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116 J. Physiol. (954) 23, 6-25 THE USE OF SACS OF EVERTED SMALL NTESTNE FOR THE STUDY OF THE TRANSFERENCE OF SUBSTANCES FROM THE MUCOSAL TO THE SEROSAL SURFACE BY T. H. WLSON* AND G.

1 116 J. Physiol. (954) 23, 6-25 THE USE OF SACS OF EVERTED SMALL NTESTNE FOR THE STUDY OF THE TRANSFERENCE OF SUBSTANCES FROM THE MUCOSAL TO THE SEROSAL SURFACE BY T. H. WLSON* AND G. WSEMAN From the Medical Research Council Unit for Research in Cell Metabolism, Department of Biochemistry, and the Department of Physiology, University of Sheffield (Received 11 July 1953) A preparation of isolated small intestine of the rat or golden hamster (Mesocricetus auratus) is described hich permits convenient measurement of respiration and glycolysis during periods of active transference of substances across the all. The difficulty of adequate oxygenation is overcome by everting a piece of intestine, tying it at both ends and filling it ith sufficient fluid to distend the all. The eversion exposes the highly active mucosa to the ell-oxygenated suspending medium, hile the distension increases the surface area of the sac and reduces the thickness of the sac all. The oxygenation of the inner layer of the serosal surface is facilitated by an oxygen bubble hich is introduced into the sac along ith the inner fluid. The relatively small volume of fluid contained in the sac (serosal side) allos a rapid rise in concentration of transferred substances. A number of adjacent segments of intestine from the same animal may be studied simultaneously. The method as tested ith glucose and methionine, both substances being knon to be transferred against a concentration gradient (Fisher & Parsons, 1949b; Wiseman, 1953). Aerobically both ere transferred against a concentration gradient in this preparation, but no active transference took place anaerobically. EXPERMENTAL Preparation of tissue. The animal (rat or hamster) as killed by a blo on the head, the abdomen opened by a midline incision, and the entire small intestine ashed out ith a solution of.9% (/v) NaCl containing 3 % (/v) glucose. The hole of the small intestine as then removed by cutting across the upper end of the duodenum and the loer end of the ileum and manually stripping the mesentery from the intestine. n the case of the hamster all the fat and mesentery ere removed by this procedure, but in the rat a small amount of fat remained on the * Exchange Fello of the American ncer Society.

MOVEMENT OF SUBSTANCES ACROSS NTESTNE 117 intestine along the line of mesenteric attachment. There as never any evidence of perforation of the intestine as a result of this treatment.

