Materials.-Glycine-2-C"4 (specific activity 2.5 me per mmole); a-aminoisobutyric

Transcription

1 INHIBITION OF AMINO ACID TRANSPORT IN RAT KIDNEY CORTEX BY PUROMYCIN* BY Louis J. ELSASt AND LEON E. ROSENBERG DEPARTMENT OF MEDICINE, YALE UNIVERSITY SCHOOL OF MEDICINE Communicated by C. N. H. Long, November 17, 1966 The kinetics of active transmembrane transport of amino acids and sugars are similar in several important respects to those of enzyme-catalyzed reactions. These similarities, which include substrate specificity, saturability, and responsiveness to competitive and noncompetitive inhibitors, have led many investigators to postulate that enzymes or "carrier" proteins in the cell membrane are responsible for the entry of amino acids and sugars into microbial and mammalian cells. This postulate has been strengthened by the recent work of Fox and Kennedy,' who identified a protein from cell membrane fragments of Escherichia coli which they consider to be a galactoside "carrier" molecule. Additional indirect evidence for a role of proteins or peptides in mediated transport has been provided by Adamson,2 Finerman,3 and their co-workers, who demonstrated inhibition of amino acid transport in embryonic or fetal bone tissues by puromycin, a known inhibitor of new protein synthesis. The present studies were designed to investigate the effects of puromycin on amino acid transport in rat kidney cortex, a nonembryonic, nonskeletal tissue in which amino acid transport mechanisms have been characterized extensively--7 This report presents evidence that puromycin is an effective inhibitor of amino acid transport in this tissue preparation and describes, in some detail, the mode of action and the potential significance of this inhibitory effect. Methods.-M ale Spraque-Dawley rats weighing gm were fed water and Purina(R) rat chow ad libitum until sacrificed by stunning and decapitation. The techniques employed for the preparation of kidney cortex slices, incubation in Krebs-Ringer bicarbonate buffer (ph 7.4), estimation of total tissue water, determination of extracellular space with inulin carboxyl-c'4, assessment of intracellular and medium concentration of labeled amino acids, and determination of efflux have been described in detail previously.4'8 Unless stated to the contrary, amino acid concentrations in the incubation medium were 0.1 mm. Incorporation of L-lysine-C14 into tissue protein was investigated by incubating this amino acid with kidney cortex slices in the presence or absence of puromycin. Trichloracetic acid-precipitable tissue protein was prepared by the method of Steinberg et al. 9 as modified by Manchester and Young. 10 Oxygen consumption was determined by standard techniques with the Gilson respirometer (GME model no. G-8). Tissue respiration was recorded every 15 minutes for a total duration of 210 minutes. Tissue sodium and potassium concentrations were obtained from aliquots of distilled water in which kidney cortex slices had been boiled for six minutes. Cation concentrations were read directly from a flame photometer against a lithium standard by standard techniques. Materials.-Glycine-2-C"4 (specific activity 2.5 me per mmole); a-aminoisobutyric acid-i-c'4 (specific activity 3.97 me per mmole); 1-aminocyclopentane- 1-carboxylic acid-carboxyl-c14 (specific activity 1.89 me per mmole); L-lysine- 371

2 ,372 B7OCHEMISTRJY: ELSA S AND ROSENBERG PROc. N. A. S. UC14 (specific activity 126 me per mmole); and inulin carboxy-c14 (specific activity 2.6 mc per gram) were obtained from New England Nuclear Corporation. Unlabeled glycinie, L-lysine, 1-aminiocyclopenitlaiie-l-carboxylic acid, puromycin dihydrochloride, and the aminonucleoside of puromycin were obtained from Nutritional Biochemicals Company. Unlabeled a-aminoisobutyric acid and 6- dimethylaminopurine were purchased from California Biochemicals Corporation. Cycloheximide was obtained from the Cancer Chemotherapy National Service Center. Results.