SODIUM MOVEMENTS ACROSS THE VASCULARLY PERFUSED ANURAN SMALL INTESTINE AND COLON

Transcription

1 Quarterly Journal of Experimental Physiology (1982) 67, Printed in Great Britain SODIUM MOVEMENTS ACROSS THE VASCULARLY PERFUSED ANURAN SMALL INTESTINE AND COLON D. S. PARSONS* AND S. A. WADE Department of Biochemistry, South Parks Road, Oxford OX] 3QU (RECEIVED FOR PUBLICATION APRIL 1981) SUMMARY Values of unidirectional Na fluxes measured across the vascularly perfused small intestine of three anuran species are higher than those found in other preparations in vitro. In Rana ridibunda and R. pipiens no net movement of Na across the small intestine can be detected. In contrast, the unidirectional fluxes of Na across the colon of R. ridibunda and R. pipiens are lower than across the small intestine and a significant net absorption is found. Apparent loading and unloading pools for Na within the small intestine, as measured with tracer Na under standard experimental conditions, consist largely ofextracellular Na presumably within the bulk phase of the lumen. The size of these pools can be greatly reduced by the rapid addition to or removal from the lumen of tracer. The loading pool appears to occupy not more than about 9% of the total tissue water, equivalent to about 20% of the extracellular water of the tissue. The washout of tracer Na from preloaded small intestine into the vascular bed is bi-exponential and appears from a pool, or pools, of apparent greater size than that of the loading pool. The results show that Na can move very rapidly across the small intestine and suggest that a high proportion of this movement occurs possibly via paracellular shunt pathways. INTRODUCTION Visscher and his colleagues used 24Na to measure unidirectional fluxes of Na across mammalian intestine in vivo (Visscher, Varco, Carr, Dean & Erickson, 1944). They were the first to show that net Na movement across the small intestine could be described by the algebraic sum of two large unidirectional fluxes and that the unidirectional fluxes were up to ten times greater than those across the colon. More recently, sheets of intestine mounted in flux chambers to which short-circuit current is applied have been used (Ussing & Zerahn, 1951; Schultz & Zalusky, 1964). Using this and other electrophysiological procedures, such as micro-electrodes, to measure membrane potentials (Rose & Schultz, 1971; Armstrong, 1976), it has been shown that the pathway between the epithelial cells involving the tight junction, or zonula occludens, and the intercellular space, represents a region of low electrical resistance (shunt pathway) in which the ionic permeability is approximately that of a neutral pore (PNa/PCl 2) (Schultz, Frizzell - & Nellans, 1974; Parsons, 1976; Schultz, 1977). The clearance of Na away from and the delivery to the mucosal epithelium thus became important factors in determining the transporting capability of the intestine. It is clearly of interest, therefore, to study Na transport across the intestine of a preparation in which these processes can take place rapidly and will not be limited by the resistance to diffusion of tissue layers deep to the epithelium that are found under in vitro conditions. We here describe measurements of Na fluxes across both the small intestine and colon of the frog in which the vascular supply to the intestine is perfused in situ. The fluxes of * To whom correspondence should be addressed.

