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Canadian Journal of Physio and Pharmacology
Journal canadien
Published by
P11bliC par
THE NATIONAL RESEARCH COUNCIL OF CANADA
1.E CONSEIL NATIONAL DE RECHERCHEB DU CANADA
Volume 57
Volume 5'7
Number 1
January 1949
et pharrnacologie numkro 1
janvier 1979
ELectrogenic amino acid transport Depnrtrnerat of Biochernisrry, Faculty ofMedicine, McGill University. Monrreal, P.Q., Canada H3G I YQ
Received September 7, 1978
Introduction This brief review aims to highlight current views on Na+-coupled nonelectrolyte transport in animal cells. Emphasis has been placed on the recent recognition that the Na+-coupled transport of electrically neutral solutes is electrogenic and thus influences, and is influenced by, the potential difference existing across the plasma rne~nbrane.This recognition is adding a new dimension to the study of the meckailisms which regulate transport activity. It raises the question whether variation of ionic conductance results iin consequences such as altered availability of nutrients like amino acids, vitamins, and sugars. A cursory glance at the current literature on nonelectrolyte transport shows the extent to which elcctrogenicity of transport processes is being recognized and studied in cells ranging from E, coli to cultured huinnan cells. The notion that movemeint of electrically neutral solutes across inembranes can be ignored with respect to effects on the membrane potential is clearly no longer tenable. Neutral molecules are translocated chiefly in association with protons in microorganisms and plants and with Na+ ions in animal cells. The result is a net transfer of charge concomitant with the neutral solute. FurtherInore, present-day views on energy-transducing mechanisms consider the membrane potential to be a major source of useable energy for ATP synthesis as well as for doing osmotic work. Although the literature surveyed for this article is not exhaustive, I hope readers will find it sufficient
to provide a bird's-eye view of the status of amino acid transport in Na+-independent Transport of Amino Acids Although amino acid transport in animal cells has been studied intensively for about 25 years, it was recognized much earlier that animal cells contain higher a-an~inonitrogen levels than the surrounding fluids. The development of a technique by Van Slyke to measure a-amino nitrogen was followed by measurements of amino nitrogen levels in animal tissues and fluids. Van Slyke and hfeyer (19 13) showed that liver could accumulate amino acids to levels well in excess of those in the plasma following an infusion of amino acids. Since those early observations, it has become evident that in all cells examined, specialized mechanisms exist for transport of amino acids. Several distinct amino acid transport systems are found in mammalian tissues and most amino acids are transported by more than a single route. Christensen and his colleagues (Thomas et al. 1971 ; Christensen 1973; Christensen et al. 1973) have made a particular study of the efIect of molecular size, group replacement, and spatial orientation on amino acid transport systems using the Ehrlich ascites tumour cell as the model cell. Although tissue differences cannot be ignored, overall tlne use of the Ehrlich ascites tumotar cell appears to be an appropriate model to study the structural characteristics of amino acid transport systems (Thomas et al. 19'71 ) .
0008-4212/79/010001-15$81.00/0 @ 1979 National Research Council of Canada/Consei%national de reeherches clu Canada
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CAN. J. PHYSIQL. PHARMACQL. VOL. 57, 1979
Despite the variety of systems, which include systems for basic amino acids, acidic amino acids, glycine, and imino acids, in addition to the major group of neutral a-amino monocarboxylic acids, transport of amino acids can be separated into two main categories, namely, Na+-dependent and Na+-independent transport. The Na+-independent systems are generally equivalent to a type of facilitated diffusion. A particularly common form of Na+-independent transfer is an exchange diffusion between internal and external a n ~ i i ~acids, o where the rate of solute transfer is enhanced by the presence of a similar (or the same) solute on the trans1 side of the membrane (Heinz and t\'alsh I95 8 ; Johnstone and Scholefield 1961 ; Qxender and Christeaasen 1963). Whether exchange diffusion is always a Na+-independent process is moot. In Ehrlich cells, it has been established that amino acid exchange is independent of Na+ (Johnstone and Scholefield 1965). Similarly in sheep reticulocytes, histidine exchanges at the same rate with and without Na+ (Benderoff, Blostein et al. 1978n). In reticulocytes from other sources, Na+ has been reported to enhance exchange of some amino acids (Winter and Christensen 1965), although exchange does occur in Na+-free media. An unresolved question is whether the Na+-independent exchange diffusion system(s) for amino acids is (are) also able to catalyze the net movement of an amino acid, i.e., whether in absence of an exchangeable solute on the trans side, net transport activity is catalyzed by the carrier responsible for the exchange activity. The evidence that the exchange carrier is capable of net uptake of an amino acid is not compelling, being based on the uptake of an isotopically labelled amino acid under special conditions, for example, in Na+-free media. Most cells however contain large pools of amino acids. Unless cells are deliberately 'emptied' of their free amino acid pools, sufficient intracellular amino acid may exist to exchange with the exogenous amino acid. The uptake of an isotopically labelled amino acid without specific preloading does not itself constitute evidence for a net increase of cellular amino acid without direct measurements to verify the conclusion. Unfortunately, many experiments which are cited as evidence for .tlet movement via the 'exchange carrier' were executed under conditions where the cellular amino acid poc~lwas not measured. In the Ehrlich cells there is some evidence that the Na+-independent exchange transport is incapable of
net transport. In the latter cells, the exclnange works best with a number of longer chain a-amino acarboxylic amino acids such as leucine and methionine (Johnstone and Scholefield 696 1 Oxender and Christensen 6963 1. (Methionine, in fact, also undergoes Na+-dependent net accumulation. ) In the absence of Na-" cells depleted of amino acids show negligible rates of [lT]methionine uptake (Potashner and Johnstone 1971 ), the cellular methionine concentratio11 remaining less than that of the incubation medium even after 64) min iracubakion. In contrast, under the same conditions but with cells containing the normal levels of internal amino acids (deliberately preloaded or not depleted) there is rapid influx of ['TC]rnethio~aine reaching cellular concentrations of 14Cin excess of that in the medium in the I st mi11 despite absence of Na+ (Potashner and Johnstone 1970). These data suggest that the rapid uptake of methionine in Na+-free media in Ehrlich cells is due to an obligatory exchange process and that the exchange mechanism is either not capable or inefficient in catalyzing unidirectional uptake of amino acids (Potashner and Jolanstone 1971 ). Until more specific experiments have bee11 executed, the capacity of other exchange system(s) to catalyze net transfer of amino acids remains unclear. It has been shown that the electrically silent Clmovement in red cells is an obligatory exchange process, largely incapable of causing unidirectional C1- movement (Gunn 1973). Thcre is little doubt, however, that the Na+-dependent transport system(s) do bring about a net accumulation. often a very extensive accumulation of amino acids a g a i ~ ~aschemical t gradient. Although evidence suggests that more than one transport system participates in Na+-dependent amino acid transport, the behaviour of the systems is very similar and for purposes of this discussion, the Na+-dependent transport for amino acids will be treated as a single system except if specific conlparisons are made.
Nag-dependent Transport Not only amino acids and sugars (for reviews see Schultz and Curran 1970; Schafer 2972; Christensen et al. 1973; Kimmich 1973; Crane 1977), but vitamins (Sharma et al. 1964; Hauser 1965; Johnstone and Sung 1967; Takenawa and Tsurnita 1974), and a number of amines such as serotonin and dopamine Born and Gillson 6959: Furano and Green 1964; Sneddon 1969; Bogdanski et al. 1970; Chong and 'Trans side refers to the side to which the flow is directed, Kay 1977; Rudnick 1977) are now known to be Lee, opposite to the side from which the measured f l u taken up and accumulated by mammalian cells via a originates.
