ARCHIVES OF BIOCHEMISTRY Vol. 191, No. 2, December,
Transport
AND BIOPHYSICS pp. 551-560, 1978
of L-Arginine
in Brush Border Vesicles Derived Kidney Cortex’ DIETRICH
BUSSE
Department of Cell Physiology, Ruhr-Universitiit Received
May,
from Rabbit
Bochum, 4630 Bochum 1, German Federal Republic
1978; revised
August
7,1978
The uptake of L-arginine by brush border vesicles from rabbit kidney cortex was investigated at 37°C and pH 7.5. The initial rate of uptake (15 s) was twice as fast in a highly purified brush border as in brush border contaminated by basal-lateral plasma membranes. The initial uptake in a mannitol medium can be best described as the sum of transfer by two systems with K, values of 0.07 and 3.5 mM and V,, values of 1.5 and 8 nmol/mg protein x 15 s, respectively. For the inhibitors of L-[‘4C]arginine, uptake (15 s at two substrate concentrations of 0.1 and 2.5 mru in a mannitol medium) the following sequence of inhibitory strength was established: L-arginine, L-ornithine, L-cyst&e, L-lysine, n-arginine, and NaCl. When a vesicular membrane potential was induced transiently by a jump of the pH in the incubation medium from 5.9 to 7.5 or by an outward movement of K+ in the presence of gramicidin D, an overshoot of L-arginine uptake was observed. Initial uptake of L-arginine was slightly faster in the presence of a Na+ gradient (outside to inside) than under a K+ gradient. Both ion gradients reduced uptake as compared to the uptake in a mannitol medium. Uptake was also studied after the membrane potential was minimized by equilibrating the vesicles in a NaCl or KC1 medium in the presence of gramicidin D. Under these conditions, L-arginine uptake in the first 30 s was faster in the NaCl than in the KC1 medium. These experiments indicate, beside a major ion-independent L-arginine transport, the presence of a transport stimulated by Na+ in isolated brush border vesicles.
In 1951, cystinuria was recognized as an inherited defect of the tubular reabsorption of cystine and the dibasic amino acids in the kidney (1). Because patients with the disorder excreted large quantities of cystine, arginine, and lysine (2), it was believed that those amino acids were reabsorbed, at least partially, by a common mechanism. Disappointingly, in vitro experiments on kidney slices and tubule segments failed to show transport competition between cystine and the dibasic amino acids (3, 4). Later micropuncture studies on the proximal kidney tubule did show an inhibition of cystine transport by arginine (5), but could not demonstrate the reverse, an inhibition of the transport of arginine by cystine (6). 1 A preliminary report of this work has been presented at the 46th meeting of the Deutsche Physiologische Gesellschaft, March 1977 in Regensburg (G. F. R.) (Busse, D. Pohl, B., and Bartel, H. (1977) Pfltigers Arch. 368, R 13). This work was supported by the Deutsche Forschungsgemeinschaft.
The inhibition of cystine transport by arginine could be reproduced on brush border vesicles recently (4). Equally puzzling was the effect of Na+ upon the transport of the dibasic amino acids. In kidney slices, lysine and arginine are actively transported despite a lack of Na+ in the incubation medium (8), whereas in recent micropuncture experiments, the transport of arginine, lysine, and ornithine was highly Na+-dependent (9, 10). It was agreed that much of the controversial results were due to the fact that in kidney slices and isolated tubules, the transport through the luminal brush border, which is the site of cystine-dibasic amino acid interaction (7,11), was obscured by uptake through the basal-lateral cell membrane (7). The transport of the dibasic amino acids and cystine through the luminal brush border is apparently an area of research lacking much definite knowledge. Observations which suggest two transport systems for
551 0003-9861/78/1912-0551$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
552
DIETRICH
the dibasic amino acids and two for cystine complicate this field even more (6, 7,12). A new technique, such as the measurement of transport in isolated brush border vesicles, might bring some insights into the reaction of these amino acids with the membrane. This study shows the inhibition of L-arginine transport by cystine, which was postulated by clinical observations, for the first time experimentally. As will be seen, the situation in vitro is quite different from that in viuo, because in vitro no active pumping of ions linked to metabolism takes place. Therefore, the accumulation of L-arginine at equilibrium seen in this study is probably caused only by a A pH in these vesicles. Na+ like K’, when added as chloride salts, inhibit initial L-arginine transfer by building up a transient lumen-positive membrane potential and reduce uptake at equilibrium by decreasing the A pH. However, when the effects of Na+ and K+ were compared, the initial transport of L-arginine was faster in the NaCl medium under conditions of an increased, as well as of a highly reduced, membrane potential. MATERIALS
AND
METHODS
L-[ U-‘%]Arginine, [Y!]methylamine, and 3H-water were purchased from New England Nuclear Co. Carbony1 cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D were purchased from Boehringer Mannheim GmbH, G.F.R. All other reagents were of the highest purity available.
Preparation of brush border vesicles and measurement of uptake. The brush border vesicles prepared from isolated tubule segments of rabbit kidney by hypotonic lysis, isolated by differential centrifugation, and purified further by a density gradient centrifugation have been described previously (13). The assays of the enzymes used as markers for various cell organelles have also been described (13). The brush border vesicles were used as soon as the isolation was completed which means that the time between the killing of the rabbits and the study of transport was never longer than 12 h. All studies of uptake were performed at pH 7.5 and a temperature of 37°C. Termination of uptake, separation of the brush border vesicles from the incubation medium by centrifugation through a layer of oil, and the measurement of uptake by a double isotope technique using 3H-water as a marker for the sediment volume and poly-[“%]ethyleneglycol as a marker for the extravesicular space of the sediment have also been described previously (14). Paper chromatography. For chromatography, the radioactive material was extracted from the vesicular
BUSSE sediment three times with 60% ethanol. Aliquots of the combined extracts and of the incubation medium were spotted on Whatman No. 1 paper and chromatographed in two chromatographic systems, descending with phenol saturated by water as a solvent, and descending with the solvent butanol:acetic acid:water (4:l:l; v/v/v). After chromatography, the chromatograms were cut into strips and analyzed for radioactivity in a liquid scintillation counter. RESULTS
The brush border prepared by hypotonic lysis and isolated by differential centrifugation has been characterized previously (13). By a density gradient centrifugation (13), this brush border was separated further into brush border contaminated by basal-lateral plasma membrane (upper part of the density gradient), highly purified brush border (central part), and brush border contaminated by mitochondria (lower part). Since all three brush border preparations derived from the density gradient centrifugation were used for the study of transport, an enzymatic characterization is given in Table I. In addition, the bottom line of Table I gives the volume enclosed by the vesicles. As can be seen, the intravesicular volume does not differ significantly in upper and central part of the density gradient, whereas the lower part contains membranes with a 40% smaller volume enclosed per mg of membrane protein, suggesting that nonvesicular brush border with a higher specific density had been concentrated in this part of the gradient. Specificity and Saturability Uptake
of L-Arginine
Figure 1 depicts the uptake of L-arginine in a mannitol and in a NaCl medium in the three brush border preparations obtained by density gradient centrifugation. Two different substrate concentrations were used, 0.1 and 2.5 mu of L-arginine (upper half and lower half of Fig. 1, respectively). The measurement of uptake in the brush border derived from the lower part of the density gradient (depicted under C) had been difficult because 70-80s of the sediment volume was extravesicular. In the upper and the central part of the density gradient, only 50-55s of the sediment volume could
TRANSPORT
OF
L-ARGININE
IN
RABBIT
TABLE SPECIFIC
ACTIVITIES
OF MARKER Kidney homogenate
BRUSH
BORDER
I
ENZYMES
AND INTRAVESICULAR
Brush horder preparation
Brush
border
upper 85 135 125
Trehalase Alkaline phosphatase Na+, K’-stimulated, Mg’+-dependent ATPase Glucose 6-phosphatase Succinate-cytochrome c reductase Intravesicular volume (@mg protein)
81 20
553
VESICLES
1376 1470 690
part
1694 419
VOLUMES separated by density centrifugation central
part
gradient
lower
part
-
-
1525 70
861 22
33 5.0
48 0.6
49 2.6
71 12.4
3.6
2.9
3.1
1.8
a Specific activities of marker enzymes and intravesicular volume in the kidney homogenate and in four different brush border preparations. Brush border was prepared by differential centrifugation and is characterized in the second column. This brush border preparation was separated further by a density gradient centrifugation (13) into brush border contaminated by basal-lateral plasma membrane (upper 8 ml of the density gradient), highly purified brush border (central 12 ml of the density gradient), and brush border contaminated by mitochondria (lower 10 ml of the density gradient). All specific activities are expressed as nanomoles product formed per min per mg of protein. For the assay of the marker enzymes, see reference 13. For the estimation of intravesicular volume. see reference 14. All values depicted are the mean of at least three observations. A
C
FIG. 1. Uptake of L-arginine in a mannitol medium (solid circles) and in a medium in which mannitol was replaced by NaCl (open circles) by the three brush border preparations obtained by density gradient centrifugation (characterized in Table I). Brush border was resuspended in 140 mM mannitol, 1.4 mM MgC12, 1.4 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-T&, pH 7.5. Fifty microliters of this suspension was injected either into 250 pl of the same medium (solid circles) containing 3H-water and L-[‘4C]arginine or into a medium in which mannitol was replaced by 70 mM NaCl and incubated for various times. A, uptake by the brush border derived from the upper part of the density gradient; B, uptake by the brush border derived from the central part; C, uptake by brush border derived from the lower part. Two substrate concentrations of 0.1 and 2.5 mu were used, upper part and lower part of the figure, respectively. Each point represents the mean of at least three observations.
