ACTIVE TRANSPORT OF SALICYLATE BY RAT JEJUNUM

Transcription

1 Quarterly Journal of Experimental Physiology (1981) 66, Printed in Great Britain ACTIVE TRANSPORT OF SALICYLATE BY RAT JEJUNUM R. B. FISHER University Laboratory of Physiology, Oxford (RECEIVED FOR PUBLICATION 3 JUNE 1980) SUMMARY Using an improved segmented flow technique the uptake of salicylate in low concentration (1-3 mm) by rat jejunum is found to be steady for ca. 30 min, and is proportional to lumen concentration. At higher lumen concentrations (6-8 mm) the rate of uptake falls from the beginning to very low levels. At low lumen concentrations the tissue fluid concentration rises to approximately three times the lumen concentration without significant effect on the uptake rate. This rise is an exponential function of the net water transport across the intestinal wall. The rate constant is of the same order of magnitude as those found earlier for equilibration of tissue fluid with glucose and wash-out of protein from the tissue fluid. The uptake of salicylate is inhibited irreversibly by 2,4-dinitrophenol (2-4 x 1O-5 M). INTRODUCTION Proportionality between lumen concentration and the rates of intestinal absorption of salicylic acid and aniline from rat small intestine was regarded by Schanker, Tocco, Brodie & Hogben (1958) as evidence that these compounds were absorbed by passive diffusion, since an active transport mechanism should show a falling off from proportionality at high lumen concentrations as saturation of the process is approached. Unfortunately it is never possible to know whether the range of lumen concentration has been extended far enough to enable incipient saturation to be detected, so the possibility remains that a linear relation between absorption rate and lumen concentration can exist over the whole of the observable range for a solute that is actively transported. The methyl red isomers afford an example of this (Fisher, 1980). It therefore seemed desirable to re-investigate the compounds studied by Schanker et al. (1958). Preliminary work showed that aniline is converted, apparently almost completely, into an amino-phenol in the course of transport. This makes it impossible to determine whether it can be transported against a concentration gradient. This paper therefore deals only with salicylic acid. METHODS Perfusion technique. For intestinal perfusion the method of Fisher (1980) was used without modification. This involves the re-circulation through the lumen of Krebs-bicarbonate-glucose segmented with bubbles of 95% 02/5 % CO2, to which salicylate is added. A similar solution is infused into the circuit at 2 ml./min. The effluent from the circuit is collected in timed periods and the fluid which drips from the serosal surface of the gut is collected at the same time. Jejunal segments from female white rats were used. Estimation of salicylate. The red colour with FeCl3 was used. The sample was mixed with approximately ten volumes of 15 mm FeCl3 in 0-2 N HCl in an Autoanalyser. The extinction was recorded at 530 nm and compared with that of standards /81/ $ The Physiological Society 4 EPH 66

