Life Sciences, Vol. 25, pp . 1497-1506 Printed in the U.S .A .
Pergamon Press
THE INFLUENCE OF GLUTATHIONE OXIDATION ON RENAL CORTEX TAURINE TRANSPORT Russell W . Chesney and Diane K . Jax Department of Pediatrics Pediatric Renal Disease laboratory The University of Wisconsin Center for Health Sciences Madison, Wisconsin 53792 (Received in final form September 11, 1979) Summary The interaction of diemide, a rapidly reversible thioloxidizing reagent, with the tsarina accumulation system was examined in rat kidney cortex slices from animals of different ages . Diamide at 10 mM lowered renal cortex glutathione content by SOx at a time that tsarina accumulation was inhibited by 65% . Although the addition of equimolar GSH overcame diamade inhibition of tsarina uptake, GSH per se inhibited tsarina accumulation at 0 .01 mM, but not at 0 .2 or 1.0 mM . Dithiothreitol (DTT) also overcame diemide inhibition of uptake . As previously shown by Pillion et al (Etiu . J . Bioch® . 79, 73, 1977) diamade inhibited gluconeogenesis by cortex slices . Diami de inhibited tourins accumulation by 85~ by the low Km tourins transport site in cortex from newborn, 2 week, 4 week and adult animals, but only 50z at the high Km site . In contrast to the situation in adult tissue, efflux of tourins from preloaded slices of immature animals was not increased by diamide . Accordingly, one maturational event identified by these studies is that diamade-enhanced efflux was found on]y in mature cortex . Diazenedicarboxylic acid bia (N,N-dimethylemide), having the trivial name diamade, is a rapidly reversible oxidant of erythrocyte intracellular glutathione (GSH) (1,2) . It can also react with reduced pyridine nucleotides and membrane bound sulfhydryl groups (3,~+), but is primarily a GSH oxidant that will both lower GSH concentrations within rat renal cortex and suppress the active accumulation of a-amino acids (5) and a-methyl-D-glucoside (6,7) in renal cortex slices . Diamide will also inhibit endogenous renal cortex protein kinase activity (6) . When GSH or other thi~ols are added, they will reverse the inhibition of this enzyme activity and the suppression of amino acid and sugar transport . Diamade will also reversibly inhibit gluconeogenesis by renal cortex (8) . We have previously examined the influence of diamade on the renal cortex slice accumulation of tsarina (9,10), the ß-amino acid that is characteristic of x'odent urine (11,12) . Diemide inhibited tsarina accumulation of the low Km (0 .01 mM) high affinity uptake system far more than the high Km (13 mM), low affinity system (9,10) . Tsarina uptake was inhibited under two experimental settings : (a) with simulteaeous incubation with diamade and (b) preincubation of slices in diamade containing medium that prevented tsarina accumulation even 0024-3205/79/171497-10$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1498
Diamide Influence on Renal Taurine Uptake
Vol. 25, No . 17, 1979
when the slices were later incubated in a diamade-free medium . Simultaneous preincubation of diamade and equimolar GSH (20 mM) largely prevented this diamade induced inhibition of taurine uptake . In addition, diamade strongly enhanced taurine efflux from tissue preloaded with this ß-amino compound at typical low Km site concentrations . These previous studies suggested that the decreased taurine accumulation may have resulted from this increased efflux in the presence of dia.mide (9,10), but failed to examine the influence of GSH alone or other oxidants on taurine uptake . We have also examined the ontogeny of taurine transport in newborn rat cortex and found a neonatal taurinuria disappearing between the second and fourth week of life (13) . In addition, in vitro cortex from immature animals had the same two uptake systems found in adult tissue, but slices from young animals demonstrated decreased efflux and reduced initial rate of taurine uptake (14) as had been shown for other a-amino acids (15,16) . The purpose of this paper is .to examine further the characteristics of diamade action on renal tubular taurine accumulation by adult Spre .gue