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REVIEW /SYNTHESE
Na+-requiring mechanism. With the e.xception of mature mammalian red cells, Na+-dependent amino acid transport has been shown to occur in most animal tissues. Until recently Na+-stimulated amino acid transport was associated only with higher organisms. Now, it has been shown that in a number of microbial systems, extracellular Na+ enhances amino acid transport and accumulation (Thsmson and MacLeod 1973; Lanyi 1977) although in microorganisms the majority of the coupled transport systems work in conjunction with protons rather than Na+ (for review see i\'ilson and Maloney 1976). In some cases such as with Halobacterium, Naf and a proton gradient are required (Lanyi 1977 ) . There is a wide variation in the capacity of mammalian cells to accun~ulateamino acids against a concentration (or electrochemical) gradient. Accumulations up to 20-fold or more of the concentration in the incubation medium have been reported (see Schultz and Curran 1970; Schafer 1972 ; Kimmich 1973 ;Crane 1977). Variability also exists with respect to the kinetic constants for transport although it can be geilerally said that most K,, values for the amino acid lie between 180plV and 5 mM for the Na+-dependent processes. Since the demonstration in the late 1950's and early 1960's that transfer of organic solutes across mammalian cell membranes occurs optimally in Na+-containing medium, there has been an intensive investigation of this phenomeizon. Although Ricklis and Quastel ( 1958) first recognized that the absence of Naf from the medium reduced the transfer of glucose across the guinea pig intestine, it was Crane and collaborators (Crane 1965 ; Bihler and Crane 1962) who showed that Naf alters the kinetic parameters for the tran5port sf sugars. In the earliest reports. it was shown that Na+ lowered the K,,, for the sugars, thus ensuring high rates of transport at physiological glucose concentrations. Subsequent experiments in other laboratories showed that the kinetic parameter altered by the presence of Na+ is not necessarily the K,,, value for the organic solute, but that V,,,,, or both K,,, and V,,:,, are affected in different systems (Tnui and Christensen 1966; Eddy et al. 1967; Goldner et al. 1969; Crane 1977). The significance of this differcnce is not clear. In a single organ (rabbit small intestine) Na+ changes the K,,, for amino acid and the V,,,, for sugar transport (Goldner et al. 1969; Schultz and Curran 1970). In his early proposals Crane (1965) outlined a scheme consistent with his initial observations which much subsequent work has supported, at least in its broadest outlines. Specifically, he proposed that ( 1 )
3
the driving force for organic solute transfer is the concentration difference for Na+ which exists across the cell membrane. (2) The influx of solute coupled to Na+ becomes largely irreversible because the K,, value for influx is much smaller than for ef'flux owing to the asynlmetric distribution of Na+. ( 3 ) The K+-ATPase maintains a low intracellular Na+ level of Na+ and high K+. High intracellular K+ effectively competes with any intracellular Na' preventing any significant backflow on the Na+-dependent carrier. (4) The dependei~ceof the organic solute transport system on low iiltracellular Na+ links together Na+-dependent solute transfer with metabolic activity. By 1965 ample experimental observations were already available that a Na+ requirement for transport was also associated with a dependence on metabolic activity. In fact Csaky (Csaky and Thale 19668;Csaky 1963) had written extensively on the association of Na+ dependence in transport systems with "active" transport or transport dependent on metabolic activity. It was then known that a number of transport processes are not affected by the presence of metabolic inhibitors such as DNP or CN- whereas others are greatly inhibited. For example, glucose transport in muscle is not affected by the metabolic state whereas amino acid transport in the same organ is significantly reduced. The presence of Na+ enhances amino acid uptake but has little effect on glucose uptake in diaphragm (Kipnis and Parrish 1965). Crane's proposal explains the requirement for Na+, the association of the Na+ requirement with metabolic activity, and most significantly that there need be no covalent modification of either carrier (as for example, the formation of a phosphorylated intermediate) or solute during energization of the transport process. This proposal contrasts with that of Csaky (1963) and his collaborators who proposed that intracellular Na+ and organic solute increase thc utilization of ATP. For some years following Crane's proposals, the inability to show quantitative relationships between the magnitude of the Na9 gradient and solute accumulation (i.e., the Na+ gradient was inadequate to account for the accumulation of solute) led to postulation of energytransducing mechanisms which involved carrier modification by ATP (Kimmich 1970; Potashner and Johnstone 1971 ; Schafer and Heinz 1971; De Cespedes and Christeilscn 1974; Johnstone 1974). Recently, as will be discussed below. most of the problems have been resolved. Na+-dependent transport has become synonymous
+
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CAN. 9. PHYSTBL. PHARMACOL. VBL. 57, 1979
with "active transport" of the older literature. The consensus that metaba8isn1-dependent traiasport of organic solutes need not use ATP directly has led to definitions of transport based on the degree of closeness of the transport event to the metabolic process. Kedenl (1961) proposed that the term "primary active" transport could be used to describc transport processes which use ATB directly, such as Na+ transport. Secondary active transport would dcscribe transport processes dependent on ion and (or) electrical gradients set up by the metabolic process. Both types of transport can lead to an accumulation of the transported solute against its own electrochemical potential.
observation is that the coupling between Na+ and solute is 1 :1 (Curran et al. 196'7; Schafer and Jacquez 196'7; Eddy 1968; Goldner et al. 1969). However a number of observations have been made where the stoichiometry is greater than 1: 1 or Bess than 1: 1 and also inconstant. For example, in pigeon red cells (Vidaver 1 9 6 4 ~ )it has been concluded that the stoichionaetry is 2 Na+ per mole of glycine. Rabbit reticulocytes however show 1 : 1 stoichiometry (Wheeler et al. 1965) for amino acids other than glycine. In a recent review, it was shown that the stoichiometry in Ehrlich cells may vary from 0.3 to 2 (Christensen et al. 1973) depending on the aislino acid and the conditioils of incubation. The variable stoichiometry (as well as other data) has led Christensen et al. (19'73) to propose energization of transport by means other than the Na+ gradient.
Coupled Flow of Na+ in the Transport of Organic Solutes The demonstration that organic solute transport is stimulated by Na+ is not synoinymous with showing that Na+ is cotraiasported with the organic solute. Direct coupling of flows was first shown in intestinal preparations (Schultz and Zalusky 1964, 1965; Curran et al. 1967). Subsequent to the demonstration that sugars and amino acids increased Na+ transport across the intestinal wall (Schultz and Zalusky 1964. 19651, Curran et al. (196'7) showed that the presence of amino acids increases the unidirectional transfer of Na+ into the cells of the intestinal epithelium and that Na+ enhances the transfer of amraillo acids. At high Na+ levels, the increase in Na+ Wow caused by the amino acid is equivalent to the increase in amino acid Mow caused by Na+ (Curran et al. 1967). Similar observations on the coupling of flows have been made with sugars as the traiasported solute (Goldner et al. 1969). Although simnultaneous measurements of Wa+ and organic solute flux have been made Zess frequently than studies om Na+-stimulated solute transfer per se, the general consensus appears to be that Na+ dependence implies that Na+ flow accompanies the flow of organic solute. However, it should be mentioned that Christensen et al. (1973) have reported that in some instaa~ces,Wa+ uptake may be severely restricted without effect on amino acid uptake wlaich is nonetheless Na+ dependent. The significance of these observations is not clear. It does raise the question whether Na+ dependence unquestionably means coupling of Na+ flow to the flow of the organic solute.
Energization of Transport In his original model, Crane proposed that the movement of Na+ (down its electrochemical potential) provided the energy for the uphill movement of the organic solute to which the Na+ movement was coupled. Qualitatively, there is good agreei~ment between the accumulation of organic solutes and the magnitude of the Na+ gradient. For example, ( a ) Bihler and Crane (1962) showed that in the intestine the extent of sugar accumuTation is a linear function of the chemical gradient for Na+ and that a decrease iia the Na+ gradient obtained by varying external Na+ concentration results in decreased accumulation. (However, as will be discussed below, Na+ per se has an effect on transport in absence of transmembrane Na+ gradients. Hence, it is less clear whether the data reported reflect only the effects of a variable gradient or the effects of variable Na+ concentration, or both.) ( b ) It was shown that in intestinal cells depleted of ATP and Na+ nearly normal rates of solute influx obtain in a Na+ medium when the Na9 gradient is similar in magnitude to that of normal cells (Hajjar et al. 1970; Goldner et al. 1972). ( c ) Vidaver (19640) showed proportionality between the Na+ gradient and uptake of glyciile in pigeon red cells at constant extracellular Na9. (d) Eddy and collaborators (1967) also showed proportionality between the magnitude of the Na+ gradient and the extent of amino acid accumulation in A T f -depleted Ehrlich cells,
Stoiehiometry of Na+-eouplledTransport The stoichiometry of Na+-coupled flows have been examined in a number of systems. The general
Reasons for the Apparent Inadeqt~aeyin Energy Qualitatively these and many other observations are consistent with the predictions of the hypothesis.
