Intestinal glycyl-L-phenylalanine and L-phenylalanine transport in a euryhaline teleost.

Intestinal transport

glycyl+phenylalanine in a euryhaline teleost AND

GREGORY

A. AHEARN

STEPHAN

J. RESHKIN

Department

of Zoology, University of Hawaii at Manoa, Honolulu, Hawaii 96822

RESHKIN, STEPHAN J., AND GREGORY A. AHEARN. Intestinal glycyk-phenylalanine and L-phenylalanine transport in a euryhaline teleost. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R563-R569, 1991.-The transport mechanisms for the dipeptide glycyl+phenylalanine (Gly-Phe) and L-phenylalanine (Phe) were characterized in fish intestinal brush-border membrane vesicles (BBMV). Gly-Phe was rapidly hydrolyzed only intravesicularly with almost total hydrolysis occurring even at 10 s. Dipeptide uptake was not stimulated by an inward gradient of Na, K, or H. Phe uptake was stimulated by an inward gradient of either Na or K but displayed an overshoot phenomenon only in the presence of an Na gradient. Kinetic analysis of the effect of substrate concentration on transport rate revealed that transport of both Gly-Phe and Phe occurred by a saturable process conforming to Michaelis-Menten kinetics. The K, for Gly-Phe was 9.8 t 3.5 mM, whereas that for Phe in the presence of Na or K, respectively, was 0.74 t 0.13 and 1.1 & 0.37 mM. Maximum uptake for Gly-Phe and for Phe in the presence of Na and K was 5.1,0.9, and 0.4 nmol mg and protein-’ -5 s-l, respectively. Gly-Phe and Phe transport displayed different patterns of inhibition by dipeptides and amino acids. These results suggest that Gly-Phe and Phe are transported via different mechanisms, with Gly-Phe being hydrolyzed during a carrier-mediated, cation-independent process and Phe being transferred via a Na’ cotransport process similar to that described in mammals. During conditions of high luminal dipeptide concentrations, the Gly-Phe pathway may make a significant contribution to total Phe uptake.

brush-border membrane vesicles; peptide transport; amino acid transport; cotransport; sodium cation dependence; intestinal physiology; membrane transport; sodium gradient hypothesis; tilapia; Oreochromis mossambicus

amino nitrogen can be absorbed from the small intestinal lumen not only in the form of free amino acids but also as di- and tripeptides (3). Just after a meal, there are as many small peptides present as free amino acids in the small intestinal lumen (23), and this peptide fraction is composed primarily of 2-4 amino-acid residue peptides (1). While some peptides are hydrolyzed by the epithelial brush-border membrane and then the component amino acids absorbed, there is evidence that others, especially those containing glycine or proline residues, are transported intact and presumably hydrolyzed intracellularly (3, 28, 33). Recent work utilizing brush-border membrane vesicles (BBMV) has demonstrated that dipeptides can be transported intact and that transport is energized by coupling to a proton gradient rather than to a sodium gradient (16, 17, 19). However, Rajendran et al. (25) recently IN

and L-phenylalanine

MAMMALS,

0363-6119/91

$1.50 Cowright

reported non-cation-stimulated facilitated transport of glycyl+proline in intestinal BBMV of mice, rabbits, and humans. Similarly, Na- and H-independent carrier transport of the tripeptide glycylglycyl+proline was also disclosed in BBMV of human jejunum (34). These results suggest that brush-border dipeptide and tripeptide transport systems may be more heterogeneous than previously thought. There may be multiple peptide uptake systems as have been described for amino acids (3). Assessment of the relative contributions of both peptides and amino acids to overall uptake of amino nitrogen, resulting from digestive activities, is a significant underinvestigated physiological field. There is some evidence in mammals that rates of di- and tripeptide uptake may be greater than those of free amino acids (2, 22). In a more recent study, Rajendran et al. (24) comparing transport rates of glycyl-L-proline and L-proline in intestinal BBMV reported dipeptide transport rates to be four times the rate of the amino acid alone. In fish, the characteristics of peptide transport and the relative importance of this process to overall amino nitrogen absorption remains unstudied (13). In the present investigation, we compare the transport mechanisms for the dipeptide glycyl-L-phenylalanine and one of its component amino acids, L-phenylalanine, in intestinal BBMV from the teleost, Oreochromis mossambicus, to evaluate the possible independence of dipeptide and amino acid absorption. MATERIALS

