J. Physiol. (1975), 250, pp. 409- 429 With 8 text-ftgures Printed in Great Britain

409

AMINO ACID MOVEMENTS ACROSS THE WALL OF ANURAN SMALL INTESTINE PERFUSED THROUGH THE VASCULAR BED

BY C. A. R. BOYD,* C. I. CHEESEMAN AND D. S. PARSONS From the Department of Biochemistry, South Parks Road, Oxford, OX1 3QU

(Received 10 February 1975) SUMMARY

1. L-leucine transfer across the wall of the small intestine has been studied in a vascularly perfused preparation from four species of frog. Some properties of the preparation are described. 2. A description is given of the endogenous amino acids appearing in the vascular bed and of the kinetic properties of this washout of endogenous L-leucine. 3. In the steady state of absorption, the transfer function relating the net flux of exogenous L-leucine into the vascular bed to the concentration in the lumen exhibits saturation. Under the conditions of the experiments the apparent concentration in the lumen for half-maximum transfer of L-leucine is found to be 2-1 + 0 4 (5) mM. 4. When Na ions are removed from the lumen the transfer of L-leucine into the vascular bed is inhibited. However, the additional removal of Na ions from the fluid in the vascular bed is further inhibitory to the transfer of the amino acid. 5. L-leucine previously absorbed from the lumen appears in the vascular bed in a biphasic fashion. Estimates are deduced of the size of the pool of L-leucine within the tissue which drains into the vascular bed. 6. These results are discussed in relation to previous work on amino acid transport undertaken with various sorts of preparation of small intestine. INTRODUCTION

When the intestinal absorption of amino acids and sugars is studied using classical in vitro techniques there is marked accumulation of the absorbed substance within the intestinal wall (see Fisher & Parsons, 1953; Agar, Hird & Sidhu, 1954; McDougall, Little & Crane, 1960; Wilson, * MRC Junior Research Fellow.

C. A. R. BOYD AND OTHERS 410 1962). An important question which has not been resolved in quantitative terms is the extent to which the properties of the intestinal mucosa with respect to absorption under in vitro conditions, in which accumulation occurs, differ from those obtained under in vivo conditions with a blood supply to the mucosal epithelium, when the accumulation might not be so extensive. In particular two questions are of interest. What is the size of the pool of transported substrate, amino acid or sugar, under conditions of vascular perfusion and to what extent does the rate of perfusion determine the size of this pool? A second important question concerns the now well known dependence of sugar and amino acid transport in the intestine upon the presence of Na ions in the intestinal lumen. There is much evidence that under in vitro conditions the dependence of transport upon Na ions is marked (for review see Schultz & Curran, 1970). Under conditions simulating those in vivo the findings are less clear-cut; see, for example Forster (1972), Fisher & Gardner (1974a). In the work reported here we have attempted to investigate these questions in the case of the amino acid L-leucine using the vascularly perfused intestine of the frog as described by Parsons & Prichard (1968). In the present experiments the original procedure has been modified in a number of ways, the most important of which is the cannulation of the portal vein to permit continuous sampling of the vascular effluent from the tissue. This has been achieved by leaving the intestine in situ in the animal. The procedure has been demonstrated to the Physiological Society (Boyd, Cheeseman & Parsons, 1973). METHODS

AnimaiB Rana pipien8, weight range 26-66 g, were supplied by Carolina Biological Supply Co., Burlington, North Carolina, U.S.A. Rana cate8beiana, weight range 150-320 g, were supplied by Mogel Ed, Osh Kosh, Wisconsin, U.S.A. Rana temporaria, weight range 20-40 g, were supplied by Gerrard and Haig Ltd, East Preston, U.K. Rana ridibunda, weight range 40-150 g, were supplied by Maved, Budapest, Hungary. R. catesbeiana, pipiens and temporaria were kept at 200 C in tanks containing a few stones and through which a trickle of tap water was run continuously. Rana ridibunda were kept in a tank with 0 3 m depth of running water and with access to a dry ledge. All frogs were fed on live maggots, usually twice a week. Operative procedure After pithing, the animal was placed on a metal plate and the anterior abdominal vein ligated at both ends before opening the abdominal wall from the pelvis to the xiphisternum. Both clavicles were cut through and the sternum removed. After making appropriate windows in the mesentery, the vessels leading to and from the stomach and pancreas were tied off. The spleen was ligated unless this compromised