REVIEW iSYNTHESE
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But attempts to show a quantitative relationship be- (unpublished). U7hetheror not sequestration of Na+ tween the magnitude of the Na+ gradient and solute by the nucleus contributes to the apparent energy accumulation have been less successful, particularly deficit, it is probable that underestimations of the in tissues where extensive accumulation of solutes potential difFerence or overestimations of the cellular Na+ activity are sufficient to account for the apparent takes place (see Discussions in Schafer ( f 972) ) . In early discussions it was coilsidered that the energy inadequacy in the Na+ gradient required for membrane potential was too small in the tissues solute accumulation. Future work will no doubt reunder study to makc a significant contribution to the solve the issue fully. overall electrochen~icalgradient for Na+ (Schultz 1968; Jacquez and Schafer 1969; Heinz 1973). Electrogenic Nature of Nag-coupled Transport Based on the energy available in tbe chemical comAs mentioned above, the earliest formulation of ponent of the Na+ gradient, accumulation of amino Na+-gradient hypothesis stressed the chemical the acids in excess of that feasible from the chemical rather than the electrical nature of the Na+-gradient gradient of Nag alone (i.e., where at steady state R T driven processes. Moreover, although it is implicit In ([Na+]o/[NaC]i) < RT In ([amino a~id]~/[amino that movement of a positive charge (Na+) in conacidlo) ) were reported (Jacquez and Schafer 1969; junction with a neutral amino acid could alter the Lin and Johnstone 197 1; Schafer and Heinz 197 1; membrane potential, compensatory movements of Johnstone 1972; Heinz 1973; Tucker and Kiminich other ions, particularly K+, could lead to an elec1973). Even when attempts were made to deduce trically silent process. Schultz and Zalusky ( 1964, the energy contribution from the electrical com1965) first showed that transport of sugars and ponent of the electrochemical potential for Na+ by amino acids coupled to Na+ increases the short-cirindirect estimates of the membrane potential, the cuit current across the intestinal wall. These elecuptake of amino acids appeared to take place against trical changes were ascribed to increased Na+ pumpan opposing driving force (Jacquez and Schafer 1969; Schafer and Heinz 197 1; Johnstone 1974). ing across the serosal barrier of the intestine as a Although the problems have not been entirely re- result of an increase in cellular sodium. Recently, solved, it appears that the membrane potential may it has been shown (Fig. I ) in a variety of systems have been appreciably underestimated, especially that the transfer of an organic solute into the cell under conditions used to elevate cell Na+ (Kimmich via a Na+-dependent route causes (i) a depolariza1970; Schafer and Heinz 1971; Johnstone 1974). tion of the cell (Rose and Schultz 1971 ; White and Thus, conditions such as cold storage which were Armstrong 1971; Heinz et al. 1975 ; Philo and Eddy 1975; Laris et a1. 1976, Fig. 1; Okata et al. 1977) used to elevate cell Na+ may have led to an increase and (ii) that changes in the membrane potential inin Na+ pump activity and hyperpolarization of the crease or decrease the Na+-coupled influx of amino cell membrane (Heinz et al. 1975). acids (Viclaver 19648; Gibb and Eddy 1972; Meinz Other factors may also contribute to the apparent et aI. 1977; Pershadsingh et al. 1978) depending energy inadequacy of the Na+ gradient, such as ason the direction of the potential change. These obsuming that the activity of cell Na+ is equivalent to servations have added support to the idea that the its concentration, whereas the Na+ activity is probNa+-coupled process is electrogenic and that the ably a fraction of the concentration (Lee and Arinmajor if not the only driving force for amino acid strong 1972). Furthermore, Pietrzyk and Heinm (and sugar) accumulation in mammalian cells is the (1974) showed sequestration of Na+ by nuclei in electrocht=rnical gradient for Na+. EhrIich cells and concluded that the unrecognized sequestration, and hence the unequal distribution of Evidence for the Electaogenic Nature Na+ in the cytoplasm, contributed to the energy of Na+-coupled Flows deficit since calculations for energy availability were based on high cytoplasmic Na+ values. In regard to Vidaver (1964b) was among the first to show the latter, it should be mentioned that in this labo- that by altering the membrane potential with conratory attempts to demonstrate preferential Na+ stant Na+ (inside and out), glycine uptake siguptake by isolated nuclei and to confirm the findings nificantly decreases as the potential difference deof Pietrzyk and Heinz were not successful. We found creases (becomes more positive inside). Gibb and no evidence for preferential sequestration of Na+ Eddy (1 972) showed that the percentage stirnulaover K+. With isolated nuclei, both ions were found tion of amino acid uptake with valinomycin at concentrations equivalent to those in the medium decreases as K f o increases (K+i was constant) in a
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CAN. J. PHYSIQL. PHARMACOL. VOL. 57, 8979
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Minutes
FIG. 1. Constant level of fluore~centintensity of dye with time for O.3(,{, suspensions of cells in Naf-Ringer incubated at room temperature. At O min, cells were diluted to 0.3',, , ancl at the times indicated aboke, dye was added to 3.2-mL aliquots The constant lekels withdrawn from the cell suspension of fluorehcent intensity are presented abow. At time marked G , glycine was added (final concentration equals 3 a1a.W) to a portion of the incubation mixture and the sannplirag was contialued ( 0 ) . An increase in fluorescent intensity indicates a depolarization and a decrease in fluorescent intensity indicates a hyperpolariration. The data show that on incubation without glycine (0) the cells gradually depolari~e.In addition, upon injection of glqcine ( 0 ) there is extensive depolariiration and gradually the cell? repolari~cto the level seen in the control. (Reprinted fro111 Larls et al. 8976, with permission of the journal. 8 .);,1(
near-linear way. With valinomycin, a Kf selective ionophore, the Kf distribution is probably near equilibrium after a few minutes incubation and thus, the membrane potential can be assessed from the Kf distribution using the Nerst equation where A$ (RTj n F ) In ([K+],j [Kf l o ) . Heinz and collaborators ( 1977) using valinomycin and variable [K+],, showed that amino acid accumulation increases as the membrane potential increases (inside negative) in a near-linear manner (Fig. 2 ) . These data were interpreted as showing that an increase in the potermtial difference (inside negative) stimulates transport. Latterly, Bershadsingh et al. ( 1978) showed that propranolol, whicla at high concen trations ( > 1O-,' M ) increases K+ permeability (Ekman et al. 1969; Pershadsingh et al. 1978), increases and decreases amino acid influx at low and high K f o respectively, when K+, is nearly constant. The stimulation and inhibition of uptake by propranolsl follow the increase and decrease respectively of the membrane potential in a near-linear manner (Bershadsingh et al. 1978) as measured with a fluorescent dye (Hoffman and Laris 1974 ) . Work with isolated membrane vesicles which show Naf -dependent solute transport has been particularly useful in showing that influx of organic solutes re-
-
_ A
50
-
.
1K!
