Metabolism Clinical and Experimental SEPTEMBER

VOL 41, NO 9

Competition

1992

for Transport of Amino Acids Into Rat Heart: Effect of Competitors on Protein Synthesis and Degradation

Armando

R. Tovar,

Jean K. Tews,

Nimbe

Torres,

David C. Madsen,

and Alfred

E. Harper

Transport of the neutral amino acids, 2-(methylamino)isobutyrate (MeAIB) and Phe, was examined in isolated rat hearts perfused by the Langendorff method. Hearts were perfused by recirculating for various time periods buffer containing [WI-MeAIB or [‘Cl-Phe plus desired additions. Uptake of MeAlB was linear for approximately 30 minutes; Phe uptake was linear for a maximum of 5 minutes, and reached a steady state after 15 minutes. K,,, and Vmaxfor MeAlB were 1.1 f 0.03 mmol/L and 37.7 + 0.4 pmol/pL intracellular fluid (ICF)/min; values for Phe were 1.6 + 0.02 mmol / L and 364 + 5 pmol / pL ICF/ minute. Uptake of MeAlB (0.2 mmol/L) was reduced 95% in the presence of Ser (10 mmol/L), and less severely by large neutral amino acids ([LNAA], 10 mmol/L) such as Phe and Leu (by 46% and 54%. respectively). Uptake of Phe (0.2 mmol/L) was reduced by LNAA such as Val, Leu, and Ile (by 51%, 76%. and 61%. respectively), or by commercial preparations used in parenteral nutrition, eg, Travasol or Travasol plus extra branched-chain amino acids (BCAA) (Branchamin); Ser had little effect (6% reduction). Insulin in the perfusion medium increased the fractional rate of protein synthesis. Individual BCAA at physiological concentrations (0.2 mmol/L) did not alter the rate of protein synthesis. Branchamin or Travasol plus Branchamin also had no effect on the rate of protein synthesis in heart, but did depress the rate of degradation. These studies suggest that amino acid transport into heart may be affected by normal levels of plasma amino acids, whereas protein synthesis is not. Copyright 0 1992 by W. 6. Saunders Company

R

ATE OF TRANSPORT of amino acids into tissues is potentially important for metabolic regulation. Amino acids are transported into cells by carriers that are generally, but not completely, specific for groups of amino acids with similar chemical characteristics, so there is overlap in the uptake of amino acids by the different transport systems. The most important systems for transport of the neutral amino acids are the L system, which transports primarily large neutral amino acids (LNAA), the A system, for small neutral amino acids (SNAA), and the ASC system, which is relatively specific for Ala, Ser, Cys, and Thr.l,* Amino acid transport has not been extensively studied in heart. Manchester & Wool3 observed that free amino acids accumulated in cells of the isolated perfused heart; the extent of accumulation depended on the amino acid itself and on the duration of the perfusion. Accumulation of Pro, Gly, or the analogue, ct-aminoisobutyric acid, increased in the presence of insulin, but generally only after prolonged perfusion with the hormone. Transport of other tested amino acids was not increased by insulin.3s4 Bafios et aI5 concluded from results of infusion studies in rats that K,,, values for transport of amino acids into heart are high compared with their concentrations in plasma, suggesting that competition between amino acids sharing a common transport system is likely to be low in heart. In Metabolism,

Vol41, No 9 (September),

1992: pp 925-933

contrast, Morgan et al6 observed a decrease in Phe concentration in hearts perfused with radioactive Phe in the presence of normal or increased concentrations of plasma amino acids, evidence that uptake of Phe may be depressed by competition from other amino acids transported by the same carrier system. Increasing the concentrations of amino acids in the perfusion medium can increase the rate of protein synthesis in the perfused hear@; thus, rates of protein synthesis in heart may be regulated by intracellular concentrations of amino acids. Protein synthesis is also enhanced in the presence of insulin.7-10 The purpose of the present study was to examine in the isolated perfused heart some characteristics of systems for the transport of 2-(methylamino)isobutyrate (MeAIB), a recognized model for the A system for amino acid trans-

From the Departments of Nutritional Sciences and Biochemistry, University of Wuconsin-Madison, Madison, WI. Supported in part byfindsfrom the College ofAgricultural and Life Sciences, University of Wuconsin-Madison; Clintec Technologies, Inc, Deerfield, IL; and the Coca-Cola Foundation, Atlanta, GA. Address reprint requests to Jean R Tews, PhD, 5445 Lake Mendota Dr, Madison, WI 53705. Copyright 0 I992 by W.B. Saunders Company 00260495/92/4109-0001$03.00/0 925