2 122 D. S. PARSONS AND S. A. WADE Na across the small intestine are found to be large and apparently passive whereas the fluxes across the colon are lower and a significant net absorption of Na is found. A preliminary account of some of these findings has been published (Wade, 1980). METHODS General The essential feature of vascular perfusion of the frog small intestine is the single pass of luminal and vascular fluids through the preparation, which can receive an adequate oxygen supply without the need to perfuse erythrocytes through the vascular bed. Details of the animals and their husbandry and the composition of luminal and vascular saline fluids are described by Boyd, Cheeseman & Parsons (1975) and Boyd & Parsons (1978). Most of the experiments were performed using Rana ridibunda, although R. pipiens and R. catesbeiana were used when R. ridibunda was not available. All frogs were fed twice weekly with live house crickets (Xenopus Ltd) or chopped ox liver. Experimental procedure Small intestine. The technique of vascular perfusion of Boyd et al. (1975) was modified by perfusing only a short (about 5 cm) length of small intestine from the region just distal to the duodenum; all blood vessels to other regions of the intestine were tied off. Colon. The lumen was perfused with fluid flowing through one cannula inserted in the rectum via the anus and the other placed in the ileal sphincter. By tying off blood vessels to selected regions of the intestine the colon could be perfused alone or simultaneously with a segment of small intestine (Wade, 1979). In all cases 1 mm sodium butyrate was added to the vascular perfusate as a metabolizable substrate and 2 g. 100 ml-' bovine serum albumin was found to produce a stable vascular recovery for periods up to 8 h. Flux measurements. Unidirectional fluxes of Na across the intestine were measured with radioactive isotopes, individually (24Na alone) or simultaneously (24Na and 22Na). The fluids were pumped through the luminal and vascular circuits from reservoirs, two of which were provided for each circuit, one containing tracer-free fluid and the other containing fluid to which isotopic-na had been added. The standard procedure for measuring fluxes of Na was to switch from the tracer-free to the tracer-containing fluid by turning a 3-way tap that connected each pair of reservoirs to its circuit. The flow rates per segment were usually about 05 ml. min-' (luminal) and 0-2 ml. min-' (vascular). The amount of tracer added to the cis side of the epithelium was such that, when isotopic equilibrium had been achieved (e.g. after 30 min), only a small fraction (2%) of the radioactivity delivered to the cis side appeared on the trans side. Size of transport pool. Sizes of the pool of tissue Na with which the isotope equilibrates were calculated from plots of the accumulated appearance of Na on the trans side during the loading or unloading phase (see Andersen & Zerahn, 1963; Cuthbert, 1971). Zero time was taken as the moment when tracer Na could first be detected in the effluent on the trans side (loading pool) or was found to decrease (unloading pool). Using standard rates of lumen flow, the size of the pool appears to be distorted by effects of unstirred layers in the lumen (see Discussion). To minimize these effects, Ringer containing 24Na was flushed rapidly (5-10 s) into the lumen of the small intestine from a syringe attached to the inflow cannula (rapid flush procedure). The portal venous cannula was shortened so that effluent containing radioactivity appeared within about 30 s. Fractions of this effluent were then collected at short time intervals (e.g. 25 s). The same procedure was adopted when flushing out the 24Na from the lumen. Counting of radioactivity. If using 24Na alone, samples were counted from the emission of Cerenkov radiation in aqueous solution in an LKB Wallac scintillation counter. Addition of the wavelength shifter 7-amino-1,3-naphthalene disulphonic acid (ANDA) (1 mm) increased the counting efficiency to 60%, from 40% with water alone. For counting 24Na with 22Na the technique used was that of