-
*-
150 ~
0
~
~
FIG.2. Effect of electrical potential on the uptake of AIB. The ordinate shows the AIR distribution ratio (K:,)after 4 (: j) and 6 ( 0 ) min incubation of Kt-depleted Ehrlich cells in the presence of varioub oaaabain concentrations in the extr:acellular n~ecliurn.The abscibsa shows the cortespoiading ratios of the distribution of tetraphenylphosphonium (TPf3+) ions, also after 4 and 6 min incubation. The K -depleted cells were preincubated w ~ t hTP13+ ( 1 0 piZf) and K t-free Krebs-Ringer pho\phate buffer, pH 7.4, for 10 rnin at 37 C before buffers , ouabain (0- 0.75 containing AIR (0.B mM), K+ (1 8 n ~ h f )and n&" were added. It is seen that beyond a certain value of TPPf distribution the transient accumulation ratio of AIR linearly rises with the ratio of TPP+. The Na+ concentrations inside and outside the cell are approximately the same in all experiments. To the extent that the T P P t distribution ratio indicates the electrical potential, the experiment shows that the transient accumulation ratio of AIR is strongly influenced by the electrical potential or by the dilTercnce in electrochemical activity of Nat. (Reprinted from Heinz et al. 1977, with permission of the journal.)
sponds to a membrane potential (Fig. 3 ) . In most of the experiments with vesicles the changes in nlembrane potential are induced with valinomycin ( ~ other ionoph~res)and variable K* levels or by using lipid-soluble anions like CNS- to replace Cl(Colombini and Johnstone 1974; Murer and Hopfer 1974; Sigrist-Nels~net al. 1975; Evers et al. 1976; Quinlan et al. 1976; Han~mermanand Sacktor 1977; Lever 1977a; Bardin and Jshrastone 4 978). Since these vesicles are generally devoid of metabolic activity, addition of valinomycin and other is~aophoresis not likely to affect metabolic parameters which might aEect the responses in intact cells. The observation that amino acid (or sugar) influx is afiected by the magnitude of the membrane potential is consistent with the idea that the Naf-co~mpledWows are electrsgenic. In conjunction with evidence that cells becsme depslarized during influx of solutes by Naf dependent routes (Rose and Schultz 1971; White and Armstrong 1971 ; Heinz et al. 1975; Bhilo and
7
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REVIEW /SYNTHESE
0
I
i 5
1:
kk-
T~riie, r ~ l ~ r c
FIG. 3. lonophores and transport of [I-14C]AIB in membrane vesicles from Ehrlich cells. Nat- outside and Kf inside control, (V)2 pg/ml, gramicidin the vesicles throughout. (a) D, and (e)5 pg,/m[i valinornycin. The level of AIR uptake when Na" is present on both sides is shown by the broken line. (Reprinted from Colombini and Johnstone 1973, with permission of the journal.)
Eddy 1975; Laris et al. 1976; Bkata et al. 1977). the observations attest to the electrical nature of Nag-dependent transport processes. Since organic solutes are frequently transported by Na+-dependent routes, the question also arises about possible changes in membrane potential when these solutes leave the cell. If they exit by the same or a similar Na+-dependent route. then loss of salutes would result in hyperpolarization. In a recent study sf cllangcs in the membrane potential of Ehrlich cells using cyanine dyes (Laris et al. 1976) we observed that the membrane potential of freshly isolated cells was greater (more negative inside, lower fluorescence) than after 30 to 6 0 min incubation (see Fig. 1 ). To perform the fluorescence n~casurements.it is necessary to dilute the cells to a cytocrit of 0,396. Freshly isolated cells when diluted fro111 the normal suspension of 3-596 (used for transport studies) to 0.395, for the fluorescence measurements were hyperpolarized at first and in 30 min gradually depolarized to a level that remained constant for the next hour or SO. If the cells were prcincubated for 3 0 min prior to measuring fluorescence, only the stable, relatively depolarized fluorescence was observed. On examination of the possible reasons for the shift in potential, it became apparent that the cells lose considerable quantities of endogenous amino acids upon dilution to 0.3% cytocrit (Laris et nl. 2978). Many of the amino acids lost are those known to be transported by Na+-dependent routes, such as alanine, glycine, serine, and threonine. Thus, the question arose whether increased amino acid efflux upon dilution
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l-I(;. 4. Fluorescent intensity and cellular AIR level. Cells were diluted to 1 :320 in Na '-Ringer in the presence ( c, ) and absence of 3 mM a-an~inoisokutyric acid (AIB) and incubated at 37 C. At intervals aliquots were taken; dye was added; and the steady level of fluorescent intensity recorded. After 36 min the cells were spun clown and resuspended at a dilution of 1 :20 :_ 3 rnM All3 (same as initial suspension). Aliquot\ (0.2 anl,) of the suspension were taken and diluted to 1 :320 with 3.0 mL of various concentrations of AIB (see graph) in Na+-Ringer; dye was added; and the steady level of fluorescent intensity recorded. The position of the symbols underneath the medium AIB concentration indicates the fluorescence attained with the given conce~atration of AIB. (Reprinted from Laris et al. 1978, with permission of the journal.)
(e)
contributes to the initial hyperpolarized condition of the cells. Experiments show that cclls become hyperpolarized when introduced into a medium into which there is net loss of amino acid (Fig. 4 ) . Conversely, depolarization occurs when there is net uptake of amino acids (Fig. 4 ) . No change in potential is seen if the amino acid level remains constant. Moreover, hyperpolarization of cells associated with net loss of amino acids takes place only in cells containing intracellular Nar (Table 1 ) . If cells depleted of Nag but containing high amino acid levels are introduced into an amino acid free medium, there is no associated hyperpolarization with amino acid loss. All these data are consistent with the conclusion that amino acid movements alter the membrane potential. Hyperpolarizatioil of cells by Na+-dependent amino acid efflux raises the possibility that influx
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8
CAN. J. PHYSLOL. PHARMACOL. V6E. 54, 1979
of amino acids from the medium may be stimulated by emux of an endogenotas amino acid. Earlier in this article reference h7as made to the phenoinenon of exchange diffusion of amino acids. It is now oiler 20 years since Heiilz and Walsh ( 1958) denlonstrated a stimulatioil of influx of [14C]glycine by preincubating Ehrlich cells with nonradioactive glycine. This trans stimulation was ascribed to be due to an exchangc of cellular with extracella~lar amino acid, the exchange rate being greater than the net, unidirectional flux. Since then it has been established that a Na+-independent exchange process does occur (see above) in many types of cells. The question which arises here is whether under conditions of net loss of cellular amino acid in a sodium medium (as occurred in the Heinz and Walsla report), the increase in [lT]glycine uptake was due to exchange diffusion or due to a hyperpolal-ization of the cell by loss of alniilo acid. If the latter is true. the increase in membrane potential would the11 be respoilsible for the enhanced glycine uptake. In addition, the question is also raised whether the Na+-dependent "exchange diffusion" reported in some systems (Winter and Christensen 1965) is actually due to hyperpolarization of the cells by loss of endogenous amino acids. The present observations necessitate a reexamination of data showirmg stimulatioil of amino acid influx in cells preloaded with amino acids to distinguish between a truc exchange effect and the effect of a membrane potential on trans stimulation of amino acid influx. Another interesting problem which needs further iilvestigation is whether a decrease in nleinbra~acpotential will enhar~ccNa+-coupled amino acid efflux along the concentratioan gradient at constant cell Na+. This question may be relevant in systems which release solutes, such as putative transmitters, which are normally transported into, and presumably, out of the cell, by Na+-dependent routes. There have been few studies directly addressed to this qucstion since in most cases cell Na+ increases along with depolarization (for example see Ham~nerstad and Cutler 1972). Generally, conditions which cause depolarization lead to an elevatiora of cell Na+ (for example, inhibitors of the Na+ pump or general metabolic inhibitors). Therefore, unless precautioils are taken to keep cell Wa+ constant or to compensate for the efiects of elevated cell Na+, the action of the potential per se is not clear. In a recent study on the effects c ~ fcell Wa+ on efflux, it was shown that in Ehrlich cells efflux of glycine is greater in cells extensively depolarized with gramicidin (Johnstone 1975) than in cells with comparable Na+ levels
TABLL:I. Fluorescent intensity as a function of cellular n-aminoiwbutyrate (AIB), Na+, and K concentrations* +-
--
-
Cellular concn., n.nmol;/Lcell water