2 MOVEMENT OF SUBSTANCES ACROSS NTESTNE 117 intestine along the line of mesenteric attachment. There as never any evidence of perforation of the intestine as a result of this treatment. To evert the gut, a stainless steel rod (3 mm long, 1*5 mm diameter) as used to push the ileal end of the gut into the gut lumen until it appeared at the duodenal opening of the intestine, and the eversion as completed by rolling the proximal half of the intestine on the rod. The everted intestine as then slipped off the steel rod and placed in glucose-saline at room temperature in a flat porcelain dish. A thread ligature as tied around one end of the everted intestine to facilitate subsequent identification. Measurement of initial and final volumes. A length of everted intestine (2-3 cm in most experiments) as tied off at one end by a thread ligature and a second ligature as placed loosely around the other end (ready for tying). After being blotted ith a piece of filter-paper the tissue plus the ligatures ere eighed on a torsion balance. A blunt needle, attached to a 1 ml. syringe (tuberculin type), as introduced into the intestinal lumen and the loose ligature pulled tight over the needle. The appropriate fluid along ith a small bubble of, (approximately -5 ml.) as then injected into the sac and as the needle as ithdran the ligature as tied tight. This distended sac as eighed and the increase in eight taken as a measure of the initial volume. The volume of the fluid in the sac at the end of an experiment as determined by eighing the sac before and after draining it of its contents. Chemical estimations. Lactic acid, glucose and methionine ere determined colorimetrically by the methods of Barker & Summerson (1941), Nelson (1944), and Mcrthy & Sullivan (1941) respectively. Materials. The glucose and L-methionine ere of A.R. standard. The purity of the L-methionine as checked by the use of the L-amino-acid oxidase prepared from Neurospora crassa and as found to be 1% pure ithin the limits of error (± 3 %). Manometry. Conical Warburg flasks of 25 or 125 ml. size ere used. When determining the rate of oxygen uptake, phosphate-saline (Krebs, 1933) as used as the medium, ith KOH and filter-paper in the centre-ell. n anaerobic experiments bicarbonate-saline (Krebs & Henseleit, 1932) gassed ith 5% C in 95% N2 as used. Some aerobic experiments ere performed in bicarbonate-saline hich as gassed ith 5% CO2 in 95% 2. The 25 ml. flasks ere shaken at 9 oscillations/min (total excursion 4 cm), the 125 ml. flasks at 7/min. Transference and metabolic quotients. To express the results the folloing quotients are used: QO,=pl. s uptake (indicated by minus sign) in phosphate-saline/mg dry t./hr. Qci' =. CO2 evolved anaerobically in bicarbonate-saline/mg dry t./hr. Q1iticacidd=Jsl lactic acid produced aerobically (estimated colorimetrically)/mg dry t./hr. Qglucoe metabolized =41- glucose disappearing from the medium/mg dry t./hr. Qglucose transferred and Qmethionine transferred =1.d of the material on the serosal side at the end of an experiment in excess of that initially present/mg dry t./hr. Glucose, methionine and lactate are expressed as pl. of gas (1,umole =22 4,u.). Positive transference values indicate passage of the substrate from mucosal to serosal side; negative values indicate passage in the opposite direction. Water transferred is not given in Q units (i.e. pl. gas) but as,ul. of fluid/mg dry t./hr. Dry eight. The sacs ere emptied at the end of the experimental period and the surfaces gently blotted ith Whatman no. 4 ifiter-paper. n the case of the rat the et tissue as extracted ith ether to remove fat. The eight as determined after drying for 2 hr at 11 C. RESULTS Hamster small intestine Five sacs ere prepared from consecutive segments of everted small intestine of the hamster and shaken at 9 oscillations/min in 25 ml. Warburg flasks at 37 C for 1 hr. The rate of oxygen uptake of the first sac as measured and

118 T. H. WLSON AND G. WSEMAN found to be -29,ul./mg dry t./hr. The other four served for the study of the movement of methionine, glucose and ater under aerobic and anaerobic conditions.

3 118 T. H. WLSON AND G. WSEMAN found to be -29,ul./mg dry t./hr. The other four served for the study of the movement of methionine, glucose and ater under aerobic and anaerobic conditions. Tables 1 and 2 sho the initial and final concentrations of glucose and methionine respectively as ell as the total amounts transferred in these sacs. The initial concentrations of glucose and of methionine ere the same inside and outside the sac. Aerobically the glucose concentration rose on the serosal side to more than tice the initial concentration, hile it fell on the mucosal side. n contrast, there as a net loss of glucose on the serosal side anaerobically. The net transference of glucose, methionine and ater and the utilization of glucose ere calculated from the initial and final concentrations and volumes. Aerobically there as a net gain of methionine and ater on the serosal side, hile there as a loss anaerobically. Because ater passed out of the sac more rapidly than methionine under anaerobic conditions there as a slight rise in methionine concentration on the serosal side. There as a small net loss of methionine, corresponding to -2 mg/mg dry t./hr, probably representing diffusion into the tissue. QN,o as 17f2 in sac no. 4 and 19-3 in sac no. 5. When the initial concentration of methionine is reduced to X1 % the net transference is decreased but the final concentration gradient becomes greater. n experiments ith loer substrate concentrations larger sacs ere incubated in 125 ml. Warburg flasks, giving larger volumes of fluid for analysis. Fig. 1 shos the concentration gradients developed in 1-5 hr. Oing to the large difference in the volume of fluid inside (about 2 ml.) and outside (about 1 ml.) the sac, the fall in glucose and methionine concentrations outside the sac are not as great as the rise inside the sac. The calculated transference values and the glucose metabolism values are shon in Table 3. The transference of glucose in phosphate-saline as studied ith jejunal sacs. n one such experiment the Qo2 as found to be - 2, the Qglucose transferred as 17, the Qgluose metabolzed as 8-2, and the ater transferred as 1,l. fluid/mg dry t./hr. A large amount of glucose as transferred ith little movement of ater. When such sacs ere greatly distended at the beginning of an experiment they lost fluid during incubation under aerobic conditions but there as alays a net gain of glucose ithin the sac. The largest glucose concentration gradients ere obtained ith to sacs placed in bicarbonate-saline (no methionine being added). The concentration of glucose on the serosal side rose from an initial value of 2 % to over 1%, hile that on the mucosal side fell to 1 %. With these sacs the Qglucose transferred as 36 and the ater transferred as 9-6,ul. fluid/mg dry t./hr, for the jejunal sac, hile the ileal sac gave a Qglucose transferred of 17 and a ater transference of 3.9,ul. fluid/mg dry t./hr.