2 92 R. B. FISHER Terminology. Fisher & Gardner (1974) used the term 'secretion' to mean transport from the lumen into the tissue fluid (mucosal secretion) or across the whole thickness of the intestinal wall (serosal secretion). 'Secretion' has also been used widely to mean the exudation of water and electrolytes into the intestinal lumen in pathological conditions. Since the processes studied here are active processes and the pathological processes are at least in part passive, this paper continues the usage of Fisher & Gardner (1974). The term 'uptake' is used rather than 'absorption' or 'transport' to describe the disappearance of solute from the lumen, since the process may include adsorption on to the cells as well as passage into them, and because transport across the cells may be much less than transport into them due to intracellular accumulation and metabolic degradation (Fisher, 1980). RESULTS When infusates containing 15, 2 0, 3-0 or 4 0 mm salicylate were used the results were as shown in Fig. 1. At the two lower infusion rates the lumen concentration rapidly stabilized at a level appreciably below infusate concentration and the rate of uptake of salicylate from the lumen was almost constant throughout the experimental period (0 8 hr). At the two higher infusate rates the lumen concentration rose throughout the experiment and the rate of uptake showed a marked decline after an initial period of steady uptake. The rate of appearance of salicylate in the tissue fluid can be calculated from the volume and salicylate concentration of the fluid dripping from the serosal surface (serosal secretion) (Fisher, 1980). In all instances it lagged behind uptake rate initially, but became very nearly equal to it towards the end of the experiment. The concentration of salicylate in the serosal secretion rose in a sigmoidal fashion and appeared to be approaching a plateau at the end of the experiment. At each infusate concentration the terminal secretion concentration was a little over three times the terminal lumen concentration. Salicylate transport at higher infusate concentrations. Fig. 2 shows that at infusate concentrations of 6 and 8 mm the uptake rate fell off rapidly, lumen concentration rose throughout the experiment, transport rate into tissue fluid rose initially and then fell, and serosal concentration rose to about twice lumen concentration and then fell again. Effect of2,4-dinitrophenol (DNP) on salicylate uptake. Since the results obtained with the higher salicylate concentrations suggested that the mucosa was being poisoned, the effect of an independent metabolic poison was investigated. Jejunum was perfused for 0 4 hr using a 1 mm salicylate infusate, followed by 0 4 hr with DNP and salicylate, and finally 0 4 hr with salicylate alone. Salicylate uptake in experiments using 20 fm and 40 /M DNP are compared in Fig. 3 with experiments in which the intestine was perfused with salicylate alone. It is seen that DNP depresses salicylate uptake much more than does continued exposure to salicylate. This effect is dose-dependent and irreversible. Relation between lumen concentration and rate of uptake. It can be seen from Fig. 1 that during the 2nd, 3rd and 4th collection periods of 0-1 hr uptake rates and lumen concentrations vary little as long as the infusate concentration is less than 4 mm. The mean rates and lumen concentrations for these three collection periods from the experiments shown in Fig. I A, 1 B and 1 C, together with two other sets of three experiments, are plotted against the corresponding mean lumen concentrations in Fig. 4, which also shows the line of best fit. The relationship is a straight line through the origin up to ca. 3 mm. No relation between concentration and a steady uptake rate can be established for higher concentrations because of the instability seen in Fig. 2.

3 ACTIVE TRANSPORT OF SALICYLATE Time (hr) Fig. 1. Salicylate transport rates and lumen and serosal secretion concentrations at low infusion concentrations. Each figure shows lumen concentration (0 O) and serosal concentration ( 0, mm), uptake from the lumen (O---Q) and mucosal secretion rate (0---@, l. cm-' hr'1). On each figure the concentration of salicylate in the infusate is shown. Each point is the mean of three experiments. 0 Fig. 2. Salicylate transport rates and lumen and serosal secretion concentrations at higher infusion concentrations. The details are as in Fig

4 E, 400 a. I-2 =v20 X R R X E R. B. FISHER ID -0. St o\.,, 0 No DNP 20,um DNP 0 40 mm DNP Time (hr) Fig. 3. Effect of 2,4-dinitrophenol (DNP) on salicylate uptake from the lumen. DNP was infused for 0 4 hr (between the vertical lines). Each point is the mean of three experiments a: a+1 0t co / Mean lumen concentration (MM) Fig. 4. Mean rates of salicylate uptake from the lumen (3 experiments) for collection periods 2-4 inclusive plotted against mean lumen concentrations. Relation between secretion concentration and water secretion. If the concentration of salicylate in serosal secretion is plotted against the cumulative water secretion (Fisher & Gardner, 1974) for all collections after the third it fits to an exponential y = p + q e-kv, where y is the concentration, v is the cumulative water secretion, (p + q) is the initial salicylate concentration and p is its terminal concentration, i.e. when v is very large. The parameters of the fit to this equation are shown in Table 1 for the four sets of experiments used in Fig. 4. Judging from the standard errors of the k's the fits to the equation are very close. The values of k are clearly significantly different from one another, which indicates that there