M+
Na4-
BIB
Fluore%centintensity, arbitrary units
Series 1 0 33 0 27
Series 2 O 0 0 O NOTE: Cells ( 8 :320 dilution) were precquilibrated for 30 rnin in WatRinger t 3 m M A I B at 37°C. Alicluots of the calls were resuspended (I :GO dilution) in either coid (6°C) K+-free Wn*-Kinger, I
Canadian Journal of Physio and Pharmacology
Journal canadien
Published by
P11bliC par
THE NATIONAL RESEARCH COUNCIL OF CANADA
1.E CONSEIL NATIONAL DE RECHERCHEB DU CANADA
Volume 57
Volume 5'7
Number 1
January 1949
et pharrnacologie numkro 1
janvier 1979
ELectrogenic amino acid transport Depnrtrnerat of Biochernisrry, Faculty ofMedicine, McGill University. Monrreal, P.Q., Canada H3G I YQ
Received September 7, 1978
Introduction This brief review aims to highlight current views on Na+-coupled nonelectrolyte transport in animal cells. Emphasis has been placed on the recent recognition that the Na+-coupled transport of electrically neutral solutes is electrogenic and thus influences, and is influenced by, the potential difference existing across the plasma rne~nbrane.This recognition is adding a new dimension to the study of the meckailisms which regulate transport activity. It raises the question whether variation of ionic conductance results iin consequences such as altered availability of nutrients like amino acids, vitamins, and sugars. A cursory glance at the current literature on nonelectrolyte transport shows the extent to which elcctrogenicity of transport processes is being recognized and studied in cells ranging from E, coli to cultured huinnan cells. The notion that movemeint of electrically neutral solutes across inembranes can be ignored with respect to effects on the membrane potential is clearly no longer tenable. Neutral molecules are translocated chiefly in association with protons in microorganisms and plants and with Na+ ions in animal cells. The result is a net transfer of charge concomitant with the neutral solute. FurtherInore, present-day views on energy-transducing mechanisms consider the membrane potential to be a major source of useable energy for ATP synthesis as well as for doing osmotic work. Although the literature surveyed for this article is not exhaustive, I hope readers will find it sufficient
to provide a bird's-eye view of the status of amino acid transport in Na+-independent Transport of Amino Acids Although amino acid transport in animal cells has been studied intensively for about 25 years, it was recognized much earlier that animal cells contain higher a-an~inonitrogen levels than the surrounding fluids. The development of a technique by Van Slyke to measure a-amino nitrogen was followed by measurements of amino nitrogen levels in animal tissues and fluids. Van Slyke and hfeyer (19 13) showed that liver could accumulate amino acids to levels well in excess of those in the plasma following an infusion of amino acids. Since those early observations, it has become evident that in all cells examined, specialized mechanisms exist for transport of amino acids. Several distinct amino acid transport systems are found in mammalian tissues and most amino acids are transported by more than a single route. Christensen and his colleagues (Thomas et al. 1971 ; Christensen 1973; Christensen et al. 1973) have made a particular study of the efIect of molecular size, group replacement, and spatial orientation on amino acid transport systems using the Ehrlich ascites tumour cell as the model cell. Although tissue differences cannot be ignored, overall tlne use of the Ehrlich ascites tumotar cell appears to be an appropriate model to study the structural characteristics of amino acid transport systems (Thomas et al. 19'71 ) .
0008-4212/79/010001-15$81.00/0 @ 1979 National Research Council of Canada/Consei%national de reeherches clu Canada
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2
CAN. J. PHYSIQL. PHARMACQL. VOL. 57, 1979
Despite the variety of systems, which include systems for basic amino acids, acidic amino acids, glycine, and imino acids, in addition to the major group of neutral a-amino monocarboxylic acids, transport of amino acids can be separated into two main categories, namely, Na+-dependent and Na+-independent transport. The Na+-independent systems are generally equivalent to a type of facilitated diffusion. A particularly common form of Na+-independent transfer is an exchange diffusion between internal and external a n ~ i i ~acids, o where the rate of solute transfer is enhanced by the presence of a similar (or the same) solute on the trans1 side of the membrane (Heinz and t\'alsh I95 8 ; Johnstone and Scholefield 1961 ; Qxender and Christeaasen 1963). Whether exchange diffusion is always a Na+-independent process is moot. In Ehrlich cells, it has been established that amino acid exchange is independent of Na+ (Johnstone and Scholefield 1965). Similarly in sheep reticulocytes, histidine exchanges at the same rate with and without Na+ (Benderoff, Blostein et al. 1978n). In reticulocytes from other sources, Na+ has been reported to enhance exchange of some amino acids (Winter and Christensen 1965), although exchange does occur in Na+-free media. An unresolved question is whether the Na+-independent exchange diffusion system(s) for amino acids is (are) also able to catalyze the net movement of an amino acid, i.e., whether in absence of an exchangeable solute on the trans side, net transport activity is catalyzed by the carrier responsible for the exchange activity. The evidence that the exchange carrier is capable of net uptake of an amino acid is not compelling, being based on the uptake of an isotopically labelled amino acid under special conditions, for example, in Na+-free media. Most cells however contain large pools of amino acids. Unless cells are deliberately 'emptied' of their free amino acid pools, sufficient intracellular amino acid may exist to exchange with the exogenous amino acid. The uptake of an isotopically labelled amino acid without specific preloading does not itself constitute evidence for a net increase of cellular amino acid without direct measurements to verify the conclusion. Unfortunately, many experiments which are cited as evidence for .tlet movement via the 'exchange carrier' were executed under conditions where the cellular amino acid poc~lwas not measured. In the Ehrlich cells there is some evidence that the Na+-independent exchange transport is incapable of
net transport. In the latter cells, the exclnange works best with a number of longer chain a-amino acarboxylic amino acids such as leucine and methionine (Johnstone and Scholefield 696 1 Oxender and Christensen 6963 1. (Methionine, in fact, also undergoes Na+-dependent net accumulation. ) In the absence of Na-" cells depleted of amino acids show negligible rates of [lT]methionine uptake (Potashner and Johnstone 1971 ), the cellular methionine concentratio11 remaining less than that of the incubation medium even after 64) min iracubakion. In contrast, under the same conditions but with cells containing the normal levels of internal amino acids (deliberately preloaded or not depleted) there is rapid influx of ['TC]rnethio~aine reaching cellular concentrations of 14Cin excess of that in the medium in the I st mi11 despite absence of Na+ (Potashner and Johnstone 1970). These data suggest that the rapid uptake of methionine in Na+-free media in Ehrlich cells is due to an obligatory exchange process and that the exchange mechanism is either not capable or inefficient in catalyzing unidirectional uptake of amino acids (Potashner and Jolanstone 1971 ). Until more specific experiments have bee11 executed, the capacity of other exchange system(s) to catalyze net transfer of amino acids remains unclear. It has been shown that the electrically silent Clmovement in red cells is an obligatory exchange process, largely incapable of causing unidirectional C1- movement (Gunn 1973). Thcre is little doubt, however, that the Na+-dependent transport system(s) do bring about a net accumulation. often a very extensive accumulation of amino acids a g a i ~ ~aschemical t gradient. Although evidence suggests that more than one transport system participates in Na+-dependent amino acid transport, the behaviour of the systems is very similar and for purposes of this discussion, the Na+-dependent transport for amino acids will be treated as a single system except if specific conlparisons are made.
Nag-dependent Transport Not only amino acids and sugars (for reviews see Schultz and Curran 1970; Schafer 2972; Christensen et al. 1973; Kimmich 1973; Crane 1977), but vitamins (Sharma et al. 1964; Hauser 1965; Johnstone and Sung 1967; Takenawa and Tsurnita 1974), and a number of amines such as serotonin and dopamine Born and Gillson 6959: Furano and Green 1964; Sneddon 1969; Bogdanski et al. 1970; Chong and 'Trans side refers to the side to which the flow is directed, Kay 1977; Rudnick 1977) are now known to be Lee, opposite to the side from which the measured f l u taken up and accumulated by mammalian cells via a originates.