AND

METHODS

Collection and maintenance of animals. African tilapia (0. mossambicus; each -100 g wet wt) were obtained

from commercial freshwater prawn aquaculture ponds on northwestern Oahu, HI, and transported to large circular holding tanks at the University of Hawaii. Preparation of brush-border membrane vesicles. Fish were killed by a blow to the head. Intestines were removed, and the upper half was utilized for the preparation of brush-border membranes. Because the intestine was too fragile to scrape epithelial cells free from underlying muscle tissues, the entire segments were employed for the preparation of brush-border vesicles. The magnesium precipitation method developed by Kessler et al. (20) and modified by Biber et al. (10) was used for purifying brush-border membranes from these intestinal segments. Each tissue was separately homogenized with a Polytron (Brinkmann Instruments) in 30 vol of ice-cold

0 1991 the American

Phvsiological

Societv

R563

Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (137.092.098.118) on August 12, 2018. Copyright © 1991 American Physiological Society. All rights reserved.

R564

PEPTIDE

TRANSPORT

IN

FISH

BBMV

Membrane hydrolysis of glycyl-L-phenylalanine. BrushBuffer 1. This buffer was made by diluting a stock border membrane catalyzed hydrolysis of the dipeptide solution of 300 mM mannitol, 20 mM ethylene glycolwas measured in the same incubation system used for bis( ,8-aminoethyl ether)-N,N,N’,N’-tetraacetic acid analysis of uptake. Twenty-microliter aliquots were re(EGTA), and 12 mM tris(hydroxymethyl)aminomethane moved at 0.16-, l-, 3-, and 60-min intervals, injected into (Tris) l HCl, at pH 7.5 with four volumes of ice-cold distilled water and adding 0.1 mM phenylmethylsulfo1 ml of ice-cold stop solution and filtered as in a transport experiment. The filtrate was collected and concentrated nylfluoride (PMSF) to the final mixture. Magnesium chloride was added to the homogenate suspension to a gently over low heat to -50 ~1. Aliquots were used for analysis by thin-layer chromatograpy on silica gel plates final concentration of 10 mM, and the resulting solution (silica gel F-254, Merck, FRG) using n-butanol-acetic was allowed to stand on ice for 15 min. The suspension acid-water (80:20:20, vol/vol). Chromatograms were dewas centrifuged at 3,000 g for 15 min and the resultant veloped with ninhydrin spray, and the areas correspondsupernatant at 27,000 g for 30 min. The pellet from the high-speed spin was resuspended in 30 ml of ice-cold ing to authentic L-phenylalanine, L-glycine, and glycylL-phenylalanine were cut out and analyzed for radioacBuffer 2 (60 mM mannitol, 6 mM EGTA, 12 mM Tris tivity. Internal controls in which radioactive dipeptide HCl, at pH 7.4) using a Potter-Elvehjem homogenizer. The magnesium precipitation and two centrifugations was added to the filtrate were run to determine the extent of dipeptide hydrolysis due to processing. No significant were repeated on this mixture, the resulting pellet was (P > 0.05) hydrolysis was found to take place due to resuspended with the Potter-Elvehjem homogenizer in 20 ml of ice-cold transport buffer (see Figs. l-4 for processing (data not shown). Assay of intravesicular contents. Transport experiappropriate composition), and the final suspension was centrifuged at 27,000 g for 30 min. The purified memments were run as outlined above, and the filters obtained were immediately immersed in 1 ml boiling disbrane pellet was resuspended in sufficient transport buffer to provide a final vesicle protein concentration of tilled water and extracted overnight at 5OC. The filter 5-7 mg/ml. Protein content of the preparation was asand water were then centrifuged, and the supernatant concentrated as above to -50 ~1 volume. Aliquots were sessed with the Bio-Rad protein assay. Transport measurements. Transport studies using inanalyzed by thin-layer chromatography as outlined testinal BBMV were conducted at 20°C using the Milliabove. Radioactive glycyl-L-phenylalanine was added straight to boiling water and treated as a sample to pore filtration technique of Hopfer et al. (18). For transdetermine the amount of hydrolysis due to processing of port intervals of ~20 s, a Rapid Uptake Apparatus (Inthe extract. No significant (P > 0.05) hydrolysis of novativ Labor, Adliswil, Switzerland) was used, which dipeptide took place during this processing (data not allowed automatic control of the incubation period to 1 s. shown). Estimation of influx kinetics. Carrier-mediated and difL- [‘Hlphenylalanine and [ 14C]glycyl-L-phenylalanine fusional amino acid and dipeptide influx kinetics were (Du Pont-New England Nuclear) uptake was initiated determined by the following curve-fitting procedure: toby mixing 5 ,ul membrane suspension with 20 ~1 radiolabeled incubation medium containing variable concen- tal influx data were computer fitted to a Michaelistrations of unlabeled solute. Incubation medium compo- Menten kinetic equation modified to include a diffusional sition varied with the nature of the experiments and is component indicated in the legends to Figs. l-4. Solute uptake was J oi = (Jmax [S]/Km + [S]) + P [S] (1) terminated by injection of 2 ml ice-cold stop solution (same composition as the incubation medium except by the iterative, nonlinear Marquardt method. Joi is Llacking the labeled solute). The vesicle sample was im- phenylalanine or glycyl+phenylalanine influx in picomediately filtered onto a Millipore filter (0.65 pm) and moles per milligram protein per minute; [S] is external washed with another 10 ml of ice-cold stop solution. amino acid or dipeptide concentration in millimolar; Jmax Filters, containing the vesicles and their associated ra- is maximal solute influx; K, is the concentration of S diolabeled solute, were placed in Beckman Ready Solv that yields one-half J,,,; and P is the apparent diffuscintillation cocktail and counted in a Beckman LS-8100 sional coefficient in units of liters per milligrams protein scintillation spectrometer. per 5 s. For every set of influx kinetics measurements, All isotope transport values were corrected for a “ves- the same vesicle preparation was used for both L-phenicle blank” obtained by adding the incubation medium ylalanine and dipeptide in order to facilitate comparison and vesicles directly to the stop solution before filtering, of kinetic constants for the two compounds. extracting in scintillation cocktail, and counting. Each experiment was repeated at least twice using membranes RESULTS prepared from different animals. The same experimental Time course of L-phenylalanine and dipeptide transfindings were consistently obtained in the repetition of an experiment. Within a given experiment, each point port: cation dependence. To assesswhether the transport was spewas analyzed using 3-5 replicates, and experimental scat- of L-phenylalanine and glycyl+phenylalanine ter between these replicates was always ~6%. Significifically activated by the presence of a cation gradient, cance of differences between means was determined by uptake in the following experimental conditions was Student’s t test. Throughout this study, either mean measured. Vesicles were loaded with 300 mM mannitol, 12 mM N-2-hydroxyethylpiperazine-hr’-2-ethanesulvalues obtained in an individual experiment or reprefonic acid (HEPES)-Tris at pH 7.4, and incubated with sentative examples of similar replicates are displayed.