5 Table 1. ACTIVE TRANSPORT OF SALICYLATE Fit of the salicylate concentration in serosal secretion to an exponentialfunction of cumulative water transport across the intestinal wall 95 Perfusate concn. (mm) k(cm. #ul-) p(mm)* q(mm)t s Mean values from three experiments are used for the calculations: each exponential fit is based on five points. * These values are obtained by an iterative procedure which adjusts the value of p until the mean square deviation from the regression is minimized; these are the best fit values of p to the third decimal place. t The algebraic sum of p and q is not zero because the beginning of the third collection was taken as starting point of the fit. is a real inter-animal variation, but the range of values is only from 91 to 1100 of the mean, so that the mean can be taken as a reasonable estimate of the population value of k. Fate of salicylate in the mucosa. Added salicylate can be extracted quantitatively from intestinal tissue by homogenization in n-butanol containing 10% (v/v) of water. A fresh butanol extract gives a peak extinction with FeCl3 at 450 nm but negligible absorption at 530 nm, the salicylate peak. The peak at 450 nm is sharp, whereas that given by gentisic acid at the same wavelength is rounded. The absorption spectrum on the instrument used (SP8000) is unlike that of any dihydroxybenzoic acid reacting with FeCl3. As the colour with FeCl3 which is given by the butanol extract fades quite rapidly, it has not proved possible to use it to characterize the metabolites of salicylate in the mucosa. DISCUSSION Relation between lumen concentration and rate of salicylate uptake. The finding of a proportional relation between these variables by Schanker et al. (1958) has been confirmed, within limits. In this work (Fig. 1D, Fig. 2) the rate of uptake declines as the lumen concentration rises above 3 mm whereas in the work of Schanker et al. (1958) the rate was still proportional to concentration up to 10 mm. The present findings of proportionality up to 3 mm (Fig. 4) accord better with Smith (1958) who found that, when everted sacs of rat small intestine were incubated with salicylate, oxygen consumption and oxidative phosphorylation were unaffected by 1 mm but completely inhibited by 5 mm. However, Schanker et al. (1958) expressed their results in terms of the concentration of salicylate in the infusate pumped into the intestine. The concentration at the mucosal surface must have been much lower than this both because of the high proportion of the salicylate which was absorbed and because of the diffusion gradient from lumen axis to mucosa (on the existence of which Schanker et al. themselves comment). In the present work results are expressed in terms of mean lumen concentration and precautions are taken (Fisher & Gardner, 1974; Fisher, 1980) to keep the lumen contents well mixed and to reduce the unstirred layer at the mucosal surface. Thus Schanker et al.'s (1958) relation between concentration and uptake rate is almost certainly in error by a factor of about three. All other slow perfusion studies must be considered also to be in error, the factor being dependent on the magnitude of the clearance of the solute under study: the higher the clearance, the higher will be the ratio of mean

6 96 R. B. FISHER lumen concentration to the concentration at the mucosal surface. This is important in the study of saturable processes: here the clearance will fall as the lumen concentration rises so that there will be a non-linear relation between mean lumen concentration and mucosal surface concentration. This can have profound effects on the estimation of kinetic parameters. The effects of higher salicylate concentrations. The fact that at high lumen concentrations the uptake rate falls continuously during the experiment is not explicable on the hypothesis that salicylate is absorbed by passive diffusion. The fall cannot be due to the development of greater adverse serosal lumen concentration ratios than those seen at lower lumen concentrations. Fig. 2 shows that the falls in uptake rate are already established when the concentration ratio is either still favourable or much less adverse than those seen in experiments at lower concentrations in which uptake rate remains steady. On the other hand, the fall is explicable in terms of the known toxic effects of salicylate on the intestine. Smith (1958) showed that oxygen consumption, oxidative phosphorylation, and glucose and water uptake of everted sacs of rat small intestine were all completely inhibited by incubation with 5 mm salicylate. The only activity whose time-course was studied was oxygen consumption, which fell to zero over about an hour, in conformity with the decline in salicylate uptake seen here. At 1 mm, salicylate had no toxic effect. The progressive nature of the effect suggests that it depends on the accumulation of salicylate or a metabolite in the mucosa. Fig. 5 shows, as a function of time, the difference between uptake from the lumen and secretion into the tissue fluid. The filled circles indicate the times at which marked fall in uptake rate was first detectable. In all these instances the cumulative differences are similar (0S6-07,umole/cm intestine). In the remaining sets of experiments in which there was no sharp fall in uptake rate the cumulative differences had risen to only ,amole/cm at the end of the experiment. Thus it is fairly certain that the fall in salicylate uptake rate seen at higher concentration is due to metabolic poisoning consequent on exposure of the intestine to the higher concentration of salicylate, which implies that salicylate transport is dependent on cell metabolism, and is not a passive process. It seems unlikely that salicylate is directly responsible for the toxic effects since it cannot be recovered from homogenized intestine although added salicylate can be recovered quantitatively. The kinetics of appearance of salicylate in serosal secretion. Fisher & Gardner (1974) showed that the change in concentration of protein in serosal secretion followed an exponential course in relation to the cumulative volume of secretion produced. The same was true for glucose following an abrupt change in lumen concentration. This is best accounted for by assuming that the solute and water mix thoroughly in a limited submucosal compartment and that the serosal secretion is a sample of the contents of this space. If this is so, the exponential constant should be the reciprocal of the volume of the submucosal compartment. The constants derived from the glucose and protein data were similar and indicated a compartmental volume corresponding to the extracellular space. For D2O and sodium the exponential constants are 3-4 times as great as this (Bywater, Fisher & Gardner, 1975) and this is explicable in terms of the very substantial refluxes that occur from tissue fluid to lumen: the unidirectional flux from lumen to tissue fluid is 3-4 times as great as the net flux both for water (Berger, Pecikyan & Kanzaki, 1970) and for sodium (Curran & Solomon, 1957).