Dawley rats and by immature animal renal cortex . In addition the influence both of GSH per se and dithiothreitol (Di"P) on taurine accumulation will also be described to further From these studies further understanding define the action of GSH oxidation . of ß-amino acid transport can be gained . Msterials and Methods Sprague Dawley rats of Animals and tissue incubation and preparation . different s,ges were obtained from Holtzman Co ., Madison, WI . Animals were Kidneys used were from caged separately and fed a standard Lab-Blox Chow . Thin newborn (within 36 hours of birth), 2 week, 4 week and adult animals . cortex slices were prepared as previously described (14 ) . In general, tissue was incubated as described under 1004 OZ using a Tris-Ringer glucose buffer medium (14) . Slices were removed, blotted, weighed, boiled and the supernate containing [1- 1 "C] taurine counted as described elsewhere (17) . The incubation medium was altered for certain studies ; varying concentrations of GSH, diamade end DTT were used . Slices were also incubated in medium alone, or medium and 20 mM diamade or medium with diamade (20 mM) plus GSH (20 mM) on ice for 15 Slices were then removed, blotted and placed in fresh flasks conminutes . All media were taining 20 ml buffer and again incubated with taurine at 37 o C . adjusted to pH 7 .4 and gassed with 1004 02 . The efflux of intracellular taurine was determined by the method of Segal et al (18) . Slices from animals of various ages were incubated fYcm 60-90 minutes in a taurine-containing medium so as to have similar taurine levels . Tissue was then removed, blotted, weighed and placed in substrate-free medium . Some media contained diamade or diamade plus GSH . Samples of medium were removed every 2 minutes up to 16 minutes and the rate of appearance of 14 Ctaurine into the medium and the renal tissue levels were measured . The rate of gluconeogenesis was determined as described by Pillion et al (.8 ) . Briefly, the appearance of glucose in the incubation medium was measured using glucose oxidase. Tissue slices from adult rats (70-180 mg) were incubated with or without 9 mM diamide at 0-4° C for 15 m~nutes and then placed in an Glucose incubation medium containing 10 mM pyruvate at 3i C for 60 minutes . formation was measured in 0 .5 m1 aliquots of the incubation medium using protein-free supernat es after TCA precipitation. Aa sis . The concentration of GSH in homogenates of tissue slices was measured by Hewatt et al (5) . Simply, this involved the increase in absorbante measured at 412 nanameters after incubation in 64 TCA, 0 .3 M Na2POq and 0 .044 The level of glucose in the medium after slice DTNB in 14 sodium citrate .
Vol . 25, No . 17, 1979
Diamade Influence on Renal Taurine Uptake
lá99
incubation experiments was measured by the glucose oxidase method (Sigma Technical Bulletin No 510) . Distribution ratiós were determined by the method of Rosenberg et al (19) . All slices experiments were performed in duplicate and data compared using the Student's t test . Materials . [1- 1 "C] Taurine (spec act 56 .1 mCi/mMole) and [1- 14 C]-a-aminoisobutyric acid (a-AIB) (spec act 9 mCi/mMole) were obtained frcmm New England Nuclear . Diamade, dithiothreitol, glutathione, DTNB (5,5-dithio-bie-2-nitro benzoic acid) and the glucose oxidase kit were obtained from Sigma Chemical Co . Results A . Dose dependency of diamade inhibition of ß-amino acid accumulation . Maximal inhibition of taurine uptake was seen when the diamade concentration was 9 mM or higher (Fig . 