be occupied by poly-[‘4C]ethyleneglycol and, therefore, more accurate data were obtained when brush border from these areas were used. As can be seen, the uptake of L-arginine in the brush border derived from the central part of the density gradient, this is the highly purified brush border, is higher than the uptake in the brush border taken from the upper part, and about as high as in the brush border derived from the upper part, and about as high as in the brush border derived from the lower part. In all three brush border preparations, uptake was inhibited when NaCl replaced the mannitol in the medium. Paper chromatography of an extract of the vesicular sediment (the vesicles derived from the central part of the density gradient were incubated with 0.1 mM L-[ U-14C]arginine for 10 min in a mannitol medium) showed that 98% of the radioactivity had an RF identical with authentic arginine and herewith makes certain that L-arginine remained unaltered during incubation. A kinetic analysis of the initial uptake (15 s) in two different brush border preparations, which were taken from the upper and the central part of the density gradient, showed that the uptake was saturable. Double reciprocal plots of the data obtained
554
DIETRICH
with the two preparations (Fig. 2) suggest the presence of two kinds of uptake, one operating mainly at low substrate concentratians and another one at higher substrate concentrations. Assuming that the uptake is composed of the transfer by two systems obeying Michaelis-Menten kinetics, the following constants were estimated using graphical means (15): in the highly purified brush border (central part of the gradient) for the low K, system, K, = 0.07 mu and a V,,, = 1.5 nmol/mg of protein and 15 s and for the high K, system, Km = 3.5 mM and a V,, = 8 nmol/mg x 15 s; in the brush border contaminated by basallateral plasma membranes (upper part of the density gradient), the V, values were only about half as high as compared to the highly purified brush border (0.7 and 5.0 nmol/mg x 15 s, respectively), whereas the Km values remained almost the same (0.1 and 4.0 mu, respectively). The initial upA ‘; P c
3
-7 P
2
i , P 3
a
BUSSE
take of L-arginine can be inhibited by various amino acids and by NaCl at low and high substrate concentrations (0.1 and 2.5 mu) as depicted in Fig. 3. At concentrations of 0.1 mu L-[‘“C]arginine (Fig. 3, upper part), uptake is inhibited most by unlabeled L-arginine. L-ornithine, L-cystine, and L-lysine are about equally strong as inhibitors but less strong than L-arginine. n-Arginine is an inhibitor 100 times weaker than Larginine and about 10 times weaker than the group of L-ornithine, L-cystine, and Llysine, and even higher concentrations were needed for the inhibition of uptake by NaCl. When the concentration of substrate was increased to 2.5 mu (Fig. 3, lower part), inhibition occurred at higher inhibitor concentrations, except for the inhibition by NaCl, indicating that the amino acids act by a competitive inhibition. But at this substrate concentration, only about 20 times more n-arginine is needed to get the same inhibition achieved by unlabeled Larginine as compared to the lOO-fold difference obtained at low substrate concentra-
0 . 0
l
. 3
P If
10
20
FIG. 2. Double reciprocal plot of initial uptake (15 s) of L-arginine uersus the L-arginine concentration in a mannitol medium (composition see Fig. 1). A, solid circles: uptake by brush border derived from the upper part of the density gradient (characterized in Table I); B, closed circles: uptake by brush border derived from the central part of the density gradient (Table I, fourth column). Uptake is expressed as nanomoles L-arginine per mg of protein and 15 a. Uptake at zero time of incubation (14) was always subtracted. Each point represents the mean of at least four observations. The open circles were obtained by calculation under the assumption that uptake is composed of the transfer by two systems having in A the kinetic constants K,,, = O.lmMandV,.= 0.7 nmol/mg x 15 s for the low Zf, system and K,,, = 4.0 mu and V,.. = 5.0 nmol/mg x 15 s for the high K,,, system; and in B, K,,, = 0.07 rn~ x 15 s for the low K,,, system and V,, = 1.5 nmol/mg and K,,, = 3.5 nm and V,, = 8.0 nmol/mg x 15 s for the high K,,, system.
a
I
FIG. 3. Inhibition of initial uptake of L-[‘4C]arginine (15 a) by various amino acids and NaCl in the highly purified brush border (central part of the density gradient). Two substrate concentrations of 0.1 and 2.5 mu were used, upper part and lower part of the figure, respectively. Inhibitors: 0, unlabeled L-arginine; n , Lcystine; A, L-lysine; l , L-omithine; 0, n-arginine; +, NaCI; *, L-phenylalanine. Inhibition is expressed as a percentage of control (absence of inhibitor) and plotted versus the inhibitor concentration. Uptake at zero time of incubation (14) was always subtracted. To maintain the same osmolality at all concentrations of inhibitor, the concentration of mannitol in the medium (composition see Fig. 1) was adequately varied. Each point represents the mean of at least three observations.
TRANSPORT
OF
L-ARGININE
IN
tion. It will be discussed later whether these results imply a different affinity for D-arginine of the transport at low and high substrate concentrations. L-Cystine could not be tested as an inhibitor at the high substrate concentration because of its limited solubility. In addition, L-phenylalanine was tested and did not show any inhibition.
Influence of a Transiently brane Potential
Induced
Mem-
Figures 4 and 5 show that a transiently induced membrane potential highly influences the initial rate of L-arginine uptake. Brush border vesicles were preincubated in a medium containing 44 mru KzS04,53 mM mannitol, 1.4 mM MgCL, and 3.4 mM 2morpholineethanesulfonic acid-T& pH 7.5, for 1 h at 25°C and then injected into a medium in which the K&S04 was replaced by additional 132 lll~ mannitol. As can be seen in Fig. 4, an overshoot of L-arginine uptake for about 2 min after the establishment of the gradient of K2S04 was observed. The presence of gramicidin D, which selectively facilitates the movement of K+ and Na+ across biological membranes
minutes
FIG. 4. Uptake of L-arginine by the highly purified brush border (central part of the density gradient) in the presence of a gradient of KzSOd (inside to outside). Brush border was preincubated for 1 h at 25°C in a medium containing 44 mu K~S04,53 mu manmtol, 1.4 mM MgC12, and 3.4 mM morpholineethanesuIfonic acid-Tris, pH 7.5. Fifty microliters of this suspension were injected into a medium in which the K&04 was replaced by additional 132 mru mannitol and which 1 mu L-[“‘C]arginine and traces of 3H-water had been added (open circles). Solid circles, the same conditions, except that gramicidin D (2 pg/ml) was present during incubation. Each point represents the mean of at least three observations.