AMINO ACIDS AND INTESTINE

411

the coeliaco-mesenteric artery (usually in animals of body weight below 40 g). This procedure stopped contamination of the vascular effluent with blood. Displacement of the intestine exposed the systemic arches of the aorta and the coeliaco-mesenteric artery, allowing loose ligatures to be placed around the left systemic arch and the coeliaco-mesenteric artery. At this point the major part of the stomach could be removed giving better access to the blood vessels to be cannulated. Reflexion of the liver exposed the portal vein which runs through the pancreas from the mesenteric bed; a loose ligature was placed around the pancreas and vein where they are free from the liver. To facilitate the thorough flushing out of the intestinal lumen, the distal end of the colon was opened. The stomach was opened or divided and the intestine gently irrigated with a minimum of 20-40 ml. frog Ringer. Introduction of a cannula through the pylorus was aided by dividing the circular and longitudinal muscle coats. Before inserting the arterial cannula into the coeliaco-mesenteric artery via the left systemic arch, an incision was made in the portal vein at its junction with the vessels leading from the liver. The tip of the arterial cannula was now advanced to a point beyond the gastric branch of the coeliaco-mesenteric artery and then firmly tied in place. Thus at no time did the intestine have its supply of either blood or perfusate interrupted. The duodenal end of the small intestinal lumen was cannulated with a nylon tube which was firmly tied in. Similarly the distal end of the ileum was cannulated via the colon, the blood supply to the latter having been tied off with care to avoid the nearby coeliaco-mesenteric artery. Finally the portal vein was cannulated using the anterior abdominal vein ligature as an aid to opening the i rcision which had been made previously. This cannula was then positioned to give optimal portal flow. At this stage the small intestine had a closed vascular circulation separate from the rest of the animal and the small intestine was cannulated permitting luminal flow.

Perfusion of vascular bed The vascular perfusate (for composition see Table 1) previously filtered through a 14 num Millipore filter (Millipore Corporation, Bedford, Mass. U.S.A.), was continuously gassed with 95 %/5 %, v/v, 02, CO2 mixture within the reservoir RB (Fig. 1) and maintained at 250 C. A multichannel Gilson Miniplus II peristaltic pump (Anachem Ltd, Luton, Beds) passed this solution via nylon tubing (0-6 mm i.d.) to a glass bubble trap B1 and on to a second trap B2. Between B1 and B2 the tubing passed through a small bubble sensing head (BA) which was connected to the control unit of an alarm system. The arterial cannula (03) was made from drawn out nylon tubing (initial internal diameter 0-6 nun) pulled out into an arc of 1800 with the end cut obliquely. Collection of fluid from the portal vein was also by means of a nylon tube, but this was not tapered and the end, inserted into the vein, was square cut to reduce occlusion of the vein. This cannula (C4) fitted into a tube leading to a Gilson Microcol TDC 80 fraction collector set in the drop mode. The height of the vascular collection outlet was adjusted to give a negative hydrostatic pressure.

System for luminal flow (Fig. 1) In order to maintain constant conditions within the intestinal lumen, single pass flow was employed. Solutions (for composition see Table 1) from the reservoirs RL1 and RL2 (maintained at 250 C) were pumped with a Gilson Miniplus peristaltic pump via nylon tubing to a bubble trap B3 and on to the luminal cannula C,. A constant hydrostatic head of pressure within the intestinal lumen (5-10 cm saline)

0. A. R. BOYD AND OTHERS

412

Lc

C2 Vc

Fig. 1. Rv and RL, vascular and luminal reservoirs; Pv and PL, vascular and luminal pumps; B1, B2 and B3 bubble traps; BA, bubble alarm; M, Manometer; CI and C2, luminal cannulae; C3, cannula in mesenteric artery; C4, portal venous cannula; V/c, vascular collection with fraction collector; Le, luminal collection with fraction collector, at constant pressure, h, of 5-10 cm saline. Tip of C4, 5-10 cm below C1, C2 and 0a. TABLE 1. Composition of luminal and vascular fluids. The ionic composition of the vascular perfusate was that of the sodium-containing luminal fluids unless otherwise specified. In addition it contained bovine serum albumin, 0-5 g 100 ml.-' and either sodium butyrate (1 mM) or D-glucose (2 mM)

Composition of luminal fluids Frog Ringer a

NaCl KC1 MgSO4 MgCl2 CaCI2 NaHCO3

with sodium 93-0 5-0 1-0 0-8 0-5 25-0

98-0 1.0 0-8

0-5 25-0

KHCO3

Na2HPO4

2-15

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0-85

Gas (v/v)

02 95%

2-15 0-85

CO2 5 % pH (gassed at 25° C)

b sodium free

7.4

02 95%

CO2 5% 7.4

AMINO ACIDS AND INTESTINE

413

which eliminated peristalsis, was maintained by adjustment of the outlet tube C2. The outflow from this tube could be delivered to waste or to a fraction collector depending upon the experiment. Thus a constant flow rate past the mucosal tissue could be achieved throughout the duration of the experiment. The pressure within the lumen had no effect upon the portal effluent flow rate unless it was greater than 15 cm saline.