1) . Incubation of slices in diamade containing medium led to a fall in the levels of reduced glutathione within rat kidney cortex slices . Slices were incubated in Tris-Ringer glucose medium alone or containing 10 mM diamade for 15 minutes and then placed in medium at 3i oC for 60 min ., removed and washed in diamade-free medümm at 25 o C for 15 minutes and then placed in medium at 37 o C for 60 minutes . GSH levels fell gradually in the medium from 860 _+ 28 (SE) uem GSH/gm cortex tissue to k52 + 31 . A marked drop in GSH from 860 to 180 _+ 14 }1gm GSH/gm cortex tissue was noted in the diamade containing flasks . After removal of this tissue from the diamade containing medium, the level of GSH rose to 320 _+ b0 ugm/gm tissue, net weight not different from the value in the diamade-free medium incubated slices . Parallel slices incubated in [1- 14 C] taurine at 0 .01 mM had a 65~ reduction in taurine uptake by the diamade tr~ted tissue . These results confirm the findings of Hewitt et al (5) that diami de incubation with renal cortex depletes GSH, although their studies were performed with a Kreba-Ringer HCO~ buffer . Our earlier studies had suggested that incubation of cortex slices in medium containing GSH could réduce taurine uptake (9) . We examined this point more fl~lly by using several taurine concentrations and a wide range of GSH levels (Fig . 2) . The uptake of taurine at either 0 .2 or 1 .0 mM was unaffected by concentrations of GSH ranging from 0 .2 to 2 .0 mM . However, all concentratioms of GSH inhibited taurine uptake at 0 .01 mM, a representative concentraaccumulated largely by the low Km system . GSH did not alter taurine uptake at 0 .2 and 1 .0 mM, levels of taurine largely accumulated by the high Km system . GSH levels of 0 .02 through 20 mM significantly inhibited 0 .01 mM taurine uptake . Nonetheless, as we have shown previously the addition of equimolar GSH to diamade in the preincubation step will essentially prevent diamade related inhibition of taurine uptake at 0 .01 mM (9,10) . To ascertain if sulfhydryl agents other than GSH could reverse dienvide inhibition, the effect of DTT on taurine uptake was also examined (Table I) . Fairly high concentrations of daamide were used in these studies, to P~lly establish that DTT could protect GSH . These results indicated that DTT can reverse the inhibition of taurine uptake as effectively as GSH, if placed in the preincubation step . However, when incubated directly with oiaamide and taurine, DTT did not overcome diamade-induced inhibition . B. Effect of diami de on metabolism . Gluconeogenesis, measured by appearance of glucose in the incubation medium, was found when slices were incubated in pyruvate (10 mM) containing medium . The rate of formation of glucose fell in the presence of diami de and was similar to the levels found in the absence of all substrates . After removal of slices from a diami de containing solution the inhibition of gluconeogeneais was completely reversed . Whether slices were
1500
Diamide Influence on Renal Taurine Uptake
Vol. 25, No . 17, 1979
U U - W
o ~É E a n.
UU O
8
á
6
z
0
4
m O
DIAMIDE mM FIG . 1 The influence of diamide on the uptake of 0.01 mM taurine by slices Each incubated in Tris-Ringer buffer at pH 7.4 under 100 OZ for 60 minutes . point is the mean of 12 flasks .
0
ó ó
c0
[Tourine]
w0
~ 0 .01 mM n 0 .20 mM O LOmM
0
Y O á a, c
H
FIG. 2 The influence of increasing concentrations of GSH on taurine accumulation . Slices were incubated in Tris-Ringer medium with the taurine concentrations shown . Taurine is expressed as a percent of uptake at GSH concentration = 0 . Each point represents the mean of + SE of 6 determinations .
piamide Influence on Renal Taurine Uptake
Vol . 25, No . 17, 1979
1501
100 90 80 70 a 60 c 50
c c E 0
40 30 20
FIG . 3 The efflux of taurine from slices incubated in 0 .01 mM taurine for 60-90 minutes expressed as x taurine remaining in the slice . Slices were placed in : taurine-free medium alone ~ ; taurine-free medium plus diamide (0 .02 M) o ; taurine-free medium plus diamide plus GSH (0 .02 M) 1 ; taurine-free medium plus GSH (0 .02 M) ~ . Each point represents the mean of 9-12 determinations .
100 90 80 70 ~ 60 .~ 50 c v 40 E ~ 30 d~ 20
2
4
6
8
10
Time (min)
12
14
16
FIG . la
The efflux of taurine from slices incubated in taurine at 1 .0 mM for 60 to 120 minutes . Tissue taurine levels were comparable at the onset of the efflux study . Fach point represents the mean of 9 determinations . The values in neonatal slices were significantly different frc~ the values in adults slices except at 2 minutes where the percent remaining was similar at both ages in the presence of diamide and diamide + GSH .