RABBIT
BRUSH
BORDER
VESICLES
555
in a similar range (16), resulted in an increase of this overshoot. A transiently increased gradient of H+ across the vesicular membrane could also influence the initial rate of L-arginine uptake, as shown in Fig. 5. In this experiment, brush border vesicles were preincubated for 30 min at pH 5.9 in a mannitol medium buffered with 2-morpholineethanesulfonic acid-Tris, and then injected into a medium containing 2.5 mM L-arginine which, in addition, contained enough Tris to bring the pH of the mixture to 7.5. As can be seen, the pH jump was markedly stimulated during the first minute of L-arginine uptake. The presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a substance known to facilitate the movement of protons across membranes, during preincubation and L-arginine uptake gave a slightly higher stimulation. When the brush border vesicles were
FIG. 5. Uptake of L-arginine at a substrate concentration of 2.5 mM by the highly purified brush border (central part of the density gradient, see Table I), which had been preincubated at various pH values. Brush border was resuspended in 20 mM P-morpholineethanesulfonic acid-Tris, 1.4 mM MgCh, 120 mM mannitol, pH 5.9, and preincubated at 25°C for 30 min. 50 4 of this suspension were injected into 250 ~1 of a medium containing 140 mM mannitol, 1.4 mM MgCL, and enough Tris (1.9 mM) to bring the pH of the mixture to 7.5 (open squares). This experiment was repeated in the presence 0.1 mM carbonyl cyanide ptrifIuoromethoxyphenylhydrazone (solid triangles). As a control, brush border was also resuspended in a mixture of both media described above (1:5, v/v), preincubated for 30 min at 25°C and then injected into the same mixture and incubated (closed circles). In addition, brush border was resuspended in 20 mM Tris-2-morpholineethanesulfonic acid, 1.4 mM MgCL, 120 mu mannitol, pH 9.1, and preincubated for 30 min at 25°C. Fifty microliters of this suspension were injected into 250 ~1 of a medium containing 140 mM mannitol, 1.4 mru MgClz, and enough 2-morpholineethanesulfonic acid (1.9 mM) to bring the pH of the mixture to 7.5 (solid squares).
556
DIETRICH
preincubated in a mannitol medium containing Tris-2-morpholineethanesulfonic acid, pH 9.1, and were then injected into a medium which had enough 2-morpholineethanesulfonic acid to bring the pH of the mixture to 7.5, uptake in the first 15 s was lower than the uptake at zero time of incubation. For the measurement of uptake at zero time of incubation, the membrane suspension was cooled to 0°C and acidified by the addition of acetic acid, after which L-[ “C]arginine was added and brush border was separated rapidly from the incubation medium by centrifugation (14). After 3 min of incubation, the differences of L-arginine uptake observed during the first minute were almost abolished. Since the composition of the medium is the same in all experiments depicted in Fig. 5, the difference in uptake can be caused only by an uneven distribution of buffering substances, protons, and hydroxyl ions during the first minute of incubation, making the membrane potential more negative or more positive than later when equilibrium is achieved. Effects of Na+ As shown in Figs. 1 and 3, replacement of mannitol by NaCl in the incubation medium reduced initial uptake and uptake at equilibrium as well. This reduction of uptake probably does not represent a direct interaction of Na+ with the transport system because an increase of the substrate did not reduce the inhibition by NaCl (Fig. 3). Since a high concentration of m&to1 and low concentrations of salts in an incubation medium are not physiological, it would be more appropriate to compare uptake in a NaCl medium with that in a KC1 medium because the K+ is the major, biological counteracting monovalent cation. For this reason, uptake was studied in the presence of a NaCl and KC1 gradient (outside to inside) and a simultaneously increased gradient of H+ (inside to outside). As can be seen in Fig. 6, uptake in the NaCl medium was higher than in the KC1 medium during the first 3 min, whereas after 10 plin of incubation, this difference in uptake had disappeared. For comparison, uptake in a mannitol medium under the same
BUSSE
conditions and substrate concentration was also measured and reveals a considerably faster and higher uptake than in both salt gradients. Uptake in all three media shows an overshoot during the first 3 min. This phenomenon due to a membrane potential induced by the transiently increased pH gradient, was not seen in the experiments depicted in Fig. 5, probably because the concentration of the substrate there was 2.5 times higher than in this figure. The experiments shown in Fig. 6 might have deceived the investigator by showing a stimulating interaction of Na’ with the Larginine transport, which in reality was caused by a higher permeability of the membrane to K+ than to Na+ and consequently a faster reduction of the membrane potential in the KC1 medium. This possibility has to be considered because the permeability of isolated brush border to Na+ and to K* is not known. Thus, ways had to be found to eliminate this factor of uncertainty. Since it is not possible to main-
FIG. 6. Uptake of w@nine at pH 7.5 in the preeence of various ealt gradients by the highly purified brush border (central part of the density gradient, eee Table 1) after preincubation at pH 5.9. Brush border wae preincubated for 30 min at 25°C in a medium containing 185 mu mannitol, 1.4 mu MgCL, and 20 mu 2-morpholineethanesulfonic acid-T&, pH 5.9. Fifty microlitere of thie suspension were injected into 250 4 of a medium containing either 185 mu mannitol (solid circles) or 53 mu mtitol and 66 mu NaCl (open circles), or 53 mu mannitol and 66 mM KC1 (solid triangles). In addition, each of the three media contained 1.4 mw M&h, 1 mr+sL-[“C]arginine, traces of 3H-water, 0.1 mr+f carbonylcyanide p-tritluoromethoxyphenylhydrane and enough Trie (1.9 mu) to bring the mixture to a pH of 7.5. Each point represents the mean of at least three observations. When four or more obeervatione were available, a standard deviation wae calculated and included in the figure.
TRANSPORT
OF
L-ARGININE
IN
tain the membrane potential in brush border vesicles at a specific level for a longer period of time as it is done in electrophysiology using the voltage clamp technique, the potential of the brush border membrane was reduced as far as possible by preincubation in media containing high concentrations of NaCl or KC1 and traces of gramicidin D (Fig. 7). As could be expected, no overshoot of L-arginine uptake was observed, since the concentration of Na+ on both sides of the membrane can be considered to be equal. But even under these conditions unfavorable for transport, initial uptake was faster in the NaCl medium than in the KC1 medium and, after 3 min of incubation, uptake was equal in both media. The standard deviations depicted in this figure and in Fig. 6 are rather broad. But regarding the values obtained after 5 and 30 s of incubation in Fig. 7 in 12 out of 14 estimations, uptake was higher in the NaCl medium when the data of each experimental day were compared. Only in one estimation was uptake faster in the KC1 medium and, in another one, uptake in both media was equal.
RABBIT
BRUSH
BORDER
557
VESICLES
Demonstration of a Proton Gradient Across the Brush Border Membrane So far no attempt was made to explain the g-fold accumulation of L-arginine over a period of 40 min and its reduction by NaCl (Fig. 1). The reduction of a transiently induced membrane potential by a NaCl gradient (outside to inside) was described before in another work on transport in brush border vesicles (17), but in this work, NaCl did not affect the uptake at equilibrium. In Fig. 8, the uptake of [‘*C]methylamine was used to demonstrate a proton gradient (inside to outside) (18) in the brush border vesicles which probably causes the high accumulation of the positively charged L-arginine. In the low-buffered manmtol medium, [‘*C]methylamine reached a distribution ratio of 2 within 15 s and stayed constant throughout the time points measured. But when the vesicles were resuspended in the mannitol medium and then injected into the NaCl medium, the distribution ratio decreased from 1.5 to 1.15 between 15 s and 3 min of incubation. DISCUSSION
Specificity
and Saturability
of Uptake
In the brush border preparation contaminated by basal-lateral plasma membranes,
FIG. 7. Uptake of L-arginine at pH 7.5 in a NaCl and in a KC1 medium by the highly purified brush border (central part of the density gradient, see Table I) after a period of equilibration of 40 mm in the presence of gramicidin D. Brush border was resuspended in a medium containing 66 mu NaCl or KCl. In addition, both media contained 53 mM manmtol, 1.4 mu MgC12, 3.4 mM morpholineethanesulfonic acidTris, pH 7.5, traces of 3H-water, and gramicidin D (2 pg/ml). After 30 mm of preincubation at 25’C, 50 jd of these suspensions were injected into 250 ~1 of the same media and preincubated for a second time at 37°C for 10 mm. Then incubation was started by the addition of 10 fi L-[‘4C]arginine (final concentration, 1 mM). Each point represents the mean of at least seven observations. For the significance of the differences of uptake in the two incubation media at 5 and 30 s of incubation, see Results.