Materials Bovine Serum Albumin, Fraction V, was supplied by Miles Laboratories Ltd, Stoke Poges, England. Dextran and D- and L-leucine, sigma grade, were obtained from Sigma, London, and the PVP (polyvinylpyrrolidone) from B.D.H., Poole, England. 'Bio-Solve' BBS3 was from Beckman, Croydon, England, and the [1-14C]L-leucine and [5-3H]D-leucine were from the Radiochemical Centre, Amersham, England. The nylon tubing of various sizes was obtained from Portex Ltd, Hythe, Kent, England. Analytical methods (a) L-leucine was routinely estimated by the L-amino acid oxidase-peroxidase technique (Fujita, Parsons & Wojnarowska. 1972). Standards were made up in vascular solutions; deproteinization was not necessary. (b) For the estimation of fifteen amino acids, including L-leucine, liberated during the washout of endogenous a-amino nitrogen from the intestine, an amino acid analyzer was used (Locarte Bench Model). After deproteinization with tungstic acid (Sardesai & Provido, 1970) the supernatant was loaded directly on to the analyzer as the pH of the solution was the same as that of the loading buffer (pH 2.2). (c) For the few experiments in which both the D and L stereoisomers of leucine were to be estimated, liquid scintillation counting by routine methods was used. The scintillant was toluene based and contained 10% 'Bio-Solve' thus avoiding deproteinization (Carter & van Dyke, 1971). Quenching was measured by channels ratio. The counting was performed on a Wallac liquid scintillation counter with an efficiency of greater than 90 % for 14C and of greater than 40 % for 3H.

Estimation of tissue length, wet and dry weight The intestine was opened along its antimesenteric surface, blotted well with absorbent paper and its linear dimensions measured at its resting length. It was then weighed in a tared vessel before and after drying to constant weight in a hot air oven.

Calculation and expression of results These were as described by Parsons & Prichard (1968). The conduct of the experiment was such that each animal acted as its own control. All results with respect to transport into the vascular bed were calculated on the basis of 100 % portal recovery of the arterial infusate from the portal vein, as it was found that the composition of any small amount of fluid escaping around the intestine was very similar to that appearing in the portal vein. Those few experiments in which portal recovery fell appreciably were abandoned. The rates of vascular and luminal inflow were determined before and after each experiment, and in subsequent calculation the mean of the two values used (the difference between the two values was never greater than 1 % of the flow rate). Leucine refers to L-leucine unless otherwise stated.

414

C. A. R. BOYD AND OTHERS RESULTS

Gross properties of the intestine used are shown in Table 2 Properties of perfumed vascular bed We have found that the failure to achieve consistent and high portal effluent flow reported by Parsons & Prichard (1968) is largely due to mechanical factors. Occlusion of the portal vein, resulting in portal hypertension, rapidly reduces the rate of collection from the vein. This may be overcome by careful and accurate positioning of the portal cannula within the vessel. For this reason the intestine was left in situ, and the portal cannula was mounted on a magnet (see Parsons & Powis, 1971). Thus, accurate alignment of the two was established and small adjustments to the cannula were readily made if required. Two further features were found necessary for the achievement of high rates of portal recovery; the tip of the cannula was square cut (not bevelled) minimizing the problem of occlusion against the wall of the vein; a negative hydrostatic pressure of -5 to -10 cm saline was exerted by having the tip of the collecting tube below the portal cannula, this pressure being sufficient to overcome the frictional resistance associated with the flow within the narrow tube. The problems associated with embolism, both particulate and gaseous, of the capillary bed were avoided by careful filtering of all arterial solutions, and by use of bubble traps. A 14 /tm Millipore filter was used (the capillaries of the frog are some 20 ,tm in diameter, see Intaglietta & Zweifach, 1974), and two bubble traps were included in the vascular system. As an aid to detection of small gas bubbles, in later experiments a 'bubble alarm' (a photo-electric bubble detector coupled to a tone generator) was mounted between bubble traps B. and B2; this reduced the degree of 'supervision' that the preparation required when established. With use of these precautions, the fractional recovery of the arterial infusate from the portal vein ('portal recovery') was consistently better than 0 95, often approaching 1 00, over a wide range (1-15 ml. min- g dry wt.-') of the arterial flow rates. Routinely, however, a vascular flow of approximately 5.0 ml. min- g dry wt.-' was used for all species, this being based on an estimate of portal flow rate in vivo of Rana temporaria (see Parsons & Prichard, 1968). The vascular perfusate (see above) was well gassed with 95 % 02/5 % CO2 (v/v). Calculation showed that enough 02 in physical solution was delivered to the tissue to account for the maximal rate of glucose utilization observed (Parsons & Prichard, 1968). Although we found no evidence that the presence or absence of metabolizable substrate affected portal recovery, steady rates of transport across the epithelium were found when

AMINO ACIDS AND INTESTINE

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416 C. A. R. BOYD AND OTHERS either glucose or butyrate was present, and one of these was used depending upon the experimental situation. In order to maintain the initial high values for portal recovery over a period of several hours it was found that the addition of albumin to the vascular infusate was essential. Other colloids, PVP (polyvinylpyrrolidone molec. wt. 36,000 and 44,000) and Dextran (molec. wt. 86,900) were markedly less effective at concentrations up to 3-5 g 100 ml.-'. The effect of albumin upon portal recovery was tested over a range of concentrations 1.0 oo 0~ 00000 0o 0000000000000 00 0 0 00

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Time (hr) Fig. 2. Portal vein collection rate expressed as a fraction of the arterial infusion rate which was 3-84 ml. min-' g dry wt-'. At time 6 hr 30 min the albumin (1 g 100 ml.-') was removed from the arterial infusate and at time 7 hr it was replaced. R. temporaria.

and from Fig. 2 it is clear that 0'5 g 100 ml.-' can maintain a constant recovery. The complete removal of the albumin from the infusate resulted in an immediate drop in the portal recovery which, over a short time course, was completely reversible (Fig. 3): after longer periods of perfusion with albumin-free Ringer the recovery upon reintroducing an albumin containing perfusate was only partial. Subsequent experiments showed that the addition of albumin 0-5 g 100 ml.-' could maintain the portal recovery for periods lasting up to 9 hr. Analysis of vascular effluent Examination of the blood-free fluid recovered from the portal vein showed that despite the absence of addition to the luminal fluid or vascular infusate, appreciable quantities of amino acid appeared. The washout of endogenous amino acids was investigated, analysis being performed on an amino acid analyser.