3 INTESTINAL SODIUM FLUXES A A = 0-6 * >* 0 A * A A AAA A A A 3A A A A A 0-2A A Time (min) Fig. 1. The appearance of tracer Na in the luminal or vascular effluents. This experiment shows the time course of the changes in the specific activity of 24Na in the vascular effluent (A) and of the specific activity of 22Na in the luminal effluent (A). The lumen flow rate was 044 ml. min-1 and the vascular flow rate was 0-18 ml. min-'. After washing out the radioactivity, Ringer containing 24Na of the same specific activity as that used earlier was flushed rapidly into the lumen (see Methods) and the change in specific activity in the vascular effluent followed (0). R. ridibunda, small intestine. Quay & Armstrong (1969). The two isotopes were counted in a Packard channel gamma spectrometer setting one channel to count 24Na at a high efficiency relative to 22Na. After allowing the short-lived 24Na to decay (at least 9 d) a second count was taken with the second channel set to bracket the major photon peak of 22Na. The 24Na counts could then be corrected for decay and spillover of 22Na. Calculation and expression of results Fluxes of Na were calculated from knowledge of the rates of arterial or luminal inflow and of the counts appearing in the respective effluents. For Na fluxes into the vascular bed, 100% recovery of arterial infusate from the portal vein was assumed. When expressing net fluxes, a positive sign indicates net transfer from lumen to blood and a negative sign net transfer from blood to lumen. Values of Na fluxes are given usually in units of,umol. cm-2. h-1, the serosal area of the gut segment being measured from its linear dimensions at resting length. Source of materials Tracer 24Na and 22Na were obtained as sterile isotonic solutions from the Radiochemical Centre, Amersham, Bucks., England. ANDA was obtained from Aldrich Chemical Co. Ltd, Gillingham, Dorset, England. Other materials were obtained as described by Boyd et al. (1975) and Boyd & Parsons (1978). RESULTS Tracer Na movements across the small intestine Fig. 1 shows the time course of 24Na appearance in the vascular effluent and 22Na in the luminal effluent. With standard flow rates through the preparation, it takes some 4 min after switching from tracer-free to tracer-containing fluid in the reservoirs for radioactivity to appear in the effluent from the lumen or portal vein, and the isotope takes at least 30 min to achieve equilibrium within the intestine. Most of this time is due to the filling of the dead

4 124 D. S. PARSONS AND S. A. WADE Table 1. Unidirectional and net Na fluxes across small intestine Species Lumen-blood Blood-lumen Net R. ridibunda* *26 (36) *89 (17) R. ridibunda (22) R. pipiens (8) R. catesbeiana (5)t Values, #mol.cm-2.h-1, are of means+s.e. of means of n animals and observations. Fluxes measured simultaneously except *. For net fluxes, P for net absorption > 0 1 except, 0-02 > P > space consisting of tubing (1 6 ml, luminal circuit; 1 2 ml, vascular circuit) and intestinal lumen, since if 24Na is flushed rapidly into the lumen, isotopic equilibrium is reached in only 5 min. Magnitude of Nafluxes across the small intestine Unidirectional and net Na fluxes across the small intestine of R. ridibunda, R. pipiens and R. catesbeinana are shown in Table 1. In earlier work with R. ridibunda, 24Na alone was used to measure the Na fluxes separately. The difference in the mean of these pooled values gives a net flux not significantly different from zero (P> 01). In later work the unidirectional fluxes were measured simultaneously as described in Methods. The mean of the differences between the pairs of fluxes across the small intestine of both R. ridibunda and R. pipiens also reveals no significant net movement of Na (P > 0 1). In the bullfrog R. catesbeiana, the species most commonly used for in vitro studies, a large net absorption is found, although among the five animals used there was wide variation in the value of the net flux (range ,umol. cm-2. h-1). It can be seen from Table 1 that the mean values of Na fluxes across R. ridibunda small intestine measured simultaneously are lower than the values from the earlier work. This seems to be a seasonal effect, for if account is taken of the time of year in which the studies were performed (Table 2), clear differences in the magnitude of Na fluxes can be observed. Thus during the period from 1978 to 1979 the mean of the Na fluxes measured during the spring and winter months were lower than when measured during the months from July to October (P < 0 05). Similarly fluxes measured during the summer months of 1980 were greater than when measured during February and March (P < 0.02). Size of transport pools for Na Values of the size of the pool required to be filled by tracer Na to achieve the steady state of isotopic transfer (loading pool), together with those of the pool from which the tracer unloads during the washout period, are given in Table 3. When measured under standard conditions (lumen flow 0 5 ml. min-. segment-') the pool sizes are found not to depend upon the side from which the tissue is loaded or into which side it unloads (P > 0 1). When 24Na is rapidly added or removed from the lumen (rapid flush procedure) the values of pool sizes are up to ten times less then those obtained with the standard procedure. The unloading pool, however, is significantly greater than the loading pool whichever experimental procedure is adopted (P < 0-05). Washout of tracer Na The bi-exponential pattern of washout of preloaded tracer Na into the vascular bed enables two rate constants to be derived by curve stripping (see Fig. 2 for an individual

5 INTESTINAL SODIUM FLUXES 125 Table 2. Seasonal variation in Na fluxes Months Apr.-June July-Oct. Nov.-Mar. Feb.-Mar. May-Sept. Na flux n P for difference < 0 05 < 0 01 < 0-02 Values of unidirectional fluxes,,umol cm-2. h-1, are means + S.E. ofmeans of n animals and observations. During the months shown, experiments were performed regularly on R. ridibunda small intestine. The values shown for 1980 are means of lumen-blood Na fluxes only. Table 3. Loading and unloading pool sizes for Na Loading Unloading Experimental procedure From lumen From blood Into blood Into lumen Standard (50) (37) (18)* (13)t Rapid flush (4) (4) Values,,umol. cm-2 serosal area, are of means+s.e. of means of n animals and observations. For differences between lumen and blood sides, P > 0 1. For increased unloading pool size with the standard procedure, 0 01 > P > 0 001*; 0-05 > P < 0-02t. For increased unloading pool size with the rapid flush procedure, 0 02 > P > Details ofexperimental procedures are in Methods. 1 cm2 ofsmall intestine contains approximately 22,ul water. R. ridibunda, small intestine. experiment). The maximum rate of washout is delayed until the dead space is filled with tracer-free perfusate similar to the delay seen during the loading of the intestine with 24Na (Fig. 1). The fast component (rate constant, kl, mean = min-', n = 10) accounts for about 80% of the washout and can be altered, e.g. k1 is significantly increased if 10 mm-glucose is present in the lumen (D. S. Parsons & S. A. Wade, unpublished observations): the slow component (rate constant, k2, mean = min-', n = 10) is found to be independent of such conditions. Both rate constants, however, can be greatly increased by flushing the 24Na rapidly out of the lumen (k' and k'2 in Fig. 2). Magnitude of Nafluxes across the colon Values of unidirectional Na fluxes, measured simultaneously, and the corresponding net fluxes across the colon of R. ridibunda and R. pipiens are shown in Table 4. For R. ridibunda, the lumen-blood Na flux is up to eight times less than the lumen-blood flux across the small intestine, while for R. pipiens, the lumen-blood Na flux is about half of that across the small intestine. For both species, however, the lumen-blood Na flux across the colon is at least twice the corresponding blood-lumen flux, so there is significant net absorption of Na. DISCUSSION Fluxes of Na across small intestine In comparison with other species. When compared with values ofna influx across isolated, non-perfused intestine of other animals (Table 5), the Na fluxes are up to seven times higher

6 126 D. S. PARSONS AND S. A. WADE o * 0 10D 0 O S0 Time (min) Fig. 2. The disappearance of tracer Na from the vascular effluent. This experiment shows the time course of the changes in the specific activity of 24Na in the vascular effluent (0) during washout from the preloaded small intestine. The vascular flow rate was 0 15 ml. min-'. After reloading the tissue with Ringer containing 24Na of the same specific activity as that used earlier, the radioactivity was flushed rapidly out of the lumen and samples collected approximately every 45 s (0). The vascular flow rate was unchanged. The rate constants, but not the initial pool sizes, were determined by curve stripping using a computer programme described by Cheeseman (1979). R. ridibunda. For (0): k, = 0-35 min-', ti = 2-0 min; k2 = 0-05 min-', t1 = 13-6 min. (0): k' = 