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The organic cation (OC) transporters OCT1 and OCT2 are suspected of mediating substrate entry from the blood into proximal tubule cells as the first step in renal secretion of OCs. We examined the contribution of each process in different rabbit renal proximal tubule (RPT) segments, taking advantage of the fact that rabbit orthologs of OCT1 and OCT2 can be distinguished by the high affinity of the former for tyramine (TYR) and of the latter for cimetidine (CIM). We verified that TEA uptake, for which both transporters share a similar affinity, is relatively constant in all three segments (apparent inhibitory constant of 33, 74, and 30 microM and maximal rate of mediated TEA uptake of 0.8, 1.0, and 1.2 pmol x mm(-1) x min(-1) in S1, S2, and S3, respectively). In the S1 segment, TYR was a more effective inhibitor of TEA uptake than CIM (IC50 values of 39 and 328 microM, respectively), implicating OCT1 as the predominant pathway for TEA transport. The opposite profiles were noted in the S2 segment (IC50 values of 302 and 20 microM for TYR and CIM, respectively) and S3 segment (IC50 values of 2,900 and 54 microM for TYR and CIM, respectively), suggesting that OCT2 is the predominant TEA transporter in the later portion of RPT. TEA sufficient to saturate OCT1 and OCT2 blocked only 37% of mediated amantadine transport in the S2 segment, confirming the functional presence of at least one additional OC transporter (perhaps OCT3). These data indicate that renal OC transport involves the concerted activity of a suite of transport processes.

In renal proximal tubules, the organic cation transporters rOCT1 and rOCT2 are supposed to mediate the first step in organic cation secretion. We investigated whether previously described differences in amantadine and tetraethylammonium (TEA) uptake into isolated renal proximal tubules could be explained by differences in their transport by rOCT1 and rOCT2. By expressing rOCT1 and rOCT2 in Xenopus oocytes and HEK 293 cells, we demonstrated that both transporters translocated amantadine. In Xenopus oocytes, the inhibitory potency of several rOCT1/2 inhibitors was similar for amantadine compared to TEA uptake and supports amantadine transport by rOCT1 and rOCT2. In proximal tubules, procainamide, quinine, cyanine(863), choline, and guanidine in concentrations that inhibit rOCT1/2-mediated TEA or amantadine uptake in Xenopus oocytes exhibited no effect on amantadine uptake. At variance, these inhibitors blocked TEA uptake into proximal tubules. Amantadine and TEA transport were sensitive to modulation by 25 mM bicarbonate. The effect of bicarbonate on organic cation transport was dependent on substrate (amantadine or TEA), cell system (oocytes, HEK 293 cells, or proximal tubules), and transporter (rOCT1 or rOCT2). In proximal tubules, only amantadine uptake was stimulated by bicarbonate. The data suggested that rat renal proximal tubules contain an organic cation transporter in addition to rOCT1 and rOCT2 that mediates amantadine uptake and requires bicarbonate for optimal function. TEA uptake by the basolateral membrane may be mediated mainly by rOCT1 and rOCT2, but these transporters may be in a different functional or regulatory state when expressed in cells or oocytes compared with expression in vivo.

The purpose of this study was to determine if the inhibitory effect of lactate on the tubular transport of amantadine would extend to a demonstrable sex difference between male and female rats. Enriched fractions of renal proximal and distal tubules were incubated with [(3)H]-amantadine in the presence and absence of racemic lactate. Lactate buffer unmasked a sex difference in proximal tubular transport capacity. Compared to bicarbonate, lactate also decreased the affinity for amantadine uptake by both proximal and distal tubule fragments in both male and female rats. No difference occurred between male and female rats in terms of the inhibitory constant (K(i)) for lactate. These data suggest that female rats have decreased amantadine transport efficiency and potentially increased susceptibility to amantadine toxicity due to transport inhibition by lactate. Our data may help to explain the mechanism for increased amantadine toxicity observed in elderly female patients., (Copyright 2001 S. Karger AG, Basel)

