AMERICAN

JOURNAL

OF

PHYSIOLOGY

Vol. 231, No. 6, December

1976.

Prdnted

in USA.

Characterization liver in vivo

of leucine

ROGER PERSELL AND AUDREY Department of Biological Sciences, New York City 10021; and Marine

AND AUDREY E.V.

of leucine transport by tuadfish liver in vivo.

liver transport mechanisms; synthesis; kinetic analysis

A METHOD

leucine

uptake;

HAS BEEN INTRODUCED

mannitol;

reCent1y

by toadfish

E. V. HASCHEMEYER Hunter College, City University of New York, Biological Laboratory, Woods Hole, Massachusetts

HASCHEMEYER. CharAm. J. Physiol. 231(6): 1817-1823. 1976. -Kinetic analysis of Lleucine uptake by toadfish liver at 20°C in vivo has been carried out after pulse injection of L-[14C]leucine into the hepatic portal vein. n-[3H]mannitol, which is taken up slowly by toadfish liver, is used as a marker for extracellular space and space accessible by simple diffusion. At normal plasma leucine concentration (0.1 mM), leucine uptake occurs rapidly (tl,* = 0.25 min), representing a flux of 0.6 pmol/min for the liver as a whole. Analysis of the distribution of radioactive leucine among intracellular and extracellular free pools and proteinbound form at times of 30 s to 5 min after injection is consistent with operation of a concentrative or uphill transport system accounting for 40% of uptake at normal plasma concentration. Saturation of uptake occurs at increasing leucine loads; calculation of intracellular pool dilution from protein synthesis data indicates that 20-30% of liver intracellular space is occupied by incoming leucine during the first 2 min after portal injection, Maximal flux IV,,,) is 4.1 pmol/min per 7-g liver as a whole with K,, = 0.6 mM. Competitive inhibition of leucine uptake is afforded by isoleucine and phenylalanine with lesser effects by aspartic acid, cysteine, methionine, threonine, tyrosine, and valine. No effect is observed with alanine, glycine, histidine, lysine, and proline. PERSELL,ROGER,

acterization

transport

protein

fbr eXpWi-

mental analysis of amino acid uptake by liver in vivo (1 I), In this procedure, distribution of the substrate of choice and a suitable marker are determined in various compartments of liver and in blood draining from liver at short times after hepatic portal vein injection in the anesthetized animal. A related approach to the study of hepatic transport systems has recently been described in rat in vivo (21) based on methods developed in brain (1.9). A marine teleost, the toadfish (Opsanus tau), has been used in the present study in order to take advantage of reduced body temperature for slowing of reaction rates. Considerable kinetic data concerning the protein synthetic pathway of liver in the toadfish in vivo are available (7-9). This paper presents a simple multicompartmental kinetic analysis for assessment of the uptake, saturation, and competition properties characteristic of L-leucine transport by toadfish liver in vivo. Preliminary results have been reported (10).

MATERIALS

02543

AND METHODS

Animals. Adult toadfish Opsanus tau, body weight 220 t 20 g, liver weight 7.0 t 1.0 g, about 80% male, were collected at the Marine Biological Laboratory in June (1973, 1974, 1975) and utilized during the following 2 mo. Fish were maintained in running seawater aquariaat *ambient temperature (22°C) and fed live killifish or mussel meat to appetite until 2 days before experiment. Experimental procedure. Anesthesia and hepatic portal vein injection in the toadfish were carried out as previously described (11). The injection solution contained 2 &i of L-[U-14C]leucine, 4 &i of ~-[13H(N)]mannitol, or 4 &i L- [3-3H(N)]phenylalanine (New England Nuclear Corp.), unlabeled L-amino acids (Sigma Chemical Co.) as indicated, and salts in the proportions given by Hoare (13) to yield a fmal estimated osmolarity of 0.29. The standard kinetic experiments were carried out at concentration of 0.1 mM Lleucine and 0.3 mM n-mannitol. The fish were kept well oxygenated by a vigorous flow of O,-saturated seawater over the gills. At various times after the 5- to 10-s injection, livers were rapidly excised, blood collected, and assay of radioactivity in various fractions carried out as previously described (11). In experiments in which high doses of [12C]leucine were administered, incubation time was well below that at which metabolic disturbances would be expected to occur (2). The fraction of administered dose recovered in the total free pool of liver (A&,) and in protein-bound form (Am protein ) was determined from assay of acid-soluble anh acid-insoluble fractions, respectively. The distribution ratio of amino acid and marker for extracellular space was determined from plasma free radioactivity [(A/W plasma]as previously described (11). The fraction of dose associated with the free intracellular pool of liver was calculated from A -free 1

=

A free tota

-

CBliver)

(A/B)plasma

(1)

where Bliver is the fraction of dose of radioactive mannito1 recovered in the liver. The last term on the right represents the fraction of amino acid radioactivity estimated to occupy extracellular space (A,). Control experiments indicated that blood cells trapped in liver (toadfish he matocrit = 16%) do not make a significant contribution to the radioactivity recovered in the various fractions.