1-2 min-', ti = 0-6 min; k'2 = 0-2 min-', ti = 3-5 min. than across rat and three times higher than across rabbit small intestine. Moreover, the influx of Na across perfused bullfrog small intestine is some fifteen times higher than across the non-perfused tissue showing the influence of vascular perfusion upon the magnitude of the Na fluxes. Yet the values of the Na fluxes in the present studies lie within the range of Na fluxes measured across small intestine in vivo. In comparison with the other epithelia. A characteristic feature of some epithelia is that the transepithelial unidirectional fluxes ofna seem to be higher than the unidirectional fluxes across some plasma membranes. In fact the Na fluxes across the frog small intestine are some two orders of magnitude greater than the efflux of Na across the plasma membrane of cell types such as the human erthrocyte and squid giant axon (see Cereijido & Rotunno, 1968). Differences in the Na permeability of various epithelia have been related to the resistance of the tight junction to Na flow (Schultz, 1977). From the examples shown in Table 5, it is evident that Na fluxes across the anuran small intestine are much greater than across so-called 'tight' epithelia such as frog skin and toad urinary bladder but resemble those across 'leaky' epithelia (rabbit gall bladder and renal proximal tubule). The values we find are equivalent to a partial Na-conductance across the epithelium of about

7 INTESTINAL SODIUM FLUXES 127 Table 4. Unidirectional and net Na fluxes across colon Species Lumen-blood Blood-lumen Net R. ridibunda (12)* R. pipiens ±0 97 (6)t Values,,umol. cm-2. h', are of means +S.E. of means of n animals and observations. For net absorption, 0-01 > P > 0.001*; 0 05 > P > 0 02t. Of the twelve experiments on R. ridibunda, nine were performed during the autumn of All experiments on R. pipiens were performed during the summer of Table 5. Transepithelial influx of Na Small intestine-in vitro Bullfrog Tortoise Rat Guinea pig Rabbit Small intestine-in vivo Rat Dog Human Small intestine-present work R. pipiens R. ridibunda R. catesbeiana Other epithelia-in vitro Skin (frog) Urinary bladder (toad) Proximal renal tubule (rabbit) Gall bladder (rabbit) Influx (pmol. cm-2. s-') Reference Armstrong (1976) Gilles-Baillien, Havaux & Chapelle (1979) Curran (1960) Lauterbach (1976) Frizzell (1976) Curran & Solomon (1957) Visscher, Fetcher, Carr, Gregor, Bushey & Barker (1944) Atwell & Duthie (1964) Biber & Mullen (1980) Lipton (1972) Andreoli, Schafter & Patlak (1978) Frizzell, Dugas & Schultz (1975) Where necessary, values recalculated from the authors' data. Influx is in the direction lumen to blood and is referred to unit serosal area except for small intestine in vivo where the units are of mucosal area. Small intestinal mucosal area per cm length is taken as 48 5 cm2 (dog) and mucosal area/serosal area = 25 (human) (Unpublished data of D. S. Parsons). 1/35 S. cm-2. The electrical resistance of straight renal tubules of rabbit cortex is about 5 Q1 cm2 (Andreoli, Schafer & Patlak, 1978). Absence of net Na flux. Many studies on salt transport across short-circuited small intestine have shown a small but significant net absorption of Na and this has been attributed to active transcellular Na movement (Schultz & Curran, 1968). No significant nor consistent net Na movement could be detected across the small intestine of R. ridibunda or R. pipiens, and even with R. catesbeiana the value of the net absorption varied widely. The calculation of the net flux involves the substraction of two large unidirectional fluxes, so any net Na movement may be too small to be detected by the methods employed for measuring the fluxes. It is possible that the increased magnitude of Na fluxes across