150 2
Vol. 25, No . 17, 1979
Diamide Influence on Renal Taurine Uptake
preincubated in diamide or not, the rates of glucose formation from 10 mM pyruvate were comparable to those found by Pil~ion et al (8) . Furthermore, performance of the final incubation step at 3~ C in a diamide containing medium either with or without pyruvate prevented glucose formation . TABLE I The Influence of Dithiothreitol (DTT) on the Distribution Ratio of Taurine in Diamide-Treated Slices A
B
Taurine
6 .6 + 0 .1
3 .0 + 0.1
Taurine + DTT
6 .0 + 0 .1
3 .1 + 0.2
Taurine + DTT + D
1 .7 + 0 .2*
3 .4 + 0.4
Taurine + D
1 .1 + 0 .1*
2 .1 + 0.2*
*p < .O1
Slices were incubated with Tris-Ringer glucose medium under 2 experimental settins : (A) Incubation in 0 .11 mM taurine at 3~ oC for 60 minutes with or without the addition of 30 mM diamide (D) and/or 20 mM DTT . (B) Slices were preincubated at 4° C for 15 minutes with or without diamide (D) and/or DTT at the same concentration (incubation 1). . The tissues were then incubated for 60 minutes at 3Z o C in the presence of 0.11 mM taurine . Values represent the mean ratio _+ S .E . from 12 experiments . Since diamide led to a reduction in gluconeogensis in kidney cortex slices it is possible that this metabolic effect could be related to the diamide induced inhibition of taurine uptake . Nonetheless, since slice experiments were performed in the presence of 5 mM glucose in the medium, it is important to Were this know whether this exogenous glucose is metabolized by cortex slices . glucose utilized, then gluconeogenesis might be unnecessary . Accordingly, we examined the metabolism of glucose by kidney cortex slices not exposed to Approximately 60 to 90 mg of tissue ws.s incubated diamide from adult animals . The medium was filtered through a millipore in medium at 3T C for 60 minutes . (0 .45 u) filter prior to these studies to preclude bacterial contamination . The medümm concentration of glucose before and after incubation was measured in protein-free filtrates . The utilization of glucose by tissue slices was 4 .1 _+ 1 .5 ~tmoles/gm tissue wet weight/60 minutes (mean + SE), n = 40, or approximateSince the samples in these stuly 0 .24 umoles utilized per 60 mg of tissue . dies were incubated with 10 Umoles of glucose, this represented a use of only 2 .4~ the available glucose . The utlization of glucose was not measured in the presence of diamide . C. Diamide influence in immature kidney cortex . The addition of diamide to the incubation medium was examined in tissue fra~m immature animals (newborn, 2 week and 4 week old) and adults . The uptake of both 0.01 and 1 .0 mM tsarina vas examined since the lower concentration is largely taken up by the first
150 3
Diamade Influence on Renal Taurine Uptake
Vol . 25, No . 17, 1979
transpa~rt site (apparent Km~ = 0 .1 mM) and the higher concentration by the second site (apparent KmZ =~12 mM) (Table II) . In almost every case diamade significantly inhibited the concentratioe accumulation of taurine ; only at 1 .0 mM taurine in 4 week old animals did diamade not inhibit taurine accumulation . Under those conditions, diamade behaved in immature tissue as it did in adult tissue : Diamade inhibited taurine accumulation at 0 .01 mM by ~5 to 85 percent and inhibited 1 .0 mM taurine accumulation in newborn by 50 percent . Where diami de was added to the medium in a preincubation step and tissue was then washed free and placed in taurine containing medium diamade again prevented uptake (Table III) . Accordingly, diami de acts to impair taurine uptake in tissue from animals of all ages but may be slightly more active in tissue from adult animals, hence higher diamade concentrations were used in these studies in immature animals . TABLE II Taurine Isotopic Distribution Ratio Taurine Concentration
Age of Animal
0 .01 mM
Newborn
9 .0 _+ 0 .5 (n = 10)
1 .6 _+ 0 .1 (n = 10)
< .001
2 week
8 .4 + 0 .6
2 .2 + 0 .1
< .001
4 week
6 .8 + 0 .1 (n --5)
1 .3 + 0 .1 (n=5)
< .001
10 .7 + 0 .4
2 .1 + 0 .1
< .001
Pergamon Press