L 0
1
3
10
FIG. 8. Distribution of [‘4C]methylamine in the highly purified brush border preparation incubated in a mannitol medium (solid circles) and in a NaCl medium (open circles) f SD. Brush border derived from the central part of the density gradient (characterized in Table I) was resuspended in the mannitol medium (for composition, see Fig. 1). Fifty microliters of this suspension were injected into 250 al of the same medium containing 3H-water and 4 pM [%!]methylamine or into a medium in which mannitol was replaced by 70 mM NaCl.
558
DIETRICH
the initial rate of L-arginine uptake is only half as fast as in the highly purified brush border derived from the central part of the density gradient centrifugation (Figs. 1 and 2). This suggestsa location of the L-arginine transport system(s) mainly in the brush border. But because this study does not include the measurement of uptake in a preparation of basal-lateral plasma membranes, this suggestion remains to be proven. In the subsequent study, only the highly purified brush border derived from the central part of the density gradient was used. Since the brush border microvilli have retained their tube-like form (19) and since there is some evidence that they have also retained a part of their soluble high molecular content (13), one can assume that the brush border was not inverted when the vesicles were formed. For this reason, one should be able to compare to a certain extent the vesicular uptake of L-arginine with in uiuo studies. The molecular specificity of L-arginine transport found in this study confirms this view. In a microperfusion study on kidney tubules, the reabsorption of both labeled L-arginine and labeled L-lysine was more depressed by the addition of cold L-arginine than by the addition of cold L-lysine (20). Silbernagl and Deetjen (6) extended this study using an improved micropuncture technique and found L-ornithine to be an inhibitor about as strong as L-lysine, whereas D-aiginine was only a weak inhibitor of L-arginine transport. This sequence of inhibition by the amino acids was also found in the vesicular uptake (Fig. 3). In addition, it was shown for the first time in this study that L-cystine is as strong an inhibitor as L-lysine and L-ornithine (Fig. 3). Considering the kinetic constants obtained by in viva and in vitro studies, however, one should not expect a close resemblance of the data because the conditions of transport were too different regarding the presence of inorganic ions and the electrical potential of the membrane. In a micropuncture study, Silbernagl and Deetjen estimated a K,,, of 1.2 mM for L-arginine reabsorption (21) and found some evidence for a second, kinetically distinct mechanism of reabsorption working primarily at low substrate concentrations (6). Also in this
BUSSE
study, two kinds of uptake could be distinguished in a kinetic analysis (Fig. 2). Whether these data reflect the presence of two different transport systems, a cooperative effect within a single system, or transport at high substrate concentrations and transport plus binding at low substrate concentrations, cannot be decided at this moment. Binding of L-arginine to the outer surface of the brush border has to be considered, although the adjustment of the brush border suspension to pH 4 after incubation (see Materials and Methods) probably abolishes most of this binding. Binding to the brush border inside would be less critical, because it includes a transfer through the brush membrane. At high substrate concentrations (1 mM), binding to the brush border must be very low, since uptake at equilibrium in media with high concentrations of salts reached a distribution ratio of 1.1 (Fig. 7). Therefore, a saturable transport process can be established at these substrate concentrations. At low substrate concentrations (0.1 mM), however, binding to the outer brush border surface cannot be excluded, because uptake at equilibrium in the presence 70 mru NaCl reached a distribution ratio of 2.7 (Fig. 2B). This excess of uptake could be due to binding to the outside or to the inside, or due to a A pH, which is not completely abolished by the presence of NaCl (Fig. 8). Therefore, the data depicted in Fig. 2 do not prove the presence of two kinds of transport.
Influence of a Transiently Induced Membrane Potential and Effects of Na+ A A pH, probably due to a difference in buffering capacity between the brush border inside and the medium, as shown in Fig. 8, can explain the high accumulation of the positively charged L-arginine and its reduction by NaCl (Fig. 1). A metabolic conversion of the L-arginine taken up could be excluded by paper chromatography. A membrane potential generated by a metabolic process, causing the uneven distribution of L-arginine, is considered less likely because a metabolic process without exhaustion of endogenous fuel substrate can probably not be maintained over the long period of 40 min (Fig. 1). But when a lumen-negative, membrane
TRANSPORT
OF
L-ARGININE
IN
potential, was induced transiently, the initial rate of L-arginine transfer was accelerated (Figs. 4 and 5). Na+, when added as NaCl, exerted two effects upon the transport of L-arginine which counteracted each other: they stimulated the transfer by interacting with the transport system and they simultaneously diminished transfer by building up a lumenpositive membrane potential, (Figs. 6 and 7). This peculiar situation is quite characteristic for an in vitro study because in uiuo ion pumps linked to metabolism maintain the membrane potential at a certain level. The latter reduction of L-arginine transfer by Na+ in isolated brush border vesicles was reported before (22). Unfortunately, the author could not detect a difference between the inhibition of uptake by unlabeled L- and n-arginine. At the high concentration of inhibitors used (25 mM), much of the inhibition by L- and D-aI@Ik? was probably caused by building up a lumenpositive membrane potential. This mixed inhibition by high concentrations of dibasic amino acids, partly an inhibition by interaction at the substrate binding site of the carrier and partly by inducing a membrane potential, could explain why the difference between L- and n-arginine inhibition is smaller at high substrate concentration than at low substrate concentration (Fig. 3). The Na’ dependency of r.,-arginine transfer observed in in viva in micropuncture studies (9, 10) is confirmed by this study. The stimulation by Na+ shown here is small in relation to the Na+-independent transport (Fig. 6), but might be crucial for the net flux of L-arginine across the epithelial wall. That this stimulation by Na+, in relation to K+, was not a deception by the way of varied membrane potential is shown in Fig. 7 which depicts the transport in a NaCl and KC1 medium after the membrane potential was minimized. The Na’-independent transfer was observed before in a study on isolated tubule segments (8), but at that time, uptake through the luminal brush border and the basal-lateral plasma membrane could not be distinguished. In micropuncture studies, this Na’-independent transport was not noticed, probably because only the net fluxes
RABBIT
BRUSH
BORDER
559
VESICLES
across the epithelial wall are observed by this technique (9, 10). Therefore, we consider the following model feasible: the Na’independent transfer is located in both the luminal and the antiluminal plasma membrane, whereas the Na+-dependent transfer is located only in the luminal brush border and shifts the balance of the transfers towards the direction of reabsorption. This study also confii the Na+-dependency of amino acid transport observed in isolated brush border vesicles using Lalanine (23), L-proline (24), and L-phenylalanine (25). In addition, it describes a Na’independent transport for L-arginine located in the brush border membrane. ACKNOWLEDGMENTS I am grateful to Professor H.Ch. Liittgau for support and helpful criticism during this work. I thank Mrs. B. Pohl and Mr. H. Bartel for their skillful assistance. I also feel indebted to Dr. L.E. Rosenberg, in whose laboratory I was introduced to the problems of cystinuria. REFERENCES 1. DENT,
C. E., AND ROSE,
G. A. (1951)
Q. J.
Med.
20,205-207. 2. STEIN, W. H. (1951) Proc. Sot. Exp. Biol. Med. 78, 705-708. 3. Fox, M. S., THIER, S., ROSENBERG, L. E., KISER, W., AND SEGAL, S. (1964) N. Engl. J. Med. 270, 556-559. 4. ROSENBERG, L. E., DOWNING, S. J., AND SEGAL, S. (1962) J. Biol. Chem. 237,2265-2270. 5. SILBERNAGL, S., AND DEETJEN, P. (1972) Pfliigers Arch. 337.277-284. 6. SILBERNAGL, S., AND DEETJEN, P. (1973) Pfliigers Arch. 340,325-334. 7. SEGAL, S., MCNAMARA, P. D., AND PEPE, L. M. (1977) Science 197,169-171. 8. SEGAL, S., SCHWARTZMAN, L., BLAIR, A., AND BERTOLI, D. (1967) B&him. Biophys. Acta 135, 127-135. 9. ULLRICH, K. J., RUMRICH, G., AND KLGSS, S. (1974) Pfliigers Arch. 351,49-60. 10. SAMARZIJA, J., AND FR~MTER, E. (1976) Pfliigers Arch. 369, R 119. 11. PFALLER, W., AND SILBERNAGL, S. (1975) PfZiigers Arch. 360, 189-192. 12. ROSENBERG, L. E., ALBRECHT, I., AND SEGAL, S. (1967) Science 155, 1426-1428. 13. BUSSE, D., WAHLE, H. U., BARTEL, H., AND POHL, B. (1978) Biochem. J. 174,509-515. 14. BUSSE, D., JAHN, A., AND STEINMAIER, G. (1975) Biochim. Biophys. Acta 401.231-243.