926

TOVAR ET AL

port, and of Phe, a typical LNAA that is not degraded by heart6 and that is normally transported primarily via the L system. We have also examined the potential physiological importance of competition between these and other natural amino acids, especially the branched-chain amino acids (BCAA), for transport into heart. In particular, we have tested the effects of commercial amino acid solutions in concentrations used for parenteral nutrition, including those enriched with the BCAA. In addition, protein synthesis and degradation in the presence of exogenous Phe have been examined under conditions shown to depress Phe transport into heart. MATERIALS

AND METHODS

Heart Preparation Male Sprague-Dawley rats weighing 250 to 300 g were fed a commercial rat diet ad libitum. Rats were housed in wire cages with a 1Zhour light/dark cycle; they were fasted overnight before experiments were performed. The rats were anesthetized with an intraperitoneal (IP) injection of sodium pentobarbitaI(55 mg/kg body weight); they also received heparin (2.5 mg per rat, IP). Hearts were removed and placed in 10 mL ice-cold, modified Krebs-Ringer bicarbonate buffer (pH 7.4) containing NaCI, 118 mmol/L; KCl, 4.7 mmol/L; CaC12. 2Hz0,3.0 mmol/L, KH2P04, 1.2 mmol/L; MgS04 . 7H20, 1.2 mmol/L, NaHC03, 25.0 mmol/L; EDTA, 0.55 mmol/L; and glucose, 11 mmol/L.” Surface blood was removed by washing with fresh buffer. Extra tissue surrounding the heart was removed and the aorta was exposed without removing the heart from the buffer. Heart Perfusion Hearts were perfused by the Langendorff technique.12 Isolated hearts were cannulated and, before tests were performed, were immediately perfused without recirculation for 5 minutes at 37°C with the buffer, which was continuously gassed with Oz:CO2 (955). For measurement of rates of transport of amino acids, hearts were perfused for different times by recirculating 125 mL buffer containingcold Phe or MeAIB at the desired concentration and either 1.25 PCi [U-14C]-Phe (specific activity, 499 mCi/mmol) or [l-14C]MeAIB (specific activity, 48.4 mCi/mmol). The perfusion system contained a filter (SM, 5-p,rn pore, Millipore, Bedford, MA) to eliminate from the medium small particles that could obstruct the coronary capillaries. The perfusion rate was approximately 12 to 13 mL/min. After perfusion was complete, hearts were quickly flushed with fresh, nonradioactive buffer, and then immediately removed from the cannula and blotted. The apex was removed, weighed ( - 750 mg), and homogenized with 6% perchloric acid. After centrifugation, 1-mL portions of the supernates were pipetted into scintillation vials, 10 mL scintillant (Biosafe, Mount Prospect, IL) was added, and the vials were counted with an external standard for quench correction (LKB Rackbeta, Model No. 1217, Gaithersburg, MD). Competition Studies Individual L-amino acids were added as competitors to the perfusion medium at a concentration of 10 mmol/L each; Phe or MeAIB were present at 0.2 mmol/L, a concentration within the physiological range for most of the amino acids in plasma. In some experiments, commercial solutions used for parenteral nutrition (Travasol, a 10% solution of natural LNAA; Branchamin, a 4% equimolar solution of BCAA; Clintec, Deerfield, IL) were added