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1818

R. PERSELL

Liver and plasma free amino acid pools were determined with a Beckman automatic amino acid analyzer after extraction with ethanol and chloroform or with 10% trichloroacetic acid. Norleucine and standard amino acid mixtures were added to liver homogenates and plasma for calculation of recoveries. Plasma to be used for amino acid analysis was obtained from blood drawn from the heart after dissection to expose the bulbus arteriosus. Polypeptide chain assembly time was determined as previously described (7, 16). Kinetic anaZysis. Movement of amino acid (A) and diffusible marker (B) was evaluated in terms of reversible monomolecular reactions. For the marker B body

5, K3

e

K3

K, e * K4

A free i

l

A. E. V. HASCHEMEYER

r

08 l

06 l

Be 04.

(2)

I

where B, represents the fraction of B recovered in liver and attributed to extracellular and diffusion-accessible space. Bbodv represents that fraction of the injected dose of B that is lost to the rest of the body. For the amino acid KS A body --A

10

AND

ff:

A*protein 1

I

I

I

I

I

I

1

2

3

4

5

6

Time (mid

(3)

where the terms from left to right represent the fraction of A in the body excluding the liver, in te liver extracellular space, in the liver intracellular free pool, and in liver protein-bound form. Kinetic analysis was performed for two time periods: in the first time period, t = 0 to t = 0.25 min, representing the average time for passage of the injected bolus through the liver, & and K5 were set to zero; in the second period, t > 0.25 min, values of & and Kg were fitted to equation 2 on the basis of the time course of mannitol disappearance from the liver. Initial values of & and B, were unity; all other compartments are given initial values of zero. No term for protein turnover has been included because of the short time of these experiments, i.e., less than that required for complete polypeptide chain synthesis. Metabolic degradation of leucine, which is minimal in liver (5), is also neglected. Solution of the set of simultaneous first-order differential equations describing the reaction scheme of equation 3 for the two time periods indicated above was carried out with a program in the FOCAL language for graphical output using a PDP8/M computer with Tektronix 4010-l cathode-ray graphics terminal (22) or with a program in FORTRAN-IV for use with an IBM 370 computer. Numerical integration was performed by the simple Euler method, after the approach of Moore and Ramon (18). RESULTS

Time course of mannitol recovery in liver. Averaged data for the recovery of n-mannitol in toadfish liver at ZO-22OC as a function of time after hepatic portal vein injection of a 0.1 ml bolus are presented in Fig. 1. A satisfactory fit to equation 2 was found for the following values of the constants: & = K, = 0.0 for 0 5 t 5

0.25 min (flow-through time); & = 0.9 min-’ and K3 = 0.1 min-l for subsequent values of t up to 6 min where recovery roughly equilibrates at a level of 10% of dose. Previous studies (11) suggested that n-mannitol would constitute a suitable marker for extracellular space, which is rapidly occupied by small molecules, and for intracellular space accessible by diffusion, Although Dmannitol occupies liver intracellular space completely within 1-3 h after injection, its half-time for uptake (t 112= 20 min) is slow compared with the time course of the present experiments. Initial n-mannitol space (by extrapolation to zero time) is 0.31 ml/g, comparable to inulin space (11). The behavior of mannitol in toadfish liver contrasts strikingly with findings in rat liver. Pardridge and Jefferson (21) report that mannitol uptake is 80% that of water during an 18-s circulation period. Experiments in rat liver comparable to those reported here for toadfish similarly show a very rapid uptake of Dmannitol into intracellular space (unpublished observations). Time course of leucine uptake. The levels of free intracellular radioactive leucine ( AifPee), determined by means of equation 1, reach a maximum at about 1 min after injection and subsequently decline (Fig. 2>, The proportion of Aitotal recovered in protein (Fig. 3) continuously increases during the same period. Although protein synthetic rate as indicated by average polypeptide chain assembly time [about 6 min in 20”-acclimated fish measured at 20°C (7)] is relatively constant among different individuals, the fractional rate of leutine incorporation also depends on leucine pool specific activity. The data of Fig. 3 yield an initial estimate for K2 in equation 3 of 0.20 min-l in this experimental series. Further analysis requires estimation of K1 and K4 in equation 3. Evaluation of KJK, for simde leucine exchange. If