560
DIETRICH
15. BUSSE,
D., ELSAS,
L. J., AND ROSENBERG,
L. E.
(1972) J. Biol. Chem. 247, 1188-1193. 16. HENDERSON, P. J. F., MCGIVAN, J. D., AND CHAPPELL, J. B. (1969) Biochem. J. 111,521~535. 17. EVERS, J., MURER, H., AND KINNE, R. (1976)
Biochim. Biophys. Acta 426,598-615. 18. JOHNSON, R. G., CARLSON, N. J., AND SCARPA, A. (1978) J. Biol. Chem. 253, 1512-1521. 19. BUSSE, D., AND STEINMAIER, G. (1974) Biochim.
Biophys. Acta 345.359-372.
BUSSE 20. BERGERON,
M.,
AND MOREL,
F. (1969)
Amer. J.
Physiol. 216, 1139-1149. 21. SILBERNAGL,
S., AND DEETJEN,
P. (1972)
Pfltigers
Arch. 336,79-86. 22. HAMMERMAN, M. R. (1975) Fed. Proc. 34, Abstr. 513. 23. FASS, S. J., HAMMERMAN, M. R., AND SACKTOR, B. (1977) J. Biol. Chem. 252,583-590. 24. HAMMERMAN, M. R., AND SACKTOR, B. (1977) J.
Biol. Chem. 252,591-595.
Transport
AND BIOPHYSICS pp. 551-560, 1978
of L-Arginine
in Brush Border Vesicles Derived Kidney Cortex’ DIETRICH
BUSSE
Department of Cell Physiology, Ruhr-Universitiit Received
May,
from Rabbit
Bochum, 4630 Bochum 1, German Federal Republic
1978; revised
August
7,1978
The uptake of L-arginine by brush border vesicles from rabbit kidney cortex was investigated at 37°C and pH 7.5. The initial rate of uptake (15 s) was twice as fast in a highly purified brush border as in brush border contaminated by basal-lateral plasma membranes. The initial uptake in a mannitol medium can be best described as the sum of transfer by two systems with K, values of 0.07 and 3.5 mM and V,, values of 1.5 and 8 nmol/mg protein x 15 s, respectively. For the inhibitors of L-[‘4C]arginine, uptake (15 s at two substrate concentrations of 0.1 and 2.5 mru in a mannitol medium) the following sequence of inhibitory strength was established: L-arginine, L-ornithine, L-cyst&e, L-lysine, n-arginine, and NaCl. When a vesicular membrane potential was induced transiently by a jump of the pH in the incubation medium from 5.9 to 7.5 or by an outward movement of K+ in the presence of gramicidin D, an overshoot of L-arginine uptake was observed. Initial uptake of L-arginine was slightly faster in the presence of a Na+ gradient (outside to inside) than under a K+ gradient. Both ion gradients reduced uptake as compared to the uptake in a mannitol medium. Uptake was also studied after the membrane potential was minimized by equilibrating the vesicles in a NaCl or KC1 medium in the presence of gramicidin D. Under these conditions, L-arginine uptake in the first 30 s was faster in the NaCl than in the KC1 medium. These experiments indicate, beside a major ion-independent L-arginine transport, the presence of a transport stimulated by Na+ in isolated brush border vesicles.
In 1951, cystinuria was recognized as an inherited defect of the tubular reabsorption of cystine and the dibasic amino acids in the kidney (1). Because patients with the disorder excreted large quantities of cystine, arginine, and lysine (2), it was believed that those amino acids were reabsorbed, at least partially, by a common mechanism. Disappointingly, in vitro experiments on kidney slices and tubule segments failed to show transport competition between cystine and the dibasic amino acids (3, 4). Later micropuncture studies on the proximal kidney tubule did show an inhibition of cystine transport by arginine (5), but could not demonstrate the reverse, an inhibition of the transport of arginine by cystine (6). 1 A preliminary report of this work has been presented at the 46th meeting of the Deutsche Physiologische Gesellschaft, March 1977 in Regensburg (G. F. R.) (Busse, D. Pohl, B., and Bartel, H. (1977) Pfltigers Arch. 368, R 13). This work was supported by the Deutsche Forschungsgemeinschaft.
The inhibition of cystine transport by arginine could be reproduced on brush border vesicles recently (4). Equally puzzling was the effect of Na+ upon the transport of the dibasic amino acids. In kidney slices, lysine and arginine are actively transported despite a lack of Na+ in the incubation medium (8), whereas in recent micropuncture experiments, the transport of arginine, lysine, and ornithine was highly Na+-dependent (9, 10). It was agreed that much of the controversial results were due to the fact that in kidney slices and isolated tubules, the transport through the luminal brush border, which is the site of cystine-dibasic amino acid interaction (7,11), was obscured by uptake through the basal-lateral cell membrane (7). The transport of the dibasic amino acids and cystine through the luminal brush border is apparently an area of research lacking much definite knowledge. Observations which suggest two transport systems for
551 0003-9861/78/1912-0551$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
552
DIETRICH
the dibasic amino acids and two for cystine complicate this field even more (6, 7,12). A new technique, such as the measurement of transport in isolated brush border vesicles, might bring some insights into the reaction of these amino acids with the membrane. This study shows the inhibition of L-arginine transport by cystine, which was postulated by clinical observations, for the first time experimentally. As will be seen, the situation in vitro is quite different from that in viuo, because in vitro no active pumping of ions linked to metabolism takes place. Therefore, the accumulation of L-arginine at equilibrium seen in this study is probably caused only by a A pH in these vesicles. Na+ like K’, when added as chloride salts, inhibit initial L-arginine transfer by building up a transient lumen-positive membrane potential and reduce uptake at equilibrium by decreasing the A pH. However, when the effects of Na+ and K+ were compared, the initial transport of L-arginine was faster in the NaCl medium under conditions of an increased, as well as of a highly reduced, membrane potential. MATERIALS
AND
METHODS
L-[ U-‘%]Arginine, [Y!]methylamine, and 3H-water were purchased from New England Nuclear Co. Carbony1 cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D were purchased from Boehringer Mannheim GmbH, G.F.R. All other reagents were of the highest purity available.
