ionic requirements for amino acid transport in the rat kidney cortex slice: i. influence of...

BIOCHIMICA ET BIOPHYSICA ACTA 167

BBA 4300

I O N I C R E Q U I R E M E N T S F O R A M I N O ACID T R A N S P O R T

I N T H E RAT K I D N E Y C O R T E X S L I C E

I. INFLUENCE OF E X T R A C E L L U L A R IONS

MAURICE FOX*, SAMUEL THIER*, LEON ROSENBERG**, AND STANTON SEGAL*

Metabolic Diseases Branch and Clinical Endocrinology Branch*, National Institute of Arthritis and Metabolic Diseases and Metabolism Branch, National Cancer

Institute ~*, National Institutes of Health, Bethesda, Md. (U.S.A.)

(Received July 9th, 1963)

SUMMARY

I. Active transport of amino acids in rat kidney cortex slices diminished as the Na + concentration of the medium was decreased below physiologic levels. In Na+-free media, active transport of glycine and ~-amino[I-14Clisobutyric acid was abolished, but active transport of lysine persisted.

2. Lysine transport was found to be mediated by two mechanisms--one Na + dependent and ouabain sensitive, and the other independent of Na + and insensitive to ouabain.

3. Maximal transport of amino acids occurred over a narrow range of medium K+ concentrations, falling off at higher and lower K + levels.

4. Substitution of other ions for Na + in the medium caused significant alterations of the intracellular and extracelhilar fluid spaces of the tissues.

5. Replacement of medium Na + by K+ resulted in tissue swelling and suppression of amino acid transport beyond that caused by the absence of Na +.

6. The common ionic requirements of kidney ATPase systems and of the mechanisms for active transport of certain amino acids suggest that these processes may be intimately related.

INTRODUCTION

Recent investigations of the mechanisms of intracellular accumulation of amino acids by various tissues have implicated a relationship with Na+ and K + concentrations in the intra- and extracelhilar fluids. RIGGS et al. 1 have suggested that amino acid influx in Ehrlich ascites-tumor cells is regulated by the intracellular K + level, while IffEINZ 2 has called attention to the regulatory role of extracellular Na + in amino acid transport. LEVINSKY et al. s have reported that prolonged incubation of rat diaphragm in media rich in lysine resulted in loss of up to one-third of the cellular K +, with apparent replacement of the K+ deficit by lysine.

Abbreviation: AIB, a-amino[i-14Clisobutyric acid.

Biochim. Biophys. Acta, 79 (1964) 167-176

168 M. FOX, S. THIER, L. ROSENBERG, S. SEGAL

Previous studies from this laboratory have demonstrated that ~*C-labelled amino acids are actively accumulated against a concentration gradient in rat kidney cortex slices 4. The processes involved conform to Michaelis-Menten kinetics and are inhibited by anaerobiasis and uncoupling of oxidative phosphorylation 4. Under standard con- ditions, the ratio of amino acid concentration in the intracellular fluid to that in the medium (the distribution ratio) is highly reproducible. This report presents data con- cerning the influence of changes in medium ion concentrations on amino acid accumulation in the rat kidney cortex slice iu vitro.

The results indicate that the extracellular ion exerting the greatest influence is Na +. In Na+-free media active transport of the neutral amino acids c~-aminoisobutvric acid and glycine is completely inhibited, and that of lysine and histidine is reduced. Intracellular accumulation of the amino acids tested increased as the Na + concentration of the medium was increased. In this role, Na + could not be replaced by Li+, K +, Tris +, or lvsine + ions.

METHODS

Male Sprague-Dawley rats weighing 12o-16o g were used in all experiments and were fed a Purina R chow diet and water ad libitum until sacrificed by stunning and de- capitation. The techniques employed for the preparation of kidney cortex slices, aerobic incubation in Krebs-Ringer bicarbonate buffer (pH 7.4) at 37 °, and assess- ment of intracellular accumulation of the 14C-labelled amino acids have been described in detail previously 4,5. Distribution ratios were calculated as follows:

Coun t s [min /ml in t race l lu /a r fluid --

ne t t i ssue counts / ra in ! (medium counts /min/ rn l ) (ml 714C]inulin space)]

ml t issue wa te r ml [14C~jinulin space

c o u n t s / m i n / m l in t r ace l lu l a r fluid D i s t r i bu t i on ra t io -- -

c o u n t s / m i n / m l m e d i u m

Extracellular space was calculated using distribution of [14CTinulin and total tissue water was determined by the difference between tissue weight before and after drying in a crucible at lO5 ° in a vacuum oven for 24 h (ref. 6). All radioactive samples were counted in a liquid-scintillation spectrometer with an efficiency of 57 % for 14C. Na + and K + were determined by Li+ internal s tandard flame photometry.

