uptake was markedly biphasic with maximum stimulation of transport occurring

Transcription

1 J. Phy"iol. (1980), 304, pp With 7 tex-figure Printed in Grea Britain EFFECTS OF METABOLIC INTERMEDIATES ON SUGAR AND AMINO ACID UPTAKE IN RABBIT RENAL TUBULES AND BRUSH BORDER MEMBRANES BY IAN KIPPEN, JAMES R. KLINENBERG AND ERNEST M. WRIGHT From the Department of Medicine, 502 Halper Building, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA and the Department of Physiology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. (Received 10 October 1979) SUMMARY 1. The effects of tricarboxylic acid cycle intermediates on the renal transport of a-methyl-d-glucoside and ac-amino-isobutyric acid were examined using separated renal tubules of the rabbit. 2. The effect of citrate on a.-methyl-d-glucoside and x-amino-isobutyric acid uptake was markedly biphasic with maximum stimulation of transport occurring at a citrate concentration of 0-64 mm. Biphasic effects were also apparent for L-malate, succinate, fumarate, ox-ketoglutarate and oxaloacetate. 3. The route of uptake of a-methyl-d-glucoside into separated renal tubules is primarily across the brush border (luminal) membrane. 4. Tricarboxylic acid cycle intermediates produced significant stimulation of renal 02 consumption; however, the effects on 02 consumption were not biphasic suggesting that reduced stimulation of transport at high substrate concentration was not caused by a reduction in the supply of metabolic energy. 5. In purified renal cortical brush border membrane vesicles, citrate and a- ketoglutarate inhibited the uptake of cx-methyl-d-glucoside and a-amino-isobutyric acid indicating that inhibition of their transport in respiring renal tubules by high concentrations of tricarboxylic acid cycle intermediates occurs via an effect at the membrane level. INTRODUCTION The renal proximal tubule is known to transport a wide variety of organic solutes against an electrochemical gradient (Ullrich, 1976). The nature of the energy sources and of the coupling between energy production and solute uptake across the basallateral (peritubular) and brush border (luminal) aspects of the tubule are only incompletely understood. Detailed studies on the effects of metabolic substrates on the renal transport of sugars and amino acids have not been reported to our knowledge. Studies in our laboratory were initially directed toward the relationship between renal energy production and the uptake of organic anions such as p-aminohippurate. The consistent finding that tricarboxylic acid cycle intermediates both stimulate /80/ $ The Physiological Society