Preparation of brush border vesicles and measurement of uptake. The brush border vesicles prepared from isolated tubule segments of rabbit kidney by hypotonic lysis, isolated by differential centrifugation, and purified further by a density gradient centrifugation have been described previously (13). The assays of the enzymes used as markers for various cell organelles have also been described (13). The brush border vesicles were used as soon as the isolation was completed which means that the time between the killing of the rabbits and the study of transport was never longer than 12 h. All studies of uptake were performed at pH 7.5 and a temperature of 37°C. Termination of uptake, separation of the brush border vesicles from the incubation medium by centrifugation through a layer of oil, and the measurement of uptake by a double isotope technique using 3H-water as a marker for the sediment volume and poly-[“%]ethyleneglycol as a marker for the extravesicular space of the sediment have also been described previously (14). Paper chromatography. For chromatography, the radioactive material was extracted from the vesicular
BUSSE sediment three times with 60% ethanol. Aliquots of the combined extracts and of the incubation medium were spotted on Whatman No. 1 paper and chromatographed in two chromatographic systems, descending with phenol saturated by water as a solvent, and descending with the solvent butanol:acetic acid:water (4:l:l; v/v/v). After chromatography, the chromatograms were cut into strips and analyzed for radioactivity in a liquid scintillation counter. RESULTS
The brush border prepared by hypotonic lysis and isolated by differential centrifugation has been characterized previously (13). By a density gradient centrifugation (13), this brush border was separated further into brush border contaminated by basal-lateral plasma membrane (upper part of the density gradient), highly purified brush border (central part), and brush border contaminated by mitochondria (lower part). Since all three brush border preparations derived from the density gradient centrifugation were used for the study of transport, an enzymatic characterization is given in Table I. In addition, the bottom line of Table I gives the volume enclosed by the vesicles. As can be seen, the intravesicular volume does not differ significantly in upper and central part of the density gradient, whereas the lower part contains membranes with a 40% smaller volume enclosed per mg of membrane protein, suggesting that nonvesicular brush border with a higher specific density had been concentrated in this part of the gradient. Specificity and Saturability Uptake
of L-Arginine
Figure 1 depicts the uptake of L-arginine in a mannitol and in a NaCl medium in the three brush border preparations obtained by density gradient centrifugation. Two different substrate concentrations were used, 0.1 and 2.5 mu of L-arginine (upper half and lower half of Fig. 1, respectively). The measurement of uptake in the brush border derived from the lower part of the density gradient (depicted under C) had been difficult because 70-80s of the sediment volume was extravesicular. In the upper and the central part of the density gradient, only 50-55s of the sediment volume could
TRANSPORT
OF
L-ARGININE
IN
RABBIT
TABLE SPECIFIC
ACTIVITIES
OF MARKER Kidney homogenate
BRUSH
BORDER
I
ENZYMES
AND INTRAVESICULAR
Brush horder preparation
Brush
border
upper 85 135 125
Trehalase Alkaline phosphatase Na+, K’-stimulated, Mg’+-dependent ATPase Glucose 6-phosphatase Succinate-cytochrome c reductase Intravesicular volume (@mg protein)
81 20
553
VESICLES
1376 1470 690
part
1694 419
VOLUMES separated by density centrifugation central
part
gradient
lower
part
-
-
1525 70
861 22
33 5.0
48 0.6
49 2.6
71 12.4
3.6
2.9
3.1
1.8
a Specific activities of marker enzymes and intravesicular volume in the kidney homogenate and in four different brush border preparations. Brush border was prepared by differential centrifugation and is characterized in the second column. This brush border preparation was separated further by a density gradient centrifugation (13) into brush border contaminated by basal-lateral plasma membrane (upper 8 ml of the density gradient), highly purified brush border (central 12 ml of the density gradient), and brush border contaminated by mitochondria (lower 10 ml of the density gradient). All specific activities are expressed as nanomoles product formed per min per mg of protein. For the assay of the marker enzymes, see reference 13. For the estimation of intravesicular volume. see reference 14. All values depicted are the mean of at least three observations. A
C
FIG. 1. Uptake of L-arginine in a mannitol medium (solid circles) and in a medium in which mannitol was replaced by NaCl (open circles) by the three brush border preparations obtained by density gradient centrifugation (characterized in Table I). Brush border was resuspended in 140 mM mannitol, 1.4 mM MgC12, 1.4 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-T&, pH 7.5. Fifty microliters of this suspension was injected either into 250 pl of the same medium (solid circles) containing 3H-water and L-[‘4C]arginine or into a medium in which mannitol was replaced by 70 mM NaCl and incubated for various times. A, uptake by the brush border derived from the upper part of the density gradient; B, uptake by the brush border derived from the central part; C, uptake by brush border derived from the lower part. Two substrate concentrations of 0.1 and 2.5 mu were used, upper part and lower part of the figure, respectively. Each point represents the mean of at least three observations.
be occupied by poly-[‘4C]ethyleneglycol and, therefore, more accurate data were obtained when brush border from these areas were used. As can be seen, the uptake of L-arginine in the brush border derived from the central part of the density gradient, this is the highly purified brush border, is higher than the uptake in the brush border taken from the upper part, and about as high as in the brush border derived from the upper part, and about as high as in the brush border derived from the lower part. In all three brush border preparations, uptake was inhibited when NaCl replaced the mannitol in the medium. Paper chromatography of an extract of the vesicular sediment (the vesicles derived from the central part of the density gradient were incubated with 0.1 mM L-[ U-14C]arginine for 10 min in a mannitol medium) showed that 98% of the radioactivity had an RF identical with authentic arginine and herewith makes certain that L-arginine remained unaltered during incubation. A kinetic analysis of the initial uptake (15 s) in two different brush border preparations, which were taken from the upper and the central part of the density gradient, showed that the uptake was saturable. Double reciprocal plots of the data obtained
554
DIETRICH
with the two preparations (Fig. 2) suggest the presence of two kinds of uptake, one operating mainly at low substrate concentratians and another one at higher substrate concentrations. Assuming that the uptake is composed of the transfer by two systems obeying Michaelis-Menten kinetics, the following constants were estimated using graphical means (15): in the highly purified brush border (central part of the gradient) for the low K, system, K, = 0.07 mu and a V,,, = 1.5 nmol/mg of protein and 15 s and for the high K, system, Km = 3.5 mM and a V,, = 8 nmol/mg x 15 s; in the brush border contaminated by basallateral plasma membranes (upper part of the density gradient), the V, values were only about half as high as compared to the highly purified brush border (0.7 and 5.0 nmol/mg x 15 s, respectively), whereas the Km values remained almost the same (0.1 and 4.0 mu, respectively). The initial upA ‘; P c
3
-7 P
2
i , P 3
a
BUSSE
take of L-arginine can be inhibited by various amino acids and by NaCl at low and high substrate concentrations (0.1 and 2.5 mu) as depicted in Fig. 3. At concentrations of 0.1 mu L-[‘“C]arginine (Fig. 3, upper part), uptake is inhibited most by unlabeled L-arginine. L-ornithine, L-cystine, and L-lysine are about equally strong as inhibitors but less strong than L-arginine. n-Arginine is an inhibitor 100 times weaker than Larginine and about 10 times weaker than the group of L-ornithine, L-cystine, and Llysine, and even higher concentrations were needed for the inhibition of uptake by NaCl. When the concentration of substrate was increased to 2.5 mu (Fig. 3, lower part), inhibition occurred at higher inhibitor concentrations, except for the inhibition by NaCl, indicating that the amino acids act by a competitive inhibition. But at this substrate concentration, only about 20 times more n-arginine is needed to get the same inhibition achieved by unlabeled Larginine as compared to the lOO-fold difference obtained at low substrate concentra-
0 . 0
l
. 3
P If
10
20
FIG. 2. Double reciprocal plot of initial uptake (15 s) of L-arginine uersus the L-arginine concentration in a mannitol medium (composition see Fig. 1). A, solid circles: uptake by brush border derived from the upper part of the density gradient (characterized in Table I); B, closed circles: uptake by brush border derived from the central part of the density gradient (Table I, fourth column). Uptake is expressed as nanomoles L-arginine per mg of protein and 15 a. Uptake at zero time of incubation (14) was always subtracted. Each point represents the mean of at least four observations. The open circles were obtained by calculation under the assumption that uptake is composed of the transfer by two systems having in A the kinetic constants K,,, = O.lmMandV,.= 0.7 nmol/mg x 15 s for the low Zf, system and K,,, = 4.0 mu and V,.. = 5.0 nmol/mg x 15 s for the high K,,, system; and in B, K,,, = 0.07 rn~ x 15 s for the low K,,, system and V,, = 1.5 nmol/mg and K,,, = 3.5 nm and V,, = 8.0 nmol/mg x 15 s for the high K,,, system.
a
I
FIG. 3. Inhibition of initial uptake of L-[‘4C]arginine (15 a) by various amino acids and NaCl in the highly purified brush border (central part of the density gradient). Two substrate concentrations of 0.1 and 2.5 mu were used, upper part and lower part of the figure, respectively. Inhibitors: 0, unlabeled L-arginine; n , Lcystine; A, L-lysine; l , L-omithine; 0, n-arginine; +, NaCI; *, L-phenylalanine. Inhibition is expressed as a percentage of control (absence of inhibitor) and plotted versus the inhibitor concentration. Uptake at zero time of incubation (14) was always subtracted. To maintain the same osmolality at all concentrations of inhibitor, the concentration of mannitol in the medium (composition see Fig. 1) was adequately varied. Each point represents the mean of at least three observations.
TRANSPORT
OF
L-ARGININE
IN
tion. It will be discussed later whether these results imply a different affinity for D-arginine of the transport at low and high substrate concentrations. L-Cystine could not be tested as an inhibitor at the high substrate concentration because of its limited solubility. In addition, L-phenylalanine was tested and did not show any inhibition.
