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Therapeutic value of the alkylating agent ifosfamide has been limited by major side effects including encephalopathy. Although the underlying biochemical processes of the neurotoxic side effects are still unclear, they could be attributed to metabolites rather than to ifosfamide itself. In the present study, the effects of selected ifosfamide metabolites on indices of neuronal activity have been investigated, in particular for S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA). Because of structural similarities of SCMC with glutamate, the Ca(2+)(i) response of single mouse cortical neurons to SCMC and TDGA was investigated. SCMC, but not TDGA, evoked a robust increase in Ca(2+)(i) concentration that could be abolished by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but only partly diminished by the N-methyl-D-aspartate receptor antagonist 10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK=801). Cyclothiazide (CYZ), used to prevent AMPA/kainate receptor desensitization, potentiated the response to SCMC. Because activation of AMPA/kainate receptors is known to induce proton influx, the intracellular pH (pH(i)) response to SCMC was investigated. SCMC caused a concentration-dependent acidification that was amplified by CYZ. Since H(+)/monocarboxylate transporter (MCT) activity leads to similar cellular acidification, we tested its potential involvement in the pH(i) response. Application of the lactate transport inhibitor quercetin diminished the pH(i) response to SCMC and TDGA by 43 and 51%, respectively, indicating that these compounds may be substrates of MCTs. Taken together, this study indicates that hitherto apparently inert ifosfamide metabolites, in particular SCMC, activate AMPA/kainate receptors and induce cellular acidification. Both processes could provide the biochemical basis of the observed ifosfamide-associated encephalopathy.

MDCK-I cells, grown on collagen-coated filters, were used as a model of the distal nephron to study the nonionic diffusion of salicylic acid. The apparent transcellular permeability (PSal, centimeters per second) and intracellular content (ContSal, picomoles) of [14C]salicylic acid (ionized and nonionized) were measured from both apical-to-basolateral and basolateral-to-apical unidirectional fluxes. During the measurement of apical-to-basolateral fluxes at 21 degrees C, both PSal and ContSal correlated directly with the proportion of nonionized salicylic acid in the apical medium over the pH range 6.0-7.4. This indicated that the observed permeability was due to pure nonionic diffusion. However, in the basolateral-to-apical direction, the correlation was less evident and ContSal was 10 to 20 times higher, although PSal was of the same magnitude. These observations could not be explained by facilitated transport of anionic salicylic acid, at 21 degrees C, because 1 mM probenecid, 1 mM p-aminohippurate and 1 mM cold salicylic acid did not abolish the higher intracellular content obtained during basolateral-to-apical fluxes. Raising the temperature produced only a gradual increase of PSal measured in the basolateral-to-apical direction and an unexpected decrease of ContSal. Because basolateral-to-apical facilitated transport does not play a significant role, the most plausible reason for the higher ContSal during basolateral-to-apical fluxes is that the apical membrane possesses a much lower (10- to 20-fold less) salicylic acid permeability than the basolateral membrane and constitutes the rate-limiting step for transcellular salicylic acid flux.

LLC-PK1 cells were used as a model of renal proximal epithelium to study the nonionic diffusion of salicylic acid (SAL). The apparent [14C]SAL transcellular permeability (PSal) and intracellular content were estimated at 20-21 degrees C from fluxes measured across cell monolayers grown on filters, in both apical-to-basolateral and basolateral-to-apical directions. The medium pH of the cis-side was varied from 6.0 to 7.4, and the medium pH of the trans-side was kept at 7.4. In the apical-to-basolateral direction, PSal increased linearly with the calculated concentration of nonionized SAL, indicating that SAL permeability was essentially the result of nonionic diffusion. In the basolateral-to-apical direction, PSal was about 2.5-fold higher than in the apical-to-basolateral direction and was not linearly related to the concentration of nonionized SAL molecules (0-4.5 nM), suggesting that besides nonionic diffusion, SAL was transported in its ionized form by a facilitated mechanism still active at 21 degrees C. This was confirmed by measuring basolateral-to-apical fluxes at 37 degrees C and observing that probenecid, an inhibitor of organic anion secretion, and cold SAL decreased PSal. Interestingly, at 37 degrees C, PSal in the apical-to-basolateral direction was also decreased by probenecid and cold SAL, suggesting the existence of a facilitated transport in this direction. These data demonstrated that the secretory transport of SAL is present in LLC-PK1 cells. The facilitated transport observed in the apical-to-basolateral direction suggests that in proximal tubule, SAL reabsorption might occur by facilitated mechanism and nonionic diffusion.

The role of nonionic diffusion in the transport of salicylic acid across rabbit proximal S2 and S3 segments was investigated using the in vitro isolated perfused tubule technique. The [14C] salicylic acid apparent reabsorptive permeability (P'I-b, 10(-5) cm/s) was measured at 19 degrees C with luminal solutions kept at different pH and bath maintained at pH 7.4. In S2 tubules, P'I-b was 25.0 +/- 3.5 when luminal pH was 6.0; P'I-b decreased to 8.1 +/- 1.4 and to 4.4 +/- 1.2 at a luminal pH of 6.5 and 7.0, respectively. In S3 tubules, P'I-b was 17.6 +/- 2.4, 5.3 +/- 1.1 and 3.4 +/- 1.1 at a luminal pH of 6.0, 6.5 and 7.0, respectively. There was a close correlation between P'I-b and the calculated proportion of nonionized salicylic acid present at each pH, indicating that only the nonionized molecule could diffuse in our conditions. We calculated the apparent permeability of nonionic salicylic acid and found 0.248 +/- 0.032 cm/s for S2 and 0.176 +/- 0.022 cm/s for S3 tubules. These calculated permeabilities were independent of pH.

The renal excretion of 3'-azido-3'-deoxythymidine (AZT) was investigated in clearance experiments in anesthetized rats. AZT was filtered freely and subsequently secreted, reabsorption being of minor importance. Probenecid, the classical inhibitor organic anion secretion, decreased AZT secretion. AZT, on the other hand, decreased p-aminohippurate secretion. These observations demonstrate that AZT is secreted by the organic anion secretory mechanisms. Cimetidine, an organic cation, also decreased AZT secretion at a concentration which did not inhibit the secretion of the organic anion, p-aminohippurate. This suggests that AZT might also be transported by the mechanisms secreting organic cations. However, as AZT did not inhibit the secretion of the organic cation tetraethylammonium, it cannot be concluded that AZT is transported by the mechanisms secreting organic cations.

The mechanisms involved in the transport of the organic cation N1-methylnicotinamide (NMN) were investigated in the isolated perfused rabbit S2 proximal tubule. NMN underwent a small reabsorptive transport which appeared to be passive. NMN secretory transport was saturable, inhibited by mepiperphenidol (10(-4) M) and presented a relatively low apparent affinity (apparent Km of 852 microM) for the organic cation transport system, with transport against a concentration gradient being observed only at low flow rates. Acidification of the perfusate, by either buffering it at pH 6.8 with MES, or by bubbling the bath with a mixture of 80% O2-20% CO2, resulted in a decrease, rather than an increase, of NMN secretion. Carbonic anhydrase inhibition with ethoxyzolamide (10(-4) M in the bath) did not modify NMN secretion. Addition of 20 to 40 microM NMN in the perfusate also did not change NMN secretion. Proton or organic cation counterexchange seemed therefore not to play a major role in NMN apical step of secretion, the basolateral step appearing to be the general organic cation system of transport for which NMN shows a low affinity.