2 374 I. KIPPEN, J.R. KLINENBERG AND E. M. WRIGHT and inhibit organic anion uptake had led to some confusion- in interpreting the connexion between transport and oxidative metabolism (Weiner, 1973). However, we recently determined that, under appropriate conditions, all tricarboxylic acid cycle intermediates, as well as other renal fuels, stimulate organic anion uptake indicating a direct link with renal oxidative metabolism (Kippen & Klinenberg, 1978). We subsequently determined that metabolizable substrates produce significant stimulation of both amino acid and sugar uptake in suspensions of separated renal tubules of the rabbit. This observation was followed up by detailed studies on the effects of tricarboxylic acid cycle intermediates on the uptake of ac-amino-isobutyric acid and a-methyl-d-glucoside as a representative amino acid (Rosenberg, Blair & Segal, 1961) and sugar (Segal, Rosenhagen & Rea, 1973), respectively. As part of these experiments, we studied the route of uptake of ac-methyl-d-glucoside into separated tubules. Also, as a corollary to these studies, we determined the effects of tricarboxylic acid cycle intermediates on renal oxygen consumption and, to aid in discriminating between effects on uptake mediated via metabolic processes and those at the membrane level, we examined the effects of citrate and ac-ketoglutarate on the uptake of x-methyl-d-glucoside and x-amino-isobutyric acid in highly purified renal brush border membrane vesicles. We conclude from our experiments that exogenous metabolic substrates increase rates of sugar and amino acid uptake, and that the increase may be a consequence of increased rates of oxidative metabolism by respiring renal tubules. METHODS The animals used in all experiments were New Zealand white rabbits weighing 2-3 kg. Separated renal cortical tubules were prepared by the previously described method (Kippen, Nakata & Klinenberg, 1977). The buffer solution used in the experiments with separated renal tubules had the following composition: NaH2PO4-Na2HPO4 buffer, ph 7 4, 10-0 mm; NaCl, 120 mm; KCl, 16-2 mm: MgSO4.7H2O, 1-2 mm; and CaCl2, 1I0 mm. In addition, the uptake solutions contained various concentrations of the tricarboxylic acid cycle intermediates, as indicated, plus 0-4 mm-a-methyl- D-glucoside or a-amino-isobutyric acid and tracer amounts of the corresponding 14C-labelled compound. Solutions were readjusted to ph 7-4 after addition of all constituents. In the experiments with separated renal tubules, 100jd. of the tubule suspension containing 4-8 mg of tubule protein were added to 1 ml. of uptake medium. The tube was gassed with 100% 02 for 30 sec, capped tightly, and shaken gently. After the appropriate time, usually 15 min, a 0 5 ml. aliquot was removed and filtered through an 8-0 jsm pore size Nuclepore filter (Nuclepore Corp., Pleasanton, Ca, U.S.A.). The filter was washed once with 4 ml. ice cold buffer and placed in a liquid scintillation vial with 10 ml. Aquasol (New England Nuclear, Boston, Ma., U.S.A.). The length of time of filtering and washing each sample was under 10 sec. The vials were then counted in a Searle Analytic Mark III liquid scintillation spectrometer. Tubule protein was determined by the Biuret method (Gornall, Bardawill & David, 1949). We examined the effects of one or more tricarboxylic acid cycle intermediates on both a-amino-isobutyric acid and ac-methyl-d-glucoside uptake in tubules from the same animal so that the results would be directly comparable. 02 consumption by separated renal tubules was determined with an 02 monitor (Model 53, Yellow Springs Instrument Company, Yellow Springs, Ohio, U.S.A.) equipped with a Clark type O. electrode fitted to a 1-6 ml. water-jacketed cell. To 1*44 ml. of the above buffer containing the appropriate concentration of tricarboxylic acid cycle intermediate were added 160 d4. tubule suspension containing approximately 10 mg tubule protein. The suspension was stirred with a magnetic stirrer and the temperature maintained constant by a circulating water bath.

