preliminary account of some of the findings has been given [Parsons and

Transcription

1 FLUID AND SOLUTE TRANSPORT ACROSS RAT COLONIC MUCOSA. By D. S. PARSONS and C. R. PATERSON.* From the Department of Biochemistry, University of Oxford, South Parks Road, Oxford, Great Britain. (Received for publication 2nd October 1964) A method is described of preparing everted sacs of rat colonic mucosa free of the smooth muscle layers. Fluid movement across the wall of the sacs may be maintained for periods of up to 4 hr. in vitro under aerobic conditions in the presence of a substrate. Glucose, mannose, acetate, butyrate and to a lesser extent, pyruvate, are effective substrates. Although taken up from the mucosal fluid, glucose is most effective as a substrate for fluid movement when present in the transmucosal fluid. IIn the presence of glucose, the rate of lactate formation is low under aerobic conditions in vitro, but increases some eight-fold under anaerobic conditions. With glucose present, lactate appears at a higher concentration in the transmucosal fluid than in the mucosal fluid. The colon can transport water from mucosal fluid into the transmucosal fluid against an osmotic gradient; reasons are given for supposing that this fluid movement is secondary to the functioning of an active solute transport mechanism. Glucose, galactose and 3--methylglucose are not subjected to active transport (translocation) across the colonic mucosal layers against a concentration gradient. STUDIES in the mammal have indicated that an important function of the colon is the terminal absorption of fluid and electrolytes. Thus, water, Na and Cl are rapidly absorbed and HCO3 secreted when salt solutions isotonic with plasma are recirculated through rat colon in vivo [Parsons, 1956]. Goldschmidt and Dayton [1919] have shown that in the dog colon, fluid absorption continued from solutions in the lumen which were hypertonic with respect to blood; more recently Curran and Schwartz [196] concluded that water transport across rat colon in vivo is a passive process and secondary to active solute transport. Davidson and Garry [1939] have found no evidence for glucose absorption from isotonic solutioxvs instilled into rat colon in vivo, but studies of sugar transport by colon surviving in vitro appear to have been neglected. The purpose of the present experiments was to examine the conditions under which fluid absorption occurred in a new preparation of rat colon in vitro and to look for evidence of net water transport across the colonic mucosa against an osmotic gradient, as has been found to occur in rat small intestine [Parsons and Wingate, 1961]. The capacity of rat colon to transport sugars 'actively' against a concentration gradient has also been examined. A preliminary account of some of the findings has been given [Parsons and Paterson, 196]. Everted sacs have been used in studies of solvent and solute exchanges in frog skin and stomach and in the mammalian small intestine [Huf, 1935; Davies, 1948; Wilson and Wiseman, 1954 a]. The preparation of rat colon * M.R.C. Training Scholar. Present address: Department of Chemical Pathology, The University of Leeds, Leeds 2. 22

2 Absorption in Rat Colon described here is similar to the everted small intestinal sac described by Crane and Wilson [1958] except that it has proved possible to strip off the muscle layer leaving viable colonic mucosa supported only by a thin layer of Muscularis Mucosa. With this preparation it is possible to measure independently the fluid uptake from the mucosal side, the changes in the tissue fluid content and the amount of fluid appearing in the interior of the everted sac. METHODS Unfasted male albino rats (125-2 g.) were killed by decapitation; the entire descending colon was removed, dropped into ice-cold