to the perfusion medium to determine if they might affect transport of Phe. Total and Extracellular Water Content Total water content was determined by drying weighed samples to constant weights in a vacuum oven. In the present experiments, total water content of heart tissue was similar at different perfusion times (mean, 82.3% + 0.1%; n = 20). Extracellular fluid content was determined with [14C]-sucrose to be 28.7% t 0.2% overall (n = 20); values of 28.7% -C0.4% and 29.1% ? 0.5% were obtained after perfusion times of 5 and 60 minutes, respectively (n = 4). These values are in agreement with those reported previously by others.3,4 Transport was expressed as pmol/kL intracellular water (ICF) for a given time by the equation {[(dpmlmg heart - dpm/pL medium)(ECF/100)/(TWC/100 - ECF/lOO)Jl[dpm/~L medium]] x concentration (kmol/L) in medium, where ECF represents the extracellular space and TWC is the total water content of the heart. Protein Synthesis Hearts were perfused for 20 minutes with a recirculating buffer volume of 75 mL for measurement of protein synthesis. Hearts were perfused with unlabeled Phe (0.2 mmol/L) plus 3.75 PCi [14C]-Phe. Phe was selected because it is not degraded in the heart, is used only for the formation of cardiac proteins, and equilibrates rapidly with the intracellular pool of amino acids.s,6 This level of Phe (0.2 mmol/L) is within the range of concentrations associated with rates of protein synthesis comparable to those observed with slightly higher Phe concentrations, where specific activities of extracellular and intracellular free Phe and tRNA-bound Phe were identical.g After perfusion was terminated, hearts were quickly flushed with fresh buffer before removal from the cannula; the apex was removed, blotted, weighed, and frozen in liquid nitrogen. The procedure for measurement of Phe incorporation was similar to that described by Garlick et al. l3 Briefly, pieces of heart weighing approximately 500 mg were homogenized in 2 mL of 2% perchloric acid. Homogenates were centrifuged, supernates were neutralized with 350 p,L of 20% KOH, and the mixtures were centrifuged to remove perchlorate. The resulting supernates were mixed with 160 p,L of 50% sulfosalicylic acid and centrifuged again. A portion (0.5 mL) was counted, as described above, to measure radioactivity in acid-soluble Phe; another portion was adjusted to pH 2.2 with lithium citrate buffer before the Phe concentration was analyzed by ion-exchange chromatography (Beckman Model 119CL, Palo Alto, CA), by a method devised for rapid analysis of only the Phe area of the chromatogram.14 Norleucine was used as the internal standard. The precipitate from the heart homogenate was washed with 10 mL of 2% perchloric acid and centrifuged; this procedure was repeated twice more, and the precipitate was then resuspended in 10 mL of 0.3 mol/L NaOH and incubated for 60 minutes in a water bath at 37°C. Proteins were reprecipitated with 2 mL of 20% perchloric acid, centrifuged, and washed twice with 5 mL of 2% perchloric acid. The final precipitate was suspended in 5 mL of 6 mol/L HCI, transferred to tubes, and gassed for 30 seconds with nitrogen to prevent oxidation of some amino acids. The tightly capped tubes were placed in a heating block for 17 hours at 106°C to hydrolyze the proteins. The resulting solution was filtered through Whatman no. 57 filter paper. A portion (0.5 mL) was counted as described above. Another portion (0.1 mL) was diluted with 4.9 mL water, and 2 mL of this solution was evaporated to dryness; the residue was again dissolved in water and evaporated. The dry sample containing free amino acids was redissolved in a mixture of 1.92 mL lithium citrate buffer and 0.08 mL 2.5 mmol/L norleucine, and analyzed for Phe content as debcribed above.

AMINO ACID TRANSPORT AND PROTEIN METABOLISM

IN HEART

927

The results were expressed as fractional rates of protein synthesis (KS),calculated as follows: KS= Sg x 100/S~ x t, where Sa is the specific radioactivity of Phe bound in the protein (@/mmol), SA is the specific radioactivity of free intracellular Phe in tissue (KG/ mmol), and t is the time of incorporation expressed in days. K, units are percent per day.13 Protein Degradation Limited studies on protein degradation were performed by determining the rate of appearance of [14C]-Phe in the perfusion medium, after proteins had been previously labeled15J6 by perfusing hearts for 20 minutes with Phe (0.2 mmol/L plus 5 FCi [14C]-Phe) in a recirculating volume of 75 mL buffer. The hearts were then perfused for 2 hours with nonrecirculating buffer containing Phe (0.2 mmol/L), cycloheximide (0.02 mmol/L) to prevent continuing protein synthesis,17J8 and amino acid mixtures as desired. Radioactivity and Phe concentrations were determined as described above in perfusate samples taken at several intervals during the following 2-hour perfusian period. Perfusate samples from before 30 minutes were discarded,15J6J* as specific activity of Phe declined rapidly during this portion of the perfusion period, presumably because of rapid efflux of free [14C]-Phe from heart tissue. Rate constants of degradation (Kd) were based on the exponential decline in specific activities of Phe during the last 90 minutes of perfusion, and were determined from semi-log plots by linear regression analysis.lg

20

4

Materials

7. 2

L-amino acids amino acid analogues, and insulin (porcine, 26.2 IU/mg) were obtained from Sigma, St Louis, MO. Radioactive MeAIB and Phe were obtained from New England Nuclear (DuPont, Boston, MA). Travasol and Branchamin solutions were gifts from Clintec Technologies, Inc, Deerfield, IL.