THE INFLUENCE OF GLUTATHIONE OXIDATION ON RENAL CORTEX TAURINE TRANSPORT Russell W . Chesney and Diane K . Jax Department of Pediatrics Pediatric Renal Disease laboratory The University of Wisconsin Center for Health Sciences Madison, Wisconsin 53792 (Received in final form September 11, 1979) Summary The interaction of diemide, a rapidly reversible thioloxidizing reagent, with the tsarina accumulation system was examined in rat kidney cortex slices from animals of different ages . Diamide at 10 mM lowered renal cortex glutathione content by SOx at a time that tsarina accumulation was inhibited by 65% . Although the addition of equimolar GSH overcame diamade inhibition of tsarina uptake, GSH per se inhibited tsarina accumulation at 0 .01 mM, but not at 0 .2 or 1.0 mM . Dithiothreitol (DTT) also overcame diemide inhibition of uptake . As previously shown by Pillion et al (Etiu . J . Bioch® . 79, 73, 1977) diamade inhibited gluconeogenesis by cortex slices . Diami de inhibited tourins accumulation by 85~ by the low Km tourins transport site in cortex from newborn, 2 week, 4 week and adult animals, but only 50z at the high Km site . In contrast to the situation in adult tissue, efflux of tourins from preloaded slices of immature animals was not increased by diamide . Accordingly, one maturational event identified by these studies is that diamade-enhanced efflux was found on]y in mature cortex . Diazenedicarboxylic acid bia (N,N-dimethylemide), having the trivial name diamade, is a rapidly reversible oxidant of erythrocyte intracellular glutathione (GSH) (1,2) . It can also react with reduced pyridine nucleotides and membrane bound sulfhydryl groups (3,~+), but is primarily a GSH oxidant that will both lower GSH concentrations within rat renal cortex and suppress the active accumulation of a-amino acids (5) and a-methyl-D-glucoside (6,7) in renal cortex slices . Diamide will also inhibit endogenous renal cortex protein kinase activity (6) . When GSH or other thi~ols are added, they will reverse the inhibition of this enzyme activity and the suppression of amino acid and sugar transport . Diamade will also reversibly inhibit gluconeogenesis by renal cortex (8) . We have previously examined the influence of diamade on the renal cortex slice accumulation of tsarina (9,10), the ß-amino acid that is characteristic of x'odent urine (11,12) . Diemide inhibited tsarina accumulation of the low Km (0 .01 mM) high affinity uptake system far more than the high Km (13 mM), low affinity system (9,10) . Tsarina uptake was inhibited under two experimental settings : (a) with simulteaeous incubation with diamade and (b) preincubation of slices in diamade containing medium that prevented tsarina accumulation even 0024-3205/79/171497-10$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1498
Diamide Influence on Renal Taurine Uptake
Vol. 25, No . 17, 1979
when the slices were later incubated in a diamade-free medium . Simultaneous preincubation of diamade and equimolar GSH (20 mM) largely prevented this diamade induced inhibition of taurine uptake . In addition, diamade strongly enhanced taurine efflux from tissue preloaded with this ß-amino compound at typical low Km site concentrations . These previous studies suggested that the decreased taurine accumulation may have resulted from this increased efflux in the presence of dia.mide (9,10), but failed to examine the influence of GSH alone or other oxidants on taurine uptake . We have also examined the ontogeny of taurine transport in newborn rat cortex and found a neonatal taurinuria disappearing between the second and fourth week of life (13) . In addition, in vitro cortex from immature animals had the same two uptake systems found in adult tissue, but slices from young animals demonstrated decreased efflux and reduced initial rate of taurine uptake (14) as had been shown for other a-amino acids (15,16) . The purpose of this paper is .to examine further the characteristics of diamade action on renal tubular taurine accumulation by adult Spre .gue Dawley rats and by immature animal renal cortex . In addition the influence both of GSH