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REVIEW /SYNTHESE
Na+-requiring mechanism. With the e.xception of mature mammalian red cells, Na+-dependent amino acid transport has been shown to occur in most animal tissues. Until recently Na+-stimulated amino acid transport was associated only with higher organisms. Now, it has been shown that in a number of microbial systems, extracellular Na+ enhances amino acid transport and accumulation (Thsmson and MacLeod 1973; Lanyi 1977) although in microorganisms the majority of the coupled transport systems work in conjunction with protons rather than Na+ (for review see i\'ilson and Maloney 1976). In some cases such as with Halobacterium, Naf and a proton gradient are required (Lanyi 1977 ) . There is a wide variation in the capacity of mammalian cells to accun~ulateamino acids against a concentration (or electrochemical) gradient. Accumulations up to 20-fold or more of the concentration in the incubation medium have been reported (see Schultz and Curran 1970; Schafer 1972 ; Kimmich 1973 ;Crane 1977). Variability also exists with respect to the kinetic constants for transport although it can be geilerally said that most K,, values for the amino acid lie between 180plV and 5 mM for the Na+-dependent processes. Since the demonstration in the late 1950's and early 1960's that transfer of organic solutes across mammalian cell membranes occurs optimally in Na+-containing medium, there has been an intensive investigation of this phenomeizon. Although Ricklis and Quastel ( 1958) first recognized that the absence of Naf from the medium reduced the transfer of glucose across the guinea pig intestine, it was Crane and collaborators (Crane 1965 ; Bihler and Crane 1962) who showed that Naf alters the kinetic parameters for the tran5port sf sugars. In the earliest reports. it was shown that Na+ lowered the K,,, for the sugars, thus ensuring high rates of transport at physiological glucose concentrations. Subsequent experiments in other laboratories showed that the kinetic parameter altered by the presence of Na+ is not necessarily the K,,, value for the organic solute, but that V,,,,, or both K,,, and V,,:,, are affected in different systems (Tnui and Christensen 1966; Eddy et al. 1967; Goldner et al. 1969; Crane 1977). The significance of this differcnce is not clear. In a single organ (rabbit small intestine) Na+ changes the K,,, for amino acid and the V,,,, for sugar transport (Goldner et al. 1969; Schultz and Curran 1970). In his early proposals Crane (1965) outlined a scheme consistent with his initial observations which much subsequent work has supported, at least in its broadest outlines. Specifically, he proposed that ( 1 )
3
the driving force for organic solute transfer is the concentration difference for Na+ which exists across the cell membrane. (2) The influx of solute coupled to Na+ becomes largely irreversible because the K,, value for influx is much smaller than for ef'flux owing to the asynlmetric distribution of Na+. ( 3 ) The K+-ATPase maintains a low intracellular Na+ level of Na+ and high K+. High intracellular K+ effectively competes with any intracellular Na' preventing any significant backflow on the Na+-dependent carrier. (4) The dependei~ceof the organic solute transport system on low iiltracellular Na+ links together Na+-dependent solute transfer with metabolic activity. By 1965 ample experimental observations were already available that a Na+ requirement for transport was also associated with a dependence on metabolic activity. In fact Csaky (Csaky and Thale 19668;Csaky 1963) had written extensively on the association of Na+ dependence in transport systems with "active" transport or transport dependent on metabolic activity. It was then known that a number of transport processes are not affected by the presence of metabolic inhibitors such as DNP or CN- whereas others are greatly inhibited. For example, glucose transport in muscle is not affected by the metabolic state whereas amino acid transport in the same organ is significantly reduced. The presence of Na+ enhances amino acid uptake but has little effect on glucose uptake in diaphragm (Kipnis and Parrish 1965). Crane's proposal explains the requirement for Na+, the association of the Na+ requirement with metabolic activity, and most significantly that there need be no covalent modification of either carrier (as for example, the formation of a phosphorylated intermediate) or solute during energization of the transport process. This proposal contrasts with that of Csaky (1963) and his collaborators who proposed that intracellular Na+ and organic solute increase thc utilization of ATP. For some years following Crane's proposals, the inability to show quantitative relationships between the magnitude of the Na9 gradient and solute accumulation (i.e., the Na+ gradient was inadequate to account for the accumulation of solute) led to postulation of energytransducing mechanisms which involved carrier modification by ATP (Kimmich 1970; Potashner and Johnstone 1971 ; Schafer and Heinz 1971; De Cespedes and Christeilscn 1974; Johnstone 1974). Recently, as will be discussed below. most of the problems have been resolved. Na+-dependent transport has become synonymous
+
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4
CAN. 9. PHYSTBL. PHARMACOL. VBL. 57, 1979
with "active transport" of the older literature. The consensus that metaba8isn1-dependent traiasport of organic solutes need not use ATP directly has led to definitions of transport based on the degree of closeness of the transport event to the metabolic process. Kedenl (1961) proposed that the term "primary active" transport could be used to describc transport processes which use ATB directly, such as Na+ transport. Secondary active transport would dcscribe transport processes dependent on ion and (or) electrical gradients set up by the metabolic process. Both types of transport can lead to an accumulation of the transported solute against its own electrochemical potential.
observation is that the coupling between Na+ and solute is 1 :1 (Curran et al. 196'7; Schafer and Jacquez 196'7; Eddy 1968; Goldner et al. 1969). However a number of observations have been made where the stoichiometry is greater than 1: 1 or Bess than 1: 1 and also inconstant. For example, in pigeon red cells (Vidaver 1 9 6 4 ~ )it has been concluded that the stoichionaetry is 2 Na+ per mole of glycine. Rabbit reticulocytes however show 1 : 1 stoichiometry (Wheeler et al. 1965) for amino acids other than glycine. In a recent review, it was shown that the stoichiometry in Ehrlich cells may vary from 0.3 to 2 (Christensen et al. 1973) depending on the aislino acid and the conditioils of incubation. The variable stoichiometry (as well as other data) has led Christensen et al. (19'73) to propose energization of transport by means other than the Na+ gradient.
Coupled Flow of Na+ in the Transport of Organic Solutes The demonstration that organic solute transport is stimulated by Na+ is not synoinymous with showing that Na+ is cotraiasported with the organic solute. Direct coupling of flows was first shown in intestinal preparations (Schultz and Zalusky 1964, 1965; Curran et al. 1967). Subsequent to the demonstration that sugars and amino acids increased Na+ transport across the intestinal wall (Schultz and Zalusky 1964. 19651, Curran et al. (196'7) showed that the presence of amino acids increases the unidirectional transfer of Na+ into the cells of the intestinal epithelium and that Na+ enhances the transfer of amraillo acids. At high Na+ levels, the increase in Na+ Wow caused by the amino acid is equivalent to the increase in amino acid Mow caused by Na+ (Curran et al. 1967). Similar observations on the coupling of flows have been made with sugars as the traiasported solute (Goldner et al. 1969). Although simnultaneous measurements of Wa+ and organic solute flux have been made Zess frequently than studies om Na+-stimulated solute transfer per se, the general consensus appears to be that Na+ dependence implies that Na+ flow accompanies the flow of organic solute. However, it should be mentioned that Christensen et al. (1973) have reported that in some instaa~ces,Wa+ uptake may be severely restricted without effect on amino acid uptake wlaich is nonetheless Na+ dependent. The significance of these observations is not clear. It does raise the question whether Na+ dependence unquestionably means coupling of Na+ flow to the flow of the organic solute.
Energization of Transport In his original model, Crane proposed that the movement of Na+ (down its electrochemical potential) provided the energy for the uphill movement of the organic solute to which the Na+ movement was coupled. Qualitatively, there is good agreei~ment between the accumulation of organic solutes and the magnitude of the Na+ gradient. For example, ( a ) Bihler and Crane (1962) showed that in the intestine the extent of sugar accumuTation is a linear function of the chemical gradient for Na+ and that a decrease iia the Na+ gradient obtained by varying external Na+ concentration results in decreased accumulation. (However, as will be discussed below, Na+ per se has an effect on transport in absence of transmembrane Na+ gradients. Hence, it is less clear whether the data reported reflect only the effects of a variable gradient or the effects of variable Na+ concentration, or both.) ( b ) It was shown that in intestinal cells depleted of ATP and Na+ nearly normal rates of solute influx obtain in a Na+ medium when the Na9 gradient is similar in magnitude to that of normal cells (Hajjar et al. 1970; Goldner et al. 1972). ( c ) Vidaver (19640) showed proportionality between the Na+ gradient and uptake of glyciile in pigeon red cells at constant extracellular Na9. (d) Eddy and collaborators (1967) also showed proportionality between the magnitude of the Na+ gradient and the extent of amino acid accumulation in A T f -depleted Ehrlich cells,
Stoiehiometry of Na+-eouplledTransport The stoichiometry of Na+-coupled flows have been examined in a number of systems. The general
Reasons for the Apparent Inadeqt~aeyin Energy Qualitatively these and many other observations are consistent with the predictions of the hypothesis.