MATERIALS

AIB, specific activity 16.8 mC/mmole was obtained from Isotope Specialties Co., Burbank, Calif. [2-14C]Glycine, specific activity 2.31 mC/mmole and L-E14C6~histidine, specific activity 1.o86 mC/mmole, were obtained from Volk Radiochemical Company. L@4C6]Lysine, specific activity 146 mC/mmole and Ecarboxy-laCJinulin (molecular weight 3ooo-4ooo ), specific activity 2.1 mC/mg were obtained from New England Nuclear Corporation. Each amino acid was shown to be chromatographically pure using a descending one-dimensional paper system in butanol-acetic acid-water (4: I : 2, v/v). The labelled amino acids were added to the media in the following initial concentrations: 0.065 mM AIB; 0.03 mM glycine; o.18 mM L-histidine; 0.065 mM L-lysine. After incubation, aliquots of the aqueous tissue extract were chromato-

Biochim. Bioph),s..qcta, 79 (1904) I67-17 °

AMINO ACID TRANSPORT IN THE KIDNEY CORTEX 169

graphed and over 9 ° % of the radioactivity was recovered at the appropriate RF for the specific amino acid studied.

Incubation media

Changes in the ionic constituents of the media were made by substituting in an isomolar fashion for the contents of Krebs-Ringer bicarbonate buffer. Thus, Tris was used in place of Na + in "Tris buffer", and K+ was used in place of Na + in "all K + buffer". Substitution for Na + could be complete or partial, depending on the experi- mental design. Tris was useful as a substitute for Na + because it served as a buffer as well as a source of cations. Osmotic studies in erythrocytes have shown that Tris does not affect the integrity of the membrane, though some intracellular penetration by the un-ionized molecule does occur 7. At physiological pH, Tris chloride solution was 75- 80 % ionized and served adequately to maintain the osmolarity of the extracellular fluid.

The "K+-free ' ' buffers contained no K + initially, but after 9 ° min incubation, enough K + leaked out of the slices to raise the medium concentration significantly. By leaching the slices in "K+-free ' ' medium for 2o min at 25 ° prior to incubation, the final medium K + concentration was kept below 1 mequiv/1. Buffers with K + concentrations above 7 mequiv/1 were made by adding the required amount of KC1 to Krebs- Ringer bicarbonate buffer.

RESULTS

Total tissue water and extracelluIar fluid space

Total tissue water and extracellular fluid space for three buffers are plotted in Fig. I. In Krebs-Ringer bicarbonate buffer the total tissue water remained constant at 80.0 ± 0.09 % of the total wet tissue weight from 15-12o min incubation. The distribution of inulin reached equilibrium at 30 rain and remained constant through

~ 8o

~, 75

°oi 3 ,NULIN SPACE

I /Me.x SP.CE .~22oI- - -

140 . . . . ,ool 60]- r - -

~ , i , , i J , i , , i , , i ,

0 3 O 60 90 120 150 INCUBATION TIME (MIN)

Fig. I. Changes in tissue fluid spaces of rat kidney cortex slices on incubation in "al l -K +'' ( • - - 0 ) , "Tr is" ( • - - • ) and " K rebs -R i nge r b icarbonate" ( • - - • ) buffers. Total tissue water is expressed as per cent of wet tissue weight. The inulin spaces are plotted as per cent of wet and of dry tissue

weight. Each point represents 3 4 determinations.


17o M. FOX, S. T H I E R , L. R O S E N B E R G , S. S E G A L

12o min. This inulin space at equi l ibr ium is defined as the ext race l lu lar space and comprises 25.7 % of the wet t issue weight.