oxygenated saline and washed out at least twice with the same fluid to remove ftecal matter. Preparation of Sacs. - A glass cannula, 7-5 mm. o.d. and 1 cm. long with a constriction just short of one end and tapered to 2-5 mm. o.d. over the terminal 1 cm. at the other, was cooled and the tapered end inserted into a segment of colon of about 5 cm. length. A longitudinal incision was gently made with a blunt scalpel through the muscle layer of the whole length of the distended colon; the retracted muscle layer could then be peeled off with the fingers leaving behind the mucosa supported by a thin layer of muculari8s mucosce (fig. 1). One end of the segment was tied with linen thread at the constriction in the cannula; the rest of the segment was then gently drawn over the ligature and the open end tied to give an everted sac suspended from the cannula ml. of incubation medium was introduced into the interior of the sac (transmucosal side) through thin polythene tubing attached to a syringe; the transmural hydrostatic pressure (sac interior - sac exterior) amounted to some 2 cm. saline. The sac was immersed in incubation medium, gassed with 95 per cent 2, 5 per cent CO2 at 38 C. (fig. 2a). For some experiments it was necessary to increase the volume of the transmucosal fluid. In sacs where the initial transmucosal volume was of the order of 1 ml. it was found that during incubation the osmotic activity of the transmucosal fluid always increased, sometimes by as much as 5 m.osm./kg. water, while the activity of the mucosal fluid always fell. Thus in cases where the mucosal fluid was initially hypertonic to that in the transmucosal compartment, the gradient of osmotic activity existing initially across the mucosal layers fell markedly as absorption proceeded. These changes in the gradient of osmotic activity were reduced by employing larger initial volumes for the transmucosal fluid. In experiments to examine the effects of transmural gradients of osmotic activity on fluid movements, the sacs were therefore each suspended from a cannula provided with a bulb at the lower end so that the initial volume of fluid on the transmucosal side was increased to about 5 ml. The segment of colon was first stripped of its muscle coat and then everted on a tapered glass rod of similar dimensions to the plain cannula usually used. The lower end of the bulb type cannula was now inserted into the open end of the everted sac and tied in; the sac was cut free from the glass rod and the lower end closed with a further ligature. The contents of these larger cannulae were stirred by a stream of gas bubbles (fig. 2b). Incubation Media. - The basic medium used contained (mm) NaCl, 113; Na2HPO4, 1-8; NaH2PO4, -2; NaHCO3, 25; CaCl2, 1-25; MgSO4, 1-; KCI, 4-5; sodium benzyl-penicillin, 2 units/ml. and streptomycin sulphate, 5,ug./ml. In some experiments the composition of the incubation media was altered as indicated below. Determination of Fluid Movements. - Fluid 'uptake' is defined as the fluid removed from the mucosal side; fluid 'transport', the fluid appearing in the interior of the sac (transmucosal side). Both of these quantities were measured using a weighing 221

3 gas inlet tiii I go-s inlet.-- as inlet ~polythene tube ~~mucosaisac-.- -sintered--- glass disk a FIG. 2. Arrangement for incubation of everted sacs of colonic mucosa. (a) Sac suspended from plain cannula, initial volume of transmucosal fluid,.5-1.o ml. (b) Sac suspended from cannula with bulb, initial volume of transmucosal fluid, 5 ml. In both instances volume of mucosal fluid, 5 ml. b FIG. 3. Linear relationship between fluid uptake (U) from lumen and fluid transport into transmucosal compartment (T) in sacs of colonic mucosa incubated for 2 hr. See text. Slope of regression line,.94±.4 (23 degrees of freedom).