8 128 D. S. PARSONS AND S. A. WADE vascularly perfused small intestine, compared with non-perfused tissue, is due to a higher proportion of passive Na movement. Any active component of Na transfer must, therefore, form only a very small fraction of the total. Seasonal effect. The magnitude of the Na fluxes measured across the small intestine is dependent upon the time of year in which the experiments were performed (Table 2). Thus fluxes measured during the summer and autumn months are higher than those measured in the winter and spring months. All fluxes measured during 1980 were lower than when measured during the period from 1978 to 1979 although it is not known why this was so. Such seasonal effects have also been reported by Gilles-Baillien, Havaux & Chapelle (1979) for Na transport across the small intestine of the tortoise, by Csaiky & Galluci (1977) for amino acid and sugar transport and for (Na + K)-ATPase activity in the small intestine of R. pipiens, and by Boyd & Perring (1980) for the lumen to blood transfer of a-amino isobutyric acid across the small intestine of R. ridibunda. It is not known how these changes in transport activity are induced but it does highlight a feature to be taken into account when working with these animals. Loading and washout of tracer Na from the small intestine Loading. It is apparent that during the loading phase, the time taken to reach isoptic equilibrium within the small intestine (Fig. 1) and the values obtained for pool size (Table 3) are largely determined by the experimental conditions. Under standard conditions of loading isotopic equilibrium is reached in at least 30 min and the values of pool sizes are equivalent to a substantial fraction of the total tissue water. If the tracer is flushed rapidly into the lumen it takes about 5 min to achieve isotopic equilibrium and the pool size (0 23,tmol.cm-2) is about one tenth of that obtained with standard lumen flow rates. Thus some 90% of the apparent loading pool under standard conditions appears to be tracer Na equilibrating with the bulk phase of the lumen. Presumably by flushing the tracer rapidly into the lumen turbulence is created thereby increasing stirring adjacent to the epithelial border. Moreover, the results obtained with this technique show how quickly Na moves across the intestinal epithelium; the time from tracer starting to enter the lumen to its appearance from the portal venous cannula (30-40 s) is the same as the time for fluid to clear the dead space occupied by the portal vein and the tubing leading to the fraction collector. Thus the time of transit of Na across the epithelium cannot be more than a second or so. A transport pool of 0-23,mol cm2 of Na within the intestinal wall represents a volume of 1I 9 l.cm-2 if the Na is present in the tissue water at the same concentration as in the Ringer (120,cmol ml-'). Since 1 cm2 serosal area of small intestine contains approximately 22 #u1 of water, this transport space represents about 9% of the total tissue water. Furthermore, we have previously reported (Parsons & Wade, 1981) that the extracellular fluid within the wall of the small intestine is about 15 ml. g dry wt.-', equivalent to 9 1At cm-2, at the vascular flow rate of about 5 ml. g dry w.-r min-' that was used in these experiments. Thus the transport pool for Na occupies 20% of the total extracellular water of the whole tissue. These estimates must all be maximum values. Washout. The washout of tracer Na from the preloaded tissue is bi-exponential, a feature also seen in the washout of sugars and amino acids from the frog small intestine (Boyd & Parsons, 1978; Parsons & Sanderson, 1980). If the tracer is flushed rapidly out of the lumen, the unloading into the vascular bed is still bi-exponential but the initial rate of washout is much faster (see Fig. 2 where k' = 1 16min-';tq = 0-6 min) and the unloading pool size

9 INTESTINAL SODIUM FLUXES Table 6. Fluxes of Na across colon 129 Na influx Flux pmol. cm-2. S-1 ratio Reference Bullfrog Cooperstein & Hogben (1959) Rabbit Frizzell & Schultz (1978) Human Grady, Duhamel & Moor (1970) Pig Smith (1976) Rat Curran & Lindemann (quoted by Schultz & Curran, 1968) R. ridibunda present work R. pipiens present work Where necessary, values recalculated from the authors' data. Influx is in the direction lumen to blood and is referred to unit serosal area. Flux ratio = Na influx/na efflux. (0-41,umol. cm-2, Table 3) is about one ninth of that obtained with standard flow rates. Using a preparation of stripped guinea-pig small intestine mounted in a flux chamber especially modified for studying washout kinetics, Lauterbach (1976) reported similar values for half-times of 22Na washout. This suggests that the initial washout of tracer Na into the vascular bed under standard conditions is a reflexion of the decreasing specific activity of the tracer in the lumen as well as in the tissue wall itself. Differences in the velocity of linear flow through the lumen may then explain why