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LEUCINE

TRANSPORT

IN

1819

LIVER

no compartmentalization of leucine within these spaces. On the basis of previous liver space determinations (ll), and neglecting the small contribution of trapped blood cells, one obtains

1.0

0.8 E

K,lKd = (0.47 ml/g)/(0.31 ml/g) = 1.5

I

I

2

1

I

Estimation of KJK, from liver and plasma Leucine pools. Although leucine pools vary widely among individuals, particularly in relation to nutrition and time of year, liver concentrations, in general, slightly exceeded those of plasma in untreated fish or those injected at low leucine doses (Table I), If leucine is freely exchangeable and if plasma leucine concentration is taken to apply to all liver extracellular space, intracellular free leucine may be calculated

I

3 4 Time (mid

I

I

5

6

2. Recovery of L-[‘YJleucine, as fraction of administered dose, in intracellular free pool of liver, determined by means of equation. 1. Data are presen’ted as standard error bars; total number of animals is 48, Theoretical curves for Aifree, based on equation 3, are shown for following sets of parameters: (- -) R = 1.0, K2 = 0.25; 9 R = 0.75, Kz = 0.23; I---- ) R = 0.5, Ka = 0.21; (0 - 0) R = 0.25,KQ = 0.21; (- - -) R = O,O, KP = 0.18. Other rate constants are: K, = 2.7, KS = 0.9, and K:, = 0.1, all in units of min? K4 is calculated according to equation 5. FIG.

(.

l

l

(4)

.

0.12 Fmol/g - [(0.31 ml/g)(O.lO pmol/ml)] [Leuli = 0.47 ml intracellular H20/g = 0.19 pmollml intracellular

(5)

H,O

The resultant concentration ratio for intracellular and extracellular space together with the volume ratio yields the following estimate of K1/K4

WK, = (Vi/V,> ([Ledi/ Led,> = (1.5)

0.19 pmol/ml 0.10 pmol/ml

= 28 (6)

l

Net loss of leucine through plasma protein synthesis [20% of Kz (S)] is small compared to K, and K4 and has been neglected in this calculation. Experimental determination of K, and K,. Determination of the best fit of the rate constants of equation 3 to the experimental data was carried out by selection of values for Kr within the range suggested by previous studies of amino acid uptake by toadfish liver (11) and by calculation of Kq for various values of a parameter, R, defined by

08l

E I’0 Ic 0.6.-\c aI “0 & 0.4z02.

K 4 = K1 (1.0 - R)/1.5

I

2

3 4 Time (mid

5

1 6

FIG. 3. InCOrpOratiOn Of L-[%]leucine into liver protein, presented as ratio of fraction of dose recovered in protein to total intracellular radioactivity (Aitotal = Aifree + AiProtein)m Theoretical curves are shown for following sets of rate constants: ( - -) R = 1.0, Kg = 0.25; (->R = 0.5, K2 = 0.21; (---) R = 0.0, K2 = 0.18.

the observed uptake process is assumed to represent an exchange of radioactive leucine between intracellular and extracellular compartments of the same concentration (i.e., that no concentrative uptake occurs), the theoretical value of KJK, can be calculated from the ratio of volumes associated with the two compartme&s. Since Aifree and A, are referred to the whole liver, concentration equilibrium is achieved when A.free/V. = A,/& where Vi and Ve are the volumes of i&aceilular and extracellular space, respectively. Thus, K1/K4 = VJV, for exchange diffusion, assuming

(7)

where R = 0.0 corresponds to the case of pure exchange diffusion and R = 1.0 represents concentrative uptake only. The theoretical curves of Fig. 2 illustrate the effect of variation ofR on the predicted time course of Aifree based on equation 3 The value of K, chosen for these calculations is 2.7 min+ based on previous determinations in 20°C toadfish (11). No significant improvement in fit l

TABLE

1. Uptake of L-[14C]Leucine by liver in viva yG

AproteIn

L-Leucine Injected, pm01

0.01 1.5

10.0

Avg A,luta’

Avg ~ Wta1

0.43 2 0.05 (30) 0.22 4 0.05 (10) 0.10 2 0.04 (4)

18 k 9 (30) 6.0 2 3.0 (10) 2.0 f 0.8 (5)

L-Leucine Liver, pmollg

Concn Plasma, fimollml

0.12 A 0.04 (8)

0.10 +- 0.02 (4)

ND 0.70 2 0.10 (61

1.65 2 0.15 (4)

ND

~ Values,

shown for three concentrations of leucine injected into hepatic portal vein, are calculated as explained in the text for t = 2 min and include corresponding SD and number of samples. Analyses of plasma L-leucine concentrations are made from blood drawn from the heart at t = 2 min after injection without liver excision. T = 20-22°C. ND, not determined.