-Inhibition of amino acid uptake by puromycin: The nonutilizable amino acid, a-aminoisobutyric acid (AIB),"I was chosen as the substrate for most of these experiments because it is actively transported in kidney cortex slices, but is not incorporated into protein.4 Therefore, the effect of puromycin on the transport mechanisms for this substrate could be dissociated from its influence on ribosomal protein synthesis. The time course of AIB uptake in the presence and absence of puromycin (0.55 mmi) is shown in Figure 1. No inhibitory effect of puromycin on AIB uptake was noted during the initial 90 minutes of incubation. After 120 and 180 minutes of incubation, a difference between control and puromycin-containing flasks was seen, but these differences were not statistically significant. To determine if this inhibitory effect of puromycin was reproducible and significant, tissues were preincubated in medium containing puromycin for periods up to 180 minutes prior to 45-minute incubations with AIB. As shown in Figure 2, AIB uptake fell progressively as the duration of preincubation with puromycin increased. Uptake was inhibited by 36 and 43 per cent after 150 and 180 minutes of preincubation, respectively. These differences were significant from control uptake values (p < 0.01). The half time of this inhibitory effect was estimated to be about 210 minutes. 7 0 U AIB -CONTROL 5- / FI e c eon pre cn ISO MINUTES OF INCUBATION FIG. 1. Time course of AIB uptake in the presence and absence of puromycin (0.55 mm). Rat kidney cortex slices were incubated aerobically (95% 02 and 5570 CO2) in 2.0 ml of Krebs-Ringer bicarbonate buffer at ph 7.4 at 370C. The data represent the mean of triplicate observations. The "p" values for the differences between control and puromycin-containing flasks were >0.8 and >0.5 at 120 and 180 min, respectively. Uptake is defined in this and other figures as the distribution ratio of cpm per ml of intracellular fluid to cpm per ml of incubation medium,

3 VOL. 57, BIOCHEMISTRY: ELSAS AND ROSENBERG 373 -Jo so zi- z 0~~~~~~~~~~~~~ X. 40., MINUTES OF PRE-INCUBATION WITH PUROMYCIN FIG. 2.-Inhibitory effect of preincubation with puromycin (0.55 mm) on AIB uptake. All tissues were preincubated for 180 mmn prior to addition of AIB, and puromycin was added at appro-e priate intervals. AIB uptake was determined after 45 mmn of incubation and control values were derived from flasks containing no puromycin or from those to which puromycin was added simultaneously with AIB. Each point, plotted semilogarithmically, represents the mean of six separate observations. Differences from control value are significant for preincubation intervals of 120, 150, and 180 mmn (p <0.01). The inhibitory effect of puromycin on amino acid uptake was not restricted to AIB (Table 1). Significant inhibition of uptake of glycine and 1-aminocyclopentane-l-carboxylic acid (ACPC),"l a nonutilizable cyclic amino acid, was noted after 120 and 180 minutes of preincubation, respectively. Additional studies revealed that uptake of AIB and glycine was inhibited maximally at puromycin concentrations as low as mm. In subsequent experiments, the concentration of puromycin used was mmd. TABLE 1 INHiBITION OF AMINO ACID UPTAKE BY PUROMYCIN Distribution Ratio* Significance of difference Amino acid Control Puromycin from controlt AIB-1-C14 (12)t 4.30±i ±+0.23 p<o0.01 ACPC-carboxyl-C"4 (11) 3. 10±i ± p < 0.01 Glycine-2C14 (6) 5.35±i ±i0. 11 p<o0.01 All1 tissules were preincubated in Krebs-Ringer bicarbonate buffer (ph 7.4) in a Dubnoff metabolic shaker with an atmosphere of 95% 02 and 5% 002 at 370 for 120 mmn (AIB and glycine) or 150 min (ACPC) prior to addition of the labeled amino acid. Duration of incubation was 45 min Pulromycin concentration was mm. * Results expressed as the mean + one standard error. t From student's "t" test. t Values in parentheses denote number of observations. The inhibitory effect of puromycin did not depend on the presence of the drug during the final incubation with labeled amino acid. Tissue slices were preincubated with puromycin for 120 minutes and were then transferred to fresh buffer containing AIB. The data in Table 2 demonstrate that AIB uptake was inhibited equally in the presence or absence of puromycin during the final 45-minute incubation.