417 AMINO ACIDS AND INTESTINE Table 3 shows the relative rates of appearance of some of these amino acids during the initial 30 min of vascular and luminal flow. Of those tested, only alanine comes out at a rate faster than glycine, while serinc, threonine, glutamate, and leucine also appear at appreciable rates. To 1.0

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3 4 5 Time (hr) Fig. 3. Influence of albumin concentration in the arterial infusate upon the rate of collection of fluid from the portal vein. Arterial infusion rate was 2-80 ml. min- g dry wt-'. Values are of albumin concentration, g 100 ml-'. infusate. R. catesbeiana. 1

2

TABLE 3. Relative rates of appearance of endogenous amino acids in vascular bed during initial 30 min of vascular perfusion. Values are of means + S.E. of mean of washout rates relative to glycine = 1l00. Mean rate of glycine washout = 2X75 + 0-8 (ten animals) smole g dry wt.-1 hr-1. R. temrporaria

Amino acid Alanine Glycine Serine Threonine Glutamate* Leucine Valine Isoleucine Tyrosine Phenylalanine Cysteine Aspartate Proline Methionine *

Rate of appearance 1-54 + 0O11 1l00 0 61 +0-08 0.59 + 010 0-59 + 0-05 0-56 + 0-08 0-40 + 0-05 030 + 0 04 0-27 + 0-02 0-27 + 0-02 0-22 + 0-22 0-18 + 0 04 Trace Trace

May include small amount of citrulline.

No. of observations 10 9 9 9 6 6 6 6 5 2 6 6 6

418 C. A. R. BOYD AND OTHERS examine more fully the kinetics of the endogenous L-leucine washout, the vascular effluent samples were analysed using L-amino acid oxidase. Initial experiments showed that over 90 % of the reagent positive reaction was accounted for by the presence of L-leucine. In Fig. 4a the accumulated total amount of endogenous leucine appearing in the vascular effluent is shown over a period of more than 4 hr. 8 .0

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419 AMINO ACIDS AND INTESTINE The time course of the leucine washout may be closely described by the monoexponential function, Qt ekt -

QOD

(Atkins, 1969, p. 32), where Qt is the total quantity of leucine that has appeared by time, t; QOO is the total amount of leucine that would appear 0

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after an infinite time (i.e. the asymptote) and k is equal to the rate constant for exit from this compartment (see Fig. 4b). Thus the leucine appears to be coming from a single compartment, with a rate constant k, equal to the negative slope of the semilogarithmic plot. The dependence of this rate constant upon both vascular flow and upon vascular albumin concentration is shown in Figs. 5 and 6. Estimation of Q.,, (the initial amount of amino acid in the pool) and of tissue water enables a 'tissue concentration' to be derived. For L-leucine this was 2-2 + 0.5 (6) mM; for glycine it was 3*4 + 0*7 (3) mm.

Properties and analysis of luminal effluent In these experiments a single pass system was employed; however recirculation by a gas lift as described by Parsons & Prichard (1968) may be used. The composition of the luminal fluid was the same as that of the

C. A. B. BOYD AND OTHERS concentration is shown in Fig. 7a and is clearly linear, showing saturation vascular infusate (Table 1) except for the omission of albumin and substrate. Gassing ensured an alternative supply Of 02 to the tissue. It was found that the maintenance of a constant luminal flow was dependent upon a sufficient distension pressure (hydrostatic head of 5-10 cm saline) to eliminate peristalsis. Luminal flow rates covered a wide range 420

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Fig. 6. The effect of changing the albumin concentration in the arterial infusate upon the rate of appearance of endogenous leucine in the portal venous effluent. K = rate constant of washout. Numbers represent g albumin 100 ml.-' infusate. R. cate8beiana.

(5-50 ml. min- g dry wt.-') depending upon the experimental situation. Provided that the lumen was initially well irrigated to remove debris, analysis of the subsequent luminal effluent showed negligible washout of amino acids, despite the appearance in appreciable amounts of amino acids in the vascular effluent. Transfer of leucine from the lumen (a) Steady state. Under these conditions leucine appears in the vascular effluent at a rate which is greater than that from endogenous amino acids. For a given luminal concentration this rate of appearance was found to be the same as that up to 6 hr later. The dependence of the steady-state rate of leucine transfer upon luminal

AMINO ACIDS AND INTESTINE

421 Lumen Na+ - present (control)

Lumen Na+ free

Luminal concentration L-leucine (mM) 140

120

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V/s (ml. g dry wt.-1 hr-') Fig. 7. a, influence of sodium in lumen on steady state of appearance of leucine in the vascular effluent. Control curve (open circles) derived from five experiments, sodium-free curve (filled circles) derived from three experiments. Values are of means + 1 s.E. of mean, b, Hanes plot of the data presented in Fig. 7a. 8, concentration of L-leucine in lumen (mM). Open circles are control data, triangles are sodium-free data. R. pipiens.