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1820

R. PERSELL

was obtained with larger or smaller values of K, in the range of LO-5 0 min. K3 and KS have the values obtained from Fig. 1. Kz has been adjusted in each calculation in order to maintain a fit of the incorporation data, &Protein/&total ( F ig. 3). As shown, increasing values of R necessitate small increases in K2 to maintain agreement with the data. Figure 4 illustrates the theoretical curves for A, at various values of R, superimposed on the experimental A, calculated from BIiver and (A/B),,,,,, (see equation I). Another test for the KJ& ratio is provided by the experimental data for (A/B)pl,,,a as a function of Biiver, obtained at times less than one full circulation period (2 min). As shown in Fig. 5, the theoretical curves of &I B e3 calculated according to equation 2 and equation 3, are strongly influenced by the relati ve proportion of concentrative uptake (R = 1.0) versus exchange uptake (R = 0.0). The experimental data fall between the two extremes. These data show that during the time mannito1 is washed out of the liver, the movement of L-leucine is such that a constant plasma ratio is maintained in blood leaving the liver. A value of R = 0.4 or K1/Kd = 2.5, similar to the result suggested by the leucine pool data (equation 6), gives satisfactory agreement and falls within the range of R indicated by Figs. 2 and 4. The value of 2.7 min-l for K, represents a leucine influx rate of 0.6 pmol/min for the liver as a whole at normal plasma leucine concentration (0.1 mM) or 0.18 pmol/min per ml intracellular H,O at ZOOC. At the same extracellular concentration, leucine uptake by Ehrlich cells in culture at 37°C is 0.33 pmol/min per ml [from Km = 0.5 mM and V,a, = 2 pmol/min per ml (ZO)]; at ZO°Ca value of 0.07 pmol/min per ml would be expected, based on a temperature coefficient for uptake (Q& of 2.5 (20). Saturation of leucine uptake. Increasing the concentration of leucine in the injection pulse produces a continuous decline in the recovery of radioactive leucine in the liver (Fig. 6). A concomitant increase in the ratio of

AND

A.

E. V.

HASCHEMEYER

r

1.0

0/-

08t l

0 /

m” \ 2

0.6

0.5

0.4

0,3

0.2

0.1

Be FIG. 5. Plasma ratio of L-[ 14c]leucine: ized) as a function of mannitol recovery times up to 2 min after injection. Six animals each point, and standard errors are shown. 8, vs. B, based on rate constants given in

n-[3H]mannitol (normalin liver (B,) obtained at have been averaged for Theoretical curves of AJ Fig. 2 are shown.

03 . 1 Ai . 02 I 1

. 01

I

/

0

1

2

3

Leucine Dose (pmolelO.1 ml) 6. Dose dependency of total intracellular rJ14C]leucine recovery in toadfish liver at t = 2 min, temperature = ZOOC. Data presented as fraction of injected radioactivity with bars indicating standard error. n = 4-M at each dose. FIG,

Time (mid FIG. 4. Recovery of L-[14C]leucine in liver extracellular space, as fraction of administered dose, calculated according to equation A, = Theoretical curves for A, are shown based on paramef3, (Am,,,,ln,. ters given in Fig. 2.

leucine to mannitol [(A/B),,,,,,] in blood leaving the hepatic circulation is observed (e.g., from 0.40 at the lowest dose to 0.65 at 1.5 pmol). A double reciprocal plot of uptake at t = 2 min as a function of leucine dose, treated as a one-way process only, yields a straight line corresponding to a Michaelis-Menton relationship. At infinite concentration, maximal uptake is 0.6 pmol per 7-g liver; half-maximal uptake is achieved at a dose of 1.5 pmol. Taking into account dilution with portal blood, the average concentration of leucine during passage of the bolus at the half-maximal dose is estimated to be about 0.8 mM. Dilution with arterial blood, about 30% of liver blood flow in other species (6), would further reduce the value to 0.6 mM. Chemical analysis of liver leucine levels at the highest injection dose confirmed the results of radioactivity measurement. Data for livers analyzed at t = 2 min after injection are summarized in Table 1. After a lo-