In "Tr is buffer" the to ta l water and inulin spaces were both reduced ini t ia l ly when compared wi th K r e b s - R i n g e r b ica rbona te buffer. Both spaces g radua l ly ex- p a n d e d wi th fur ther incubat ion. The ini t ia l effect of exposing the tissue to this Na +- free med ium appears to be dehydra t ion . The g radua l reaccumula t ion of wate r by the t issue m a y be a t t r i bu t ab l e to the re la t ive ly slow rate of pene t ra t ion of the Tris molecule into the t issue spaces of the k idney sliceL

In "al l K + buffer" the to ta l wate r was cons iderably increased and the slices incuba ted in this med ium appea red swollen and moist even af ter blot t ing. The d a t a in Fig. I indicate t ha t this increase in tissue wate r was p r imar i ly intracel lular . The inulin space expressed as per cent of d ry tissue weight was ac tua l ly increased over the inulin space in K r e b s - R i n g e r buffer. When expressed as per cent of wet tissue weight , however, the inulin space in "al l K + buffer" appea red to diminish, due to the compa r a t i ve ly grea ter rise in the wa te r content of the in t race l lu la r space. The increased inulin space at 15o rain is p robab ly ar t i fac tual . By t ha t t ime the in tegr i ty of the cell membranes was p robab ly d a m a g e d enough to allow inulin to en ter some in t race l lu la r compa r tmen t s .

When NaC1 was added to K r e b s - R i n g e r b ica rbona te buffer to raise the concen- t r a t ion of Na + to 24o mequiv/1, there was a concurrent rise in the ex t race l lu la r space to 34 .1% wi th a sl ight reduct ion in the to ta l wate r to 78.7 °o of the wet t issue weight. I t is appa ren t t ha t nei ther K + nor Tris + was capable of ma in ta in ing the in t race l lu la r and ext racelhf iar fluid w)lumes in as cons tan t a manner as physiological concentra- t ions of Na +. W i t h every change in the ionic content of a buffer, one mus t know the to ta l t issue wate r and ex t race l lu la r space of the k idney slices in tha t buffer for any t ime of incubat ion , in order to be able to calculate meaningful d a t a on accumula t ion of amino acids.

Effect of total N a + deprivation on amino acid accumulation

The effect of Na + depr iva t ion on the ab i l i ty of the ra t k idney cor tex slice to t r anspo r t amino acids is out l ined in Tabte I. The d i s t r ibu t ion rat ios of AIB, glycine, lysine and his t idine in K r e b s - R i n g e r b ica rbona te buffer were all above one, ind ica t ing act ive accumula t ion agains t a concentra t ion gradient . When the Na + ion was r emoved

T A B I . E i

E F F E C T OF N a 4 - F R E E M E D I A ON A M I N O A C I D A C C U M U L A T I O N IN RAT K I D N E Y C O R T E X SLICI*S

T h r e e s l ices w e i g h i n g a t o t a l of 80 i o o m g w e r e i n c u b a t e d in 2 ni l b u f f e r ( p H 7.4) fo r 9o r a i n a t 37:. c o u n t s / m i n / m l i n t r a c e l l u l a r f lu id

D i s t r i b u t i o n r a t i o s . . . . . r e p r e s e n t t h e m e a n + S .E . of t h e m e a n . c o u n t s / m i n / m l m e d i a

T h e n u m b e r of d e t e r m i n a t i o n is g i v e n b e t w e e n p a r e n t h e s e s .

Distribution ratios

3led ium Krebs Ringer bufJor Tris buffer Tris K + free buffi~r A l l K + buffrr

A I B 4 .48 ! O. l i (24) 0 .90 :± 0 .05 (15) 0 .92 ~ O.Ol (3) 0 .86 ~: 0 .02 (12)

G l y c i n e 9 .3 ° :~ 0 .44 ( t l ) 1 .14 ~ 0 .04 (6) I.O9 @ O.Ol (3) t . l 3 ! O.Ol (3)

L y s i n e 5.Ol ~: o .24 (9) 2 .46 ~ o .o0 (12) 2 .73 ± o . o t (3) 1.36 2:: o .o4 (5)

H i s t i ( l i n e 3 . i 5 - O.l 4 (6) 1.45 ~ 0 .o2 (9) 1.46 4 o .o2 (3) 1.14 ~ o .o 5 (6)