REVIEW iSYNTHESE
5
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But attempts to show a quantitative relationship be- (unpublished). U7hetheror not sequestration of Na+ tween the magnitude of the Na+ gradient and solute by the nucleus contributes to the apparent energy accumulation have been less successful, particularly deficit, it is probable that underestimations of the in tissues where extensive accumulation of solutes potential difFerence or overestimations of the cellular Na+ activity are sufficient to account for the apparent takes place (see Discussions in Schafer ( f 972) ) . In early discussions it was coilsidered that the energy inadequacy in the Na+ gradient required for membrane potential was too small in the tissues solute accumulation. Future work will no doubt reunder study to makc a significant contribution to the solve the issue fully. overall electrochen~icalgradient for Na+ (Schultz 1968; Jacquez and Schafer 1969; Heinz 1973). Electrogenic Nature of Nag-coupled Transport Based on the energy available in tbe chemical comAs mentioned above, the earliest formulation of ponent of the Na+ gradient, accumulation of amino Na+-gradient hypothesis stressed the chemical the acids in excess of that feasible from the chemical rather than the electrical nature of the Na+-gradient gradient of Nag alone (i.e., where at steady state R T driven processes. Moreover, although it is implicit In ([Na+]o/[NaC]i) < RT In ([amino a~id]~/[amino that movement of a positive charge (Na+) in conacidlo) ) were reported (Jacquez and Schafer 1969; junction with a neutral amino acid could alter the Lin and Johnstone 197 1; Schafer and Heinz 197 1; membrane potential, compensatory movements of Johnstone 1972; Heinz 1973; Tucker and Kiminich other ions, particularly K+, could lead to an elec1973). Even when attempts were made to deduce trically silent process. Schultz and Zalusky ( 1964, the energy contribution from the electrical com1965) first showed that transport of sugars and ponent of the electrochemical potential for Na+ by amino acids coupled to Na+ increases the short-cirindirect estimates of the membrane potential, the cuit current across the intestinal wall. These elecuptake of amino acids appeared to take place against trical changes were ascribed to increased Na+ pumpan opposing driving force (Jacquez and Schafer 1969; Schafer and Heinz 197 1; Johnstone 1974). ing across the serosal barrier of the intestine as a Although the problems have not been entirely re- result of an increase in cellular sodium. Recently, solved, it appears that the membrane potential may it has been shown (Fig. I ) in a variety of systems have been appreciably underestimated, especially that the transfer of an organic solute into the cell under conditions used to elevate cell Na+ (Kimmich via a Na+-dependent route causes (i) a depolariza1970; Schafer and Heinz 1971; Johnstone 1974). tion of the cell (Rose and Schultz 1971 ; White and Thus, conditions such as cold storage which were Armstrong 1971; Heinz et al. 1975 ; Philo and Eddy 1975; Laris et a1. 1976, Fig. 1; Okata et al. 1977) used to elevate cell Na+ may have led to an increase and (ii) that changes in the membrane potential inin Na+ pump activity and hyperpolarization of the crease or decrease the Na+-coupled influx of amino cell membrane (Heinz et al. 1975). acids (Viclaver 19648; Gibb and Eddy 1972; Meinz Other factors may also contribute to the apparent et aI. 1977; Pershadsingh et al. 1978) depending energy inadequacy of the Na+ gradient, such as ason the direction of the potential change. These obsuming that the activity of cell Na+ is equivalent to servations have added support to the idea that the its concentration, whereas the Na+ activity is probNa+-coupled process is electrogenic and that the ably a fraction of the concentration (Lee and Arinmajor if not the only driving force for amino acid strong 1972). Furthermore, Pietrzyk and Heinm (and sugar) accumulation in mammalian cells is the (1974) showed sequestration of Na+ by nuclei in electrocht=rnical gradient for Na+. EhrIich cells and concluded that the unrecognized sequestration, and hence the unequal distribution of Evidence for the Electaogenic Nature Na+ in the cytoplasm, contributed to the energy of Na+-coupled Flows deficit since calculations for energy availability were based on high cytoplasmic Na+ values. In regard to Vidaver (1964b) was among the first to show the latter, it should be mentioned that in this labo- that by altering the membrane potential with conratory attempts to demonstrate preferential Na+ stant Na+ (inside and out), glycine uptake siguptake by isolated nuclei and to confirm the findings nificantly decreases as the potential difference deof Pietrzyk and Heinz were not successful. We found creases (becomes more positive inside). Gibb and no evidence for preferential sequestration of Na+ Eddy (1 972) showed that the percentage stirnulaover K+. With isolated nuclei, both ions were found tion of amino acid uptake with valinomycin at concentrations equivalent to those in the medium decreases as K f o increases (K+i was constant) in a
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CAN. J. PHYSIQL. PHARMACOL. VOL. 57, 8979
I
1
20
40
60
88
108
128
Minutes
FIG. 1. Constant level of fluore~centintensity of dye with time for O.3(,{, suspensions of cells in Naf-Ringer incubated at room temperature. At O min, cells were diluted to 0.3',, , ancl at the times indicated aboke, dye was added to 3.2-mL aliquots The constant lekels withdrawn from the cell suspension of fluorehcent intensity are presented abow. At time marked G , glycine was added (final concentration equals 3 a1a.W) to a portion of the incubation mixture and the sannplirag was contialued ( 0 ) . An increase in fluorescent intensity indicates a depolarization and a decrease in fluorescent intensity indicates a hyperpolariration. The data show that on incubation without glycine (0) the cells gradually depolari~e.In addition, upon injection of glqcine ( 0 ) there is extensive depolariiration and gradually the cell? repolari~cto the level seen in the control. (Reprinted fro111 Larls et al. 8976, with permission of the journal. 8 .);,1(
near-linear way. With valinomycin, a Kf selective ionophore, the Kf distribution is probably near equilibrium after a few minutes incubation and thus, the membrane potential can be assessed from the Kf distribution using the Nerst equation where A$ (RTj n F ) In ([K+],j [Kf l o ) . Heinz and collaborators ( 1977) using valinomycin and variable [K+],, showed that amino acid accumulation increases as the membrane potential increases (inside negative) in a near-linear manner (Fig. 2 ) . These data were interpreted as showing that an increase in the potermtial difference (inside negative) stimulates transport. Latterly, Bershadsingh et al. ( 1978) showed that propranolol, whicla at high concen trations ( > 1O-,' M ) increases K+ permeability (Ekman et al. 1969; Pershadsingh et al. 1978), increases and decreases amino acid influx at low and high K f o respectively, when K+, is nearly constant. The stimulation and inhibition of uptake by propranolsl follow the increase and decrease respectively of the membrane potential in a near-linear manner (Bershadsingh et al. 1978) as measured with a fluorescent dye (Hoffman and Laris 1974 ) . Work with isolated membrane vesicles which show Naf -dependent solute transport has been particularly useful in showing that influx of organic solutes re-
-
_ A
50
-
.
1K!