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LEUCINE

TRANSPORT

IN

1821

LIVER

pmol dose, intracellular free leucine may be estimated at about 0.40 pmol/ml intracellular H,O after correction for extracellular leucine based on plasma levels in these fish by means ofequation 5. Net uptake is thus 0.7 pmol per 7-g liver, or 7% of dose. Uptake of radioactivity from a lO+mol dose is 10%. Although full kinetic analysis of the saturation behavior is difficult, an estimate of maximal flux (V,,,) can be made from the extrapolated uptake value (0.6 pmol/liver) in conjunction with the data at 0.1 mM and at the high (10 pmol) dose If saturation did not occur, uptake at the latter dose, representing an average extracellular concentration of 4.9 mM (after dilution with 2.0 ml extracellular fluid and plasma at 0.1 mM), would be about 50 times that at the lower dose, or 30 pmol/min per 7-g liver based on the 0.1 mM flux rate calculated from K1. Uptake, however, is reduced due to saturation by the factor 0.0610.43 compared to the 0.1 mM level; hence V,,, (taken here as identical to uptake at the lopmol dose) is found to be 4.1 pmol/min per 7-g liver or 1.2 pmol/min per ml intracellular H,O. Calculation of Km from the 0.1 mM uptake data by means of the equation l

v (leucine

flux) =

xnaxILmmna Km2 + ~Leulplasma

(8)

yields a value of 0.58 mM, similar to that estimated above for the effective extracellular concentration at the half-maximal dose of 1.5 pmol. Incorporation into protein at high leucine concentrutions. Examination of the proportion of intracellular radioactivity incorporated into protein at various leutine doses provides a picture of the fate of the injected amino acid relative to preexisting intracellular pools. With hepatic portal vein injection, uptake would not be expected to be uniform throughout the entire tissue, particularly at low doses. Dissection of the lobes before analysis has indicated variations in uptake per unit weight of tissue of about fivefold at the O.Ol-pmol dose and about twofold at the 1.5~pmol dose (unpublished data). The uptake pattern generally follows the circulatory anatomy of the toadfish liver; the highest levels occur in the distal portions of the two larger lobes. High levels of incorporation were obtained at tracer doses (Table l), indicating good tissue viability. Previous studies of liver protein synthesis in toadfish using hepatic portal vein injection have shown that although polypeptide chain assembly time is relatively invariant, levels of amino acid incorporation into protein are strongly affected by oxygen supply to the animal (7). The latter observation has also been made in the hypoxic rat (25). This factor is important in transport studies, since an energy-requiring component of the transport process is likely to be similarly affected bY hypoxia. At increased leucine doses, L- [ 14C]leucine incorporation into protein relative to radioactivity taken up is reduced (Table 1). No effect, however, was observed on the incorporation of [“Hlphenylalanine administered simultaneously, nor on polypeptide chain assembly time measured with [ i4C]leucine at doses up to 1.5

pmol. Thus, protein synthesis was not affected within this time period by the leucine load. The change in incorporation rate, therefore, may be attributed to reduced specific radioactivity resulting from leucine uptake and consequent expansion of the intracellular pool. The data for AiprOt”in/AitOtalshow a threefold and ninefold effect at the 1.5- and lo-pmol doses, respectively. To achieve this increase over the normal intracellular concentration (equation 5) would require an uptake of 1.3 and 5.0 pmol of leucine, respectively, for the entire liver (intracellular space = 3.3 ml). The data for Aitotal, however, indicate that net uptake cannot be more than 0.33 and 1.0 pmol at the two doses. If the value for uptake is divided by the change in concentration calculated from the dilution of the pool for protein synthesis, one obtains the volume occupied. The result is 0.87 ml or 26% of intracellular space at the 1.5~pmol dose and 20% at the IO-C_cmoldose. Competition experiments. Table 2 presents the effect of various L-amino acids at 15 mM concentration (1.5 pmoI/O.l ml) on the fractional uptake of [14CJleucine into intracellular space and on the observed leucine:mannitol ratio of plasma. Both isoleucine and phenylalanine produced reductions of leucine uptake comparable to that found with leucine itself at that concentration. No effect was found with alanine, glytine, histidine, lysine, and proline. Variable effects ranging from 15 to 40% inhibition were found with the other six amino acids tested. Levels of incorporation of [ 14C]leucine into protein (Aiprotein/Aitotal) were not signiflcantly affected by the presence of the other amino acids. The experiment was reversed in the case of phenylalanine in order to evaluate the effect of leucine load on [“Hlphenylalanine uptake and incorporation into protein. At a leucine dose of 1.5 pmol, phenylalanine uptake was reduced to one-half the level observedin the absence of leucine, an effect comparable to the phenylalanine inhibition of leucine uptake. In spite of reduced uptake, the proportion of phenylalanine radioactivity incorporated into protein remained high and was not affected by leucine doses up to 10 pmol. TABLE 2. Uptake of L-[T]leucine L-amino acids at 15 mM Amino Acid