Hiochim. Biophys. Acta, 79 (19t)4) 1°7- ~ 7 ('


from the medium and replaced by Tris + ion, the distribution ratios of AIB and glycine were reduced to I (P ~ o.ooi), and their penetration into the cell could be explained by simple diffusion alone. Lysine and histidine accumulation was considerably diminished (P < o.ooi), but some concentrative transfer persisted in spite of the absence of Na + from the medium. Removal of K+ from the medium, as well as Na ÷, caused no further reduction in amino acid accumulation. Active transport of lysine and histidine was virtually abolished by substitution of K + for the extracellular Na ÷ (P < o.ooi). This inhibition of non-Na+-dependent lysine and histidine transport illustrates that high extracellular K + concentrations per se have a deleterious effect on the active transport of some amino acids. High medium K + also impeded accumulation of AIB by the slices even in the absence of concentrative transfer. This is demonstrated in Fig. 2 in which the uptake of AIB is plotted in two Na+-free media. The slices bathed in the high K + medium accumulated AIB at a slower rate than those bathed in the normal K + medium.

o'it / ~o2

~oo~ o r5 ~0 ~'0 ~0 ,~0 INCUBATION TIME{ MIN)

Fig. 2. I n t r ace l l u l a r a c c um ula t i on of A I B in two Na+-free med ia : "Tris'" ( ~ k - - A ) and "a l l -K +'' ( O - - O ) . At each t ime po in t the d a t a were pai red and de te rmina t ions were done in t r ip l ica te . I n these media , a ccumula t i on of A I B aga ins t a concen t ra t ion g rad i en t does no t occur and the

d i s t r ibu t ion ra t io does not exceed i.

The possibility must be considered that the inhibition of amino acid transport by "Tris buffer" was due to some property of Tris itself rather than the actual lack of Na +. Data bearing on this point are shown in Table II. AIB transport after 9 ° min incubation was tested with three buffers containing 25 mequiv/1 Na ÷, with the osmotic deficit of 12o mM made up, respectively, by Li +, Tris + and lysine + ions. The use of a

TABLE If

EFFECT OF SEVERAL EXTRACELLULAR CATIONS ON TRANSPORT OF AIB

Dis t r i bu t i on ra t ios for each expe r imen t represent the mean + S.E. of the mean of t r ip l i ca te pa i red de te rmina t ions . A s t a t i s t i c a l l y s ignif icant difference separa tes the d i s t r i bu t ion ra t ios of A I B in

K r e b s - R i n g e r buffer from those in the o ther buffers used.

Distribution ratios

Expt . No. Krebs-Ringer L i + Tris Lysine K + Free K + Free K + free bicarbonate buffer z2o* z~o* s2o* p lus lysine** p lus NH4+, **

I 4.22 ± 0.30 2.93 zF 0.04 2.18 ! 0.22 1.6 9 i o.12 2 4.49 ± o.21 3-30 ± 0.23 3 4.55 ± o.15 3.22 ± 0.29 2.75 ± o.17 4 4.22 ~z 0.30 3.47 ~- o.12

* Krebs- lRinger buffer modified so t h a t 120 mM Na + was replaced by equ imola r concen t ra t ions of Li +, Tris or lysine.

** Krebs- t~ inger buffer modified so t h a t 6 mM K + was replaced by equ imola r concen t ra t ions of lys ine or N H , +.

Biochim. Biophys. Acta, 79 (1964) 167 176

1.72 M. FOX, S. THIER, L. ROSENBERG, S. SEGAL

modes t Na + concentra t ion p e r m i t t e d uniform p H control wi th the b ica rbona te sys tem, and assured d is t r ibu t ion rat ios above I , ind ica t ing some concent ra t ion agains t a chemical gradient . A t 9 ° min incubat ion the to ta l wate r and ext race l lu lar fluid spaces in all of these media were comparable . As can be seen, op t imal AI~B t r anspor t d id not occur in the presence of any of the cat ions tes ted except Na +. Li+ suppor t ed more t r anspo r t t han d id Tris or lysine bu t d id not app roach Na+ in effectiveness. I t seems reasonable to conclude t h a t the inab i l i ty of the slices to achieve op t ima l amino acid t r anspor t in Tris and Li + media was due to the fact t ha t bo th med ia lacked Na +.