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5 12 T. H. WLSON AND G. WSEMAN TABLE 3. Glucose and methionine transferred by sacs of golden -hamster small intestine (125 ml. Warburg flasks containing sacs, bicarbonate-saline and gas phase 5% CO. in 2. Experimental period 1-5 hr. 37 C. See Fig. 1 for concentrations of glucose and methionine for sac no. 1.) H2 transferred Dry t. (1ld. fluid/ Q Qcethouine Qglucoe Sac no. (mg) Location of sac mg/hr) sfred tfraferred metabolized 1 61 Loer jejunum Upper ileum 9* S 4_ bo E 2 - Glucose Methionine Fig. 1. Concentration gradients developed by a jejunal sac of the golden hamster. (Sac no. 1, Table 3. nitial vol. on serosal side = 1- ml.; initial vol. on mucosal side = 15-Orml. Bicarbonatesaline. Gas phase 5% CO2 in 2. Experimental period 1-5 hr.) 8, serosal side; M, mucosal side; -.., initial concentration. Rat small intestine Tables 4 and 5 sho the results obtained ith sacs of rat everted small intestine. To pairs of adjacent sacs ere placed in large manometer flasks containing bicarbonate-saline, one of each pair being gassed ith 5 % CO2 and 95% 2, the second ith 5% CO2 and 95% N2. The Q2 as determined in a sac prepared from an area of intestine lying beteen the regions used for the pairsi just described, and the value found to be Table 4 shos that glucose is transferred against a concentration gradient aerobically but not anaerobically. The values for Qglucoe transferred are loer than those obtained ith hamster intestine, hile the values for Qgiucos metabolized are similar in both species. Table 5 shos that aerobically there is a movement of methionine from the mucosal side to the serosal side against a concentration gradlient hile anaerobically there is a slight loss from the serosal side. The QXjr- as 19-6 in sac no. 1 and 24-5 in no. 2.

6 MOVEMENT OF SUBSTANCES ACROSS NTESTNE 121 caco- -, _7 R o (M p Q o C v r d) r ;.4 co '4 a..9 o oo - 41 co -4 co P4 - D *t x CO 5.4 PA co Q p4 W S. S. p_4 2; RSe 5 O VV o e. 84 -A a; p4 C Zozo.5 -El.5.5 to q n &. CP o2 41 d o *5 :3 o ^ > co 5.4 CO _ i CX O

7 122 T. H. WLSON AND G. WSEMAN To further experiments are reported in hich no methionine as added to the suspending medium. With an initial glucose concentration of '15% on both sides of the intestinal all the Qgiucose transfered of a jejunal sac as 8-2 and in to ileal sacs of the same animal 2-8 and 1-1. With an initial glucose concentration of -3 % to adjacent sacs (jejunal) from a second animal gave transference values of 7- and 8-1. Aerobic lactic acid production n a fe experiments the temperature of incubation as reduced from 37 to 32 C in order to decrease the rate of respiration and ensure the adequacy of oxygenation. The ratio of the Qo2 to Q,ic acid in the experiments at both of these temperatures as similar (about 1) (Wilson & Wiseman, 1954), and therefore adequate oxygenation in the experiments at 37 C is assumed. Eight rat jejunal sacs ere studied at 32 C, ith an initial glucose concentration of -4 %. The average Qglucose transferred as 4-3 and the Qglucose metabolized as The Q t1 acid as Lactic acid production therefore may account for over half of the glucose metabolized. As shon in Table 6 the concentration of lactate is greater on the serosal than the mucosal side, although the total amounts on the to sides are similar (the volume on the serosal side being -5 ml., that on the mucosal side being 3- ml.). The hamster sacs sho similar gradients of lactate though the concentrations are loer. TABLE 6. Concentration gradients of lactic acid developed by sacs of rat and golden hamster small intestine during periods of glucose transference (nitial fluid inside and outside sac as bicarbonate-saline containing -3% glucose. Gas phase 5% CO2 in 2. Experimental period 1 hr. 37 C.) Serosal concn. Mucosal concn. Animal Location (mm) (mm) Rat Jejunum Hamster Jejunum Hamster leum Time course of absorption To study the development of concentration gradients across the sac all, four consecutive sacs of rat jejunum ere prepared ith the same initial concentration of glucose inside and outside the sac. At 2 min intervals after the start of incubation, sacs ere removed from the manometer flasks and the glucose concentration determined. The results are shon in Fig. 2. n the first 2 min there as a fall in concentration of about -3% on both sides of