7 ACTIVE TRANSPORT OF SALICYLATE 97 E E Z Time (hr) Fig. 5. Cumulative differences between uptake of salicylate from the lumen and its appearance in the tissue fluid. The filled circles show the times when the first substantial fall in uptake rate occurred. The figures associated with the curves show the salicylate concentrations in the infusates (mm). Salicylate might be expected to follow the same exponential pattern as protein and glucose as long as its uptake rate remains steady, and the exponential constant should indicate whether there is any substantial reflux into the lumen. The observed rate constant of 0 024,ul.-I cm is lower than the mean value obtained by Fisher & Gardner (1974) for glucose and protein, which was 0-033, and significantly different from it. However the earlier value was determined on the whole small intestine, whereas the present value is for jejunum only, and different strains of rat were employed. Preliminary results indicate that rate constants for protein, glucose and salicylate concentrations in jejunum when determined simultaneously are indistinguishable from one another. There is therefore probably no reflux of salicylate into the lumen. CON CLU S ION This work provides a second example of a solute which is taken up from the lumen at a rate proportional to lumen concentration although uptake takes place against a concentration gradient and is inhibited by a metabolic inhibitor. The first example was provided by the methyl red isomers (Fisher, 1980). These two pieces of work indicate that kinetic studies concerned solely with uptake from the lumen are not sufficiently discriminating to provide reliable guides to mechanism - especially if they extend over limited periods of time. In particular, any appearance of proportionality of uptake rate to'lumen concentration calls for the investigation of the effects of a metabolic inhibitor before it is concluded that the uptake process is passive. I am grateful to Professors D. Whitteridge and C. Blakemore for facilities provided.

8 98 R. B. FISHER REFERENCES BERGER, E. Y., PECIKYAN, R. & KANZAKI, G. (1970). Water flux across the rat jejunum and ileum. Journal of Applied Physiology 29, BYWATER, R. J., FISHER, R. B. & GARDNER, M. L. G. (1975). Transport of deuterium oxide across isolated rat small intestine. Journal of Physiology 249, CURRAN, P. F. & SOLOMON, A. K. (1957). Ion and water fluxes in the ileum of rats. Journal ofgeneral Physiology 41, FISHER, R. B. (1980). Active intestinal transport of methyl red isomers. Quarterly Journal of Experimental Physiology 65, FISHER, R. B. & GARDNER, M. L. G. (1974). A kinetic approach to the study of absorption of solutes by isolated perfused intestine. Journal of Physiology 241, SCHANKER, L. S., Tocco, D. J., BRODIE, B. B. & HOGBEN, C. A. M. (1958). Absorption of drugs from the rat small intestine. Journal of Pharmacology and Experimental Therapeutics 123, SMITH, M. J. H. (1958). Effects of salicylate on the metabolic activities of the small intestine. American Journal of Physiology 193,