PEPTIDE

TRANSPORT

either 0.36 mM [‘4C]glycyl-L-phenylalanine or 0.1 mM L-[3H]phenylalanine in a medium at pH 7.5 having a final concentration of either 1) 120 mM NaCl, 2) 120 mM KCl, 3) 300 mM mannitol, or 4) 300 mM mannitol at pH 5.5. In the dipeptide rapid-uptake experiment (Fig. 1B), an additional incubation medium was used having dipeptide at a final concentration of 100 mM at pH 7.5. Figures 1, A and B, and 2 illustrate the effects of these transmembrane cation gradients on the time course of [14C]glycyl-L-phenylalanine (Fig. 1, A and B) and L-[“Hlphenylalanine (Fig. 2) uptake by tilipia upper in-

IN

FISH

R565

BBMV

700

600

0 Y 0

80

t

. 60 -

30

n

60

90

a

Time \ 5 E n W x a

L

0

,

0

I

I

1

2

I

I

FIG. 2. BBMV of except for in Fig. 1A. of 3 values,

1 /

4

3 Time

5

c

I

I

I

I

0

2

4

6

8

Time

EWJ

Time course of L-[“Hlphenylalanine uptake into intestinal tilapia. Inside medium was same as in Fig. 1. Outside media, substitution of 0.1 mM L-[“Hlphenylalanine, were same as Time of equilibrated uptake was 60 min. Values are means and SE bars are encompassed within symbols.