-
*-
150 ~
0
~
~
FIG.2. Effect of electrical potential on the uptake of AIB. The ordinate shows the AIR distribution ratio (K:,)after 4 (: j) and 6 ( 0 ) min incubation of Kt-depleted Ehrlich cells in the presence of varioub oaaabain concentrations in the extr:acellular n~ecliurn.The abscibsa shows the cortespoiading ratios of the distribution of tetraphenylphosphonium (TPf3+) ions, also after 4 and 6 min incubation. The K -depleted cells were preincubated w ~ t hTP13+ ( 1 0 piZf) and K t-free Krebs-Ringer pho\phate buffer, pH 7.4, for 10 rnin at 37 C before buffers , ouabain (0- 0.75 containing AIR (0.B mM), K+ (1 8 n ~ h f )and n&" were added. It is seen that beyond a certain value of TPPf distribution the transient accumulation ratio of AIR linearly rises with the ratio of TPP+. The Na+ concentrations inside and outside the cell are approximately the same in all experiments. To the extent that the T P P t distribution ratio indicates the electrical potential, the experiment shows that the transient accumulation ratio of AIR is strongly influenced by the electrical potential or by the dilTercnce in electrochemical activity of Nat. (Reprinted from Heinz et al. 1977, with permission of the journal.)
sponds to a membrane potential (Fig. 3 ) . In most of the experiments with vesicles the changes in nlembrane potential are induced with valinomycin ( ~ other ionoph~res)and variable K* levels or by using lipid-soluble anions like CNS- to replace Cl(Colombini and Johnstone 1974; Murer and Hopfer 1974; Sigrist-Nels~net al. 1975; Evers et al. 1976; Quinlan et al. 1976; Han~mermanand Sacktor 1977; Lever 1977a; Bardin and Jshrastone 4 978). Since these vesicles are generally devoid of metabolic activity, addition of valinomycin and other is~aophoresis not likely to affect metabolic parameters which might aEect the responses in intact cells. The observation that amino acid (or sugar) influx is afiected by the magnitude of the membrane potential is consistent with the idea that the Naf-co~mpledWows are electrsgenic. In conjunction with evidence that cells becsme depslarized during influx of solutes by Naf dependent routes (Rose and Schultz 1971; White and Armstrong 1971 ; Heinz et al. 1975; Bhilo and
7
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REVIEW /SYNTHESE
0
I
i 5
1:
kk-
T~riie, r ~ l ~ r c
FIG. 3. lonophores and transport of [I-14C]AIB in membrane vesicles from Ehrlich cells. Nat- outside and Kf inside control, (V)2 pg/ml, gramicidin the vesicles throughout. (a) D, and (e)5 pg,/m[i valinornycin. The level of AIR uptake when Na" is present on both sides is shown by the broken line. (Reprinted from Colombini and Johnstone 1973, with permission of the journal.)
Eddy 1975; Laris et al. 1976; Bkata et al. 1977). the observations attest to the electrical nature of Nag-dependent transport processes. Since organic solutes are frequently transported by Na+-dependent routes, the question also arises about possible changes in membrane potential when these solutes leave the cell. If they exit by the same or a similar Na+-dependent route. then loss of salutes would result in hyperpolarization. In a recent study sf cllangcs in the membrane potential of Ehrlich cells using cyanine dyes (Laris et al. 1976) we observed that the membrane potential of freshly isolated cells was greater (more negative inside, lower fluorescence) than after 30 to 6 0 min incubation (see Fig. 1 ). To perform the fluorescence n~casurements.it is necessary to dilute the cells to a cytocrit of 0,396. Freshly isolated cells when diluted fro111 the normal suspension of 3-596 (used for transport studies) to 0.395, for the fluorescence measurements were hyperpolarized at first and in 30 min gradually depolarized to a level that remained constant for the next hour or SO. If the cells were prcincubated for 3 0 min prior to measuring fluorescence, only the stable, relatively depolarized fluorescence was observed. On examination of the possible reasons for the shift in potential, it became apparent that the cells lose considerable quantities of endogenous amino acids upon dilution to 0.3% cytocrit (Laris et nl. 2978). Many of the amino acids lost are those known to be transported by Na+-dependent routes, such as alanine, glycine, serine, and threonine. Thus, the question arose whether increased amino acid efflux upon dilution
0
6
12
18
24
30
36
Minutes
l-I(;. 4. Fluorescent intensity and cellular AIR level. Cells were diluted to 1 :320 in Na '-Ringer in the presence ( c, ) and absence of 3 mM a-an~inoisokutyric acid (AIB) and incubated at 37 C. At intervals aliquots were taken; dye was added; and the steady level of fluorescent intensity recorded. After 36 min the cells were spun clown and resuspended at a dilution of 1 :20 :_ 3 rnM All3 (same as initial suspension). Aliquot\ (0.2 anl,) of the suspension were taken and diluted to 1 :320 with 3.0 mL of various concentrations of AIB (see graph) in Na+-Ringer; dye was added; and the steady level of fluorescent intensity recorded. The position of the symbols underneath the medium AIB concentration indicates the fluorescence attained with the given conce~atration of AIB. (Reprinted from Laris et al. 1978, with permission of the journal.)
(e)
contributes to the initial hyperpolarized condition of the cells. Experiments show that cclls become hyperpolarized when introduced into a medium into which there is net loss of amino acid (Fig. 4 ) . Conversely, depolarization occurs when there is net uptake of amino acids (Fig. 4 ) . No change in potential is seen if the amino acid level remains constant. Moreover, hyperpolarization of cells associated with net loss of amino acids takes place only in cells containing intracellular Nar (Table 1 ) . If cells depleted of Nag but containing high amino acid levels are introduced into an amino acid free medium, there is no associated hyperpolarization with amino acid loss. All these data are consistent with the conclusion that amino acid movements alter the membrane potential. Hyperpolarizatioil of cells by Na+-dependent amino acid efflux raises the possibility that influx
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CAN. J. PHYSLOL. PHARMACOL. V6E. 54, 1979
of amino acids from the medium may be stimulated by emux of an endogenotas amino acid. Earlier in this article reference h7as made to the phenoinenon of exchange diffusion of amino acids. It is now oiler 20 years since Heiilz and Walsh ( 1958) denlonstrated a stimulatioil of influx of [14C]glycine by preincubating Ehrlich cells with nonradioactive glycine. This trans stimulation was ascribed to be due to an exchangc of cellular with extracella~lar amino acid, the exchange rate being greater than the net, unidirectional flux. Since then it has been established that a Na+-independent exchange process does occur (see above) in many types of cells. The question which arises here is whether under conditions of net loss of cellular amino acid in a sodium medium (as occurred in the Heinz and Walsla report), the increase in [lT]glycine uptake was due to exchange diffusion or due to a hyperpolal-ization of the cell by loss of alniilo acid. If the latter is true. the increase in membrane potential would the11 be respoilsible for the enhanced glycine uptake. In addition, the question is also raised whether the Na+-dependent "exchange diffusion" reported in some systems (Winter and Christensen 1965) is actually due to hyperpolarization of the cells by loss of endogenous amino acids. The present observations necessitate a reexamination of data showirmg stimulatioil of amino acid influx in cells preloaded with amino acids to distinguish between a truc exchange effect and the effect of a membrane potential on trans stimulation of amino acid influx. Another interesting problem which needs further iilvestigation is whether a decrease in nleinbra~acpotential will enhar~ccNa+-coupled amino acid efflux along the concentratioan gradient at constant cell Na+. This question may be relevant in systems which release solutes, such as putative transmitters, which are normally transported into, and presumably, out of the cell, by Na+-dependent routes. There have been few studies directly addressed to this qucstion since in most cases cell Na+ increases along with depolarization (for example see Ham~nerstad and Cutler 1972). Generally, conditions which cause depolarization lead to an elevatiora of cell Na+ (for example, inhibitors of the Na+ pump or general metabolic inhibitors). Therefore, unless precautioils are taken to keep cell Wa+ constant or to compensate for the efiects of elevated cell Na+, the action of the potential per se is not clear. In a recent study on the effects c ~ fcell Wa+ on efflux, it was shown that in Ehrlich cells efflux of glycine is greater in cells extensively depolarized with gramicidin (Johnstone 1975) than in cells with comparable Na+ levels
TABLL:I. Fluorescent intensity as a function of cellular n-aminoiwbutyrate (AIB), Na+, and K concentrations* +-
--
-
Cellular concn., n.nmol;/Lcell water
M+
Na4-
BIB
Fluore%centintensity, arbitrary units
Series 1 0 33 0 27
Series 2 O 0 0 O NOTE: Cells ( 8 :320 dilution) were precquilibrated for 30 rnin in WatRinger t 3 m M A I B at 37°C. Alicluots of the calls were resuspended (I :GO dilution) in either coid (6°C) K+-free Wn*-Kinger, I