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6 Absorption in Rat Colon technique. In general, over any period of observation, fluid uptake (U) and fluid transport (T) are related by U =T + AH, where AH = change in hydration of the tissue. Fluid uptake was deduced from increases in the combined weight of the cannula, the sac and its contents measured at intervals of 3-6 min. Before each weight determination the sac was blotted with absorbent paper using a standard technique. To prevent evaporation during weighing, the sac was enclosed in a glass test tube with a rubber bung and the increase in weight of the whole was taken as the amount of fluid taken up from the mucosal side. The period during which the sac was out of the incubation medium for weighing was not more than 3 sec. Net fluid transport was measured by a method of collection and weighing. The amount of fluid in the interior of the sac at the start of an experiment comprises (a), the fluid initially adhering to the transmucosal surface of the sac and (b), the medium introduced at the beginning of the experiment. The amount of fluid present inside the sac at the end of the incubation is (c),that quantity removed by a syringe and (d), that remaining on the serosal surface of the sac. Thus, T=c+d-(a+b) In experiments in which 5 ml. of incubation medium was added initially to the transmucosal side, this fluid was mixed by a stream of bubbles issuing from a polythene tube. Fluid lost from the interior of the sac by evaporation comprises (e), that collected by condensation on the previously dry wall of the cannula and (f), water vapour escaping to the atmosphere, with the gas stream. Some fluid (g) was lost in the procedure of removing the bubbling-tube. Then the net fluid transport is given by: T = c + d + e +f +g - (a + b) For each experiment, (b) was determined by weighing a syringe, initially containing warm medium, before and after filling the sac; (c) was determined by reweighing a tared syringe into which the sac contents had been aspirated; (d) was measured using a standard technique, by blotting the transmucosal surface with a tared filter paper which was then weighed; (e) was measured by reweighing the cannula at the end of each experiment. In addition, appropriate estimates of (a), (f) and (g) were employed, the values used being derived from dummy experiments. Relationship between ' Uptake' and 'Transport'. - It was thus possible to determine independently on each sac the fluid uptake from the mucosal side and the fluid transport into the transmucosal side. In fig. 3, the fluid transport (T) is shown related to fluid uptake (U) 'for a number of sacs on which both were measured. Uptake and transport are linearly related, the difference between them representing the increase in tissue hydration(ah); the line is thus consonant with the relation U=T+AH. Determination of Solute Movements. - Movements of solutes were deduced from the changes in concentration and volume of the incubation media present on either side of the sac wall. The osmotic activity of the experimental fluids was measured using the Fiske Osmometer (Fiske Associates Inc., Boston, Mass., U.S.A.). Each value was determined until acceptable duplicates, agreeing to better than 5 per cent, were obtained. With each analytical run, the instrument was calibrated against standard solutions of NaCl. Analytical Methods. - Reducing sugars were determined by Nelson's method as modified by Somogyi [1952] and lactate by the method of Hullin and Noble [1953]. The tissue was weighed wet and again after drying overnight on an air oven at 1-14O C, the reduction in weight being taken as the fluid content of the tissue. The dry weight was determined on the tissues after extraction of neutral fat as described for liver slices by Parsons and van Rossum [1961]. The results are expressed in terms of the fat-free dry weight (FFDW). VOL. I,, wo