Ala

ASP CYS GlY His Ile Leu LYS Met Phe Pro Thr TYr Val

Avg Body Weight, g

203 190 213 265 191 220 209 233 214 202 288 211 232 206

All results are mean the last column. Values

in presence of other

Avg Liver Weight, g

A% (A/B),l,SllM

7.0 6.8 5.3 6.8 7.8 5.8 6.8 6.6 5.5 7.2 5*3 5.7 5.6 8.2

0.40 0.48 0.46 0.36 0.43 0.56 0.65 0.36 0.46 0.61 0.42 0.48 0.37 0.54

values for the number of Aifotal include SD.

Avg Aitola’

0.46 0.28 0.31 0*51 0.45 0,20 0.21 0.43 0.35 0.21 0.52 0.35 0.26 0.29

2 f * 2 f k 5 k f 2 k * 2 2

of animals

0.10 0.08 0.07 0.07 0.03 0.06 0.07 0.03 0.06 0.10 0.04 0.06 0.06 0.04

(6) (5) (2) (3) (2) (4) (10) (2) (3) (10) (2) (2) (3) (3)

given

in

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1822

R. PERSELL

AND

A.

E. V. HASCHEMEYER

maximum rate of leucine incorporation into protein (0.06 pmol/min per 7-g liver) estimated from polypepLeucine transport. The present results provide evitide chain assembly time and ribosome concentration dence of a leucine transport system in liver in vivo (7) and 10 times the rate of loss of leucine from the free with many characteristics of the system of L-mediation pool through plasma protein synthesis (8) and growth. first described in EhrIich cells (20). The observed satu- At high leucine doses, maximal uptake is 1.2 pmol/ ration of uptake is consistent with a carrier-mediated min per ml intracellular H,O at 20°C about half the process for leucine transport, as demonstrated for eu- value of V,,, found in Ehrlich cells at 37OC(20). Comkaryotic cells in culture (12, 13, ZO), The value for Km parable results for mammalian liver in vivo are needed determined by two methods is close to that found for to distinguish differences in transport capacity associEhrlich cells, suggesting a similarity in carrier mole- ated with cell type and evolutionary factors. cules between the fish and mammalian systems. ComLeucine distribution and protein synthesis. The prespetition or cross-inhibition is observed within the ent study provides insight on the early events in the group comprising leucine, isoleucine, and phenylalaprocessing of an amino acid entering the liver by the nine. Cross-inhibition between the acidic amino acids portal circulation. The uptake of amino acids by liver has been found by a similar method in rat liver in vivo after an amino acid load, simulating a protein meal, has been described in mammals (17), and liver is coneu 1; addition to determination of uptake levels and sidered to act as a buffer for plasma amino acid levels examination of saturation and competition phenom(3). In the toadfish th e rapid uptake of radioactive ena, the present analysis of the time dependency of amino acids introduced into the portal circulation estransfer among compartments permits an estimation tablishes a high specific radioactivity in liver for the of influx and efflux rates operating in vivo. This is of study of protein synthesis (7-g), At tracer levels, retenparticular interest with regard to the mechanism of tion in the first few minutes after injection is up to 75% leucine transport. The leucine system of eukaryotes is of dose for a mixture of 15 W-labeled amino acids (11) generally considered to function by carrier-mediated and 43% for [Wlleucine alone (Table 1). These high exchange (facilitated diffusion) only (12), although levels of uptake together with subsequent uptake by early results suggested some concentrative uptake other body tissues (24) minimize uncertainty associ(20). Recent studies in perfused brain, for example, do ated with isotope recirculation in the whole animal. not show an energy requirement for leucine transport The use of a rapid pulsed system, however, intro(l), although energy dependency is well established in duces questions about the distribution of radioisotope yeast (23) and bacteria (26). The present results for in tissue at very short times. The protein synthetic toadfish liver indicate that as much as 40% of leucine data obtained at high leucine doses indicate that the isotope equilibrates with only a fraction of the total uptake may be attributable to