422 C. A. R. BOYD AND OTHERS at higher concentrations. Analysis on the basis of Michaelis-Menten kinetics plotted according to Hanes (1932) (Fig. 7b) of the function relating transfer to luminal concentration gives a maximal transfer rate (Vmax) of 151 + 12 (5) ,tmole g dry wt.-' hr-4 and a luminal half saturation concentration (Kt) of 2-1 + 0.4(5) mM. The rate of transfer was reduced by the replacement of luminal sodium by potassium (Fig. 7a). A similar kinetic analysis (Fig. 7b) shows this to be a complex effect upon the transfer of leucine across the epithelium with a reduction of Vmax to 91 + 3 (3) cmole g dry wt.-' hr-1 and an increase in the Kt to 45 + 1x5(3) mM. TABLE 4. Effects of replacing sodium in experimental fluids on transfer of leucine from luminal fluid into vascular bed. Concentration of leucine in luminal fluid, 5*0 mM. Sodium was replaced by potassium according to Table 1. Values are of means + S.E. of mean of rates of transfer relative to control values measured in the same animal with sodium present in both lumen and vascular fluids. The percent inhibition is also given. Data from three animals. R. pipiens. The mean control rate of leucine transfer found for the three animals was 79 7 + 190 molee g dry wt-'. hr-1.

Sodium in fluid in A

Lumen +

+

-

Rate of transfer of L-leucine as % Vascular bed of control rate 100 + 57+3 +

-

73+5 23±6

Inhibition of leucine transfer (%) 0

43+3 27+5 77+6

Table 4 shows that the replacement of sodium in the vascular perfusate is also inhibitory; furthermore profound yet reversible inhibition is observed when sodium is simultaneously removed from both perfusates. The transfer of leucine, measured by scintillation counting, showed stereospecificity, the L-isomer being transferred by R. catesbeiana at a rate 2-34 (range 2.3-2.46) times greater than its D-isomer when both were present at a concentration of 2 mm in the lumen. Also in R. catesbeiana the transfer of L-leucine in the steady state (1 mm in the lumen) was inhibited from 63-0 + 3.0(6) to 45-6 + 0.3(6) ,tmole g dry wt.-I hr-1 by the addition of 10 mM L-alanine in the lumen. (b) Non-steady state. In the semi-logarithmic plot shown in Fig. 8, the fall in the rate of vascular appearance of leucine (as the luminal concentration falls) appears to show two phases; an initial fast fall is followed by a slower fall. The slope of the slow component is similar to that of the endogenous washout. For a series of eight frogs (R. catesbeiana) the rate constant for endogenous leucine washout was 4-8 + 0 4 x 10-3 min- and for the slow component of leucine from the lumen it was 5-6 + 1-4 x 104 min'. The means of these results are not significantly different (P = 0.4).

423 AMINO ACIDS AND INTESTINE These data can also be used to discover an apparent size of the pool in the tissue (cf. Andersen & Zerahn, 1963; Cuthbert, 1971). Following exposure of the intestine to leucine in the lumen the 'unloading pool' was estimated to be 2-67 + 0*14 Itmole g dry wt.-'. In contrast a maximal estimate of the pool made from data obtained during the loading of the tissue from the luminal solution containing the same leucine concentration (1 mM) gave a size of 1*48 + 0*17 pmole g dry wt.-'. Thus under unloading conditions the tissue pool size appeared to be twice that found during the loading of the amino acid. 250

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Fig. 8. The rate of appearance of leucine in the portal venous effluent following exposure of the intestinal lumen to a pulse of leucine (1 mm) of 20 min duration. Semilogarithmic plot. R. catesbeiana.

DISCUSSION

Vascular perfusion Many authors have noted the problems associated with classical in vitro techniques (Parsons, 1968; Smyth, 1974). One of the major problems associated with all such techniques, the accumulation of transported substrate within the tissue, can be avoided by using a preparation in which the vascular capillary bed of the epithelium is perfused, thus allowing the experimenter to control not only the composition of the fluid bathing the luminal face of the cell, but also to control that of the fluid at the base.