7 224 Parsons and Paterson RESULTS Effect of Substrate Orientation on Fluid Uptake. - Fluid uptake by everted sacs of colonic mucosa was measured: (a) with 12.2 mm glucose initially on both sides of the sac, (b) with 12.2 mm glucose on the transmucosal side only, (c) with 12.2 mm glucose on the mucosal side only and (d) without substrate. In the media to which glucose was added, the NaCl content was reduced to TABLE I. EFFECT OF SUBSTRATE ORIENTATION ON FLUID UPTAKE BY SACS OF COLONIC MUCOSA INCUBATED AT 38 C. FOR THE TIME INDICATED Values, mg.water/mg. dry weight/hr., are means ± standard error of mean (number of observations). Conditions of incubation Fluid uptake 1st hr. 2nd hr. 3rd hr 4th hr. No substrate (13) 3*2+5 (13) 2'7±1- (4) 4± 7 (4) Glucose 12-2 mm both sides (18) 1-5±-7 (18) 6-3±8 (11) 4-6 (7) Glucose 12-2 mm transmucosal side ±1-1 (15) (15) 5-2±-8 (8) (7) Glucose 12-2 mm mucosal side ±1-1 (6) 5-7A1-5 (6) 3-8±1-3 (6) 1-9±-6 (6) 17 mm/l. The results of these experiments are given in Table I. It is seen that fluid is absorbed at a considerable rate for up to 4 hr. and that the uptake rates are substantially higher in the presence of glucose than in its absence. It is also seen that glucose supplied on the transmucosal side is much more effective in maintaining fluid uptake for 2-3 hr. than glucose supplied on the mucosal side. With glucose present initially on the mucosal TABLE II. EFFECTS OF METABOLICALLY ADVERSE CONDITIONS ON FLUID UPTAKE BY SACS OF COLONIC MUCOSA INCUBATED FOR 2 HR. Substrate 12-2 mm/1 glucose in both compartments, temperature 38 C. and oxygen present. Values, mg./mg. dry weight/hr., are means i standard error of mean (number of observations). Conditions Fluid uptake Optimal ±.7 (18) Anoxic (gas phase 95 per cent. N2 5 per cent. 2). 5± 3 (4) Fluoracetate, 1 mm/l present ±6 (4) No substrate (4) Temperature 29.5 C ±V11 (4) Temperature 22 C ±-2 (4) side only, fluid absorption is only slightly greater than with no substrate at all, and in the absence of substrate fluid absorption continues for about 2 hr. Inhibition of Fluid Uptake. - It was found that, with glucose as substrate, the rate of fluid uptake by the mucosal sacs was inhibited by reducing the temperature of incubation, by incubation in anoxic conditions, and by incubation in the presence of 1 mm/i. Na fluoroacetate (Table II). Support of Fluid Uptake by Various Substrates. - A number of experiments were performed to determine what other substances could act as substrates for fluid uptake. In each case the substrate was added at initially the same concentration to the media on both sides of the sac.

8 Absorption in Rat Colon 225 The results (Table III) show that there is marked enhancement of the rate of fluid uptake by acetate, acetoacetate, n-butyrate and to some extent by pyruvate, but with fumarate, L-glutamate and succinate there is little significant stimulation. Fructose and mannose, like glucose, stimulated fluid uptake while galactose, 3--methylglucose and L-arabinose were ineffective. TABLE III. SUPPORT OF FLUID UPTAKE OVER 2 HR. IN RAT COLONIC MUCOSA BY VARIOUS SUBSTRATES, PRESENT INITIALLY AT EQUAL CONCENTRATIONS IN FLUIDS ON EITHER SIDE OF THE SACS Values, mg. water/mg. dry weight/hr. are means ± standard error of mean (number of observations). Fluid uptake No substrate ±7 (13) Glucose 12-2 mm ± 7 (18) Mannose 12-2 mm ±-8 (4) Fructose 12-2 mm (4) 3--methylglucose 1-3 mm.. 7-2± 6 (4) L-arabinose 111 mm ±1 (6) Galactose 12-2 mm ± 7 (4) Sodium acetate 1 mm ±-8 (4) Sodium n-butyrate 1 mm.. 111±11 (4) Sodium acetoacetate 1 mm.. 9-7±1-2 (4) Sodium pyruvate 1 mm.. 87±16 (4) Sodium fumarate 1 mm.. 7-6±-8 (4) Sodium L-glutamate 1 mm.. 6-8±-3 (4) Sodium succinate 1 mm.. 6-3±9 (4) Sodium citrate 1 mm ± 9 (4) Effects of Cations on Fluid Uptake. - Fluid uptake was measured in experiments in which the NaCl of the medium bathing the mucosal surface was replaced by isomolar quantities of KCI, LiCl, RbCl or choline chloride. The fluid in the transmucosal side was Na-containing incubation medium with 12.2mM glucose. It is seen (Table IV) that with potassium, rubidium and choline, the rate of fluid uptake was almost as