active transport. This conclusion follows primarily from the analysis of leu- liver leucine pool during the first 2 min after injection. cine:mannitol ratio in blood leaving the liver, a quanAlthough the results do not support the notion that tity highly sensitive to relative rates of influx and extracellular amino acids are preferentially used in efflux of leucine within the liver (Fig. 5). These data protein synthesis [see (4) for review], a combination of suggest a ratio of influx to efflux rate constants (KJK,) direct utilization and equilibration with intracellular of about 2.5, compared with a theoretical value of 1.5 pools cannot be excluded. In addition, some indication for pure exchange diffusion (equation 4). This result of nonuniformity over different regions of the liver has falls within the range indicated by the absolute values been obtained, and it is quite possible that at tracer of Aifree and A, (Figs. 2 and 4), although it must be doses, early uptake and incorporation into protein is noted that these quantities are rather insensitive to concentrated in cells with greatest access to portal K,/Kg between R = 0 and R = 0.5. The estimate of R = blood. These cells have been suggested to have higher 0.4 derived from Fig. 5 represents a component of in- rates of protein metabolism than the average of the flux, amounting to 40% of K1, that is not counterbaltissue (15). If so, the effect appears not to be due to anced by efflux. Such a component will concentrate differences in elongation rate in protein synthesis, as leucine in liver intracellular space relative to plasma no difference in polypeptide chain assembly time was and may therefore be considered uphill or active transfound at high and low doses of [14C]leucine. Another port. The result yields a predicted steady-state concen- source of -heterogeneity in the liver system may be the tration ratio for liver and plasma very close to that result of streamlining in the portal flow (14). In syscalculated from amino acid pool measurements (see tems of this type, it is clear that caution must be equations 5 and 6). This provides a degree of confirmaexercised if protein synthetic rates are to be calculated tion, although the error associated with small differfrom incorporation data combined with specific radioences in measured leucine concentrations of liver and activity determined for the tissue as a whole. plasma (Table 1) is recognized. Perspectiues. The results of these studies indicate the The magnitude of the leucine influx in toadfish liver feasibility of assay of amino acid transport by liver in at normal body temperature and plasma leucine con- vivo for determination of both quantitative fluxes and centration compares well with data in Ehrlich cells of characteristics of transport systems as they operate (see RESULTS). Even if only 20% of the liver participates in the whole animal. The method is not restricted to in the rapid uptake process as studied here, leucine nonmetabolizable amino acids; the analysis of amino influx for the organ as a whole is substantial (0.12 acid incorporation into protein and the simultaneous timol/min per 7-g liver). This is twice the theoretical determination of aolvnentide chain assemblv time proDISCUSSION

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LEUCINE

TRANSPORT

IN

1823

LIVER

vide a measure of tissue metabolism independent of the transport process. Possible applications of pulse injection methodology to the study of classic transport problems in vivo have been discussed by Pardridge and Jefferson (21). Extension of the present work to the examination of effects of very low temperatures on transport by liver in vivo is in progress.

This study was supported by National Science Foundation Grant GB-42752 and Public Health Service Grant HD-04670. A portion of this work was presented at the General Meetings of the Marine Biological Laboratory, Woods Hole, Mass., August, 1973

and 1974f

R. Persell was a Recipient Graduate Studies in the Life Received

for publication

of the I3eatrice Sciences.

6 February

Konheim

Award

for

1976.