3 SUGAR AND AMINO ACID UPTAKE Highly purified renal brush border membrane vesicles were prepared by a procedure utilizing rate differential and density gradient centrifugation (Kippen, Hirayama, Klinenberg & Wright, 1979a). Purity of the membranes was routinely determined by assay of alkaline phosphatase (Mircheff & Wright, 1976) and trehalase (Dahlqvist, 1974), markers for the renal brush border, (Na++K+)ATPase (Fujita, Matsui, Nagano & Nakao, 1971), a marker for basal-lateral membranes, and succinic dehydrogenase (Pennington, 1961), a marker for mitochondria. Protein was determined by the BioRad protein method (BioRad Laboratories, Richmond, Ca., U.S.A.). Brush border membrane vesicles, as evidenced by assay of trehalase and alkaline phosphatase, were enriched tenfold with respect to protein. The corresponding enrichment was less than 0-3-fold for basal-lateral, mitochondrial, nuclear and endoplasmic reticular material. In the experiments with brush border membrane vesicles, 50,I. of the brush border membrane vesicle suspension containing mg of membrane protein were added to 100 PIl. uptake buffer. The uptake buffer was 1 mm-hepes-tris (1 mm-hepes (N-2-hydroxyethylpiperazine- N'-2-ethanesulphonic acid) adjusted to ph 7*5 with Tris) containing either 300 mm-mannitol or 100 mm-mannitol plus 100 mm-nacl. Also present were 0 4 mm-a-amino-isobutyric acid or a-methyl-d-glucoside (including 0-05 #uc/150 jl. of the corresponding 14C-labelled compounds) and the appropriate concentration of tricarboxylic acid cycle intermediate. Uptake was terminated by addition of 850 1d. ice cold stop buffer consisting of 154 mm-nacl in 1 mm- HEPES-Tris, ph 7-5 (Aronson & Sacktor, 1975). The suspension was then rapidly filtered through a 0-45 jam HAWP Millipore filter and washed with 4 ml. of the stop buffer. The filter was placed in 10 ml. Aquasol and counted by liquid scintillation spectrometry. The stopping, filtration and washing procedures took less than 10 sec. Correction for non-specific binding to the membranes and filters was made by subtracting from all data the value of a blank prepared routinely by adding membranes to a tube to which stop buffer had already been added. All experiments were conducted at 22 'C. Most data given represent means of at least three experiments done in duplicate. Errors are presented as standard error of the mean. [14C]a-methyl-D-glucoside, 184 mc/mmole, [14C]3-O-methyl glucose, 51-5 mci/m-mole, and 14C-oa-amino-isobutyric acid, 51-6 mc/m-mole were obtained from New England Nuclear. All other chemicals were of reagent grade purity and were purchased from Sigma (St Louis, Mo., U.S.A.). RESULTS Uptake into separated tubules The time courses of a-methyl-d-glucoside and a-amino-isobutyric acid uptake in the presence and absence of citrate are shown in Fig. 1. The stimulatory effect of citrate on both ac-methyl-d-glucoside and x-amino-isobutyric acid uptake was clearly apparent at time points as early as 4 min and was manifest throughout the entire 60 min time course. We generally obtained steady-state tissue-to-medium ratios of 6 for a-methyl-d-glucoside and for a-amino-isobutyric acid in control experiments. Since the effects of citrate were substantial at 15 min, all subsequent measurements of uptake into the separated tubules were estimated at this time point. The deviation of this estimate of transport rate from the initial rate was less than 20 %. x-ketoglutarate and The effects of various concentrations of citrate, cis-aconitate, oxaloacetate on the uptake of 0-4 mm-ac-methyl-d-glucoside and ac-amino-isobutyric acid into separated renal tubules are illustrated in Fig. 2. The curves for a-methyl-dglucoside and c-amino-isobutyric acid were similar and all showed a biphasic response, i.e. stimulation at concentrations up to about 1 mm and reduction in the degree of stimulation above 1 mm. The most striking biphasic effect was exhibited by citrate which caused 85+5 % (a-methyl-d-glucoside) and 80+7 % (ac-aminoisobutyric acid) stimulation of uptake at 0-64 mm falling to % (ac-methyl-d- 375

4 *0 376 I. KIPPEN, J. R. KLINENBERG AND E. M. WRIGHT glucoside) and % (oc-amino-isobutyric acid) at 10-2 mm. Possible explanations for the biphasic nature of these effects, including feedback regulation of transport and effects at the membrane level, were explored in later experiments. The effect of L-malate, succinate and fumarate on the uptake of cc-methyl-d-.!- C 05._ E 0 Time (min) C. _. L, 0*4 _-4 v (B) AIB ~ Aon ro 01~~~~~ /I ' Time (min) Fig. 1. Time courses of uptake of a-methyl-d-glucoside (MDG) and a-amino-isobutyric acid (AIB) in the presence or absence of 0-64 mm-citrate. The concentrations of the sugar and amino acid were 0-4 mm. Data are the mean of two experiments done in duplicate.

5 SUGAR AND AMINO ACID UPTAKE 377 glucoside and a-amino-isobutyric acid are shown in Fig. 3. As with the other tricarboxylic acid cycle intermediates, there was some indication of a decreasing effect at higher concentrations, although the sharply biphasic effect of citrate was not produced by any of these compounds o I c0) E 0 e v+ 0. I 0) Substrate (mm) Fig. 2. Effects of citrate, ci8-aconitate, a-ketoglutarate and oxaloacetate on the uptake of az-methyl-d-glucoside and a-amino-isobutyric acid into separated renal tubules. Data are for uptake at 15 min. The absolute amounts taken up were: a-methyl-dglucoside, 0-99 ± 0-05 n-mole mg protein-' and a-amino-isobutyric acid, n-mole mg protein-' (mean+ s.e. for fifty-three controls). Data are the mean of at least three experiments done in duplicate. Standard errors of the means obtained for absolute uptake values were consistently less than 10O.