high as in the presence of sodium; with lithium, the rate of fluid uptake was greatly reduced and the fluid transport was not significantly different from zero. Net Transport of Water against an Osmotic Gradient. - Other experiments were undertaken to examine the capacity of the sacs of mucosa to induce net water transport against an osmotic gradient. For this purpose we have looked for the net appearance of fluid in the transmucosal compartment under conditions such that throughout the period of observation the mucosal fluid was always hypertonic with respect to the fluid in the transmucosal compartment. In these experiments the sacs were suspended from cannulae of capacity 5 ml. (see methods) and the fluid uptake and the fluid and the solute transport into the transmucosal fluid determined over periods of 2 hr. The magnitude and the direction of the initial gradient of osmotic activity across the wall of the tissue was varied by altering the NaCl concentration in the glucose-free mucosal fluids. The transmucosal fluid was the basic VOL. L, NO *

9 226 Parsons and Paterson incubation medium containing glucose, 28 mm/1. The results are presented in Table V. It was found that, as in the small intestine [Parsons and Wingate, 1961], the net rate of water influx into the serosal fluid decreases as the adverse osmotic gradient increases; and further, that the sacs of colonic mucosa are capable of inducing a significant (p<.1) net water movement across the TABLE IV. EFFECT OF REPLACING THE NACL IN MucOsAL FLUID ON WATER MOVE- MENT IN SACS OF COLON Water Predominant Cationic content (mm) Water uptake transport cation Na+ K+ other (mg.water/mg. dry weight/hr.) Sodium ±-9 (15) 11-1±-9 (15) Potassium ±-6 (4) 8-3±-6 (4) Lithium Li ±-5 (4) -4±-5 (4) Rubidium Rb ±1-1 (4) 8-5±1-7 (4) Choline Choline ±1-6 (4) 7-1±1-7 (4) mucosal and subepithelial layers against an adverse osmotic gradient of m.osm./kg. However increasing the magnitude of the adverse osmotic gradient to above 5 m.osm./kg. arrests water movements. The data in Table V also show that under all conditions there is a net transport of solute into the transmucosal fluid. The changes in tissue hydration observed in these experiments are given in Table VI. It is found that the hydration of the sac wall increases during absorption, the increase being apparently directly related to the rate of fluid movement across the wall. TABLE V. SEGMENTS OF RAT DESCENDING COLON, INCUBATED 2 HR., 28 MM GLUCOSE PRESENT ON TRANSMUCOSAL SIDE ONLY Initial osmotic activity on serosal side ca. 3'm-osm. Osmotic gradient altered by changing NaCl concentration on nilucosal side. P=difference in osmotic activity, mulcosal-transmucosal. Fluiid trans- Solute trans- Fluid uptake port into port into AP from mucosal transmucosal transmucosal Mucosal m-osm fluid fluid side fluid Initial Final mg./mg.dw/hr. mg./mg.dw/hr. m-osm/mg.dw/hr. Hypertonic ±-4 (4) -1-±-3 (4) 1-8±-2 (4) Hypertonic ±-3 (8) 1-4±-2 (8) 2-1±-2 (8) Isotonic ±-6 (7) 6-4±-6 (5) 2-7±-2 (5) Hypotonic ±1-4 (7) 9-7±1-3 (7) 2-7±-4 (7) Net Transport of Sugars against a Concentration Gradient. - Experiments were undertaken to examine the capacity of the tissue to induce a net translocation of sugar from the mucosal fluid across the colonic wall into the transmucosal fluid against a concentration gradient as is known to occur in the small intestine, [Fisher and Parsons, 195; Wilson, 1962]. In these experiments solutions of the same composition and containing the sugar to be tested were placed on both sides of the sacs of mucosa and the changes in the quantity of sugar in the transmucosal compartment examined after 2 hr. incubation. The findings, Table VII, indicate that at the concentrations employed, there is no evidence of the net translocation into the transmucosal