the value of k, (0 35 min-', Fig. 2) is greater than the mean value of k, from ten earlier experiments. During unloading the tracer Na washes out from a pool, or pools, of apparently greater size than the loading pool whether the intestine has been loaded rapidly or under standard conditions. This phenomenon of 'up-down asymmetry' has also been observed for amino acids and sugars in the frog small intestine (Boyd et al ; Boyd & Parsons, 1978), for Na in frog skin (Fuchs, Gebhardt & Lindemann, 1972) and for K in frog skeletal muscle (Hodgkin & Horowicz, 1960). Fluxes of Na across colon Although the unidirectional fluxes of Na measured across the colon of R. ridibunda and R. pipiens are up to eight times lower than across the small intestine, a consistent and significant net absorption of Na is found (Table 4) showing that the colon is a major site Na absorption in these two anuran species. Interestingly, whereas the Na fluxes across the vascularly perfused small intestine of the frog are higher than fluxes across isolated tissue (Table 5), the fluxes across the vascularly perfused colon in these studies resemble values obtained from other in vitro preparations (Table 6). Moreover, the flux ratio, Na influx to Na effilux, is comparable with other studies. Conclusions Na moves rapidly in either direction across the small intestine of the frog and a very high proportion of the movement seems to occur by diffusion along low resistance pathways, most probably the paracellular shunt pathway. The transported Na is contained within a pool occupying not more than 900 of the total tissue water and 20% of the extracellular water of the intestinal wall. Although active Na pumping by the epithelial cells may occur at a rate which is only a very small fraction of the unidirectional rates, such pumping must be essential for the EPH 67

10 130 D. S. PARSONS AND S. A. WADE maintenance of not only the volume of the epithelial cells but also of the conditions within the low resistance pathways upon which the bulk of the Na movements depends. This work was supported by a M.R.C. Project Grant, G977/1 16/S. REFERENCES ANDERSEN, B. & ZERAHN, K. (1963). Method for non-destructive determination of the sodium pool in frog skin with radiosodium. Acta physiologica scandinavica 59, ANDREOLI, T. E., SHAFER, J. A. & PATLACK, C. S. (1978). Mechanisms for salt and water transport in the mammalian proximal straight tubule. In Membrane Transport Processes, vol. 1, ed. HOFFMAN, J. F., pp New York: Raven Press. ARMSTRONG, W. McD. (1976). Bioelectric parameters and sodium transport in bullfrog small intestine. In Intestinal Ion Transport, ed. RoBINSON, J. W. L., pp Lancaster: M.T.P. Press. ATWELL, J. D. & DUTHIE, H. L. (1964). The absorption of water, sodium and potassium from the ileum of humans showing the effects of regional enteritis. Gastroenterology 46, BIBER, T. U. L. & MULLEN, T. L. (1980). Effect of external cation and anion substitutions on sodium transport in frog skin. Journal of Membrane Biology 52, BOYD, C. A. R., CHEESEMAN, C. I. & PARSONS, D. S. (1975). Amino acid movements across the wall of anuran small intestine perfused through the vascular bed. Journal of Physiology 250, BOYD, C. A. R. & PARSONS, D. S. (1978). Effects of vascular perfusion on the accumulation, distribution and transfer of 3-O-methyl-D-glucose within and across the small intestine. Journal of Physiology 274, BOYD, C. A. R. & PERRING, V. S. (1980). Factors influencing amino acid exit from the vascularly perfused intestinal epithelium. Journal of Physiology 301, 71-72P. CEREIJIDO, M. & ROTUNNO, C. A. (1968). Fluxes and distribution of sodium in frog skin: a new model. Journal of General Physiology 51, s. COOPERSTEIN, I. M. & HOGBEN, C. A. M. (1959). Ionic transfer across the isolated frog large intestine. Journal of General Physiology 42, CHEESEMAN, C. I. (1979). Factors affecting the movement of amino acids and small peptides across the vascularly perfused anuran small intestine. Journal of Physiology 293, CSAKY, T. Z. & GALLUCI, E. (1977). Seasonal variation in the active transporting ability and in the membrane ATPase activity of the frog intestinal epithelium. Biochimica et biophysica acta 466, CUTHBERT, A. W. (1971). Neurohypophyseal hormones and sodium transport. Philosophical Transactions of the Royal Society, Series B, 292, CURRAN, P. F. (1960). Na, Cl and water transport