424 C. A. R. BOYD AND OTHERS Parsons & Prichard (1968) collected the effluent from the arterial infusate both as tissue sweat and on occasions attempted to collect portal vein effluent. They attributed the relatively low and variable fraction of fluid flowing from the vein to the extensive lymphatic network in the frog. However we have shown that provided suitable simple procedures are adopted, a consistently near-complete recovery of the arterially infused fluid may be collected from the portal vein, and we believe that this represents a significant improvement in the technique. For example it permits rapid sampling of the composition of the portal effluent in non-steady state situations (see Parsons, 1975). Other advantages resulting from the use of frog intestine are that the preparation may be run at room temperature and the metabolic requirements of the tissue can be met without the use of erythrocytes in the vascular perfusate. In addition this intestinal preparation will transport substrate at a constant rate over many hours, thus permitting both control and experimental procedures to be conducted on a single animal, a significant improvement over the frequently brief viability of vascularly perfused mammalian small intestinal preparations. The operative procedure involved is relatively simple, the preparation taking some 30 min to establish. It is emphasized that the anatomy of the aortic arch of the frog permits arterial cannulation without any interruption of blood flow. In addition the rate of intestinal fluid transport in the frog is extremely low. This is in contrast to the mammal in which allowance has often to be made for the absorption of fluid from the lumen when calculating the rate of substrate transfer. This technique may be used on a number of different species of frog varying over a wide range of body weight, although frogs below 30 g body weight are much more difficult to prepare successfully. The data in Table 2 show that over a range of species and sizes, intestinal dry weight remains a relatively constant fraction of body weight. Intestinal length and water content generally appear to be greater in the female than in the male frogs with the exception of R. ridibunda for which the male and female body weights were not equivalent. Fisher (1955) noted a similar effect of sex on intestinal length in the rat, which recently has been investigated further by Cripps & Williams (1975). The requirement of the artificially perfused organ for a plasma colloid to prevent oedema is well known (see Ross, 1972). Our results confirm those of Parsons & Prichard (1968) in showing that albumin is greatly superior to PVP or Dextran in this respect. We have investigated the effect of albumin concentration on portal recovery and that concentrations of 05 g albumin 100 ml.-' or greater are effective in retaining fluid in the vascular bed. Mason, Michel & Took (1973) investigating single capillaries in frog

AMINO ACIDS AND INTESTINE 425 mesentery have shown a minimum requirement of 0.1 g albumin 100 ml.-' necessary to maintain a normal capillary filtration coefficient. In this context it is interesting to note that Feldhoff (1971) gives a figure of 0*84 g albumin 100 ml-'. plasma as the concentration in the blood of the young adult bullfrog (R. catesbeiana). The transfer of leucine: endogenous and exogenous That ax amino-N appears in the serosal fluid bathing in vitro preparations of intestine has been described for the frog (Gagnon, 1960) and rat (Fisher & Gardner, 1974b, Parsons & Volman-Mitchell, 1974). We have shown that, similarly, amino N appears in the vascular effluent of the perfused frog intestine. The kinetics of the washout indicate that the pool from which N is derived is apparently a single compartment, but at present we have no knowledge of the location of this compartment, whether in the epithelium or muscle, whether intra- or extracellular. On the assumption that the amino acids are uniformly distributed in the tissue water, an average effective 'concentration' of 2-2 mm leucine and 3-4 mm glycine is derived: however it was noted that in recently fed animals the concentration was greater, accounting for the large scatter in the results (from 0-8 to 4-1 mm for leucine). Fern, Hider & London (1971) found by direct analysis of rat jejunum a leucine concentration of 2-4 mM. It is interesting to note that the rate constant for the washout of endogenous leucine is increased by increasing the vascular flow rate or by increasing tissue oedema (by decreasing vascular albumin concentration) (see Figs. 6 and 7). The presence in the vascular effluent of endogenous amino acids could lead to a large error in the estimation of the rate of steady-state transfer of amino acids from the lumen, particularly at luminal concentrations below 0 5 mm, and for those amino acids with relatively high washout rates (Table 3). A simple correction for this may be made by means of initial washout sampling and subsequent analysis for the relevant amino acid. Since the washout is monoexponential these results may be extrapolated (Fig. 4), and hence the contribution that will be made at any given time by the endogenous amino acids may be derived. When subtracted from the total rate of amino acid appearance at this time, the correct value of the rate of transport of exogenous, luminally derived amino acid is found.

Transfer of exogenous amino acids: steady state Using this technique, the steady-state transfer of L-leucine has been investigated. The results suggest that leucine transfer is not by simple diffusion: in particular the processes involved in its translocation across the epithelium exhibit saturation at higher luminal concentrations, stereoI6