10 Absorption in Rat Colon fluid of any of the five sugars tested; indeed in all the experiments, except in the case of some individual instances with galactose, there was a net loss of sugar from the fluid in the transmucosal compartment. This movement was always associated with a fall in the sugar concentration in that compartment. TABLE VI. CHANGES IN TISSUE HYDRATION IN SACS OF COLON FOLLOWING 2 HRm INCUBATION Tissue hydration (mg. water/mg. dry weight) Unincubated control sacs ±*9 (3) Incubated sacs: Mucosal fluid initially 85 m-osm 'hypertonic' (4) 48 m-osm 'hypertonic'. 638±-21 (7) 'isotonic' ±.25 (6) 49 m-osm 'hypotonic'. 6-98±417 (7) 227 Glucose Utilization and Lactic Acid Production by Colonic Mucosa. - In experiments in which sacs of colonic mucosa were incubated with 12.2 mm/l. glucose initially present in both fluid compartments, the glucose lost from both compartments of the system ('glucose utilization,' Fisher and Parsons, 195) was measured over 2 hr. periods and found to total 247 ± 11 (8),imoles/ kg. FFDW/hr. In other experiments the lactic acid production by the sacs of colonic mucosa was determined after incubation for 2 hr. under various TABLE VII. EXIT OF SUGAR FRtOM TRANS1xCOSAL FLUID OF SACS AFTER 2 Em. INCUBATION Initial sugar concentration on both sides of sac as indicated; values, mm/kg FFDW/hr. means E standard error of mean (number of observations). Exit rate Glucose 12-2 mm ± 56 (8) Mannose 12-2 mm ±1F13 (4) Galactose 12-2 mm i 5.6 (13) 3--methylglucose 1-3 mm. 9*5± 7 (5) L-arabinose 11-1 mm ± 4*5 (6) conditions. In estimating the tissue content of lactate at the end of each experiment it was assumed that the concentration in the tissue water was the same as that in the transmucosal compartment. It was found (Table VIII), that under aerobic conditions small amounts of lactate are formed in the absence of exogenous substrate; the addition of glucose increased the rate of lactate formation. With glucose present, the rate of lactate formation under anaerobic conditions was about eight time sthat found in the presence of oxygen. Aerobically, with glucose present, lomm fluoroacetate increased the rate of lactate production. Glutamate did not affect the basal rate of lactic acid production. In all these experiments the quantity of lactic acid appearing in the mucosal fluids was always greater than in the transmucosal fluids; however in every case the final concentration of lactate in the transmucosal

11 A Parsons and Paterson fluid was considerably greater than that in the corresponding mucosal fluid. Thus, in six experiments in which 12.2mM/i, glucose was initially present in both the mucosal and transmucosal fluids of colonic sacs, the mean lactate concentration found after 2 hr. incubation was, for the mucosal fluids.1 ±.1 mm/l. and for the transmucosal fluid, 1.9f.3 mm/l. TABLE VIII. LACTATE PRODUCTION BY SACS OF COLONIC MUCOSA MEASURED OVER 2 Hi. THE TOTAL LACTATE PRODUCTION INCLUDES LACTATE ESTIMATED TO BE PRESENT IN THE WALL OF THE SACS. Lactate appearance (millimoles/kg. DW/hr.) Krebs bicarbonate medium gassed with Trans- 95 per cent per cent. CO2 at 38 C. Mucosal mucosal Total (a) with glucose 12-2 mm on both sides ±12 (6) (b) with glucose 12-2 mm and fluoroacetate 1 mm on both sides ±24 (4) (c) with glutamate 1 mm on both sides ±4 (4) (d) without substrate ±4 (4) Anoxia: Krebs bicarbonate medium gassed with 95 per cent. N2 + 5 per cent. CO2 at 38 C. Glucose 12-2mM on both sides ±12 (4) DiscussIoN We find that the substrate requirements for fluid absorption by rat colon are very different from those which have been described for the jejunum, although fluid movement in both tissues depends upon aerobic conditions. Appreciable fluid movement occurs in the colon in vitro over periods of up to 2 hr. in the absence of exogenous substrate; the rate of fluid movement is also increased by the addition, not only of glucose and mannose, but by acetate, butyrate and pyruvate. In rat jejunum, glucose and to a small extent fructose, are the only known substrates which support fluid absorption [Smyth and Taylor, 1957]. Glucose supplied in the mucosal fluids of the jejunum is required for the support of water absorption, very much higher concentrations on the transmucosal side being required to stimulate