The Na+-free med ia used in these exper iments d id not i r revers ib ly damage tile tissues. Over 85 °o recovery of t r anspor t ing capab i l i ty was achieved when slices pre- i ncuba ted in "Tr i s" or "a l l K +'' med ia for 60 rain were t ransfer red to K r e b s - R i n g e r b ica rbona te buffer.

T A B L E i i I

E F F E C T OF O U A B A I N 0 . 8 nl.~'~ ON A M I N O A C I D A C C U M U L A T I O N

Dis t r ibu t ion rat ios of several ami no acids were de t e rmined af ter 90 rain incuba t ion in the absence and presence of ouaba in in Krebs Ringer or Tris buffer. Each d is t r ibu t ion rat io represen ts the

m e a n of the indica ted n u m b e r of separa te de te rmina t ions .

Distribution ratios

Amino acid Krebs Ringer bicarbonate buffer Tris bujJ~:r

Control Ouabain Control Ouabain

A1B 4.48. (24) 2.30* (6) 0.90 (12) - Glycine 9.3 °* (II) 3.72* (G) i.o9 (G) Lysine 5.or* (9) 2.97* (0) 2~46"* (12) 2.42** (5)

* t ) ~ O,OOI.

** No signif icant difference.

Effect of ouabain

Ouabain , which inhibi ts act ive N a + t r anspor t s, also inhibi ts act ive t r anspor t of amino acids (Table I I I ) . Approx . 50 % reduct ion in the accumula t ion of AIB, glycine and lysine was accompl ished b y o.8 mM ouaba in in K r e b s - R i n g e r b ica rbona te buffer. However , the act ive accumula t ion of lysine which occurred in Na+-free Tris buffer was unaffected b y ouaba in at this concentra t ion. The fact t ha t ouaba in inh ib i ted lysine t r anspo r t in the presence of Na +, bu t not in the absence of Na ÷, suppor ts the view tha t the inh ib i to ry effect of ouaba in on amino acid t r anspor t is not due to non-specific t issue toxic i ty , bu t to specific inhibi t ion of the Na+-dependent por t ion of amino acid t r anspor t .

Effect of varying medium Na + concentration

The t r anspor t of several amino acids was s tud ied at va ry ing med ium N a + concen t ra t ions by isomolar subs t i tu t ion of Tris chloride for NaC1 in K r e b s - R i n g e r b ica rbona te buffer. As previous ly shown in Table I, no act ive t r anspor t of A I B was seen in Na+-free media . Act ive accumula t ion occurred (Fig. 3) at med ium Na + concen t ra t ion of 3 ° mequiv/1 and increased as the Na + concent ra t ion of the med ium was raised to levels even above 15o mequiv/1. A I B d is t r ibu t ion rat ios at four concentra- t ions of med ium N a + wi th the cat ionic deficit made up wi th K + are p lo t t ed on the

Biochim. Biophys. Acla, 79 (19(i)4) t07 q 7 ii


same axes. The points fall on the Na+-Tris curve indicating that AIB transport is primarily dependent on the extracellular Na + concentration even at high extracellular K + concentrations.

6 o

c ~

g4

~ 2 o o

0 ' i 0 '4'0 '~'0' ~0' ,~0 . . . . . ,20 ,40 ,~0' ,~0'260 . . . . ' ' 220 240 260 MEDIUM SODIUM CONCENTRATION(rnEqui~/I)

Fig. 3. The effect of varying buffer Na+-concentrat ion on active accumulat ion of AIB. The closed dots represent distr ibution ratios ± S.E. of the mean of triplicate determinat ions in modified Krebs-Ringer buffer in which the Na + deficit was made up wi th Tris +. Na + levels above 144 mequiv/1 were obtained by adding the required am oun t of NaC1 to Krebs-Ringer media. The open dots represent distr ibution ratios obtained in modified Krebs-Ringer buffer with the Na + deficit

made up with K +.