4 374 BIOCHEMISTRY: ELSAS AND ROSENBERG Pptoc. N. A. S. TABLE 2 INFLUENCE OF PREINCUBATION AND INCUBATION CONDITIONS ON INHIBITION OF AIB UPTAKE BY PUROMYCIN Puromycin in Distribution Per cent Condition Preincubation Incubation ratio inhibition * Tissues were preincubated in Krebs-Ringer bicarbonate buffer (ph 7.4) for 120 min. All tissues were then transferred to a second flask containing fresh buffer and 0.1 mm AIB-1-C'4 for a 45-min incubation period. Distribution ratios (mean i standard error) were derived from at least three observations. Note that uptake of AIB was greater in fresh buffer than in same buffer used for preincubation (Table 1). * Plus or minus sign denotes presence or absence of puromycin (0.055 mm) in preincubation or incubation media. Uptake in tissues designated by conditions 2 and 3 was not significantly different from each other but was significantly different from uptake under condition 1, p < Rapid inhibition by puromycin of L-lysine-C"4 incorporation into tissue protein: The failure of puromycin to inhibit amino acid accumulation in less than two hours when added concurrently with the substrate (Fig. 1) did not represent slow entry of puromycin into the tissue. Incorporation of L-lysine-C14 into TCA-precipitable tissue protein was markedly inhibited by puromycin after only ten minutes of incubation, without any preincubation, indicating that the drug enters this tissue and inhibits new protein synthesis very rapidly. Furthermore, puromycin (0.55 mml) inhibited lysine incorporation into protein by more than 95 per cent when added concurrently or 60, 120, and 180 minutes prior to lysine addition, demonstrating that the progressive fall in AIB uptake observed with increasing duration of preincubation (Fig. 2) did not reflect a similar progressive impairment of new protein synthesis. Relationship of inhibition of protein synthesis to inhibition of AIB transport: The following experiments were conducted to determine if the inhibitory effect of puromycin on amino acid transport was indeed related to inhibition of protein synthesis, or alternatively to some other known effect of the drug such as acceleration of glycogenolysis'2 13 or inhibition of glycogen synthesis.'4 AIB uptake was determined in the presence of cycloheximide, another antibiotic known to inhibit protein synthesis, and in the presence of 6-dimethylaminopurine and puromycin aminonucleoside, structural analogues of puromycin which do not inhibit protein synthesis but which have produced the previously mentioned alterations in glycogen metabolism. As shown in Table 3, equimolar concentrations of cycloheximide inhibited AIB uptake by approximately the same extent as piromycin did, but the puromycin analogues failed to alter AIB uptake significantly. Absence of an effect of puromycin on oxygen consumption, tissue sodium and potassium, concentrations, and tissue water spaces: The following experiments indicated TABLE 3 EFFECT OF CYCLIOHEXIMIDE, PUROMYCIN, 6-DIMETHYLAMINOPURINE, AND THE AMINONUCLEoSI)E OF PUROMYCIN ON AIB TRANSPORT Significance of difference Condition Distribution ratio from control Puromycin p < 0.01 Cycloheximide p < 0.01 Control Dimethylaminopurine p > 0.50 Aminonucleoside of puromycin p > 0.40 All tissues were preincubated for 180 min with mm concentrations of the designated drug prior to a 45-min incubation with AIB. Eachdistribution ratio (mean 4 standard error) wasderived from at least four observations.