Influence of a Transiently brane Potential
Induced
Mem-
Figures 4 and 5 show that a transiently induced membrane potential highly influences the initial rate of L-arginine uptake. Brush border vesicles were preincubated in a medium containing 44 mru KzS04,53 mM mannitol, 1.4 mM MgCL, and 3.4 mM 2morpholineethanesulfonic acid-T& pH 7.5, for 1 h at 25°C and then injected into a medium in which the K&S04 was replaced by additional 132 lll~ mannitol. As can be seen in Fig. 4, an overshoot of L-arginine uptake for about 2 min after the establishment of the gradient of K2S04 was observed. The presence of gramicidin D, which selectively facilitates the movement of K+ and Na+ across biological membranes
minutes
FIG. 4. Uptake of L-arginine by the highly purified brush border (central part of the density gradient) in the presence of a gradient of KzSOd (inside to outside). Brush border was preincubated for 1 h at 25°C in a medium containing 44 mu K~S04,53 mu manmtol, 1.4 mM MgC12, and 3.4 mM morpholineethanesuIfonic acid-Tris, pH 7.5. Fifty microliters of this suspension were injected into a medium in which the K&04 was replaced by additional 132 mru mannitol and which 1 mu L-[“‘C]arginine and traces of 3H-water had been added (open circles). Solid circles, the same conditions, except that gramicidin D (2 pg/ml) was present during incubation. Each point represents the mean of at least three observations.
RABBIT
BRUSH
BORDER
VESICLES
555
in a similar range (16), resulted in an increase of this overshoot. A transiently increased gradient of H+ across the vesicular membrane could also influence the initial rate of L-arginine uptake, as shown in Fig. 5. In this experiment, brush border vesicles were preincubated for 30 min at pH 5.9 in a mannitol medium buffered with 2-morpholineethanesulfonic acid-Tris, and then injected into a medium containing 2.5 mM L-arginine which, in addition, contained enough Tris to bring the pH of the mixture to 7.5. As can be seen, the pH jump was markedly stimulated during the first minute of L-arginine uptake. The presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a substance known to facilitate the movement of protons across membranes, during preincubation and L-arginine uptake gave a slightly higher stimulation. When the brush border vesicles were
FIG. 5. Uptake of L-arginine at a substrate concentration of 2.5 mM by the highly purified brush border (central part of the density gradient, see Table I), which had been preincubated at various pH values. Brush border was resuspended in 20 mM P-morpholineethanesulfonic acid-Tris, 1.4 mM MgCh, 120 mM mannitol, pH 5.9, and preincubated at 25°C for 30 min. 50 4 of this suspension were injected into 250 ~1 of a medium containing 140 mM mannitol, 1.4 mM MgCL, and enough Tris (1.9 mM) to bring the pH of the mixture to 7.5 (open squares). This experiment was repeated in the presence 0.1 mM carbonyl cyanide ptrifIuoromethoxyphenylhydrazone (solid triangles). As a control, brush border was also resuspended in a mixture of both media described above (1:5, v/v), preincubated for 30 min at 25°C and then injected into the same mixture and incubated (closed circles). In addition, brush border was resuspended in 20 mM Tris-2-morpholineethanesulfonic acid, 1.4 mM MgCL, 120 mu mannitol, pH 9.1, and preincubated for 30 min at 25°C. Fifty microliters of this suspension were injected into 250 ~1 of a medium containing 140 mM mannitol, 1.4 mru MgClz, and enough 2-morpholineethanesulfonic acid (1.9 mM) to bring the pH of the mixture to 7.5 (solid squares).
556
DIETRICH
preincubated in a mannitol medium containing Tris-2-morpholineethanesulfonic acid, pH 9.1, and were then injected into a medium which had enough 2-morpholineethanesulfonic acid to bring the pH of the mixture to 7.5, uptake in the first 15 s was lower than the uptake at zero time of incubation. For the measurement of uptake at zero time of incubation, the membrane suspension was cooled to 0°C and acidified by the addition of acetic acid, after which L-[ “C]arginine was added and brush border was separated rapidly from the incubation medium by centrifugation (14). After 3 min of incubation, the differences of L-arginine uptake observed during the first minute were almost abolished. Since the composition of the medium is the same in all experiments depicted in Fig. 5, the difference in uptake can be caused only by an uneven distribution of buffering substances, protons, and hydroxyl ions during the first minute of incubation, making the membrane potential more negative or more positive than later when equilibrium is achieved. Effects of Na+ As shown in Figs. 1 and 3, replacement of mannitol by NaCl in the incubation medium reduced initial uptake and uptake at equilibrium as well. This reduction of uptake probably does not represent a direct interaction of Na+ with the transport system because an increase of the substrate did not reduce the inhibition by NaCl (Fig. 3). Since a high concentration of m&to1 and low concentrations of salts in an incubation medium are not physiological, it would be more appropriate to compare uptake in a NaCl medium with that in a KC1 medium because the K+ is the major, biological counteracting monovalent cation. For this reason, uptake was studied in the presence of a NaCl and KC1 gradient (outside to inside) and a simultaneously increased gradient of H+ (inside to outside). As can be seen in Fig. 6, uptake in the NaCl medium was higher than in the KC1 medium during the first 3 min, whereas after 10 plin of incubation, this difference in uptake had disappeared. For comparison, uptake in a mannitol medium under the same
BUSSE
conditions and substrate concentration was also measured and reveals a considerably faster and higher uptake than in both salt gradients. Uptake in all three media shows an overshoot during the first 3 min. This phenomenon due to a membrane potential induced by the transiently increased pH gradient, was not seen in the experiments depicted in Fig. 5, probably because the concentration of the substrate there was 2.5 times higher than in this figure. The experiments shown in Fig. 6 might have deceived the investigator by showing a stimulating interaction of Na’ with the Larginine transport, which in reality was caused by a higher permeability of the membrane to K+ than to Na+ and consequently a faster reduction of the membrane potential in the KC1 medium. This possibility has to be considered because the permeability of isolated brush border to Na+ and to K* is not known. Thus, ways had to be found to eliminate this factor of uncertainty. Since it is not possible to main-
FIG. 6. Uptake of w@nine at pH 7.5 in the preeence of various ealt gradients by the highly purified brush border (central part of the density gradient, eee Table 1) after preincubation at pH 5.9. Brush border wae preincubated for 30 min at 25°C in a medium containing 185 mu mannitol, 1.4 mu MgCL, and 20 mu 2-morpholineethanesulfonic acid-T&, pH 5.9. Fifty microlitere of thie suspension were injected into 250 4 of a medium containing either 185 mu mannitol (solid circles) or 53 mu mtitol and 66 mu NaCl (open circles), or 53 mu mannitol and 66 mM KC1 (solid triangles). In addition, each of the three media contained 1.4 mw M&h, 1 mr+sL-[“C]arginine, traces of 3H-water, 0.1 mr+f carbonylcyanide p-tritluoromethoxyphenylhydrane and enough Trie (1.9 mu) to bring the mixture to a pH of 7.5. Each point represents the mean of at least three observations. When four or more obeervatione were available, a standard deviation wae calculated and included in the figure.
TRANSPORT
OF
L-ARGININE
IN
tain the membrane potential in brush border vesicles at a specific level for a longer period of time as it is done in electrophysiology using the voltage clamp technique, the potential of the brush border membrane was reduced as far as possible by preincubation in media containing high concentrations of NaCl or KC1 and traces of gramicidin D (Fig. 7). As could be expected, no overshoot of L-arginine uptake was observed, since the concentration of Na+ on both sides of the membrane can be considered to be equal. But even under these conditions unfavorable for transport, initial uptake was faster in the NaCl medium than in the KC1 medium and, after 3 min of incubation, uptake was equal in both media. The standard deviations depicted in this figure and in Fig. 6 are rather broad. But regarding the values obtained after 5 and 30 s of incubation in Fig. 7 in 12 out of 14 estimations, uptake was higher in the NaCl medium when the data of each experimental day were compared. Only in one estimation was uptake faster in the KC1 medium and, in another one, uptake in both media was equal.