REFERENCES 1. BETZ, A. L., D. D. GILBOE, AND L. R. DREWES. Kinetics of unidirectional leucine transport into brain: effects of isoleucine, valine, and anoxia. Am. J. PhysioZ. 228: 895-900, 1975. 2. CLARK, A. J., C. YAMADA, AND M, E. SWENDSEID. Effect of Lleucine on amino acid levels in plasma and tissue of normal and diabetic rats. Am. J. Physiol. 215: 1324-1328, 1968, 3. ELWYN, D. H., H. C. PARIKH, AND W. C. SHOEMAKER. Amino acid movements between gut, liver, and periphery in unanesthetized dogs. Am. J. Physiol. 215: 1260-1275, 1968. 4. FERN, E. V., AND P. J, GABLICK, The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo. Biochem. J. 142: 413-419, 1974. 5. FISHER, M. M., AND M. KERLY. Amino acid metabolism in the perfused rat liver. J. PhysioE., London 174: 273-294, 1964. 6. GREENWAY, C. V., AND R. D. STARK, Hepatic vascular bed. PhysioE. Rev. 51: 23-65, 1971. 7. HASCHEMEYER, A. E. V. Rates of polypeptide chain assembly in liver in vivo: relation to the mechanism of temperature acclimation in Opsanus tau. Proc. NatZ. Acad. Sci., U.S. 62: 128-135, 1969. 8. HASCHEMEYER, A. E. V. Kinetic analysis df synthesis and secretion of plasma proteins in a marine teleost. J. BioZ. Chem. 248: 16433649, 9. HASCHEMEYER, A. E. V. Control of protein synthesis in the acclimation of fish to environmental temperature changes. In: Responses of Fish to Environmental Changes, edited by W. Charvin. Springfield, Ill.: Thomas, 1973, p+ 3-30. 10. HASCHEMEYER, A. E. V., AND A. P. HUDSON, Transport of Lleucine by toadfish liver in vivo. BioZ. BUZZ. 145: 439, 1973. 11. HASCHEMEYER, A. E. V., AND R. PERSELL. Kinetic studies on amino acid uptake and protein synthesis in liver of temperature acclimated toadfish. BioZ. BuZZ. 145: 472-481, 1973. 12. HEINZ, E, Transport of amino acids by animal cells. In: Metabolic Transport, edited by L, E. Hokin. New York: Academic, 1972, vol. 6, p. 455-501. 13. HOARE, D. G. The transport of L-leucine in human erythrocytes: a new kinetic analysis. J. PhysioZ., London 221: 311-329, 1972.

14. LEBOUTON, A. V. Heterogeneity of protein metabolism between liver cells as studies by radioautography. Current Mod. BioZ. 2: 111-114, 1968. 15. LEBOUTON, A. V., AND T, E. HOFFMAN. Protein metabolism among lobes of the rat liver in relation to site of radioisotope injection. Proc, Sot. ExptZ. BioZ. Med. 132: 35-19, 1969. 16. MATHEWS, R. W., A. ORONSKY, AND A, E. V. HASCHEMEYER. Effect of thyroid hormone on polypeptide chain assembly kinetics in liver protein synthesis in vivo. J. BioZ. Chem. 248: 13291333, 1973. 17. MCMENAMY, R. H., W. C. SHOEMAKER, J. E. RICHMOND, AND D. ELWYN, Uptake and metabolism of amino acids by the dog liver perfused in situ. Am. J. Physiol. 202: 407-414, 1962. 18. MOORE, J. W., AND F. RAMON. On numerical integration of the Hodgkin and Huxley equations for a membrane action potential. J. Theoret. BioZ. 45: 249-273, 1974. 19. OLDENDORF, W. H. Measurement of brain uptake of radiolabeled substances using a tritiated water internal standard. Brain Res. 24: 373-376, 1970. 20. OXENDER, D. L., AND H. N. CHRISTENSEN. Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J. BioZ. Chem. 238: 3686-3699, 1963. 21, PARDRIDGE, W. M., AND L. S. JEFFERSON. Liver uptake of amino acids and carbohydrates during a single circulatory passage. Am, J. Physiol. 228: 1155-1161, 1975. 22. PERSELL, R, A computer program for evaluation of amino acid distribution in vivo. BioZ. BUZZ. 147: 493-494, 1974. 23. RAMOS, E. H., L. C, DEBONDIANNI, M. L. CLAISSE, AND A. 0. M. STOPPANI. Energy requirements for the uptake of L-leucine by Saccharomyces cerevisiae. Biochim. Biophys. Acta 394: 470-481, 1975. 24. SMITH, M. A. K., AND A. E. V. HASCHEMEYER. Studies on protein metabolism and growth in fish. BioZ. Bull. 147: 500, 1974. 25. SURKS, M. I., AND M. BERKOWITZ. Rat hepatic polysome profiles and in vitro synthesis during hypoxia. Am. J. Physiol. 220: 16061609, 1971. 26. WOOD, J. M. Leucine transport in Escherichia coZi. J. BioZ. Chem. 250: 4477-4485, 1975.

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Characterization of leucine transport by toadfish liver in vivo.

AMERICAN JOURNAL OF PHYSIOLOGY Vol. 231, No. 6, December 1976. Prdnted in USA. Characterization liver in vivo of leucine ROGER PERSELL AND A...
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