6 378 I. KIPPEN, J. R. KLINENBERG AND E. M. WRIGHT o +20 -W c0 E 0 a) cu -c 0 0* ) CL Substrate (mm) Fig. 3. Effects of L-malate, succinate and fumarate on the uptake of oz-methyl-dglucoside and x-amino-isobutyric acid into separated renal tubules. Experimental details as in legend for Fig. 2. Route of uptake of c-methyl-d-gluco8ide As shown in Fig. 4, the intracellular accumulation of ac-methyl-d-glucoside was blocked by ouabain, phloretin and phloridzin. Phloridzin, in addition, was more potent than its aglycone, phloretin, in slowing the equilibration of the sugar with the intracellular compartment. The accumulation of ax-methyl-d-glucoside was also significantly reduced by other sugars, D-glucose being more effective than 2-deoxy-Dglucose. By way of contrast, 3-0-methyl glucose was not accumulated within the

7 SUGAR AND AMINO ACID UPTAKE c *' 0-2 E -5 acd 0) Time (min) Fig. 4. Time courses of uptake of 0-4 mm-a-methyl-d-glucoside and 3-0-methyl glucose in the presence of various inhibitors. Concentrations of glucose and 2-deoxy-Dglucose were 10 mm. Concentrations of ouabain, phloretin and phloridzin were 1 mm. Data are the mean of two experiments done in duplicate. An uptake of 0-3 n-mole mg protein-' represents a tissue-to-medium ratio of approximately 1.

8 I. KIPPEN, J. R. KLINENBERG AND E. M. WRIGHT 380 cells and there was no effect of phloridzin on either the rate of uptake or the steady state value (Fig. 4). Phloretin significantly inhibited the rate of uptake of 3-0-methyl glucose, and 2-deoxy-D-glucose was a more effective inhibitor of transport than D-glucose. Also note that the effect of ouabain on the uptake of 3-0-methyl glucose was relatively minor compared to that on the uptake of a-methyl-d-glucoside. We take this data to indicate that c-methyl-d-glucoside is accumulated within the separated renal tubules across the brush border (luminal) membrane, whereas 3-0- methyl glucose uptake occurs primarily across the peritubular membrane (see Discussion). o0 C +100 Citrate E Fumarate 0 6 Succinate ct-ketoglutarate w +60 C j +40 E C 0 '" L-Malate * Substrate (mm) Fig. 5. Effects of citrate, fumarate, succinate, a-ketoglutarate, and L-malate on 02 consumption by separated renal tubules. The 02 consumption in control tubules was #mole 100 g wet wt.-1 hr-1 (mean + s.e. for twenty-four experiments). Experimental procedures are described in the text. Data are the mean of three experiments. Standard errors of the means obtained for absolute uptake values were consistently less than 10%. 02 consumption in separated tubules It was our original hypothesis that a positive correlation should exist between stimulation of oxidative metabolism and stimulation of transport. It was thus of interest to compare tubular oxygen consumption at different concentrations of the metabolic substrates with the a-methyl-d-glucoside and ac-amino-isobutyric acid transport rates at the same concentrations of metabolic substrates. Shown in Fig. 5 are the effects of some tricarboxylic acid cycle intermediates on the rate of oxygen consumption by separated renal tubules. The relationship between stimulation of 02 consumption and substrate concentration for all of the tricarboxylic acid cycle intermediates was not biphasic as seen in the transport experiments. This suggests that the decrease in a-methyl-d-glucoside and a-amino-isobutyric acid transport at

9 SUGAR AND AMINO ACID UPTAKE 381 2*0 1*0 C a) +a._ a) 0) a, 0. X o 0@8 exr (+Na+) A-.-O 06 0*4 -Nal) Time (min) Fig. 6. Time courses of uptake of 0-4 mm-ac-methyl-d-glucoside and a-amino-isobutyric acid into purified renal brush border membranes. Membranes were prepared in 300 mmmannitol in 1 mm-hepes-tris, ph 7-5. Uptake medium was either the same buffer (0) or a buffer in which mannitol was replaced isosmotically by 100 mm-nacl (0). Data are the mean of three experiments done in duplicate. Standard errors of the means obtained for absolute uptake values were consistently less than 100%. high concentrations of the tricarboxylic acid cycle intermediates was not mediated by way of a metabolic regulatory process (i.e. reduced oxygen consumption leading to a reduction in energy utilized for transport).