(mid

60 c B

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mu

(set)

1. Time course of [‘4C]glycyl-L-phenylalanine uptake into intestinal brush-border membrane vesicles (BBMV) of tilapia (A: normal uptake; B: rapid uptake). Vesicles were loaded with 300 mM mannitol (Mann), 12 mM HEPES-Tris, pH 7.5. Outside media were 0.36 mM [‘“Cldipeptide, 12 mM HEPES-Tris, pH 7.4 plus 150 mM NaCl (filled circles), 150 mM KC1 (open circles), 300 mM mannitol (open squares) at pH 7.5, or 300 mM mannitol (closed squares) at pH 5.5. Additionally, in rapid-uptake experiments (B) there was 200 mM mannitol and 100 mM dipeptide (open triangle) at pH 7.5. Time of equilibrated uptake was 60 min. Values are means of 3-5 replicates, and SE bars are encompassed within symbols. FIG.

-

(set)

testinal BBMV. Transport of the dipeptide glycyl-Lphenylalanine (Fig. 1, A and B), was independent of all presented cation gradients, including a proton gradient. Transport of this dipeptide was probably the result of cation-independent facilitated diffusion into the vesicles. The uptake of the free amino acid L-phenylalanine was stimulated over apparent diffusive influx by the presence of either an inwardly directed gradient of Na or K (Fig. 2). However, only transport in the presence of the inwardly directed Na-gradient displayed a transient uptake “overshoot” at 1 min of incubation where vesicular Lphenylalanine concentration temporarily exceeded the equilibrium value by a factor of -1.6 (Fig. 2). Dipeptide hydrolysis. Measurements of the intravesicular and extravesicular hydrolysis of the peptide glycylL-phenylalanine were made to clarify the ligand transport process occurring in these vesicle preparations during dipeptide uptake. Table 1 presents the time course of intra- and extravesicular hydrolysis of glycyl-L-phenylalanine by the brush-border membrane preparation at 0.16, 1, 3, and 60 min. Hydrolysis is defined as the disappearance of labeled [14C]dipeptide with the concomitant appearance of labeled L- [14C]phenylalanine in thinlayer chromatograms. Outside of the vesicles there was no significant (P > 0.05) hydrolysis of the dipeptide, while analysis of intravesicular content after transport showed that dipeptide was completely hydrolyzed even by 10 s (0.16 min). These data rule out the possibility that dipeptide uptake into these vesicles was actually the uptake of the component amino acids after extravesicular hydrolysis of the dipeptide. Furthermore, these data sug-


R566

PEPTIDE

TRANSPORT

TABLE 1. Intra- and extravesicular hydrolysis of [‘“Clglycyl-L-phenylalanine by intestinal brush-border membrane vesicles of tilapia (Oreochromis mossambicus) Dipeptide Time, min

dpm

dpm as %Total Present

6.321.5 5.1k2.4 3.9-el.9 4.2t2.3

1 3 60

FISH

BBMV

n

i 16000 v) N Q a 12000 E

5 E

e

8000

Extravesicular

Intravesicular 0.16

IN

95*7 got11 9333 88k9 0

experiments. Values are means t SE; n = 3 replicate grat,ions/min of spots on thin-layer chromatogram

40

30 (mM)

FIG. 4. Effect of external dipeptide, glycyl-L-phenylalanine, concentration on L-[14C]dipeptide influx (5 s) into tilapia BBMV loaded with 300 mM mannitol, 12 mM HEPES-Tris, and 15 PM CCCP, pH 7.5. External media consisted of 300 mM mannitol, 12 mM HEPES-Tris, and 15 PM CCCP, pH 7.5, plus L-[14C]dipeptide concentrations ranging from 0.25 to 40 mM.

5000 - I $

20 DIPEPTIDE

ANDMETHODS).

l Na OK

iii

10

dpm, Disinte(see MATERIALS

4000

z ;;

2. [14C]glycyl-L-phenylalanine and L-phenylalanine influx kinetic parameters for tilapia upper intestinal BBMV TABLE

Transport System

I

I

I

I

J

1

2

3

4

5

PHENYLALANINE

(mM)

3. Effect of external L-phenylalanine concentration on L[“Hlphenylalanine influx (5 s) into tilapia intestinal BBMV loaded with 300 mM mannitol, 12 mM HEPES-Tris, 15 PM carbonyl cyanide m-chlorophenylhydrazone (CCCP), pH 7.5. External media contained 12 mM HEPES-Tris, 15 PM CCCP, and either 150 mM NaCl or 150 mM KC1 plus L-[“Hlphenylalanine concentrations ranging from 0.025 to 10 mM. Lines drawn through data were obtained by an iterative curve-fitting procedure. Values are means of 4 replicates, and SE bars are encompassed within symbols. FIG.