.5 3 % zi lz n

40

Time

(min)

Time

(min)

60

1000 -

800 -

800 -

400 -

Statistics The results were evaluated either by Student’s t test or by ANOVA followed by Fisher’s protected least significant difference (LSD) test to determine the significance of differences among groups (P < .05). Values for K, and V,, were calculated by weighted nonlinear regression20 RESULTS

Transport of MeAIB and Phe: Perfusion Time and Substrate Concentration Uptake of the amino acid analogue, MeAIB (1 mmol/L), widely recognized as a model for amino acid transport system A, was linear for 20 to 30 minutes; by 60 minutes the rate had essentially leveled off (Fig 1A). Phe (1 mmol/L) was taken up much more rapidly than MeAIB; a linear rate was maintained for a maximum of 5 minutes, and a steady state was reached after 15 minutes of perfusion (Fig 1B). Rates of uptake of graded concentrations of MeAIB and Phe were measured, and kinetic characteristics of their transport were estimated. These measurements were made with hearts perfused with MeAIB for 15 minutes and Phe for 5 minutes, times corresponding with approximately linear rates of transport (Fig 1). K, for. MeAIB was calculated to be 1.1 ? 0.03 mmol/L, with a V,, of 37.7 t 0.4 pmol/kL ICF/min, whereas for Phe, the K, was 1.8 + 0.02 mmol/L and the V,, was 364 f 5 pmol/p.L ICF/min (Fig 2). Thus, although overall affinity for Phe was relatively low, capacity for its transport into the perfused heart was

Fig 1. Effect of time on transport into perfused heart of (A) MeAlB (1 mmol/L) and (9) Phe (1 mmol/L). Values are means f SE for four hearts for each time point. Note differences in scales.

clearly greater than for MeAIB. When the constants for the two substrates were calculated with a nonsaturable component included, little evidence was obtained for diffusion under our conditions. Competition Studies To determine the extent to which other amino acids competed with MeAIB for transport into heart, uptake of MeAIB (0.2 mmol/L) was measured for 15 minutes in the presence of various competitors (10 mmol/L). LNAA such as Phe and Leu reduced uptake of MeAIB by approximately 50%, suggesting that these LNAA may be transported in part by the A system (Table 1). Ser almost completely blocked MeAIB transport. MeAIB itself (10 mmol/L) reduced transport of [*4-C]-MeAIB by 90%, thereby again demonstrating saturability of its transport.

928

TOVAR ET AL

350

i

.

300 .E E

250

2 0 _

200

7. 3i

150

2 2

100

.L

s

5o1

E ; 0 -

40 h

0. 50 0 0

2

4

6

Concentration

8

10 al

(mM)

5 Fig 2. Effect of initial concentration on rates of transport into perfused heart of MeAlB and Phe. Perfusion time was 15 minutes for MeAlB and 5 minutes for Phe. Values are means f SE for four hearts.

E Q

0 2

0

4

6

Leucine The pattern of competition by several amino acids (10 mmol/L) of Phe (0.2 mmol/L) transport into heart during the 5-minute perfusion period differed from that for MeAIB (Table 1). Uptake of Phe was not significantly reduced by Ser. An equimolar mixture of the BCAA (3.3 mmol/L each) or the individual BCAA (10 mmol/L) competed strongly with Phe for its transport. Val was the least effective competitor (51% reduction), and Ile and Leu were the most effective ( _ 70% reduction). Effects of graded concentrations of Leu on Phe transport are shown in Fig 3. There was no further significant reduction of Phe uptake when Leu was perfused at a concentration greater than 5 mmol/L. Concentrations of Leu between 0.2 and 0.5 mmol/L, within the physiological range of concentrations of the individual BCAA in plasma, significantly inhibited Phe transport. Effects of mixtures of amino acids on Phe uptake were also examined. Concentrated Travasol, a solution which consists of a mixture of amino acids adequate for nutri-

8

10

(mM)

Fig 3. Competition with Leu for transport of Phe into perfused heart. Hearts were perfused for 5 minutes with [WI-Phe (0.2 mmol/L) plus the indicated concentrations of Leu. Values are means f SE for four hearts.

tional support in humans, was diluted with buffer so that resulting concentrations of amino acids (Table 2) would not differ greatly from those found in normal human plasma.” The presence of diluted Travasol in the perfusion medium reduced Phe transport into heart by 30% (Table 3). Branchamin, an equimolar solution of BCAA, was added to the buffer-diluted Travasol (Table 2) to provide BCAA at levels comparable to the arterial concentrations resulting from infusion of Branchamin into human hearts.22 The resulting increase in the BCAA content of the perfusion Table 2. Amino Acid Concentrations

Present in Perfusion Media

Containing Diluted Commercial Preparations Amino Acid

TM3VaSOl

Travasol +

Normal Values

Branchamin

Human Plasma’

Table 1. Effects of Some Neutral Amino Acids on MeAlB or Phe Transport in Perfused Heart pmol/pL

ICFlmin

% of Control

Leu

139

559

Ile

114

534

64

LYs

79

79

192 264

133

9.0 + 0.2a

100

Val

124

544

+ Phe

4.5 k 0.9b

54

Phe

85

85

58

+ Leu

4.1 + 0.5b

46

His

77

77

94

1.o 2 0.3”

10

Thr

88

88

145

0.41 t 0.21c

5

Met

67

67

24

45.7 i 2.2a

100

Trp

22

22

MeAlB

+ MeAlB + Ser Phe + Ser

41.8 k 2.6”

92

Ala

581

581

+ Val

22.5 ? 1.9b

49

Arg

165

165

94

+ BCAA

15.7 -t 2.3c

34

W

343

343

232

+ Leu

14.6 -c l.4c

32

Pro

14%

148

+ Ile

13.3 * 0.9’

29

Ser

119

119

9

9

64

PLNAA-Phe

552

1,812

646

IBCAA

377

1,637

461

NOTE.