10 382 I. KIPPEN, J. R. KLINENBERG AND E. M. WRIGHT Uptake into brush border membrane vesicles In order to examine the effects of the tricarboxylic acid cycle intermediates on the transport of a-methyl-d-glucoside and az-amino-isobutyric acid in the absence of renal metabolism, we utilized purified renal brush border membrane vesicles. The time courses of uptake into brush border membrane vesicles are shown in Fig I I I ~~~~~~~~~~~Itr t 0-20 VA M/DG = t e _ a- Ketoglutarate 00- E -40 0, 0 0) CL -Ketoglutarate *0 Substrate (mm) Fig. 7. Concentration dependence of the effects of citrate and c-ketoglutarate on the uptake of a-methyl-d-glucoside and a-amino-isobutyric acid by purified renal brush border membranes. Uptake buffer was 100 mm-nacl, 100 mm-mannitol in 1 mm- Hepes-Tris, ph 7-5. Uptake was measured as described in the text using a 1 min time point. Data are the mean of three experiments done in duplicate. Standard errors of the means obtained for absolute uptake values were consistently less than 10. The initial rate of uptake of a-methyl-d-glucoside was stimulated thirtyfold in the presence of a gradient of NaCl ( n-mole mg protein-' min- vs *018 n-mole mg protein-' minm as estimated from uptake at 0 5 min). Maximum uptake occurred at 4 min under the conditions of these experiments. In other experiments where the ac-methyl-d-glucoside concentration was 0-1 mm the peak uptake occurred at 1 min, thus the time point of maximum overshoot depends on the substrate concentration. The uptake of a-amino-isobutyric acid was also greater in the presence of a NaCl gradient, however the initial rate of uptake was increased less than twofold ( *081 n-mole mg protein-' min'). Additionally, uptake was virtually complete at 4 min and there was no sign of any overshoot under the conditions of these experiments. The time course of c-methyl-d-glucoside uptake resembles that for D-glucose under similar conditions (Aronson & Sacktor, 1975; Kippen et al. 1979a), however the time course for x-amino-isobutyric acid uptake is unlike that for other amino acids in that no sodium gradient-dependent overshoot is seen (Evers, Murer & Kinne, 1976; Fass, Hammerman & Sacktor, 1977).

11 SUGAR AND AMINO ACID UPTAKE Using a 1 min time point to estimate uptake rates, we examined the effects of citrate and a-ketoglutarate on the uptake of oc-methyl-d-glucoside and a-aminoisobutyric acid into renal brush border membrane vesicles in the presence of a sodium gradient (Fig. 7). At 10-2 fm both citrate and a-ketoglutarate inhibited the uptake of a-methyl-d-glucoside by about 20 %, while the uptake of a-aminoisobutyric acid was inhibited by 45 % (citrate) and 31 % (a-ketoglutarate). 383 DISCUSSION This paper reports the results of four groups of experiments: (1) the effects of a number of tricarboxylic acid cycle intermediates on the uptake of a-methyl-dglucoside and x-amino-isobutyric acid by separated renal tubules of the rabbit; (2) the route of x-methyl-d-glucoside uptake into the separated tubules; (3) the effects of these compounds on tubular oxygen consumption; and (4) a-methyl-dglucoside and a-amino-isobutyric acid transport in purified renal brush border membrane vesicles. Effects of tricarboxylic acid cycle intermediates on sugar and amino acid uptake in separated tubules The effects of tricarboxylic acid cycle intermediates at various concentrations on the 15 min uptakes of a-methyl-d-glucoside and a-amino-isobutyric acid by separated renal tubules were measured. A comment is necessary on the choice of 15 min uptakes in these experiments. Generally, in transport experiments it is appropriate to obtain estimates of uptake rates from early time points, for example 1 min or less. However, in order to affect transport by a metabolic route, the tricarboxylic acid cycle intermediates are required to enter the cell and be metabolized prior to providing energy for transport. Thus the use of times as short as one minute would not have provided the type of information we wished to obtain (this is clearly seen in Fig. 1). Since it would not have been feasible to examine the entire time course of the effects of many metabolic substrates at numerous concentrations, we chose 15 min at which to compare the effects. All of the tricarboxylic acid cycle intermediates examined stimulated a-methyl-dglucoside and c-amino-isobutyric acid uptake in the concentration range from 0-01 to 10'2 mm (Figs. 2 and 3). The effect of citrate was markedly biphasic, i.e. the stimulation of uptake was reduced at concentrations greater than 1 mm. All of the other tricarboxylic acid cycle intermediates used also caused reduction in stimulation of uptake at higher concentrations. In a previous study (Kippen & Klinenberg, 1978) p-aminohippurate uptake was stimulated in a sharply biphasic manner by all of the tricarboxylic acid cycle intermediates. Route of uptake of c-methyl-d-glucoside Uptake of substances into separated renal tubules could occur across either the peritubular or luminal aspect of the renal epithelium. However, our working hypothesis for the uptake of x-methyl-d-glucoside, and probably c-amino-isobutyric acid, into these cells is that the major route of transport is by way of a Na dependent process at the brush border membrane.