gest that hydrolysis of this dipeptide may take place during the transport process or by an enzyme bound to the interior of the brush-border membrane. Kinetic characteristics of influx. L-[3H]phenylalanine and [ “C]glycyl-L-phenylalanine influxes (5-s uptake) were measured as a function of respective substrate concentration (at 120 mM Na) to further evaluate the differences between the putative dissimilar transport systems for the free amino acid L-phenylalanine (Fig. 3) and the dipeptide (Fig. 4). The concentration of dipeptide varied from 0.25 to 40 mM, whereas the concentration of L-phenylalanine varied from 0.025 to 10 mM in the respective experiments. Transport of both compounds into the vesicles was a combination of 1) a saturable Michaelis-Menten carrier component, and 2) a linear “apparent diffusion” process having a rate that was proportional to the external ligand concentration (see MATERIALS AND METHODS). Kinetic constants K,, Jmax,and P for glycyl+phenylalanine and L-phenylalanine influxes by intestinal BBMV of tilapia are displayed in Table 2. At dipeptide concentrations between 5 and 40 mM and L-phenylalanine concentrations between 1 and 10 mM, influx remained a linear function of external concentration and may represent diffusional influx of the two solutes. The linear components of the concentra-

Gly-Phe Phe NaCl KC1

J max 9.8t3.5

5,100+1,700

0.74t0.13 1.35kO.37

910t103 420t190

P 2.320.3 5.3to. 1 5.5k0.2

Values are means & SE of 3 experiments. All 3 transport systems were always measured on same vesicle preparation. K, measured in mM substrate; maximum uptake (J,,& measured in pmolmg protein-’ -5 s-‘; apparent diffusional coefficient (P) measured in lo-” 1. mg protein-‘. 5 s-l. BBMV, brush-border membrane vesicles.

tion curves for L-phenylalanine uptake in Na and K media occurred with rates that were not significantly different (Na: 5.3 t 0.1; K: 5.5 t 0.2; P > 0.05). The linear apparent diffusional component of concentrationdependent dipeptide influx was -40% of the apparent diffusion al influx of L-phenylalanine (Table 2)) indicat ing that the larger intact dipeptide was likely responsible for the observed apparent nonmediated uptake. Large differences in both the apparent half-saturation constant (I&) and the maximum rate of influx (Jmax) were found between both Na-dependent and K-dependent L-phenylalanine influx and between L-phenylalanine and dipeptide influxes (Table 2). For L-phenylalanine uptake (Fig. 3, Table 2), both the apparent affinity and Jmaxwere significantly (P < 0.05) greater in the presence of Na than in the presence of K (&: 1.5-fold; Jmax: 2.2fold). This trend of higher affinity and maximum transport for L-phenylalanine in the presence of sodium is similar to that reported in mouse intestinal BBMV (9). The kinetic constants calculated for dipeptide influx in tilapia intestinal BBMV (Table 2) were both about an order of magnitude greater than those for either Na- or K-dependent L-phenylalanine influx in the same vesicle population, providing further evidence that in these vesicles glycyl+phenylalanine was transported via a different transport system than was free L-phenylalanine. Effect of free amino acids and dipeptide on L-phenylalanine and glycyl-L-phenylalanine uptake. To further distinguish between the transport mechanism(s) involved


PEPTIDE

TRANSPORT

in tilapia intestinal L-phenylalanine and glycyl-L-phenylalanine transport, inhibition of both Na- and K-dependent L-phenylalanine and of dipeptide uptake by a variety of amino acids and glycyl-L-phenylalanine was measured (Table 3). L-Phenylalanine and dipeptide uptake were measured at 0.1 and 0.36 mM, respectively. All potential inhibitors were added to the uptake medium at a concentration of 40 mM. None of the free acids, including glycine and L-phenylalanine, was able to significantly inhibit dipeptide uptake, which was self-inhibited by ~60%. L-Phenylalanine influx inhibition by the various tested compounds displayed a complex pattern. LPhenylalanine was essentially completely inhibited in sodium uptake medium by L-phenylalanine, L-alanine, and L-methionine, whereas of these three only L-alanine and L-methionine showed reduced inhibitory capacity in the K medium. Glycine had intermediate inhibitory capacity in both uptake media, whereas L-proline displayed intermediate capacity in the sodium medium and a low inhibitory capacity in the K medium. Glycyl-L-phenylalanine only weakly inhibited L-phenylalanine influx in either medium. This pattern of L-phenylalanine influx inhibition by these various compounds was similar to that reported for L-phenylalanine influx into rabbit BBMV (30)) suggesting similar pathways in this mammal and in fish. DISCUSSION