Perfusion

buffer contained

[14C]-MeAlB or I’%]-Phe

(0.2

mmol/L) and competitors at IO mmol/L (L forms). Perfusions were for 15 minutes (MeAIB) or 5 minutes (Phe). Values are means f SE for four hearts per treatment. Different superscripts indicate significant differences within each experiment followed by the LSD test.

(P < .05). as determined

by ANOVA

Tv

360

113

NOTE. Travasol and Branchamin were diluted with the perfusion buffer to obtain the indicated concentrations. Values are +mol/L. *From Striver et al.*’

AMINO ACID TRANSPORT AND PROTEIN METABOLISM

Table 3. Phe Transport into Perfused Heart in the Presence of Commercial Amino Acid Preparations pmol/pL

Phe

ICF/min

% of Control

45.0 -c 1.70

100

+ Travasol

31.9 _t 0.7b

70

+ Travasol + Branchamin

27.9 2 0.7c

61

NOTE. Perfusion buffer contained IW]-Phe

(0.2 mmol/L) and Trava-

sol with or without Branchamin at the concentrations shown in Table 2. Perfusions were for 5 minutes. Values are means k SE for four hearts per treatment.

Different superscripts

929

IN HEART

indicate significant differences

(P < .05), as determined by ANOVA followed by the LSD test.

medium further reduced Phe uptake to a level below that observed with Travasol alone (to 40% of control, Table 3). Effect of Insulin

Addition of insulin (200 nmol/L) to the perfusion medium did not stimulate transport of either MeAIB or Phe into hearts perfused for 15 minutes with MeAIB (-9.2 pmol/kL ICF/min) or for 5 minutes with Phe (38 to 41 pmol/pL ICF/min). Pre-perfusion for approximately 10 minutes with 125 mL buffer containing insulin alone also did not stimulate subsequent uptake of Phe in the continuing presence of insulin.

Protein Degradation

The rate at which [14C]-Phe was released from heart proteins previously synthesized during a 20-minute perfusion with radioactive Phe is represented by the exponential decline (after an initial washout period) in specific activity of Phe in the nonrecirculating perfusion medium (Fig 5). In these limited studies, the greatest rate of decline in specific activity of nearly 1.2% per minute (Table 5), and hence the greatest rate of protein degradation, occurred in control hearts perfused with the medium containing no amino acids except Phe. Inclusion of diluted Travasol in the perfusion medium, as in Table 2, did not significantly reduce the rate of decline in Phe specific activity (- l%/min), whereas diluted Bran-

1501 M

Protein Synthesis

In hearts perfused with 0.2 mmol/L Phe, specific activity of intracellular Phe increased rapidly within 5 minutes and did not change during the following 55 minutes of perfusion (Fig 4A), indicating that radioactivity of the free Phe pool reached a steady state rapidly. Specific activity of Phe in proteins increased linearly during the 60-minute perfusion period (Fig 4B), showing that the rate of protein synthesis was constant during this period. Based on the control rate of synthesis during a 20-minute perfusion period, the half-life (ln 2/K,) of total heart proteins was calculated to be 6.03 days. Despite the competition for transport between Phe and BCAAs (10 mmol/L) (Table l), the addition of Leu, Ile, or Val (0.2 mmol/L each) either separately or in combination did not significantly depress the rate of protein synthesis below the control value (Table 4). However, the addition of Leu at 5 mmol/L, sufficient to reduce Phe transport by approximately 60% (Fig 3), significantly reduced the measured rate of protein synthesis by 46% with respect to the control (Table 4). The presence in the perfusion medium of Travasol or Travasol plus Branchamin also did not significantly affect the measured rate of protein synthesis in the perfused heart (Table 4). The addition of insulin (200 nmol/L) and Travasol to the perfusion medium increased the rate of heart protein synthesis by 24% over the control value, and by 37% over the average rate in the presence of Travasol alone (Table 4). Protein synthesis was stimulated 44% above the control value when the perfusion medium contained insulin, Travasol, and Branchamin; however, this stimulation was not statistically significantly greater than that occurring in the absence of Branchamin.