5 2 _ ~~~~~~~~~~~~~~tiones. VOL. 57, 1967 BIOCHEMISTRY: ELSAS AND ROSENBERG 375 that the inhibitory effect of puromycin amino acid transport was not secondary to impairment of some other parameters of cell function. Puromycin had no effect on oxygen consumption throughout a 210-minute interval, oxygen consumption in puromycin-containing and control flasks averaging 117 Al 02/100 mg tissue per hour. These studies were conducted in Krebs-phosphate buffer (ph 7.4), a medium in which the inhibitory effect of puromycin AIB uptake was also apparent. Tissue Na+ and K+ concentrations were measured to evaluate the effect of puromycin on the cation transport system in kidney cortex slices. Although intracellular Na+ was higher than normal after 165 minutes of incubation, and intracellular K+ lower, the usual intracellular to extracellular gradient for these cations persisted, and, more importantly, were unchanged by puromycin. Furthermore, total tissue water and inulin space measurements were unchanged by puromycin under the experimental conditions used in the amino acid transport studies. Effect of puromycin of AIB influx and efflux: The kinetics of amino acid uptake and egress from kidney cortex slices were studied to determine the mode of action of puromycin on AIB transport. The time course of AIB uptake after a 120-minute preincubation period is described in Figure 3. The rate of uptake was maximal during the initial 15 minutes, and equilibrium conditions were reached by 45 minutes in the presence and absence of puromycin. Uptake of AIB was significantly impaired in the puromycin-treated tissues throughout the 90-minute interval. The reduced uptake at five minutes indicates that influx of AIB was slowed in the presence of puromycin. The difference in AIB uptake between control and puromycintreated tissues was the same at 45 and 90 minutes of incubation, suggesting that puromycin was not inhibiting efflux of AIB. To verify this latter supposition, experiments were performed in which rat kidney cortex slices were incubated with AIB for 45 minutes after being preincubated for 120 minutes with and without puromycin. Efflux was then measured directly by transferring the tissues to fresh buffer containing no labeled AIB and measuring the appearance of AIB-C14 in the media at five-minute intervals. The fractional rate of AIB efflux was identical in the presence of puromycin and in control tissues (Fig. 4). S CONTROL o 4-0 PUROMYCIN Z 3- ~ /FIG. 3.-Time course of AIB o / / uptake after 120 min of preincubation with puromycin (0.055 mm). The distribution ratio zr 2- ~ / /for control flasks at 5 min of / U) Sincubation (1.33 i 0.05) was significantly greater than that for puromycin-treated tissues (0.89 ± 0.05) with a "p" value <0.01. All points were derived from the mean of three observar MINUTES OF INCUBATION

6 376 BIlOCHEMISTRFY: ELSAS A ND ROSENBRG Pitoc. o N. A. S. 100 \ FIG. 4.-Absence of aii effect of puromycin (0.055 CD BO -\o CONTROL mmi) on the efflux of AIB. i_s * 9 PUROMYC IN ~~~~~~cubated Twenty flasks for 120were miii, prein- tenl ~ with pitromycin. AIB-C'4 2 A_ W was then added to all flasks and incubation continued for v0-45 min to achieve equilibrium z conditions. At the end of w the incubation period, six O control flasks and six puro- W.l mycini-containing flasks were X. used for calculation of the distribution ratios. Tissues 20 ' ' from four flasks were trans ferred to 2 ml of AIB-free MINUTES OF EFFLUX buffer. At the intervals shown, small samples of the media were removed from the flasks and assayed for radioactivity. After 30 min the tissues were assayed for remaining radioactivity. From the distribution ratios and calculation of the cumulative counts per minute released into the media during the designated time intervals, the total cpm present in the tissue at, the beginning of the efflux study were calculated. The ordinate represents the per cent of counts remaining during the efflux period plotted on a semilogarithmic scale. 