RABBIT
BRUSH
BORDER
557
VESICLES
Demonstration of a Proton Gradient Across the Brush Border Membrane So far no attempt was made to explain the g-fold accumulation of L-arginine over a period of 40 min and its reduction by NaCl (Fig. 1). The reduction of a transiently induced membrane potential by a NaCl gradient (outside to inside) was described before in another work on transport in brush border vesicles (17), but in this work, NaCl did not affect the uptake at equilibrium. In Fig. 8, the uptake of [‘*C]methylamine was used to demonstrate a proton gradient (inside to outside) (18) in the brush border vesicles which probably causes the high accumulation of the positively charged L-arginine. In the low-buffered manmtol medium, [‘*C]methylamine reached a distribution ratio of 2 within 15 s and stayed constant throughout the time points measured. But when the vesicles were resuspended in the mannitol medium and then injected into the NaCl medium, the distribution ratio decreased from 1.5 to 1.15 between 15 s and 3 min of incubation. DISCUSSION
Specificity
and Saturability
of Uptake
In the brush border preparation contaminated by basal-lateral plasma membranes,
FIG. 7. Uptake of L-arginine at pH 7.5 in a NaCl and in a KC1 medium by the highly purified brush border (central part of the density gradient, see Table I) after a period of equilibration of 40 mm in the presence of gramicidin D. Brush border was resuspended in a medium containing 66 mu NaCl or KCl. In addition, both media contained 53 mM manmtol, 1.4 mu MgC12, 3.4 mM morpholineethanesulfonic acidTris, pH 7.5, traces of 3H-water, and gramicidin D (2 pg/ml). After 30 mm of preincubation at 25’C, 50 jd of these suspensions were injected into 250 ~1 of the same media and preincubated for a second time at 37°C for 10 mm. Then incubation was started by the addition of 10 fi L-[‘4C]arginine (final concentration, 1 mM). Each point represents the mean of at least seven observations. For the significance of the differences of uptake in the two incubation media at 5 and 30 s of incubation, see Results.
L 0
1
3
10
FIG. 8. Distribution of [‘4C]methylamine in the highly purified brush border preparation incubated in a mannitol medium (solid circles) and in a NaCl medium (open circles) f SD. Brush border derived from the central part of the density gradient (characterized in Table I) was resuspended in the mannitol medium (for composition, see Fig. 1). Fifty microliters of this suspension were injected into 250 al of the same medium containing 3H-water and 4 pM [%!]methylamine or into a medium in which mannitol was replaced by 70 mM NaCl.
558
DIETRICH
the initial rate of L-arginine uptake is only half as fast as in the highly purified brush border derived from the central part of the density gradient centrifugation (Figs. 1 and 2). This suggestsa location of the L-arginine transport system(s) mainly in the brush border. But because this study does not include the measurement of uptake in a preparation of basal-lateral plasma membranes, this suggestion remains to be proven. In the subsequent study, only the highly purified brush border derived from the central part of the density gradient was used. Since the brush border microvilli have retained their tube-like form (19) and since there is some evidence that they have also retained a part of their soluble high molecular content (13), one can assume that the brush border was not inverted when the vesicles were formed. For this reason, one should be able to compare to a certain extent the vesicular uptake of L-arginine with in uiuo studies. The molecular specificity of L-arginine transport found in this study confirms this view. In a microperfusion study on kidney tubules, the reabsorption of both labeled L-arginine and labeled L-lysine was more depressed by the addition of cold L-arginine than by the addition of cold L-lysine (20). Silbernagl and Deetjen (6) extended this study using an improved micropuncture technique and found L-ornithine to be an inhibitor about as strong as L-lysine, whereas D-aiginine was only a weak inhibitor of L-arginine transport. This sequence of inhibition by the amino acids was also found in the vesicular uptake (Fig. 3). In addition, it was shown for the first time in this study that L-cystine is as strong an inhibitor as L-lysine and L-ornithine (Fig. 3). Considering the kinetic constants obtained by in viva and in vitro studies, however, one should not expect a close resemblance of the data because the conditions of transport were too different regarding the presence of inorganic ions and the electrical potential of the membrane. In a micropuncture study, Silbernagl and Deetjen estimated a K,,, of 1.2 mM for L-arginine reabsorption (21) and found some evidence for a second, kinetically distinct mechanism of reabsorption working primarily at low substrate concentrations (6). Also in this
BUSSE
study, two kinds of uptake could be distinguished in a kinetic analysis (Fig. 2). Whether these data reflect the presence of two different transport systems, a cooperative effect within a single system, or transport at high substrate concentrations and transport plus binding at low substrate concentrations, cannot be decided at this moment. Binding of L-arginine to the outer surface of the brush border has to be considered, although the adjustment of the brush border suspension to pH 4 after incubation (see Materials and Methods) probably abolishes most of this binding. Binding to the brush border inside would be less critical, because it includes a transfer through the brush membrane. At high substrate concentrations (1 mM), binding to the brush border must be very low, since uptake at equilibrium in media with high concentrations of salts reached a distribution ratio of 1.1 (Fig. 7). Therefore, a saturable transport process can be established at these substrate concentrations. At low substrate concentrations (0.1 mM), however, binding to the outer brush border surface cannot be excluded, because uptake at equilibrium in the presence 70 mru NaCl reached a distribution ratio of 2.7 (Fig. 2B). This excess of uptake could be due to binding to the outside or to the inside, or due to a A pH, which is not completely abolished by the presence of NaCl (Fig. 8). Therefore, the data depicted in Fig. 2 do not prove the presence of two kinds of transport.
Influence of a Transiently Induced Membrane Potential and Effects of Na+ A A pH, probably due to a difference in buffering capacity between the brush border inside and the medium, as shown in Fig. 8, can explain the high accumulation of the positively charged L-arginine and its reduction by NaCl (Fig. 1). A metabolic conversion of the L-arginine taken up could be excluded by paper chromatography. A membrane potential generated by a metabolic process, causing the uneven distribution of L-arginine, is considered less likely because a metabolic process without exhaustion of endogenous fuel substrate can probably not be maintained over the long period of 40 min (Fig. 1). But when a lumen-negative, membrane
TRANSPORT
OF
L-ARGININE
IN
potential, was induced transiently, the initial rate of L-arginine transfer was accelerated (Figs. 4 and 5). Na+, when added as NaCl, exerted two effects upon the transport of L-arginine which counteracted each other: they stimulated the transfer by interacting with the transport system and they simultaneously diminished transfer by building up a lumenpositive membrane potential, (Figs. 6 and 7). This peculiar situation is quite characteristic for an in vitro study because in uiuo ion pumps linked to metabolism maintain the membrane potential at a certain level. The latter reduction of L-arginine transfer by Na+ in isolated brush border vesicles was reported before (22). Unfortunately, the author could not detect a difference between the inhibition of uptake by unlabeled L- and n-arginine. At the high concentration of inhibitors used (25 mM), much of the inhibition by L- and D-aI@Ik? was probably caused by building up a lumenpositive membrane potential. This mixed inhibition by high concentrations of dibasic amino acids, partly an inhibition by interaction at the substrate binding site of the carrier and partly by inducing a membrane potential, could explain why the difference between L- and n-arginine inhibition is smaller at high substrate concentration than at low substrate concentration (Fig. 3). The Na’ dependency of r.,-arginine transfer observed in in viva in micropuncture studies (9, 10) is confirmed by this study. The stimulation by Na+ shown here is small in relation to the Na+-independent transport (Fig. 6), but might be crucial for the net flux of L-arginine across the epithelial wall. That this stimulation by Na+, in relation to K+, was not a deception by the way of varied membrane potential is shown in Fig. 7 which depicts the transport in a NaCl and KC1 medium after the membrane potential was minimized. The Na’-independent transfer was observed before in a study on isolated tubule segments (8), but at that time, uptake through the luminal brush border and the basal-lateral plasma membrane could not be distinguished. In micropuncture studies, this Na’-independent transport was not noticed, probably because only the net fluxes
RABBIT
BRUSH
BORDER
559
VESICLES
across the epithelial wall are observed by this technique (9, 10). Therefore, we consider the following model feasible: the Na’independent transfer is located in both the luminal and the antiluminal plasma membrane, whereas the Na+-dependent transfer is located only in the luminal brush border and shifts the balance of the transfers towards the direction of reabsorption. This study also confii the Na+-dependency of amino acid transport observed in isolated brush border vesicles using Lalanine (23), L-proline (24), and L-phenylalanine (25). In addition, it describes a Na’independent transport for L-arginine located in the brush border membrane. ACKNOWLEDGMENTS I am grateful to Professor H.Ch. Liittgau for support and helpful criticism during this work. I thank Mrs. B. Pohl and Mr. H. Bartel for their skillful assistance. I also feel indebted to Dr. L.E. Rosenberg, in whose laboratory I was introduced to the problems of cystinuria. REFERENCES 1. DENT,
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