0

A

I

0

20

40

Time

Time

60

(mln)

(min)

Fig 4. Effect of time on specific activity (pCi/mmol Phe) of (A) free phenylalanine and (8) protein-bound Phe in heart. Hearts were perfused with [‘%I-Phe (0.2 mmol/L) for the indicated times. Values are means k SE for four hearts.

TOVAR ET AL

930

Table 4. Effect of BCAA and Commercial Amino Acid Preparations on

Table 5. Effect of Perfusion With Commercial Amino Acid

Protein Synthesis in Heart

Preparations on Release of [XC]-Phe From Previously Labeled Heart Proteins

K, (%/D) Treatment

11.5 k 0.5’

Phe

Slope

Kd (rate

min ‘1

+ Leu (0.2 mmol/L)

9.3 + 0.9a

Control

0.0054 -c 0.0005a

+ Val (0.2 mmol/L)

9.7 + 0.5’

Travasol

0.0042 k 0.0003a,b

0.0097

+ Ile (0.2 mmol/L)

8.5 + 0.6”

Travasol + Branchamin

0.0029 + 0.0003b~C

0.0068

Branchamin

0.0023 k 0.0002c

0.0054

10.0 + 0.8”

+ BCAA (0.2 mmol/L each)

6.2 + O.Bb

+ Leu (5.0 mmol/LJ + Travasol

10.5 k 0.6”

+ Branchamin

10.8 t 0.6”

+ Travasol + Branchamin

11.2 + 0.7’

+ Travasol + insulin (200 nmol/L)

14.4 t 0.8”

+ Travasol + Branchamin + insulin

16.6 k 1.5c

NOTE. Perfusion buffer contained

[Y]-Phe

(0.2 mmol/L)

with or

0.0124

NOTE. Hearts previously labeled by perfusing with [“C)-Phe were perfused with nonrecirculating medium containing cold Phe (0.2 mmoli L), cycloheximide (0.02 mmol/L), and amino acid mixtures as indicated. Slopes were determined

from semi-log plots (see Fig 5) by linear

regression analysis; Kd = slope x 2.303. Different superscripts indicate significant differences (P > .05), as determined by ANOVAfollowed

by

the LSD test.

without the indicated additions. Travasol and Branchamin were present at the concentrations shown in Table 2. Perfusions were for 20 minutes. Values are means

2 SE for four hearts per treatment.

Different

superscripts indicate significant differences (P < .05), as determined by

was calculated to be approximately 1 hour, and approximately 2 hours for hearts perfused with Branchamin.

ANOVA followed by the LSD test.

DISCUSSION

chamin was a more effective inhibitor of protein degradation. This amino acid mixture reduced Phe release, and thus lowered the rate of decline of Phe specific activity in the perfusate to approximately 0.5% per minute, half that seen for the control hearts (Table 5). The combination of Travasol with the Branchamin mixture did not further reduce the rate of release of [i4C]-Phe. The half-life (In 2/&) of the protein-bound [t4C]-Phe in the control hearts

heart,“,423

0.6 1 0 Control 13 Travasoi m Branchamin A Travasol + Branchamin

-0.4

p+Aj

-0.6 -I 30

The use of the isolated perfused heart provides a model that allows controlled studies of nutrient transport via the network of capillaries, the physiological mechanism by which nutrients are delivered into cells. In general, there have been few studies on characteristics of amino acid transport in heart. Transport of several individual amino acids into hearts of rats infused in vivo has been examined5; most studies have been directed toward testing the effects of insulin on amino acid transport into the perfused

60

90

120

Time (rnin) Fig 5. Decline with time in specific activity (pCi/mmol) of Phe in perfusates of hearts after prior labeling of proteins with [14C]-Phe. Hearts were perfused with buffer containing cycloheximide (0.02 mmol/L) and Phe (0.2 mmol/L) with or without other amino acids at concentrations given in Table 2. Individual values are shown for each time point; n = 2 or 3 hearts.

The most accurate estimates of transport kinetic constants can be obtained only from measurements made under conditions of initial velocity when efflux is negligible. MeAIB uptake was linear in the present experiments for 20 to 30 minutes, and Phe uptake was linear for approximately 5 minutes. The characteristics of the time-course curves for both amino acids resemble those observed in other tissues for the A and L systems.’ Transport of amino acids via the A system, as was observed for MeAIB, does not rapidly reach steady state. In contrast, transport by the L system involves rapid equilibration between the intracellular and extracellular concentrations of amino acids. Thus, in the present study, rapid equilibration of radioactivity in the free Phe pool occurred when hearts were perfused with this amino acid, results comparable to those reported by others.5*6,13However, despite similarities of characteristics for transport of Phe and of a model for the L system such as 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH),’ Phe uptake in heart is unlikely to occur solely via the L system. Thus, our observed kinetic constants will reflect the sum of Phe transport, primarily by the L system for LNAA, but also, to a small extent, by transport via systems for SNAA. Regardless of contributions by individual transport systems for Phe uptake, the relatively low Km values estimated for transport of MeAIB and Phe into heart and the results of the competition studies indicate that the likelihood of competition occurring physiologically among amino acids transported by either the A or L systems is higher for heart