water absorption from the lumen [Lifson and Parsons, 1957]. In the colon, on the other hand, the rate of fluid absorption with glucose present in the mucosal fluid only is scarcely faster than in the absence of substrate, and the effects on fluid transport depend upon the presence of the glucose on the transmucosal side. The rat ileum resembles the colon in that fluid transport can be sustained to some extent by pyruvate as well as by glucose [Gilman and Koelle, 196 b]. The poor rates of fluid absorption observed in the colon in the presence of citrate and succinate may be due to the 'inability of these acids to penetrate cellular barriers. These findings can be related to the metabolic characteristics of rat intestinal mucous membrane. Dickens and Weil-Malherbe [1941] showed

12 Absorption in Rat Colon 229 that rat duodenal and jejunal mucous membrane possessed a high rate of aerobic glycolysis and exhibited almost no Pasteur effect. They also found lower rates of aerobic glycolysis in the ileum and colon, but the Pasteur effect in this region appeared to be variable. Wilson and Wiseman [1954 b] also found a greater rate of aerobic glycolysis in rat jejunum than in the ileum, in which a large Pasteur effect was observed. We find that in the colon which is absorbing fluid in vitro, there is a low rate of aerobic glycolysis and that this rate is increased some eight times under anaerobic conditions, i.e. that there is a marked Pasteur effect. It thus appears that in the rat colon, as in the ileum [Gilman and Koelle, 196 a and b], it is oxidative metabolism which provides the source of energy upon which fluid transport ultimately depends so that under anaerobic conditions there is no fluid transport (Table IV). It appears that in rat jejunum fluid absorption requires aerobic conditions, but that aerobic glycolysis is also intimately involved as an obligatory source of energy for fluid transport [Gilman and Koelle, 196 a and b]. The distribution of the lactate produced by the metabolism of the colon is of interest when compared with the findings for small intestine [Wilson, 1954, 1956; Parsons and Wingate, 1961]. In the small intestine in vitro it is usual for most of the lactate to appear in the transmucosal fluid and for the lactate concentration to be lowest in the mucosal fluid. A similar asymmetrical distribution of lactate has been found to occur in the case of the toad bladder [Leaf, 1959]. In the present experiments on the colon there was, relative to the volume of mucosal fluid, a much smaller volume of transmucosal fluid, and a major portion of the lactate produced in the course of metabolism appeared on the mucosal side (Table VIII). However, although the transmucosal gradient of concentration of lactate at the end of these experiments was always of the order of 2: 1, the transmucosal fluid having the higher concentration, in the absence of direct measurements of the mean lactate concentration in the metabolizing colonic mucosal cells it is not possible to assess the relative permeabilities of the mucosal and transmucosal faces of these cells. Water Transport against Osmotic Gradient across Colonic Mucosa. - Our experiments have shown that the rat colon can transport water from mucosal fluid into a transmucosal fluid in which the total solute concentration is at all times less than in the mucosal fluid, i.e. the water is transported against an osmotic gradient. This finding is consistent with the observation of Goldschmidt and Dayton [1919] that fluid was absorbed from hypertonic saline solutions (ca. 4 m.osm.) in the lumen of dog colon in vivo. As in the case of rat jejunum and ileum where a similar transport of water against an adverse solute concentration gradient has been observed [Parsons and Wingate, 1961], it is possible that the water movement is ultimately a passive process closely linked to an active solute transport mechanism. With the colon operating as in the model proposed by Curran [196], (see also Curran and Macintosh, 1962) the presence of a small excess hydrostatic pressure in the interstices of the mucosal tissue would achieve the results we have observed. In the model, solute is transported from the mucosal fluid into the tissue