The effect of varying the medium Na ÷ concentration on the transport of glycine, lysine and histidine is shown in Fig. 4- Glycine has transport characteristics similar to those of AIB. At zero Na + concentration there was no active transport, and the accumulation of glycine was closely correlated with the rising medium Na + concentration. Similarities between glycine and AIB have been noted by others, in other systems 9. However, lysine and histidine could be actively transported even in Na+-free media and their dependence on the level of the medium Na + concentration was less marked. Nevertheless, the correlation of rising intracellular amino acid accumulation with rising extracellular Na + concentration persisted.

The ability of the cationic amino acid lysine to be actively accumulated by the kidney cortex slice in the absence of Na + in the medium raised the possibility that lysine may satisfy for itself the transport requirement for a positively charged moiety, as Na + satisfies that requirement for the neutral amino acids, AIB and glycine. In that case, could lysine, serving as an extracellular cation, support the transport of a neutral amino acid such as AIB? The data in Table I I indicate that it could not, for AIB transport in the low Na + medium used was not increased when lysine (12o raM) was added.

Effect of varying medium K ÷ concentration

Fig. 5 demonstrates that optimum amino acid transport occurred over a fairly narrow range of medium K + concentration. Again, there were individual differences among the different amino acids. The transport of glycine fell off precipitously as the medium K + concentration fell below 12 mequiv/1, but at higher K + levels the diminu- tion in glycine accumulation was very gradual. Optimal AIB transport occurred in the vicinity of 7 mequiv/1 K +, the physiological level, and fell off at higher and lower K +


174 M. FOX, S. T H I E R , L. R O S E N B E R G , S. S E G A L

I 0 I l I I I

! 8

7

o

! i

g

m

[ t L ~ . [ i . I ] 0 30 60 90 120 1 5 0

MEDIUM SODIUM CONCENTRATION (mEqLJiV/I )

Fig . 4. T h e e f fec t of v a r y i n g b u f f e r N a + c o n c e n - t r a t i o n o n t h e d i s t r i b u t i o n r a t i o s of g l y c i n e ( Q - - O ) , l y s i n e ( m B ) a n d h i s t i d i n e (Jk Jk) . [ n m e d i a c o n t a i n i n g less t h a n 15 ° m e q u i v / 1 N a +, t h e c a t i o n i c de f i c i t w a s m a d e u p w i t h T r i s +. E a c h p o i n t r e p r e s e n t s t h e m e a n ~ S .E . of t h e

m e a n of 3 9 d e t e r m i n a t i o n s .

I; •

9

8

/ o 7

~ 6 g g ~

~ 4

3

0 4 8 12 16 20 24 28 A6-O MEDIUM POTASSIUM CONCENTRATION

{ mEqu;v/! )

F i g . . 5 . T h e e f fec t of v a r y i n g b u f f e r K÷ c o n c e n - t r a t i o n o n t h e a c t i v e a c c u m u l a t i o n of A I B (O .... O) , g l y c i n e ( O - - - O ) , l y s i n e ( m - - m ) a n d h i s t i d i n e ( Jk - - - Jk ) . V a r y i n g K* c o n c e n t r a t i o n s w e r e o b t a i n e d b y a l t e r i n g t h e KC1 c o n t e n t of K r e b s - R i n g e r b i c a r b o n a t e bu f f e r . E a c h p o i n t r e p r e s e n t s t h e m e a n of 3 9 s e p a r a t e d e t e r -

m i n a t i o n s .

concentrations. Lysine and histidine were similarly affected. In this svstem neither lysine nor NH4+ ions could successfully substi tute for extracellular K + (Table II).

Other io~s

The role of ions other than Na + and K + was investigated. Removal of Ca 2+ and Mg 2+ from Krebs-Ringer bicarbonate buffer did not alter AIB t ranpor t at all. Neither did raising the phosphate concentrat ion to 3.6 mM. Distribution ratios of AIB in media containing zero, 78 and 144 mequiv/1 Na + were not appreciably altered by raising the Ca 2+ concentrat ion of these media to 8 mM. In fact, A IB was t ransported normally in a buffer composed of NaC1, KC1 and NaHCO a in physiologic proport ions; however, glycine t ransport was reduced to 67 % of normal and lysine t ransport to 51 °'o of normal in this medium.