AMINO

ACID TRANSPORT

AND PROTEIN METABOLISM

IN HEART

than for skeletal muscle. In isolated soleus” or epitrochlearis muscle,25 the observed K, for MeAIB is approximately twofold to threefold greater than our value of 1.1 mmol/L for heart, although a K,,, of 0.7 mmol/L has been found in extensor digitorum longus muscle.26 For BCH (the widely recognized model for the L system), the K,,, in soleus muscle is 8.7 mmol/L, more than fourfold the value of 1.8 mmol/L for Phe in heart. The reported K,,, of 19.3 mmol/L for Phe in perfused skeletal muscle*’ is lo-fold higher than our value for perfused heart. Perfusate Leu, at concentrations of 0.2 to 0.5 mmol/L, well within the range of levels found in plasma under different dietary conditions, substantially inhibited Phe transport into heart. Concentration of total BCAA rapidly reaches a level of approximately 2 mmol/L in plasma of rats fed a high-protein diet.2s*29This value is similar to the K,,, for Phe, thereby indicating the probable occurrence of strong competition for entry of Phe into heart in animals fed high levels of protein. Competition for Phe transport into heart was clearly demonstrated simply by adding to the perfusate a commercial mixture of amino acids at concentrations resembling those normally found in plasma. Phe uptake was reduced by 30% in the presence of diluted Travasol, in which the total concentration of the other LNAA (L.eu + Val + Ile + Trp + Tyr + His) was 0.55 mmol/L. Addition of Branchamin to the perfusate increased the LNAA concentration to a total of 1.81 mmol/L and further depressed Phe uptake. These observations are also consistent with the results of Bafios et a1,5who determined an apparent K,,, of 3.7 2 0.9 mmol/L for Phe in hearts of rats infused in vivo; this relatively high value presumably reflects the competition for Phe transport into heart normally occurring in vivo as a result of the continuing presence of amino acids in the circulating blood. The higher K,,, determined by Batios et al thus indicates the higher Phe concentration required to maintain a half-maximal rate of its transport under these conditions. Evidence that competition occurs for amino acid transport into heart suggests that this organ is protected against the presence of high concentrations of amino acids in the blood, and that intracellular pools of amino acids in the heart may be regulated in part by competition for transport from the blood. Thus, amino acid transport into heart may be altered by various dietary treatments. This situation is therefore somewhat analogous to that in brain, in which K,,, values are usually comparable to or even well below plasma concentrations,30,31 and in which transport of a given amino acid can often be strongly modified by dietary treatments.29.32.33

Whether insulin stimulates amino acid transport into skeletal muscle has been much debated.24 Similarly, studies on the effects of insulin on amino acid transport into heart have not always produced consistent results.3,4s23 Such variable results probably depend on the fact that, in general, insulin stimulates the activity of transport system A, but not system L.2,34Thus, transport of SNAA, but not LNAA, is generally stimulated by insulin. Therefore, it would not be expected that Phe transport into the perfused