3 OAfR 9E (LIIIIAnEDU CNENRTIN(moesItR) MC FIG. 5.-Effect of increasing medium concentration (Af) on the mediated uptake (Y) of AIB after preincubationl for 120 mmn with or without puromycin (0.055 mm). Values for Y. expressed in mmoles per liter per 15 min, were calcullated as described previously.4 The points shown represent the mean of five observations;. Michaelis-Menten kinetics of puromycin inhibition of AIB-C14 transport: Kidney slices were preincubated for 120 minutes in the presence and absence of puromycinand then incubated with increasing concentrations of AIB for 15 minutes. The data in Figure 5 show that the velocity of uptake was slowed by puromycin throughout the 50-fold range of concentrations used. The data also show that as extracellular substrate concentration was increased, the rate of increase of mediated uptake slowed, consistent with saturation kinetics. When these data were plotted using the double reciprocal method of Lineweaver and Burk, positive ordinate intercepts were obtained in the presence and absence of puromycin. Both the slope and ordi-

7 7IOCHEMIISTRY: ELSA S ANDJROSENBERG Vroi,.w nate intercept were altered by puromyciin but the extrapolated abscissa intercept was unchanged, iiidicatiiig that the Kitt for AIB (about 3 m\1i) was not altered by puromycin. Discussion. The experiments of Adamsoii and co-workers,2 demonstrating itihibition of amino acid transport by puromycin in embryonic chick bone, suggested a new way of attacking the chemical nature of transport reactions. That the observed inhibitory effect is not species, age, or tissue-specific is demonstrated by the observations of Finerman, Downing, and Rosenberg3 in fetal rat calvarium, and by the present results. Although there have been several reports which failed to demonstrate inhibition of amino acid transport in rat diaphragm by puromycin,'51-9 additional unpublished observations from this laboratory reveal that under appropriate preincubation and incubation conditions, the inhibitory effect on amino acid transport is also demonstrable in this tissue. All of these experiments show several common features: the delayed nature of the inhibitory effect, the ability of puromycin to inhibit the uptake of several utilizable and nonutilizable amino acids, and the failure to find evidence for nonspecific tissue injury. Initially, we must ask whether there is any evidence in our study to suggest that puromycin interferes with amino acid transport directly rather than by affecting the process secondarily through its effect on ribosomal protein synthesis. Several kinds of evidence suggest a negative answer to this question. These include: (1) the absence of an effect of puromycin on total tissue water or inulin spaces which might have been expected if gross cell shrinkage or swelling had been produced by direct membrane damage; (2) the failure to observe any effect on tissue Na+ or Iv+ concentrations; (3) the long preincubation interval needed to observe the inhibitory effect; (4) the observation that puromycin could be omitted from the incubation (but not the preincubation) medium without changing the inhibitory effect observed; and (5) the experiments with cycloheximide and puromycin analogues demonstrating a correspondence between inhibition of protein synthesis and impairment of amino acid transport. Thus, the present studies strongly suggest that puromycin inhibits amino acid transport by inhibiting protein synthesis. L-lysine incorporation into tissue protein was inhibited significantly by puromycin within ten minutes, while impairment of amino acid transport required minutes of preincubation with the antibiotic. These findings suggest that the delay noted in inhibition of transport is caused, not by failure of puromycin to achieve an intracellular location rapidly, but rather by the impaired synthesis and subsequent physiologic turnover of a protein or proteins responsible for mediated transport. This thesis would also explain the lack of