acetazolamide (0.2 mM) the inhibition was almost complete (93%). In addition, ouabain inhibited the sodium coupled (secondary active) transport processes ofl-histidine, α-methyl-d-glycoside, calcium and phosphate by more than 75%. It did not affect H+ (glycodiazine) transport and PAH transport was only slightly affected. When SITS (1 mM) was applied from both sides of the cell it inhibited H+ (glycodiazine) transport by 72% and reduced JNa by 38% when given from only the peritubular cell side. SITS (1 mM), however, had no significant affect on H+ secretion and sodium reabsorption if it was applied from only the luminal side. Furthermore it had no affect on the other transport processes tested, regardless of the cell side to which it was applied. When the HCO 3 − buffer or physically related buffers were omitted from the perfusate the absorption of Na+ was reduced by 66%, phosphate by 44%, andl-histidine by 15%. All the other transport processes tested were not significantly affected. The data are consistent with the hypothesis that the active transport processes of histidine, α-methyl-d-glycoside and phosphate, which are located in the brush border, are driven by a sodium gradient which is abolished by ouabain. This may also apply to the Na+-Ca2+ countertransport located at the contraluminal cell side. The residual Na+ transport remaining in the presence of ouabain is likely to be passively driven by the continuing H+ transport which probably is driven directly by ATP. SITS seems to inhibit the exit step of HCO 3 − from the cell and secondary to that, the luminal H+-Na+ exchange and consequently the Na+ reabsorption. In the absence of HCO 3 − buffer in the perfusates the luminal H+-Na+ exchange seems to be affected and the pattern of inhibition of the other transport processes is almost the same as with SITS. The different effects onP i reabsorption observed under these conditions might be explained by possible variations in intracellular pH.