DISCUSSION

These studies indicate tha t the pr imary extracellular ion implicated in the active t ransport of amino by kidney tissue is Na +. The few published studies available con- cerning the role of extracellular Na + in amino acid t ransport are in general agreement with these observations. CHRISTENSEN et al. 1° replaced 72 and 117 mequiv/1 Na + with similar amounts of choline and noted 24 % and 57 % reduction in glycine accumulation by Ehrlich ascites-tumor cells. HEINZ 2 working with similar cells, reported tha t when extracellular Na + was reduced below 9 ° mequiv/1 (with constant K + concentra-

Biochim. Biophys. Ac!a, 79 ( I964) ~67 17<~


tion) glycine influx was diminished. ALLFREY et al. n in studying amino acid uptake by calf-thymus nuclei in cell-free systems noted that the transport of amino acids into the nuclei depended upon the presence of Na + in the medium. Whereas Li + was partially effective as a Na + substitute, neither K +, Cs + nor Rb + was able to support [l*Cialanine transport. CSAKY 12 studying frog small intestine noted that when Li+ or K + replaced Na + in the medium the active transport of L-tyrosine, DL-phenylalanine and uracil were partially inhibited.

The extracellular K+ concentration also influences amino acid transport by the kidney slice. Optimal amino acid accumulation occurred over a rather narrow range of medium K ÷ concentration with impaired transport at lower and higher levels. Using Ehrlich ascites-tumor cells, RIGGS et al. 1 observed similar results on varying medium K + concentration. They found amino acid transport to be correlated best with the levels of intracellular K ÷ and suggested that K ÷ efflux in some way drives amino acid influx. The latter thesis has been questioned by HEMPLING AND HARE 13 who have measured K ÷ fluxes in ascites-tumor cells at various levels of glycine accumulation. The affinity of glycine for the inner membrane carrier was found to be 25 times that of K ÷, making a I : I stoichiometric relationship between the two unlikely. In addition, the free energy available from K + efflux is less than the free energy required for glycine influx.

It is apparent from our studies in rat kidney cortex slices that active transport of the neutral amino acids glycine and AIB is dependent upon levels of extracellular Na + and K +. Whether this dependence is ultimately due to changes in the intracelhilar concentrations of these ions, or the ratio of these ions across the membrane is un- known. Similar dependence upon buffer Na + and K ÷ concentration for glycine and AIB transport in human leukocytes has recently been reported by YUNIS et a l ) 4.

Lysine differs from AIB and glycine in that it is accumulated to a significant extent in the absence of Na + in the medium. Lysine transport is inhibited by ouabain in Krebs-Ringer bicarbonate buffer but not in Na+-free buffer. This strongly suggests that two separate pathways for lysine transport exist in this tissue--one Na + dependent and ouabain sensitive, and the other independent of Na+ and ouabain insensitive.

The dependence of intestinal sugar transport on Na + concentration in the medium has been well demonstrated15, TM. BIHLER AND CRANE 17 explored many anions and cations and found that the role of Na ÷ could not be duplicated, nor could any other ion be shown to be essential.

That media ion concentration can influence so profoundly the transport of dis- similar moieties such as sugars and amino acids, suggests that the ionic requirement may be of the energy-generating system, a process common to all active transport. Support for this hypothesis comes from recent studies of an enzyme system in trans- porting membranes which hydrolyzes ATP to ADP and inorganic phosphate. POST et al. TM have described ATPase activity in broken human erythrocyte membranes which is stimulated by Na + and K + ions. This enzyme system shares an unusual con- stellation of common properties with, and is probably involved in, the active-linked transport of Na + and K+. ATPase systems that are stimulated by Na + and K + ions have been demonstrated in many tissues TM, 20, and their ionic requirements have gener-- ally paralleled those described above for amino acid transport. SKOU 21 has reported optimal ATPase activity in kidney and brain homogenates at media Na ÷ and K+ concentrations of 145 and IO mequiv/1, respectively. At any K ÷ level, enzyme activity


176 M. FOX, S. THIER, L. ROSENBERG, S. SEGAL

i n c r e a s e d w i t h i nc r ea s ing Na+ c o n c e n t r a t i o n . K + c o n c e n t r a t i o n s lower t h a n I0

mequiv /1 or h i g h e r t h a n 4 ° mequiv /1 t e n d e d to d i m i n i s h ac t iv i ty . No o t h e r ion cou ld

a d e q u a t e l y rep lace N a +, b u t t h e f u n c t i o n of K + cou ld be a c h i e v e d b y NH4 +. T h e

e n z y m e s y s t e m was i n h i b i t e d by o u a b a i n , b u t on ly in t h e p r e s e n c e of Na+. T h e s e

f ind ings h a v e been c o n f i r m e d b y WHEELER AND \VHITTAM 22 us ing r a b b i t k i d n e y

c o r t e x h o m o g e n a t e s .