per se and dithiothreitol (Di"P) on taurine accumulation will also be described to further From these studies further understanding define the action of GSH oxidation . of ß-amino acid transport can be gained . Msterials and Methods Sprague Dawley rats of Animals and tissue incubation and preparation . different s,ges were obtained from Holtzman Co ., Madison, WI . Animals were Kidneys used were from caged separately and fed a standard Lab-Blox Chow . Thin newborn (within 36 hours of birth), 2 week, 4 week and adult animals . cortex slices were prepared as previously described (14 ) . In general, tissue was incubated as described under 1004 OZ using a Tris-Ringer glucose buffer medium (14) . Slices were removed, blotted, weighed, boiled and the supernate containing [1- 1 "C] taurine counted as described elsewhere (17) . The incubation medium was altered for certain studies ; varying concentrations of GSH, diamade end DTT were used . Slices were also incubated in medium alone, or medium and 20 mM diamade or medium with diamade (20 mM) plus GSH (20 mM) on ice for 15 Slices were then removed, blotted and placed in fresh flasks conminutes . All media were taining 20 ml buffer and again incubated with taurine at 37 o C . adjusted to pH 7 .4 and gassed with 1004 02 . The efflux of intracellular taurine was determined by the method of Segal et al (18) . Slices from animals of various ages were incubated fYcm 60-90 minutes in a taurine-containing medium so as to have similar taurine levels . Tissue was then removed, blotted, weighed and placed in substrate-free medium . Some media contained diamade or diamade plus GSH . Samples of medium were removed every 2 minutes up to 16 minutes and the rate of appearance of 14 Ctaurine into the medium and the renal tissue levels were measured . The rate of gluconeogenesis was determined as described by Pillion et al (.8 ) . Briefly, the appearance of glucose in the incubation medium was measured using glucose oxidase. Tissue slices from adult rats (70-180 mg) were incubated with or without 9 mM diamide at 0-4° C for 15 m~nutes and then placed in an Glucose incubation medium containing 10 mM pyruvate at 3i C for 60 minutes . formation was measured in 0 .5 m1 aliquots of the incubation medium using protein-free supernat es after TCA precipitation. Aa sis . The concentration of GSH in homogenates of tissue slices was measured by Hewatt et al (5) . Simply, this involved the increase in absorbante measured at 412 nanameters after incubation in 64 TCA, 0 .3 M Na2POq and 0 .044 The level of glucose in the medium after slice DTNB in 14 sodium citrate .
Vol . 25, No . 17, 1979
Diamade Influence on Renal Taurine Uptake
lá99
incubation experiments was measured by the glucose oxidase method (Sigma Technical Bulletin No 510) . Distribution ratiós were determined by the method of Rosenberg et al (19) . All slices experiments were performed in duplicate and data compared using the Student's t test . Materials . [1- 1 "C] Taurine (spec act 56 .1 mCi/mMole) and [1- 14 C]-a-aminoisobutyric acid (a-AIB) (spec act 9 mCi/mMole) were obtained frcmm New England Nuclear . Diamade, dithiothreitol, glutathione, DTNB (5,5-dithio-bie-2-nitro benzoic acid) and the glucose oxidase kit were obtained from Sigma Chemical Co . Results A . Dose dependency of diamade inhibition of ß-amino acid accumulation . Maximal inhibition of taurine uptake was seen when the diamade concentration was 9 mM or higher (Fig . 1) . Incubation of slices in diamade containing medium led to a fall in the levels of reduced glutathione within rat kidney cortex slices . Slices were incubated in Tris-Ringer glucose medium alone or containing 10 mM diamade for 15 minutes and then placed in medium at 3i oC for 60 min ., removed and washed in diamade-free medümm at 25 o C for 15 minutes and then placed in medium at 37 o C for 60 minutes . GSH levels fell gradually in the medium from 860 _+ 28 (SE) uem GSH/gm cortex tissue to k52 + 31 . A marked drop in GSH from 860 to 180 _+ 14 }1gm GSH/gm cortex tissue was noted in the diamade containing flasks . After removal of this tissue from the diamade containing medium, the level of GSH rose to 320 _+ b0 ugm/gm tissue, net weight not different from the value in the diamade-free medium incubated slices . Parallel slices incubated in [1- 14 C] taurine at 0 .01 mM had a 65~ reduction in taurine uptake by the diamade tr~ted tissue . These results confirm the findings of Hewitt et al (5) that diami de incubation with renal cortex depletes GSH, although their studies were performed with a Kreba-Ringer HCO~ buffer . Our earlier studies had suggested that incubation of cortex slices in medium containing GSH could réduce taurine uptake (9) . We examined this point more fl~lly by using several taurine concentrations and a wide range of GSH levels (Fig . 2) . The uptake of taurine at either 0 .2 or 1 .0 mM was unaffected by concentrations of GSH ranging from 0 .2 to 2 .0 mM . However, all concentratioms of GSH inhibited taurine uptake at 0 .01 mM, a representative concentraaccumulated largely by the low Km system . GSH did not alter taurine uptake at 0 .2 and 1 .0 mM, levels of taurine largely accumulated by the high Km system . GSH levels of 0 .02 through 20 mM significantly inhibited 0 .01 mM taurine uptake . Nonetheless, as we have shown previously the addition of equimolar GSH to diamade in the preincubation step will essentially prevent diamade related inhibition of taurine uptake at 0 .01 mM (9,10) . To ascertain if sulfhydryl agents other than GSH could reverse dienvide inhibition, the effect of DTT on taurine uptake was also examined (Table I) . Fairly high concentrations of daamide were used in these studies, to P~lly establish that DTT could protect GSH . These results indicated that DTT can reverse the inhibition of taurine uptake as effectively as GSH, if placed in the preincubation step . However, when incubated directly with oiaamide and taurine, DTT did not overcome diamade-induced inhibition . B. Effect of diami de on metabolism . Gluconeogenesis, measured by appearance of glucose in the incubation medium, was found when slices were incubated in pyruvate (10 mM) containing medium . The rate of formation of glucose fell in the presence of diami de and was similar to the levels found in the absence of all substrates . After removal of slices from a diami de containing solution the inhibition of gluconeogeneais was completely reversed . Whether slices were
1500
Diamide Influence on Renal Taurine Uptake
Vol. 25, No . 17, 1979
U U - W
o ~É E a n.
UU O
8
á
6
z
0
4
m O
DIAMIDE mM FIG . 1 The influence of diamide on the uptake of 0.01 mM taurine by slices Each incubated in Tris-Ringer buffer at pH 7.4 under 100 OZ for 60 minutes . point is the mean of 12 flasks .
0
ó ó
c0
[Tourine]
w0
~ 0 .01 mM n 0 .20 mM O LOmM
0
Y O á a, c
H
FIG. 2 The influence of increasing concentrations of GSH on taurine accumulation . Slices were incubated in Tris-Ringer medium with the taurine concentrations shown . Taurine is expressed as a percent of uptake at GSH concentration = 0 . Each point represents the mean of + SE of 6 determinations .
piamide Influence on Renal Taurine Uptake
Vol . 25, No . 17, 1979
1501
100 90 80 70 a 60 c 50
c c E 0
40 30 20
FIG . 3 The efflux of taurine from slices incubated in 0 .01 mM taurine for 60-90 minutes expressed as x taurine remaining in the slice . Slices were placed in : taurine-free medium alone ~ ; taurine-free medium plus diamide (0 .02 M) o ; taurine-free medium plus diamide plus GSH (0 .02 M) 1 ; taurine-free medium plus GSH (0 .02 M) ~ . Each point represents the mean of 9-12 determinations .
100 90 80 70 ~ 60 .~ 50 c v 40 E ~ 30 d~ 20
2
4
6
8
10
Time (min)
12
14
16
FIG . la
The efflux of taurine from slices incubated in taurine at 1 .0 mM for 60 to 120 minutes . Tissue taurine levels were comparable at the onset of the efflux study . Fach point represents the mean of 9 determinations . The values in neonatal slices were significantly different frc~ the values in adults slices except at 2 minutes where the percent remaining was similar at both ages in the presence of diamide and diamide + GSH .