requirement for puromycin in the final incubation medium. The data with AIB suggest that the catabolic "half life" of the protein(s) whose impaired synthesis is reflected in the transport inhibition is about 3'/2 hours. These comments lead to the fundamental question raised by these studies. Is puromycin inhibiting the synthesis of a specific "carrier" protein or enzyme needed for transmembrane amino acid transport or, alternatively, the synthesis of an enzyme or enzymes which catalyze reactions providing energy for the transport reactions? The results suggest that puromycin is inhibiting the synthesis of a protein(s) which selectively mediates amino acid influx. The failure to detect changes in oxygen consumption or tissue cation concentrations by puromycin suggests that aerobic

8 378 BIOCHEMISTRY: ELSA S AND ROSENBERG Pitoc. N. A. S. metabolism and active cation transport, two systems known to control amino acid transport, depend on enzymes whose rates of turnover are too long to be affected by the conditions of these experiments. These characteristics certainly are compatible with those of a postulated carrier protein or enzyme, but additional studies of substrate and group specificity, cation sensitivity, and rate constants for specific flux measurements are necessary to define with greater precision the specific proteins involved. Summary. Puromycin inhibited the uptake of a-aminoisobutyric acid, glycine, and 1-aminocy(lopeiitaiie-l-carboxylic acid by rat kidney cortex slices in vitro. Protein synthesis was inhibited within ten minutes by puromycin, but transport of amino acids was significantly impaired only after 120 minutes of preincubation. Cyclohexinmide also impaired a-aminoisobutyric acid uptake, but 6-dinmethylaminopurine and the aminonucleoside of puromycin did not. Puromycin did not affect oxygen consumption, tissue water spaces, or tissue cation concentration, and reduced a-aminoisobutyric acid accumulation by slowing influx. Efflux was not altered nor was the apparent affinity of this amino acid for its proposed carrier mechanism. These results suggest that puromycin inhibits the synthesis of a rapidly turning-over peptide(s) which catalyzes mediated amino acid transport. * This work was supported by grants from the USPHS (AM 09527) and from the John A. Hartford Foundation. t Trainee in 1\etabolism of the USPHS (AM ). t Recipient of Research Career Development Award, USPHS (AM 28, 087). 1 Fox, L. F., and E. P. Kennedy, these PROCEEDINGS, 54, 891 (1965). 2 Adamson, L. F., S. C. Langelluttig, and C. S. Anast, Biochim. Biophys. Acta, 115, 345 (1966). 3 Finerman, G. A. M., S. J. Downing, and L. E. Rosenberg, Biochim. Biophys. Acta, in press. 4 Rosenberg, L. E., A. Blair, and S. Segal, Biochim. Biophys. Acta, 54, 479 (1961). 5 Rosenberg, L. E., S. J. Downing, and S. Segal, J. Biol. (Chem., 237, 2265 (1962). 6 Rosenberg, L. E., M. Berman, and S. Segal, Biochim. Biophys. Acta, 71, 664 (1963). Fox, M., S. Thier, L. Rosenberg, and S. Segal, Biochim. Biophys. Acta, 79, 167 (1964). Segal, S., A. Blair, and L. E. Rosenberg, Biochim. Biophys. Acta, 71, 679 (1963). 9 Steinberg, D., M. Vaughan, C. B. Anfinson, J. D. Gorry, and J. Logan, Liquid Scintillation Counting (New York: Pergamon Press, 1958), p Manchester, K. L., and F. G. Young, Biochem. J., 70, 297 (1958). 11 The abbreviations used are: AIB, a-aminoisobutyric acid; ACPC, 1-aminocyclopentane-1- carboxylic acid. 12 Hofert, J. F., and R. K. Boutwell, Arch. Biochem. Biophys., 103, 338 (1963). 13 Garren, L. D., W. W. Davis, R. M. Crocco, and R. L. Ney, Science, 152, 1386 (1966). 14 Sovik, O., Acta Physiol. Scand., 66, 307 (1966). 15 Castles, J. J., and I. G. Wool, Biochem. J., 91, 116 (1964). 16 Fritz, G. R., and E. Knobil, Nature, 200, 682 (1963). 17 Burrows, G. N., and P. K. Bondy, Endocrinol., 75, 455 (1964). 18 Carlin, H., and 0. Hechter, Proc. Soc. Exptl. Biol. Med., 115, 127 (1964). 19 Eboue-Bonis, 1)., A. M. Chambaut, P. Volfin, and 11. Clauser, Nature, 199, 1183 (1963).