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heart would be increased by insulin even after pretreatment of the heart with the hormone. Conversely, our failure to demonstrate an effect of insulin on MeAIB transport into heart is likely due to the short 15-minute period of exposure to insulin; for example, Manchester & Wool3 noted stimulation of a-aminoisobutyrate transport, but primarily only after 20 to 80 minutes of perfusion. Despite competition for the transport of Phe in the presence of various concentrations of BCAA (Table 1, Fig 3), no effect on heart protein synthesis from Phe in vitro was observed when hearts were perfused with physiological concentrations of BCAA (0.2 mmol/L each). This was presumably because we observed no change in specific radioactivity of intracellular Phe under these conditions. Similarly, Morgan et al6 found absolute values for specific activities of Phe to be comparable in perfusate and heart when perfusions were performed for as long as 3 hours with Phe alone (0.08 mmol/L), or with other amino acids at normal or fivefold normal plasma levels (Phe at 0.08 or 0.4 mmol/L, respectively). Although intracellular Phe concentrations were reduced, presumably by competition for transport, when hearts were perfused with amino acids at twofold to lo-fold normal plasma concentrations, Phe incorporation into heart did not decline, but did reach a plateau$ increasing Phe concentrations in the perfusate increased tissue Phe content and its incorporation into protein. However, we found that perfusion of a high concentration of Leu (5 mmol/L) did reduce the specific activity of intracellular Phe in the heart, possibly because the uptake of exogenous cold and labeled Phe was reduced enough that the endogenous free Phe comprised a relatively large portion of the free pool. Under such conditions, there may be a falsely low value for the rate of protein synthesis, only because of the low specific activity of the labeled precursor. In our experiments, brief perfusions with an amino acid mixture (Travasol), tested only at concentrations resembling human plasma levels, with or without the addition of the BCAA mixture (Branchamin), did not significantly alter the rate of protein synthesis in heart. These observations might appear to differ from those of Morgan et al,‘j who reported stimulation of heart protein synthesis in the presence of added amino acids. However, they found that perfusion with amino acids at normal plasma levels for as long as 3 hours increased heart protein synthesis by only 13% or 14% in two separate experiments; in contrast, perfusion with amino acids at fivefold normal concentrations significantly stimulated protein synthesis by 13% and 40% at 90 and 180 minutes, respectively. Thus, differences in perfusate amino acid concentrations and duration of perfusions probably account for the apparent discrepancies in these studies. Despite the lack of effect of amino acid mixtures on protein synthesis in our studies, the addition of insulin did increase the rate of Phe incorporation into protein. This effect of insulin has been well demonstrated, and has been attributed to increased formation of poiysomes and decreased release of monosomes.‘JO Previous studies have often indicated that BCAA, primarily Leu, stimulate pro-

TOVAR ET AL

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in perfused heart.17.35x36 In contrast, McNurlan et a137observed no stimulation of protein synthesis in heart (and other tissues) after infusing rats with Leu in vivo. When plasma insulin was increased as a result of including glucose in the infusion mixture, protein synthesis in heart and other muscle tissue in vivo increased; if a “complete” amino acid mixture was included with glucose, the increase in protein synthesis was more pronounced. A BCAA/ glucose mixture further stimulated protein synthesis in skeletal muscle, but, again, did not increase synthesis in heart beyond that seen with glucose alone.38 Despite our failure to demonstrate stimulation of protein synthesis in the perfused heart by physiological concentrations of amino acids in general or by BCAA specifically (as in solutions used for parenteral nutrition), the observed rates of protein synthesis were similar to those observed by others in the Langendorff preparation* or in starved rats in vivo.37 Numerous studies on protein degradation in heart have been reported. In general, insulin effectively inhibits release of protein-bound amino acids (eg, Phe) into the perfusion medium. 17~18*36 BCAA (probably due to Leu alone) have been found to decrease the rate of protein degradation in both perfused heartr7J6J9 and in skeletal muscle.s9Az In general, inhibition of degradation has usually been found with Leu concentrations of 1 mmol/L or greater,*7.40 levels above those in our medium containing diluted Travasol or Travasol plus Branchamin (140 or 560 umol/L, respectively; Table 2). Our limited results suggest that the degradation rate decreased when hearts were perfused with Branchamin alone, even though its Leu concentration was only 420 pmol/L (total BCAA, 1.26 mmol/L). Perfusion with the mixture of Branchamin and Travasol did not significantly lessen protein degradation further, despite the somewhat higher concentration of Leu. tein synthesis

Protein degradation is often measured by the appearance of label in cold medium after release from perfused tissue of radioactive amino acid from previously labeled protein, or by dilution of radioactive medium by release of cold amino acid from unlabeled tissue. However, such measurements may be affected by certain transport phenomena. Thus, the presence in the perfusate of a high concentration of an appropriate transport competitor might reduce the release of free amino acids from tissue to medium, thus implying a greater depression of protein degradation than has actually occurred. However, perfusion with the combined Travasol and Branchamin (total amino acids, 1.81 mmol/L) did not reduce Phe release more than did Branchamin alone (1.26 mmol/L). Conversely, rapid exchange across the plasma membrane occurs for amino acids that are transported primarily via the L system’; thus, the release of radioactive Phe into the perfusate might be accelerated because of exchange with certain of the amino acids added to the perfusion medium. Despite this possibility, the greatest rate of Phe release occurred when the perfusion medium contained only Phe. These studies show that, at concentrations of amino acids normally present in plasma, competition can occur among neutral amino acids for entry into heart. Despite this, heart protein synthesis appears to be protected against such reduced transport of precursor amino acids, possibly because of the very low K,,, values for amino acyl-tRNA synthetases noted for a variety of preparations (eg, 6 umol/L for leucyl-tRNA synthetase).43 ACKNOWLEDGMENT We thank William Merlau of the Veterans Administration Hospital, Madison, WI, for advice concerning the LangendorfI procedure, and Joyce Repa for determining Phe concentrations.

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