This report describes basic transport properties of the dipeptide glycyl-phenylalanine and the amino acid Lphenylalanine in purified BBMV of upper intestinal epithelia of the euryhaline teleost 0. mossambicusacclimated to seawater. The results demonstrate that the dipeptide and its component amino acids are transported by very different mechanisms and provide some insight into the variety of mechanisms and relative contribution of dipeptide transport to total dietary amino nitrogen uptake in fishes. General characteristics of uptake. Previous work from our laboratory and elsewhere has shown that purified 3. Effects of free amino acids and dipeptide on [14C]glycyl-L-phenylalanine and Na- and K-dependent L-[3H]phenylalanine influxes by tilapia intestinal BBMV TABLE

%Inhibition Inhibitor

(40 mM)

Phe Gly-Phe Na

None (mannitol) MeAIB Phe GlY Gly-Phe Ala Met Pro

0 lOOt6 41t8 27t3 96k9 91t10 44t5

K

5&Z 100t4 4955 32t6 60t6 76t5 27t3

12t3 St2 64k7 6k2 924 15k5

Values represent means t SE (n = 3 experiments) of %inhibition of control influx rate (i.e., in presence of 40 mM mannitol), which was 230 k 6, 260 k 3, and 370 & 15 pmolmg protein-‘*5 s-’ for K- and Na-dependent L-phenylalanine (Phe) (0.1 mM) and dipeptide (0.36 mM), respectively. MeAIB, cy- (methylamine) isobutyric acid.

IN

FISH

BBMV

R567

BBMV isolated from fish intestine exhibit Na-dependent nutrient transport where entry of the organic solutes is believed to be energetically linked to the transmembrane sodium gradient created and maintained by a basolateral Na-K-adenosinetriphosphatase (14, 26, 29, 32). In the present investigation, 0. mossambicus also exhibited a strong Na-dependency for apical membrane L-phenylalanine transport in upper intestines (Fig. 2). Furthermore, in this fish species, K was also able to potentiate Lphenylalanine transport into the vesicles but at a lower initial rate than in Na medium and without displaying the overshoot phenomenon. In general, this mechanism is similar to the L-phenylalanine uptake system reported by Berteloot et al. (9) for mouse intestinal BBMV. These data, together with those from others (14,29,31), suggest that the same basic coupled transport mechanisms for amino acid transfer appear to be present in fish from a variety of environments, as has been described for nutrient absorption in mammal gut (12, 27). Transport of the dipeptide glycyl-L-phenylalanine into tilapia intestinal BBMV was, on the other hand, independent of all inwardly directed cation gradients (Fig. 1, A and B), suggesting that uptake occurred by a facilitated diffusion mechanism. Dipeptide transport by such a mechanism runs counter to the currently accepted proton-gradient cotransport mechanism for dipeptides reported from mammalian intestine and kidney (16, 17, 19). However, the facilitated diffusive transport of glycylL-phenylalanine in the present report is similar to the mechanism reported by Rajendran et al. (25) for glycylL-proline in mice, rabbit, and human intestinal BBMV, for glycylsarcosine in rabbit proximal tubules (6), and for glycylglycyl+proline in human jejunum (34). That a similar cation-independent mechanism has been observed for different di- and tripeptides in two dissimilar vertebrate groups provides strong evidence for a heterogeneous nature of peptide transport mechanisms, some being energized by cation gradients and others apparently lacking coupled transport. The basic transport characteristics described in this paper suggest that in tilapia intestine there is a carrier process for the dipeptide glycyl+phenylalanine that is independent of the transport systems of the constituent amino acids. This conclusion is supported by both influx kinetics of the two solutes and by the pattern of cis inhibition of their influxes by a variety of nitogenous compounds. Kinetic characteristics. Both dipeptide and L-phenylalanine influxes by tilapia intestinal BBMV occurred by the sum of at least one carrier process exhibiting Michaelis-Menten kinetics and apparent simple diffusion (Figs. 3 and 4 and Table 2). The kinetic constants for both Na- and K-dependent L-phenylalanine influx (Table 2) were similar to the kinetic constants reported for L-phenylalanine in a number of mammalian tissues (21, 30). The present results provide further support to previous reports on the similarity of teleost and mammalian apical amino acid transport systems (14,31). The kinetic constants observed for glycyl+phenylalanine influx (Table 2) were significantly different than those for L-phenylalanine, the former displaying a much lower affinity and higher maximal influx rate. These values for