8 MOVEMENT OF SUBSTANCES ACROSS NTESTNE 123 the intestine. After 4 min the glucose concentration on the serosal side as slightly above the initial value. This as folloed by a rapid rise in concentration at the 6 and 8 min periods, and as associated ith a marked fall in concentration on the mucosal side. The 3 min lag period may represent the 6 D E 5 m jjn.4 nitial concn. E 3 Serosal side Mucosal side 2-1 2min 4min 6min 8min Fig. 2. Time course of concentration gradients of glucose developed by sacs of rat everted jejunum. (Bicarbonate-saline containing -38% glucose as used for initial serosal and mucosal fluids. nitial vol. on serosal side=-3 ml.; initial vol. on mucosal side =3- ml. Gas phase 5% CO. in 2. Temp. 32 C.) time required for the development of a high concentration of glucose ithin the tissue and for diffusion through the submucosa and muscularis. A similar fall in concentration and lag period as found by Wiseman (unpublished observations) hen studying amino-acid transference ith intestine in vitro (Wiseman, 1953). DSCUSSON The results reported in this paper sho that the sacs of everted intestine of the rat and hamster are suitable for the study of intestinal transference. Both test substances, glucose and methionine, are transferred against concentration gradients and the quantities transferred are of the same order of magnitude as in other preparations of isolated intestine (Fisher & Parsons, 1949b; Wiseman, 1953; Darlington & Quastel, 1953). An essential condition for full activity of the tissue is adequate oxygenation, and the high aerobic glycolysis of the rat intestine raises the question hether oxygenation is in fact satisfactory. The vie that oxygenation is adequate is supported by the results obtained at 32 C. f the glycolysis ere due to lack

9 124 T. H. WLSON AND G. WSEMAN of oxygen it should fall markedly hen the temperature is decreased, but the ratio Q 2Q 1,acid as the same (about 1) at 32 C. Also the fall of Qo2 accompanying the decrease in temperature, being about 4 % for a change of 5 C, is of the order expected for a system here the diffusion of oxygen is not a limiting factor. The amounts of glucose hich disappear in the rat intestine are considerably greater than the amounts hich are transferred across the all. t is very remarkable that about half of the glucose hich disappears from the mucosal side is converted into lactic acid and that this substance appears in much higher concentration on the serosal side than on the mucosal side. A similar lactate concentration gradient as observed (Wilson, 1953) in the preparation of Fisher & Parsons (1949. This suggests that the conversion of glucose to lactic acid may play an important part in the absorption of glucose by rat intestine. Hestrin-Lerner & Shapiro (1953) have recently found that during transference of radioactive glucose by the rat and cat intestine an unidentified radioactive substance is produced. t ould seem likely that this unidentified radioactive material is lactic acid. n the case of the hamster aerobic lactic acid production is considerably loer than in the rat, but a similar concentration difference is observed. Aerobically, fluid moved into the sacs against a hydrostatic pressure. When the sacs ere only moderately distended at the start of the experiment distension increased under aerobic conditions; anaerobically the reverse occurred. When the sacs ere greatly distended at the start by the injection of a larger amount of fluid, some fluid left the sac even aerobically. Thus fluid passes from the mucosal to the serosal side until a critical degree of distension is reached. Since the concentration of the inorganic ions as not estimated the full explanation for the movement of ater is uncertain. One explanation is that ater moves across the intestine passively ith the glucose until the critical distension is reached, after hich time glucose continues to be transferred but ithout a further movement of ater. McDougall & Verzair (1935) have shon that during absorption of glucose from a 5 % glucose solution, ater is absorbed along ith the glucose and that ater transference stops hen glucose absorption from such a solution is inhibited. SUMMARY 1. A method is described by hich respiration and active transference by sacs of intestine of the rat and golden hamster can be measured simultaneously. To secure adequate oxygenation, the intestine is everted and the all distended by filling the sac ith fluid at a slight pressure. 2. Aerobically glucose and methionine are transferred against a concentration gradient across the all of the sacs; anaerobically no active transference occurs.