Tetraethylammonium (TEA) and amantadine are two organic cations that are secreted by the kidney. It appears that each cation may characterize distinct renal tubule organic cation transport pathways. To test this hypothesis, we investigated the renal proximal and distal tubule energy-dependent transport properties of TEA and amantadine. Isolated tubules were incubated at 25 degrees C in bicarbonate buffer (Krebs-Henseleit solution) and nonbicarbonate buffer (Cross-Taggart) with varying concentrations of [14C]TEA or [3H]amantadine to determine initial rates of energy-dependent uptake of TEA and amantadine, respectively. The uptake of TEA could best be described by two transport sites, a high-affinity site and a lower affinity site. TEA uptake was not influenced by the presence of bicarbonate. Consistent with our previously reported data, amantadine uptake could also be described by two transport sites, a high-affinity-capacity site that is bicarbonate-dependent and a lower-affinity-capacity transport site that is bicarbonate-independent. The renal tubule uptake of amantadine into proximal and distal tubules, in Krebs-Henseleit solution or Cross-Taggart buffers, was not inhibited by 10 to 1000 microM of TEA. However, tubule accumulation of TEA could be inhibited (>90%) by amantadine in proximal and distal tubules in Krebs-Henseleit solution and Cross-Taggart buffers. In proximal tubules, N1-methylnicotinamide was not able to inhibit amantadine uptake but it reduced TEA uptake by 60 to 70% at similar concentrations. These data support the existence of multiple renal tubule organic cation transporters that have different substrate affinity and controlling mechanisms. It is also apparent that amantadine characterizes organic cation transporters that are distinct from those characterized by TEA.

The effect of L(+)-, D(-)- and racemic (DL)-lactate on the energy-dependent renal uptake of the achiral organic cation amantadine was determined with purified proximal and distal cortical tubule fragments isolated from rat kidneys. Kinetic parameters for uptake of amantadine were measured, under constant pH, in bicarbonate buffer (Krebs-Henseleit [KHS]), and in lactate buffers (5 mM) with different proportions of the enantiomers. Km for amantadine uptake increased in all lactate buffers compared with KHS for both proximal and distal tubules. Km for uptake in DL-lactate was similar to that in D(-)-lactate for proximal tubules and to L(+)-lactate in distal tubules, but Km in L(+)-lactate was higher than in D(-)-lactate for both tubules. Maximal transport capacity (Vmax) in DL-lactate and mixtures of enantiomers were similar to KHS but higher than in pure L(+)- and D(-)-lactate. In KHS, lactate inhibited energy-dependent amantadine uptake in a biphasic manner. Graded competitive inhibition of amantadine uptake was observed between 1 and 15 mM lactate for both proximal and distal tubules. This first phase (1-15 mM) inhibited 60% of amantadine uptake. The second phase (15-20 mM lactate) showed a much steeper slope and inhibited the remaining amantadine uptake. There were no differences in inhibitory potencies of the lactate enantiomers for either proximal tubules or distal tubules amantadine tubule uptake. Our present studies suggest that L(+)- and D(-)-lactate modulate amantadine transport by interacting directly with the bicarbonate-dependent transport mechanism(s).

The mechanism for bicarbonate enhancement of amantadine (A+) uptake was studied further. A selective modulatory effect of bicarbonate on the stereoselectivity of inhibition of A+ uptake by the stereoisomers, quinine (Q) and quinidine (QD), as reflected by the reversal of potency (Q > QD in KHS vs. QD > Q in phosphate), is reported. Studies were performed in bicarbonate (KHS) and phosphate buffers using purified cortical proximal (PT) and distal tubules (DT) from rat kidneys. Variations of extracellular K+ and Ca++ were used to assess the effect of membrane depolarization and calcium on A+ uptake by the tubules. K+ concentration manipulation (1.5-100 mM) did not change A+ uptake by PT or DT in KHS buffer. High Ca++ (5.0 mM) decreased A+ uptake by PT in KHS (P < .001), whereas in phosphate buffer, both 2.5 and 5.0 mM Ca++ decreased uptake (P < .001). In DT, 0.25 mM Ca++ enhanced A+ uptake (P < .05) in KHS, but 2.5 and 5.0 mM Ca++ decreased it in both buffers. Inhibition studies with Q and QD were performed to characterize the bicarbonate-dependent and bicarbonate-independent A+ transport sites further. Stereoselectivity of inhibition was observed in PT in all buffers used. Potency of Q was higher than QD in KHS but lower than QD in phosphate-buffered PT. QD potency remained unchanged. In phosphate-plus-bicarbonate-buffered PT, the inhibitory potencies for Q and QD increased, with the potency of QD being greater than in KHS (P < .001). For DT, Q and QD were equipotent in all buffers used.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of the achiral cationic drug amantadine by purified renal cortical rat tubules was investigated in bicarbonate [Krebs-Henseleit solution (KHS)] and nonbicarbonate buffers. Values of the apparent Km for amantadine accumulation were higher (P < .001, mean +/- S.E., n = 5) for all nonbicarbonate buffers compared with those in KHS in both proximal (76 +/- 5 in KHS, 277 +/- 65 in phosphate, 190 +/- 11 in Tris-HEPES, 268 +/- 46 in acetate and 620 +/- 81 microM in lactate) and distal tubules (68 +/- 4 in KHS, 162 +/- 27 in phosphate, 247 +/- 45 in Tris-HEPES, 281 +/- 50 in acetate buffer and 482 +/- 85 microM for lactate). There was a decrease in Vmax for amantadine transport (P < .001) in phosphate, Tris-HEPES and acetate buffers in both proximal (4.83 +/- 0.43 in KHS, 2.38 +/- 0.43 in phosphate, 1.77 +/- 0.31 in Tris-HEPES and 1.47 +/- 0.40 nmol mg protein-1 min-1 in acetate) and distal (4.58 +/- 0.13 for KHS, 1.37 +/- 0.27 for phosphate, 1.82 +/- 0.37 for Tris-HEPES and 1.76 +/- 0.20 nmol mg protein-1 min-1 for acetate) tubules. However, Vmax was not depressed (P < .09) in lactate buffer for proximal (2.87 +/- 0.24 in KHS and 3.12 +/- 0.34 nmol mg protein-1 min-1 in lactate) or distal tubules (2.42 +/- 0.26 in KHS and 3.21 +/- 0.31 nmol mg protein-1 min-1 in lactate), although a slight increase was observed in distal tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