R568

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teleost dipeptide concentration-dependent kinetic constants were also similar in magnitude to those reported in mammals for a number of nonhydrolyzed dipeptides or for hydrolyzed dipeptides after the vesicles were treated with papain to inhibit peptidases. This suggests that similar intestinal nutrient conditions and transport systems between the various vertebrate groups also seem to apply to dipeptides. The approximately twofold greater apparent diffusional permeability of tilapia intestinal BBMV to L-phenylalanine than to glycyl+phenylalanine (Table 2) is consistent with the larger size and complexity of the dipeptide and supplies additional evidence that these two compounds move across this membrane as dissimilar entities. The transport mechanisms for L-phenylalanine and the dipeptide were further distinguished from each other by their behavior in the presence of a variety of nitrogencontaining compounds in the extravesicular uptake medium. L-Phenylalanine influx (Table 3) was inhibited by various cis amino acids and dipeptides to an extent and in a pattern similar to what has been reported for this amino acid in mammals (30). Importantly, the amino acids glycine and L-phenylalanine exhibited very little capacity to inhibit glycyl+phenylalanine influx, while being able to inhibit L-phenylalanine influx by 40 and 100%) respectively. Furthermore, the dipeptide was able to self-inhibit its influx approximately twofold more effectively than it could inhibit L-phenylalanine influx. This pattern suggeststhat for cis inhibition of dipeptide influx either the peptide bond or some stereochemical specificity of the ligand-carrier interaction is critical for recognition. This specificity prevented significant interaction of the constituent amino acids (glycine, L-phenylalanine) with the dipeptide carrier to the extent that they exhibited only about a 10% inhibitory capacity. Inhibition of L-phenylalanine influx by the dipeptide was to a lesser extent than even inhibition by glycine, suggesting again that some aspect of the structure of the integral dipeptide was important for its function. These inhibition data support the hydrolysis data (Table I), which indicated that little hydrolysis occurred extravesicularly. In summary, the data from the present study suggest that in intestinal BBMV of the teleost 0. mossambicus glycyl+phenylalanine is transported intact by a cationindependent facilitative diffusion mechanism during which the dipeptide is hydrolyzed to its component amino acids. The dipeptide transport system was independent of those for L-phenylalanine, demonstrating different ion dependencies, kinetic characteristics, and interactions with other nitrogenous compounds. The relevance of facilitated diffusion for the transport of glycylL-phenylalanine is supported by the fact that this dipeptide was rapidly hydrolyzed into its component amino acids only intravesicularly (Table l), thereby maintaining a steep downhill concentration gradient necessary to keep transport energized in a manner analogous to group translocation, as described for many bacterial transport systems (5). In mouse intestinal BBMV where there was extensive extravesicular hydrolysis, glycyl+phenylalanine was found to be transported predominately via the compo-

IN

FISH

BBMV

nent amino acid pathways (7). However, papain treatment, which inhibited only dipeptidase activity, permitted observation of an intact dipeptide pathway similar to that reported in the present study. These data suggest that the predominant dipeptide pathway(s) present in a particular tissue may depend on the pattern of existing dipeptidase activity. This supports the conclusions of Cheeseman and Parsons (11) and Silk et al. (28), postulating that dipeptide uptake may be cation dependent only when it is concentrative and that when going down its electrochemical gradient (i.e., after rapid internal hydrolysis) facilitated diffusion is a sufficient mechanism. Quantification of the proportion of protein-derived nitrogen absorbed by the intestine as free amino acids and as peptides is one of the remaining problems in the study of peptide absorption. The present paper supports an earlier report (22) that physiologically intact peptide absorption is comparable to, if not surpassing, the uptake of amino acids (Fig. 4, Table 2). Both the low dipeptide transport affinity (Km) and the high maximal uptake rate (Jmax) could support very high rates of intact glycyl-1;phenylalanine uptake in early stages of digestion when concentrations of peptides in the intestinal lumen are high (23) with the peptide fraction consisting mainly of 2-4 amino acid residues (1). The higher affinity and lower maximal uptake in this study for L-phenylalanine and for intestinal L-phenylalanine transport in general (21) suggest that amino acid absorption may be a scavenger activity after the bulk of nitrogenous digestive products have already been absorbed as peptides. This relatively significant role in absolute absorption, together with the diversity shown in transport and hydrolysis mechanisms, highlights the necessity of further work in elucidating dipeptide transport pathways and their relationships to hydrolysis, diet, and nutrition. This investigation Grants PCM83-19973 Address for reprint The Mall, University Received