13 23 Parsons and Paterson spaces of the mucosa. Fluid enters these tissue spaces by passing through the cells from the mucosal fluid and is moved under a hydrostatic pressure gradient into the transmucosal fluid compartment. That the fluid movement in rat colon depends upon solute movement has been shown by Curran and Schwartz [196] who demonstrated that water transport is related to net Na transport. In the present experiments fluid transport from Na-containing mucosal fluids was always associated with solute movement (Table V). The existence of a hydrostatic pressure in the tissue spaces of the sacs of colonic mucosa may follow the fact that during absorption the hydration of the tissue increases (Table VI). A similar increase in the tissue hydration is shown in the data presented in fig. 3. Histological preparations of rat colon made after incubation in vitro frequently show distended tissue spaces see e.g., fig. ID. Active Transport of Sugars by Colonic Mucosa. - It is necessary to distinguish between absorption from the lumen, i.e. net uptake from the mucosal fluid and translocation, the net movement through the epithelial cells into the tissues spaces and beyond. Defining 'active absorption' as the translocation through the layer of mucosal cells and against a concentration gradient, then the colonic mucosa does not possess the capacity to absorb sugars 'actively.' Thus the findings for the colon again stand in contrast to those for the jejunum of the rat which exhibits this capacity to a marked extent. Net glucose translocation against a concentration gradient can be achieved by the terminal cm. of rat ileum [Fisher and Parsons, 195; Parsonis and Wingate, 1961] but Baker et al. [1961] were unable to demonstrate glucose translocation in the terminal 1 cm. of ileum although as in the case of the colon, the sugar can be absorbed from the mucosal fluid. The terminal (aboral) portion of the ileum of the rat may thus resemble the colon not only in its metabolic characteristics and in its capacity to secrete HCO3 into the lumen [Parsons, 1956], but also in its inability to subject sugars to translocation.

14 Absorption in Rat Colon 231 REFERENCES BAKER, R. D., SEARLE, G. W. and NuNN, A. S. (1961). Amer. J. Phy8iol. 2W, 31. CRANE, R. K. and WILSON, T. H. (1958). J. appl. Phy8iol. 12, 145. CUiRRAN, P. F. (196). J. gen. Phy8iol. 113, CutRRAN, P. F. and MACINTOSH, J. R. (1962). Nature, Lond. 193, CuRRAN, P. F. and SCHWARTZ, G. F. (196). J. gen. Physiol. 43, 555. DAVIDSON, J. N. and GARRY, R. C. (1939). J. Physiol. 96, 172. DAVIES, R. E. (1948). Biochem. J. 42, 69. DICKENS, F. and WEIL-MALHERBE, H. (1941). Biochem. J. 35, 7. FISHER, R. B. and PARSONS, D. S. (195). J. Physiol. 11, 281. GITMAN, A. and KOELLE, E. S. (196 a). Circulation, 21, 948. GILMAN, A. and KOELLE, E. S. (196 b). Amer. J. Physiol. 199, 125. GoLDSCHMIDT, A. and DAYTON, A. B. (1919). Amer. J. Physiol. 48, 433. HuIF, E. (1935). Pfltug. Arch. ges. Physiol. 235, 655. HULLIN, R. P. and NOBLE, R. L. (1953). Biochem. J. 55, 289. LEAF, A. (1959). J. Cell. Comp. Physiol. 54, 13. LIFSON, N. and PARSONS, D. S. (1957). Proc. Soc. exp. Biol. N.Y. 95, 532. PARSONS, D. S. (1956). Quart. J. exp. Physiol. 41, 41. PARSONS, D. S. and PATERSON, C. R. (196). Biochim. Biophys. Acta. 41, 173. PARSONS; D. S. and VAN ROSSuM, G. V. R.- (1961). Quart. J. exp. Phy8iol. 46, 353. PARSONS, D. S. and WINGATE, D. L. (1961). Biochim. Biophys. Acta. 46, 17. SOMOGYI, M. (1952). J. biol. (Jhem. 16, 61. SMYTH, D. H. and TAYLOR, C. B. (1957). J. Physiol. 136, 632. WISON, T. H. (1954). Biochem. J. 56, 521. WILSON, T. H. (1956). J. biol. Chem. 222, 751. WILSON, T. H. (1962). In Intestinal Absorption. Philadelphia and London: W. B. Saunders Co. Chapter 4. WILSON, T. H. and WISEMAN, G. (1954 a). J. Physiol. 123, 116. WISON, T. H. and WISEMAN, G. (1954 b). J. Physiol. 123, 126.