12 384 I. KIPPEN, J. R. KLINENBERG AND E. M. WRIGHT Evidence in support of the brush border route for a-methyl-d-glucoside uptake is as follows: (i) a-methyl-d-glucoside is accumulated within the tissue against its concentration gradient. The intracellular concentration rose above 2 mm when the incubation media contained 0 4 mma-methyl-d-glucoside. (ii) Ouabain eliminated the uphill accumulation of the sugar. (iii) Phloridzin was a more effective inhibitor of a-methyl-d-glucoside uptake than phloretin. After 4 min phloridzin reduced uptake by 90% whereas phloretin only reduced uptake by 60 %. In addition, the sugar equilibrated with the intracellular compartment about 6 times faster with phloretin than phloridzin. In isolated brush border membranes phloridzin is a more effective inhibitor of Na dependent sugar transport than phloretin (Kinne, Murer, Kinne-Saffran, Thees & Sachs, 1975; Turner & Silverman, 1977, 1978). (iv) Glucose is a more effective inhibitor of a-methyl-d-glucoside uptake than 2-deoxy-D-glucose. This is consistent with the observations in isolated brush border membrane vesicles that the rates of sodium dependent glucose and a-methyl-d-glucoside uptake are comparable and that 2-deoxy-D-glucose does not significantly inhibit the rate of D-glucose uptake (Aronson & Sacktor, 1975; Turner & Silverman, 1977, 1978). (v) It has been shown (Kinne et al. 1975) that purified renal brush border membranes but not basal-lateral membranes, contain a specific sodium dependent sugar transporter. Also, in the intestine there is no evidence for carrier mediated transport of a-methyl-d-glucoside across basal-lateral membranes (Bihler & Cybulsky, 1973; Wright, van Os & Mircheff, 1980). Evidence in support of uptake of 3-0-methyl glucose across the basal-lateral membrane is as follows. (i) The sugar appeared to only equilibrate with the intracellular fluid space, i.e. the tissue-to-medium ratio was close to 1 even after 60 min. (ii) Phloretin was a much more potent inhibitor of 3-0-methyl glucose uptake than was phloridzin, consistent with the known effects of these inhibitors on the facilitated diffusion of sugars in other cells and tissues. (iii) The Na dependent sugar transport system in isolated brush border membranes does not handle 3-0- methyl glucose (Turner & Silverman, 1977, 1978). (iv) 2-deoxy-D-glucose was a more potent inhibitor of 3-0-methyl glucose uptake than was glucose. Effects of tricarboxylic acid cycle intermediates on tubular 02 consumption There was a linear relationship between 02 consumption and concentration of tricarboxylic acid cycle intermediates (Fig. 4), i.e. no biphasic effects were seen. Thus the biphasic effects of the tricarboxylic acid cycle intermediates on the transport of the sugar and amino acid were not the result of feed-back regulation of transport mediated via the metabolic process. Of further interest was the lack of a precise correlation between stimulation of uptake and stimulation of metabolism. For example, 0-16 mm-l-malate stimulated a-methyl-d-glucoside transport by nearly 100 % while stimulating 02 consumption by only about 20 %. On the other hand fumarate stimulated a-methyl-d-glucoside transport and 02 consumption to approximately the same extent. However, because of the many factors involved (see later discussion) it is difficult to estimate precisely the degree of stimulation of uptake to be expected from a given concentration of metabolite. Uptake into brush border membrane vesicles The experiments with purified brush border membrane vesicles indicate that inhibition of a-methyl-d-glucoside and a-amino-isobutyric acid uptake at high concentrations of tricarboxylic acid cycle intermediates was probably due to an effect at the membrane level. Citrate and a-ketoglutarate at 10 mm inhibited the initial rates of a,-methyl-d-glucoside and a-amino-isobutyric acid transport into the brush border vesicles by % (Fig. 7). Studies in this laboratory (Kippen, Hirayama, Klinenberg & Wright, 1979b) have shown that citrate is transported by rabbit renal brush border membranes by a sodium dependent mechanism similar to that for sugars and amino acids (Aronson & Sacktor, 1975; Evers et al. 1976; Fass et al. 1977), and that many of the metabolic intermediates used in this study share