by rat ileum in vitro. Journal of General Physiology 43, CURRAN, P. F. & SOLOMON, A. K. (1957). Ion and water fluxes in the ileum of rats. Journal of General Physiology 41, FRIZZELL, R. A. (1976). Coupled sodium chloride transport by small intestine and gallbladder. In Intestinal Ion Transport, ed. ROBINSON, J. W. L., pp Lancaster: MTP Press. FRIZZELL, R. A., DUGAS, M. C. & SCHULTZ, S. G. (1975). Sodium chloride transport by rabbit gallbladder: direct evidence for a coupled NaCl influx process. Journal of General Physiology 65, FRIZZELL, R. A. & SCHULTZ, S. G. (1978). Effect of aldosterone on ion transport by rabbit colon in vitro. Journal of Membrane Biology 39, FucHs, W., GEBHARDT, U. & LINDEMANN, B. (1972). Delayed voltage responses to fast changes of [Na]l at the outer surface of frog skin epithelium. In Biomembranes, ed. KREUZER, F. & SLEGERS, J. F. G., chap. 3, pp New York: Plenum. GILLES-BAILLIEN, M., HAVAUX, J. F. & CHAPELLE,S. (1979). Seasonal variation in Na+ transport and (Na+-K+)-ATPase activity in tortoise intestinal epithelium. Journal of Comparative Physiology 129, GRADY, G. F., DUHAMEL, R. C. & MOORE, E. W. (1970). Active transport of sodium by human colon in vitro. Gastroenterology 59,

11 INTESTINAL SODIUM FLUXES 131 HODGKIN, A. L. & HOROWICZ, P. (1960). The effect of sudden changes in ionic concentration on the membrane potential of single muscle fibres. Journal of Physiology 153, LAUTERBACH, F. 0. (1976). Ion fluxes in isolated guinea-pig intestinal mucosa. In Intestinal Ion Transport, ed. ROBINSON, J. W. L., pp Lancaster: MTP Press. LIPTON, P. (1972). Effect of changes in osmolarity on sodium transport across isolated toad bladder. American Journal of Physiology 222, PARSONS, D. S. (1976). Closing summary. In Intestinal Ion Transport, ed. ROBINSON, J. W. L., pp Lancaster: MTP Press. PARSONS, D. S. & SANDERSON, 1. R. (1980). Influence of vascular flow on amino acid transport across frog small intestine. Journal of Physiology 309, PARSONS, D. S. & WADE, S. A. (1981). Sodium fluxes across the vascularly perfused intestine of the frog. In Epithelial Ion and Water Transport, ed. MACKNIGHT, A. D. C. & LEADER, J. P. New York: Raven Press. QUAY, J. F. & ARMSTRONG, W. McD. (1969). Sodium and chloride transport by isolated bullfrog small intestine. American Journal of Physiology 217, ROSE, R. C. & SCHULTZ, S. G. (1971). Studies on the electrical potential profile across rabbit ileum: effects of sugars and amino acids on transmural and transmucosal electrical potential differences. Journal of General Physiology 57, SCHULTZ, S. G. (1977). Some properties and consequences of low-resistance paracellular pathways across the small intestine: the advantages of being 'leaky'. In Intestinal Permeation, ed. KRAMER, M. & LAUTERBACH, F., pp Amsterdam and Oxford: Excepta Medica. SCHULTZ, S. G. & CURRAN, P. F. (1968). Intestinal absorption of sodium chloride and water. In Handbook of Physiology, section 6, Alimentary Canal, vol. 3, ed. CODE, C. F., Washington, D.C.: American Physiological Society. SCHULTZ, S. G., FRIZZELL, R. A. & NELLANS, H. N. (1974). Ion transport by mammalian small intestine. Annual Review of Physiology 36, SCHULTZ, S. G. & ZALUSKY, R. (1964). Ion transport in isolated rabbit ileum. I. Short-circuit current and sodium fluxes. Journal of General Physiology 47, SMITH, M. W. (1976). Sodium transport by the newborn pig intestine: functional changes during the first few days of postnatal life, In Intestinal Ion Transport, ed. ROBINSON, J. W. L., pp Lancaster: M.T.P. Press. USSING, H. H. & ZERAHN, K. (1951). Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta physiologica scandinavica 23, VISSCHER, M. B., FETCHER, E. S., JR, CARR, C. W., GREGOR, H. P., BUSHEY, M. S. & BARKER, D. E. (1944). Isotopic tracer studies on the movement of water and ions between intestinal lumen and blood. American Journal of Physiology 142, VISSCHER, M. B., VARCO, R. H., CARR, C. W, DEAN, R. B. & ERICKSON, D. (1944). Sodium ion movement between the intestinal lumen and the blood. American Journal ofphysiology 141, WADE, S. A. (1979). Simultaneous comparison of sodium fluxes in different regions of vascularly perfused intestine of Rana ridibunda. Journal of Physiology 296, 1-2P. WADE, S. A. (1980). Sodium fluxes across the vascularly perfused small intestine and colon of Rana ridibunda and Rana pipiens. Journal of Physiology 303, 75P. 5-2