MOVEMENT OF SUBSTANCES ACROSS NTESTNES 125 3. Data are given for the amounts of glucose and methionine hich are transferred by the intestinal all. 4.

10 MOVEMENT OF SUBSTANCES ACROSS NTESTNES Data are given for the amounts of glucose and methionine hich are transferred by the intestinal all. 4. Aerobically ater moved into the sac ith the glucose and methionine, hile anaerobically fluid as lost from the sac. 5. n the rat, a major portion of the glucose metabolized appears as lactate hich accumulates on the serosal side of the intestine. n the hamster a smaller amount of lactate is produced but accumulation on the serosal side is also observed. 6. t is suggested that the conversion of glucose to lactate by the intestine may play a part in the absorption of glucose. Part of the expenses of this research as defrayed by a grant to one of us (G. W.) from the Medical Research Fund of the University of Sheffield, and by a grant from the Medical Research Council to Prof. D. H. Smyth. The authors are indebted to Dr M. Hokin and Dr L. E. Hokin for stimulating discussion. They also ish to thank Prof. D. H. Smyth and Prof. H. A. Krebs, F.R.S., and Dr R. E. Davies for much helpful advice and interest in this ork. Thanks are also due to K. H. Curtis for technical assistance. REFERENCES BARKER, S. B. & SUMMERSON, W. H. (1941). The colorimetric determination of lactic acid in biological material. J. biol. Chem. 138, DARLNGTON, W. A. & QUASTEL, J. H. (1953). Absorption of sugars from isolated surviving intestine. Arch. Biochem. 43, FSHER, R. B. & PARSONS, D. S. (1949. A preparation of surviving rat small intestine for the study of absorption. J. Physiol. 11, FSHER, R. B. & PARSONS, D. S. (1949b). Glucose absorption from surviving rat small intestine. J. Physiol. 11, HESTRiN-LERNER, S. & SLPRo, B. (1953). Active absorption of glucose from the intestine. Nature, Lond., 171, KREBS, H. A. (1933). Untersuchungen uber den Stoffechsel der Aminosauren im Tierkorper. Hoppe-Seyl. Z. 217, KREBS, H. A. & HENSELET, K. (1932). Untersuchungen uiber die Harnstoffbildung im Tierkorper. Hoppe-Seyl. Z. 21, McCARTY, T. E. & SUrLVAN, M. X. (1941). A ne and highly specific colorimetric test for methionine. J. biol. Chem. 141, McDoUGATT, E. J. & VERZAR, F. (1935). Die Resorption von Wasser aus Kochsalz- und Zuckerlosungen. Pflug. Arch. ge8. Physiol. 236, NELSON, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. J. biol. Chem. 153, WLsoN, T. H. (1953). Lactate and hydrogen ion gradients developed across the rat intestine in vitro. Biochim. biophys. acta (in the Press). WLSON, T. H. & WSEMAN, G. (1954). Metabolic activity of the small intestine of rat and golden hamster. J. Physiol. 123, WSEMAN, G. (1953). Absorption of amino-acids using an in vitro technique. J. Physiol. 12,

J. Physiol. (I956) I33,

626 J. Physiol. (I956) I33, 626-630 ACTIVE TRANSPORT OF AMINO ACIDS BY SACS OF EVERTED SMALL INTESTINE OF THE GOLDEN HAMSTER (MESOCRICETUS AURATUS) BY G. WISEMAN From the Department of Physiology, University