150 2
Vol. 25, No . 17, 1979
Diamide Influence on Renal Taurine Uptake
preincubated in diamide or not, the rates of glucose formation from 10 mM pyruvate were comparable to those found by Pil~ion et al (8) . Furthermore, performance of the final incubation step at 3~ C in a diamide containing medium either with or without pyruvate prevented glucose formation . TABLE I The Influence of Dithiothreitol (DTT) on the Distribution Ratio of Taurine in Diamide-Treated Slices A
B
Taurine
6 .6 + 0 .1
3 .0 + 0.1
Taurine + DTT
6 .0 + 0 .1
3 .1 + 0.2
Taurine + DTT + D
1 .7 + 0 .2*
3 .4 + 0.4
Taurine + D
1 .1 + 0 .1*
2 .1 + 0.2*
*p < .O1
Slices were incubated with Tris-Ringer glucose medium under 2 experimental settins : (A) Incubation in 0 .11 mM taurine at 3~ oC for 60 minutes with or without the addition of 30 mM diamide (D) and/or 20 mM DTT . (B) Slices were preincubated at 4° C for 15 minutes with or without diamide (D) and/or DTT at the same concentration (incubation 1). . The tissues were then incubated for 60 minutes at 3Z o C in the presence of 0.11 mM taurine . Values represent the mean ratio _+ S .E . from 12 experiments . Since diamide led to a reduction in gluconeogensis in kidney cortex slices it is possible that this metabolic effect could be related to the diamide induced inhibition of taurine uptake . Nonetheless, since slice experiments were performed in the presence of 5 mM glucose in the medium, it is important to Were this know whether this exogenous glucose is metabolized by cortex slices . glucose utilized, then gluconeogenesis might be unnecessary . Accordingly, we examined the metabolism of glucose by kidney cortex slices not exposed to Approximately 60 to 90 mg of tissue ws.s incubated diamide from adult animals . The medium was filtered through a millipore in medium at 3T C for 60 minutes . (0 .45 u) filter prior to these studies to preclude bacterial contamination . The medümm concentration of glucose before and after incubation was measured in protein-free filtrates . The utilization of glucose by tissue slices was 4 .1 _+ 1 .5 ~tmoles/gm tissue wet weight/60 minutes (mean + SE), n = 40, or approximateSince the samples in these stuly 0 .24 umoles utilized per 60 mg of tissue . dies were incubated with 10 Umoles of glucose, this represented a use of only 2 .4~ the available glucose . The utlization of glucose was not measured in the presence of diamide . C. Diamide influence in immature kidney cortex . The addition of diamide to the incubation medium was examined in tissue fra~m immature animals (newborn, 2 week and 4 week old) and adults . The uptake of both 0.01 and 1 .0 mM tsarina vas examined since the lower concentration is largely taken up by the first
150 3
Diamade Influence on Renal Taurine Uptake
Vol . 25, No . 17, 1979
transpa~rt site (apparent Km~ = 0 .1 mM) and the higher concentration by the second site (apparent KmZ =~12 mM) (Table II) . In almost every case diamade significantly inhibited the concentratioe accumulation of taurine ; only at 1 .0 mM taurine in 4 week old animals did diamade not inhibit taurine accumulation . Under those conditions, diamade behaved in immature tissue as it did in adult tissue : Diamade inhibited taurine accumulation at 0 .01 mM by ~5 to 85 percent and inhibited 1 .0 mM taurine accumulation in newborn by 50 percent . Where diami de was added to the medium in a preincubation step and tissue was then washed free and placed in taurine containing medium diamade again prevented uptake (Table III) . Accordingly, diami de acts to impair taurine uptake in tissue from animals of all ages but may be slightly more active in tissue from adult animals, hence higher diamade concentrations were used in these studies in immature animals . TABLE II Taurine Isotopic Distribution Ratio Taurine Concentration
Age of Animal
0 .01 mM
Newborn
9 .0 _+ 0 .5 (n = 10)
1 .6 _+ 0 .1 (n = 10)
< .001
2 week
8 .4 + 0 .6
2 .2 + 0 .1
< .001
4 week
6 .8 + 0 .1 (n --5)
1 .3 + 0 .1 (n=5)
< .001
10 .7 + 0 .4
2 .1 + 0 .1
< .001