PPHY 250

426 C. A. R. BOYD AND OTHERS specificity, the L-isomer being transported faster than the D, competition by a second neutral amino acid (L-alanine) and an apparent requirement for sodium. Thus the properties of the leucine transfer system are similar to those found for a variety of amino acids with different preparations of small intestine from a number of species (Schultz & Curran, 1970; Wiseman, 1974). In this connexion Bronk & Parsons (1968) have shown the accumulation of leucine by rings of rat jejunum to be strongly inhibited when the bulk of the sodium ions in the incubation medium is replaced by potassium. In a 4 min incubation, the replacement of sodium by potassium inhibits the leucine transported by about 66 % (Bronk & Parsons, 1968, Table 1). Using a different preparation Bronk & Leese (1974) found that the uptake of leucine by mucosal slices was reduced by about 29 % when the sodium ions in the incubation medium were replaced by potassium (Bronk & Leese, 1974, Table 5). In both of these cases the leucine accumulation is measured from an amino acid mixture, but it is interesting that replacement of the sodium with potassium results in a substantial inhibition of leucine uptake in each of two preparations which are rather different from the one used by us. The data shown here in Table 4 indicate that removal of the sodium from the luminal fluid resulted in a 43 % inhibition of the leucine transfer. However some of the effects of replacing sodium (by potassium) are not exactly similar to those found in the small intestine by other authors. In particular, the replacement of sodium in the vascular fluid appears to be inhibitory, and this inhibition is additive with that resulting from sodium replacement in the lumen (Table 4). Csaky & Thale (1960) using an in vitro preparation of frog intestine did not find any inhibition of 3*0-methyl glucose transport when sodium was replaced by lithium in the serosal compartment for up to 4 hr. Cohen & Huang (1964) studying L-tryptophan transport using everted sacs of hamster intestine also found that sodium replacement with either lithium or potassium in the serosal fluid had no effect on the transfer of the amino acid. This discrepancy may well be explained by methodological considerations. We believe that replacement of sodium in the vascular solution in our preparation exposes the serosal pole of the epithelial cells to a sodium-free medium; when the serosal fluid is sodium free in a simple in vitro preparation without vascular perfusion this may well not be the case, since in such preparations the serosal compartment is separated from the epithelium by a layer of muscle and other tissue. Removal of sodium from the lumen alone has a mixed effect on the kinetics of leucine transfer; not only is the apparent Kt increased but the Vmax is lowered. Studies measuring unidirectional fluxes in mammalian intestine indicate that following sodium removal the Kt for the uptake at

AMINO ACIDS AND INTESTINE 427 the brush border of amino acids is increased but the Vmax is unaffected (Schultz & Curran, 1970). Again this may be a methodological discrepancy; we are measuring transfer across the epithelium and not only uptake by the cells, but a species difference cannot be ruled out. Parsons & Prichard (1968) have previously noted a similar inhibitory effect on glucose transfer from the lumen of the frog intestine when sodium was removed from the luminal Ringer. They also noted the greater inhibition of glucose translocation from maltose when sodium was replaced with lithium in the vascular infusate as well as in the luminal fluid (Parsons & Prichard, 1971). Transfer of exogenous amino acids: non-steady-state kinetics The kinetics of the washout into the vascular bed of leucine initially presented to the lumen is biphasic. An initial fast washout is followed by a slower prolonged rate of appearance, as if the leucine appearing is derived from two pools, the larger emptying faster and the smaller more slowly. With 1 mM leucine in the lumen the combined size of the washout pools is 2*67 jmole g dry wt.-'. Of some interest is the relationship between the slowly emptying pool and the endogenous amino acid pool. The rate constant for both is similar, suggesting that the compartment from which the endogenous amino acids appear may be the same as the second slowly emptying pool which can be filled from the lumen. It is apparent from Figs. 5 and 6 that the rate of appearance of endogenous leucine is somewhat increased by raising the vascular flow rate, and it is markedly increased by making the tissue oedematous (achieved by reducing the albumin concentration). This suggests that the rate limiting step for exit from this pool may be diffusion through a restricted extracellular space, possibly in the submucosa and muscle. The unloading pool is consistently found to be larger than the loading pool. We suggest that this can be explained by the presence, during the luminal washout, of leucine trapped within the interstices of the villus ridges, villi and microvilli (collectively these may be referred to as the 'unstirred layer'). This leucine continues to appear in the vascular bed, although the bulk luminal concentration of the amino acid may be extremely low. Thus the difference between the loading and the unloading pool size could be accounted for by the quantity of leucine trapped within this layer. Such an explanation requires that during loading not all of the unstirred layer has to be filled before the steady state is reached. However, during the unloading extra amino acid entrained in the layer can contribute to the total quantity of amino acid appearing in the vascular bed. We are grateful to the M.R.C. for financial support.

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C. A. R. BOYD AND OTHERS REFERENCES