16 October

was supported by National Science Foundation and DCB87-15278. requests: G. A. Ahearn, Dept. of Zoology, 2538 of Hawaii at Manoa, Honolulu, HI 96822. 1990; accepted

in final

form

5 November

1990.

REFERENCES 1. ADIBI, S. A., AND D. W. MERCER. Protein digestion in human intestine as reflected in luminal, mucosal and plasma amino acid concentrations after meals. J. Clin. Inuest. 52: 1586-1594, 1973. 2. ADIBI, S. A., AND Y. S. KIM. Peptide absorption and hydrolysis. In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1981, p. 1073-1095. 3. ALPERS, D. H. Uptake and fate of absorbed amino acids and peptides in the mammalian intestine. Federation Proc. 45: 22612267, 1986. D. H. Digestion and absorption of carbohydrates and 4. ALPERS, proteins. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 1469-1487. 5. ALBERTS, B., D. BRAY, J. LEWIS, M. RAFF, K. ROBERTS, AND J. D. WATSON. Molecular Biology of the Cell. New York: Garland, 1983, p. 297-298. 6. BARFUSS, D. W., V. GANAPATHY, AND F. H. LEIBACH. Evidence for active dipeptide transport in isolated proximal straight tubules. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F177F181, 1988. 7. BERTELOOT, A., A. H. KHAN, AND K. RAMASWAMY. Characteristics of dipeptide transport in normal and papain-treated brush border


PEPTIDE

8.

9.

10.

11.

12. 13.

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Population Genomics of the Euryhaline Teleost Poecilia latipinna.

Isolation and mRNA expression analysis of aquaporin isoforms in marine medaka Oryzias dancena, a euryhaline teleost.

K+-ATPase in the Euryhaline Teleost, Tetraodon nigroviridis.

Factors affecting in vitro activity of prolactin cells in the euryhaline teleost Sarotherodon mossambicus (Tilapia mossambica).

Endocrine effects on nitrogen excretion in the euryhaline teleost, Salmo gairdnerii.

Ion transport of marine teleost intestine.

Correlation of structure and active transport in the teleost nephron.

WNK1 and p38-MAPK distribution in ionocytes and accessory cells of euryhaline teleost fish implies ionoregulatory function.

Effects of environmental salinity and of hormones on urinary bladder function in the euryhaline teleost, Gillichthys mirabilis.

Cloning, functional characterization and nutritional regulation of Δ6 fatty acyl desaturase in the herbivorous euryhaline teleost Scatophagus argus.

The effect of prolactin on kidney structure of the euryhaline teleost Gasterosteus aculeatus during adaptation to fresh water.

Salt-induced changes in sodium transport across the skin of the euryhaline toad, Bufo viridis.

Freshwater adaptation in the euryhaline teleost, Chelon labrosus. II. Effects of continuance of adaptation, cortisol treatment, and environmental calcium on water influx in isolated gill.

Teleostean urophysis: urotensin II and ion transport across the isolated skin of a marine teleost.

The inner opercular membrane of the euryhaline teleost: a useful surrogate model for comparisons of different characteristics of ionocytes between seawater- and freshwater-acclimated medaka.

Methods of investigating intestinal transport.

Intestinal electrolyte transport and diarrheal disease.

Regulation of intestinal glucose transport.

Intestinal transport and metabolism of bile acids.

Intestinal transport of tryptophan and its analogs.

Human intestinal ion transport in vitro.

Intestinal phosphate transport in familial hypophosphatemic rickets.

Intestinal uptake and transport of proteins in the adult rat.

Neural blockade in basal and postreceptor-stimulated intestinal transport.