13 SUGAR AND AMINO ACID UPTAKE 385 the same transport system. In addition, glucose and alanine inhibited the uptake of citrate and a-ketoglutarate suggesting interactions between the sugar and amino acid and metabolic substrate transport systems. Mutual inhibition between sugar and amino acid transport has been observed in both kidney and intestinal brush borders (Murer, Sigrist-Nelson & Hopfer, 1975; Fass et al. 1977) and it has been suggested that the various solutes compete for the electrochemical potential gradient driving uphill transport. We propose that similar interactions occur between the transport of the metabolic intermediates and the transport of sugars and amino acids. Conclusions Based on the above considerations, we conclude that the effects of the exogenous metabolic substrates on the uptake of x-methyl-d-glucoside and possibly of x-aminoisobutyric acid, are mainly on the sodium co-transport system in the brush border membranes. We postulate the following series of events to explain the stimulation of uptake seen in the present experiments. The tricarboxylic acid cycle intermediates are transported into the cell and enter the renal metabolic pool. This causes increased production of ATP leading to increased turnover of the Na+/K+ exchange pump located at the basal-lateral surface of the epithelium. This results in an increase in the sodium electrochemical potential gradient across the cell leading to increased Na-coupled solute uptake across the renal brush border membrane in response to the Na gradient (Beck & Sacktor, 1978a) and membrane potential (Kimmich, Carter-Su & Randles, 1977; Beck & Sacktor, 1978b). There is evidence to support such a series of events. (i) The present experiments show that the exogenous metabolic substrates (tricarboxylic acid cycle intermediates) stimulate renal oxidative metabolism. (ii) Prior experiments (Mudge, 1951) clearly showed that addition of metabolic intermediates leads to a decrease in intracellular Na. (iii) Addition of ATP has been shown to increase sugar uptake by rat kidney slices (Weidemann, Hems & Krebs, 1969). In summary, it is clear that, under the conditions of the experiments described here, addition of tricarboxylic acid cycle intermediates increases renal metabolism and the transport of oc-methyl-d-glucoside and a-amino-isobutyric acid by separated renal tubules. Inhibition of uptake by high concentrations of the tricarboxylic acid cycle intermediates may be explained either by competition for energy provided by Na or electrical gradients, or by competition for shared transport mechanisms. However, the precise relationship between the extracellular concentration of metabolic substrate and the transport process is exceedingly complex. Whether an increase or decrease in the rate of sugar or amino acid transport occurs will necessarily depend on a number of factors including the concentration of sugar or amino acid and the actual kinetics of their uptake, the concentrations of the metabolic intermediates and the kinetics of their uptake and, in addition, the kinetics of the substrate metabolism. Further studies are clearly required in order to elucidate the quantitative interrelationships between these transport phenomena and cellular metabolism. This work was supported by grants from the National Institutes of Health (AM18261, AM25588, AM19567 and RR05468) and the Arthritis Foundation. 13 P HY 304

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