The c o m m o n ionic r e q u i r e m e n t s of t h e s e A T P a s e s y s t e m s , a n d of t he m e c h a n i s m

for ac t ive t r a n s p o r t of ce r t a in a m i n o ac ids in k i d n e y sugges t s t h a t t h e s e p rocesses m a y

be i n t i m a t e l y r e l a t ed , S imi la r conc lus ions b a s e d on s t u d i e s of i n t e s t i n a l s u g a r t r a n s -

p o r t h a v e r e c e n t l y b e e n p u b l i s h e d b y CSAKY 23.

REFERENCES

1 T. I~. [~IGGS, 1~, M. WALKER AND }-l. N. CHRISTENSEN, J. Biol. Chem., 233 (1958) I479. 2 E. HEINZ, in J. T. HOLDEN, Amino Acid Pools, Elsevier, Amsterdam, 19(32 , p. 539. a N. G. LEVINSKY, I. TYSON, R. B. MILLER AND 2\. S. RELMAN, J. Cl[ll. Invest , 41 (1902) 480. 4 L. E . ROSENBERG, A. BLAIR AND S. SEGAL, Biochim. Biophys..;tcla, 54 (19(311 479- 5 L. E. ROSENBERG, S. J. DOWNING AND S. SEGAL, J. Biol. Chem., 237 (1902) 2205 . 6 L. E. ROSENBERG, S. J. DOWNIN/; AWl1 S. SEriAL, .4m. J. Physiol., 2o2 (1902) Soo.

G. C. N~.HAS, Pharmacol. Rev., t 4 (19021 447. s H. J. SCHATZS~ANN, Helv. Physiol. ,4cta, l I (I9531 340. 9 H. N. CHRISTENSEN, Federalion Proc., 21 (19(12) 37.

10 H. N. CHRISTENSEN, T. R. RIGGS, H. FISCHER AND [. M. PALATINE, J. Biol. Chem., I91 (1952) I. 11 V. (; . ALLFREY, J. \V. HOPKINS, J. H. [:RJ6NSTER AND A. E. MIRSKY, ~'JI~l. N.Y. , .4cad. Sci., 88

(I96O) 722. 12 T. Z. CSaKY, Federatio~z Proc., 20 ([90I) I39. 1:~ Iq. (;. HEMPLING AND 11. HARE, J. Biol. Chem., 23(~ (19(311 2498. 1.1 A. A. YUNIS, (;. ARIMK;RA AND 11. M. [(IPNIS, J. Lab. Clin. Med., Oo (19(32) io28. 15 E. I{IKLIS AND J. H. QUASTI'.'L, Can. J. Biochem. Physiol., 36 (I95 S) 347. 16 T. Z. CSAKY ANO M. THALE, J. Ph.l'siol., 151 (196o) 59. 17 I. BIHLER AND R. 1{. CRANE, Biochim. Biophys..qcla, 59 (I9(32) 78. is 1{. L. POST, C. R. MERRITT, C. R. I<INSOLVlNG AND C. D. ALBRI~HT, .[. Biol. Chem., 235 (190o)

1796. 19 S. L. BONTING, f'~. :\. SIMON AND N. ~'I. HAWKINS, .4rch. l-~iochem. Biophys., 95 (19(311 4 l(3" 21 j . V. AUDITORE AND I.. MURRAY, Arch. Biochem. Biophys., 99 (19621 372. 2I j. C. SKOU, Biochim. Biophys. Acla, 58 (1902) 314 • 22 1<. P. WHEELER AND R. \VHITTAM, Biochen2. J., 85 (19(32) 495. 2a T. Z. CSAKV, l:ederalion Proc., 22 (I9(33) 3.

Biochim. Biophys..4cta, 79 (19041 167-170

ionic requirements for amino acid transport in the rat kidney cortex slice: i. influence of...

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