AGAR, W. T., HIRD, F. J. R. & SIDHU, G. S. (1954). The uptake of amino acids by the intestine. J. Physiol. 14, 80-84. ANDERSEN, B. & ZERAHN, K. (1963). Method for non-destructive determination of the sodium transport pool in frog skin with radiosodium. Acta physiol. scand. 59, 319-329. ATKINS, G. L. (1969). Multicompartment Models for Biological Systems, 1st edn, pp. 30-35. London: Methuen. BOYD, C. A. R., CHEESEMAN, C. I. & PARSONS, D. S. (1973). Transport and metabolism of peptides and amino acids by frog intestine perfused through vascular bed in situ. J. Physiol. 234, 10-lIP. BRONK, J. R. & LEESE, H. J. (1974). The absorption of a mixture of amino acids by rat small intestine. J. Physiol. 241, 271-286. BRONK, J. R. & PARSONS, D. S. (1968). Amino acid accumulation and incorporation in rat intestine in vitro. J. Physiol. 184, 950-963. CARTER, G. W. & VAN DYKE, K. (1971). A superior counting solution for water soluble tritiated compounds. Clin. Chem. 17, 576-580. COHEN, L. L. & HUANG, K. C. (1964). Intestinal transport of tryptophan and its derivatives. Am. J. Physiol. 206, 647-652. CRIPPS, A. W. & WILLIAMS, V. J. (1975). The effect of pregnancy and lactation on food intake, gastrointestinal anatomy and the absorptive capacity of the small intestine in the albino rat. Br. J. Nutr. 33, 17-32. CSAKY, T. Z. & THALE, MARGRETHE (1960). Effect of ionic environment on intestinal sugar transport. J. Physiol. 151, 59-65. CUTHBERT, A. W. (1971). Neurohypophyseal hormone and sodium transport. Phil. Trans. R. Soc. B 262, 103-109. FELDHOFF, R. C. (1971). Quantitative changes in plasma albumen during bullfrog metamorphosis. Comp. Biochem. Physiol. 40 B, 733-739. FERN, E. B., HIDER, R. C. & LONDON, D. R. (1971). Studies in vitro of free amino acid pools and protein synthesis in rat jejunum. Eur. J. clin. Invest. 1, 211-215. FISHER, R. B. (1955). The absorption of water and some small solute molecules from the isolated small intestine of the rat. J. Physiol. 130, 655-644. FISHER, R. B. & GARDNER, M. L. G. (1974a). Dependence of intestinal glucose absorption on sodium, studied with a new arterial infusion technique. J. Physiol. 241, 235-260. FISHER, R. B. & GARDNER, M. L. G. (1974b). A kinetic approach to the study of absorption of solutes by isolated perfused small intestine. J. Physiol. 241, 211-234. FISHER, R. B. & PARSONS, D. S. (1953). Glucose movements across the wall of the rat small intestine. J. Physiol. 119, 210-223. FORSTER, H. (1972). Views dissenting with the 'gradient hypothesis'. Intestinal sugar absorption, studies in vivo and in vitro. In Na-linked Transport of Organic Solutes, ed. HEINZ, E. Berlin: Springer-Verlag. FUJITA, M., PARSONS, D. S. & WOJNAROWSKA, FENELLA (1972). Oligopeptidases of brush border membranes of rat small intestine mucosal cells. J. Physiol. 227, 377-394. GAGNON, A. (1960). The absorption of amino acids by the isolated intestine of the green frog Rana clamitans. Revue can. Biol. 20, 7-14. HANES, C. S. (1932). Studies of plant amylases. I. The effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. Biochem. J. 26, 1406-1421.

AMINO ACIDS AND INTESTINE

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INTAGLIETTA, M. & ZWEIFACH, B. W. (1974). Microcirculatory basis of fluid exchange. In Advances in Biological and Medical Physics, vol. 15, eds. LAWRENCE, J. H. & HAMILTON, J. G. New York and London: Academic Press. McDouGAL, D. B. JR., LITTLE, KATHERINE D. & CRANE, R. K. (1960). Studies on the mechanisms of intestinal absorption of sugars. IV. Localisation of galactose concentration within the intestinal wall during active transport in vitro. Biochim. biophys. Acta 45, 483-489. MASON, J. C., MICHEL, C. C. & TOOKE, J. E. (1973). The effect of plasma proteins and capillary pressure upon the filtration coefficient of frog mesenteric capillaries. J. Physiol. 229, 15-16P. PARSONS, D. S. (1968). Techniques for studying intestinal absorption. In Handbook of Physiology, section 6, Alimentary Canal, vol. 3, ed. CODE, C. F. Washington, D.C.: American Physiological Society. PARSONS, D. S. (1975). Energetics of intestinal transport. In Intestinal Absorption and Malabsorption, ed. CsAKY, T. Z. New York: Raven Press (in the Press). PARSONS, D. S. & PowIs, G. (1971). Some properties of a preparation of rat colon perfused in vitro through the vascular bed. J. Physiol. 217, 641-663. PARSONS, D. S. & PRICHARD, J. S. (1968). A preparation of perfused small intestine for the study of absorption in amphibia. J. Physiol. 198, 405-434. PARSONS, D. S. & PRICHARD, J. S. (1971). Relationships between disaccharide hydrolysis and sugar transport in amphibian small intestine. J. Physiol. 212, 299-319. PARSONS, D. S. & VOLMAN-MITcHELL HETTY (1974). The transamination of glutamate and aspartate during absorption in vitro by small intestine of chicken, guinea-pig and rat. J. Physiol. 239, 677-694. Ross, B. D. (1972). Perfusion Techniques in Biochemistry. Oxford: Clarendon Press. SARDESAI, V. M. & PROVIDO, H. S. (1970). The determination of glycine in biological fluids. Clin. chim. Acta 29, 67-71. SCHULTZ, S. G. & CUxRAN, P. F. (1970). Coupled transport of sodium and organic solutes. Physiol. Rev. 50, 637-718. SMYTH, D. H. (1974). Methods of studying intestinal absorption. In Biomembranes, vol. 4A, ed. SMYTH, D. H. London: Plenum Press. WILSON, T. H. (1962). Intestinal Absorption. London: Saunders. WISEMAN, G. (1974). Absorption of protein digestion products. In Biomembranes, vol. 4A, ed. SMYTH, D. H. London: Plenum Press.

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