The present studies compared male and female rat renal proximal and distal tubular uptake of amantadine, in relation to their transport kinetics and enantioselective inhibition by two diastereoisomers, 8S,9R-(-)-quinine and 8R,9S-(+)-quinidine. Under control conditions, amantadine was concentrated by both tubule fractions with a gender difference for distal tubules (tissue:medium ratio, 18.0 +/- 1.4 for males and 11.0 +/- 0.6 for females; mean +/- S.E.M., P < .05). This was reflected by a higher Km value only in female distal vs. proximal tubular tissue (153 +/- 8 vs. 108 +/- 9 microM; P < .01) but decrease in Vmax values in distal compared to proximal tubules (P < .01) showed no gender-related difference. In proximal tubules, 8S,9R-(-)-quinine and 8R,9S-(+)-quinidine competitively inhibited amantadine transport with apparent inhibitory potency of 2- to 3-fold in favor of 8S,9R-(-)-quinine (P < .01) and without gender preference. Conversely in distal tubules, competitive inhibition of amantadine transport was also elicited by either 8S,9R-(-)-quinine or 8R,9S-(+)-quinidine at a similar concentration range (10-1000 microM), with absence of chiral or gender preference. The present transport data have demonstrated an apparent absence of stereoselectivity in distal tubular uptake inhibition of amantadine, and are suggestive of disparate pathways and/or rate limiting steps involved between renal proximal and distal tubular handling of chiral organic cations for both genders, and between genders for distal tubular transport of amantadine.

The renal organic cation transport system was examined. The accumulation of a nonchiral cation, amantadine, by rat renal proximal tubules and cortical slices was investigated, together with the effects of two diastereoisomers, quinine and quinidine. The proximal tubules actively concentrated amantadine with a tissue/medium ratio of 96.3 +/- 1.7 (mean +/- S.E.M., n = 18). Apparent Km was 85 +/- 2 microM and Vmax was 8.0 +/- 0.2 nmol/mg of tubular protein per min. Amantadine accumulation was inhibited competitively by quinine and quinidine with Ki values of 32 +/- 3 and 84 +/- 11 microM, respectively (n = 4). Amantadine was also concentrated by renal cortical slices with tissue/medium ratio of 3.3 +/- 0.3 (n = 4). Apparent Km and Vmax were 94.0 +/- 5.2 microM and 1.27 +/- 0.08 nmol/mg of tubular protein per min, respectively (n = 10). Quinine and quinidine again inhibited amantadine accumulation competitively by the slices, with Ki values of 368 +/- 28 and 780 +/- 84 microM, respectively (n = 4). A similar affinity (Km) for amantadine was observed in both preparations. However, the lower Vmax value in the slice system may be due to additional amantadine transport sites with lower capacity, lesser luminal accumulation and/or limited substrate(s) penetration in the cortical slices. In either preparation, quinine and quinidine functioned as competitive inhibitors and stereoselectivity was observed for the (-)-isomer, quinine, over the (+)-isomer, quinidine. Additional transport sites, reduced luminal substrate accumulation and/or diffusional restraints in the slices are also feasible mechanisms in explaining the differences in Ki values between the two preparations, and their relative contributions await further investigation.