bathing medium caused a decrease in short circuit current without (Received 3 February 1977)

Transcription

1 J. Phyaiol. (1977), 27, pp With 9 text-figurew Printed in Great Britain SHORT CIRCUIT CURRENT AND TOTAL CONDUCTANCE MEASUREMENTS ON RAT ILEUM M. GRAQA EMfLIO AND C. HENRIQUES DE JESUS From(Centro de Biologia, Instituto (ulbenkian de Ciencia, Oeira8, Portugal (Received 3 February 1977) SUMMARY 1. Short circuit current and total conductance measurements were made on preparations of rat terminal ileum, using either the intact tissue or the isolated epithelium. 2. The effects of D-glucose, 3--methyl-D-glucose, and 3-deoxy-3-fluor- D-glucose, added in suitable concentrations to the external bathing medium, were studied. 3. An increase in both short circuit current and total conductance was detected when these sugars were used. It was possible to determine a linear relationship between the two variables and to study the kinetics of the interaction of glucose and 3--methyl-D-glucose with the sodium sugar carrier by using increasing concentrations of these substances. 4. The increases in short circuit current and total conductance were also verified in isolated epithelium. 5. Phlorizin, or the withdrawal of glucose, reversed the increase in short circuit current, but the total conductance remained unchanged. 6. Increasing concentrations of 2-deoxy-D-glucose added to the mucosal bathing medium caused a decrease in short circuit current without changing the total conductance, either in preparations of intact ileum or in isolated epithelium. 7. The meaning of these variations in the electrical parameters of the preparations is discussed. INTRODUCTION The interaction between sodium and sugar influx across the intestinal epithelium has been extensively studied (Schultz & Curran, 197), and it is generally accepted that there is a carrier mediated mechanism accounting for the entry of glucose and other sugar molecules across the intestinal brush borders against a chemical gradient, and dependent on the presence of sodium. At the same time, the sodium sugar carrier is activated by the presence of sugar in the external medium, so that the entry of sodium

2 21 M. GRAQA EMILIO AND C. HENRIQUES DE JESUS into the cells is a function of the external sugar concentration. This is reflected in the net sodium influx, which is equivalent to the short circuit current in the preparations we are studying (Schultz & Zalusky, 1964a; Clarkson & Toole, 1964). In these circumstances, the simple measurement of the electrical parameters on the preparations gives useful information about the transport characteristics of the carrier. Previous studies on new-born pig ileum and rabbit ileum (Henriques de Jesus, 1974, 1977) have shown that the increase in short circuit current caused by adding glucose to the mucosal side of the preparation is accompanied by an increase in the total conductance in such a way that the relationship between both variables follows a straight line with a positive slope. A different relationship was found for another sugar molecule, 2-deoxy-D-glucose, which causes a decrease in short circuit current and an increase in total conductance. These findings led to the postulation of an electrical model different from the classical model of Ussing & Windhager (1964) for epithelia. The present study set out to see whether the same effects on short circuit current and total conductance would be found with a different intestinal preparation and for different sugar molecules, and to determine the kinetics of the action of various substances on the sodium sugar carrier of the rat ileum. This preparation proved to be very suitable for these types of experiments, as the electrical parameters show a good stability for the time required to follow the protocols. METHODS Unstarved male Wistar rats (15-25 g) were used in all experiments. The animals were anaesthetized with chloroform and a piece of terminal ileum of length ca. 1 cm was dissected and divided into two parts which were mounted in paired chambers. These chambers have been described previously (Henriques de Jesus, 1977) and were designed to minimize edge damage and to keep good conditions of oxygenation and temperature of the preparations. The half-chambers were filled with Krebs phosphate solution (5 ml.) kept at 37 C and aerated with pure oxygen. The experimental substances were added as concentrated solutions, so that the total volume added never exceeded 2 #sl. All the chemicals were A.R. and the electrolytic composition of the solutions was checked regularly. The area of exposed tissue was 2-4 cms. The preparations were permanently short circuited through an automatic system of voltage clamp (Henriques de Jesus & Smith, 1974) and the short circuit current was monitored by a multichannel Philips recorder. The total conductance was determined by applying a 2 mv pulse at regular intervals or by measuring the open circuit potential with a Keithley electrometer. A period of equilibrium of 15-3 min was allowed after mounting the arrangement, before the experimental conditions were introduced. Preparations with low control values of short circuit current (< 25 /esa/cm2) were discarded. After each addition of the substance under study, a lapse of time was allowed for the preparation to reach a new steady state. In a group of experiments a nutrient solution was used to bathe the

3 SOC AND ST MEASUREMENTS ON RAT ILEUM 211 serosal side of the tissue. This solution was composed of Eagle's minimum essential medium used in tissue culture techniques (Autopow, Flow Lab.), buffered with tricine and compensated for osmolality and sodium chloride concentration to match the usual Krebs solution. Previous work with amphibian epithelia had shown that the conditions of the preparations were considerably improved, in the long term, by using a similar medium (Emilio, Balls & Menano, 1975). In the present case, however, no significant difference was noticed in the general behaviour of the preparations bathed either by minimum essential medium or by Krebs solution, at least during the relatively short time of the experiments (2-3 hr). For this reason, Krebs solution was used throughout most of the work. In a group of experiments, which will be described separately, the preparations were stripped of the serosal and muscular layers by blunt dissection before setting up the chambers. Further details of protocol followed in this study will be given in the next section. 12r l l l l C-) 11 F E E a) -C c 1 1- C. 9 8 i I Short circuit current (isa/cm2) 8 1 Fig. 1. Effects of glucose additions on short circuit current and total conductance measurements. Glucose concentrations are indicated at the top of the graph. Means and S.E. of means of 17 experiments. Slope (calculated by the method of least mean squares) O54 ±.1 mmhos pta-; x2 =.959.

4 212 M. GRAQA EMfLIO AND C. HENRIQUES DE JESUS A. Glucose effects RESULTS Successive additions of glucose in steps of 1 32 mm, up to the total concentration of 6-6 mm, were followed by stepwise increases of the short circuit current and total conductance. The rise of short circuit current was very steep with the first addition of glucose and somewhat slower with the last ones. These transient characteristics, however, were not analysed in detail and only steady-state values will be presented. Two adjacent pieces of terminal ileum were used. It was noticed that the segment next to the ileo-caecal valve had a slightly higher response to the addition of glucose than the one further away. Nevertheless, all the experiments with reasonable control values of short circuit current ( > 25,uA/cm2) were considered together for statistical purposes. Mean values and s.e.'s of means of short circuit current and total conductance are presented in the graph of Fig. 1, showing that there is a good linear relationship between the two variables. 5r E 11.1 N I-- VI' 21 -' /s (mm)-' Fig. 2. Double reciprocal plot of glucose concentrations (8) against increase in short circuit current (v) in intact ileum preparations. Vm = 69 #ua. Km = 2*8 mm. Slope (calculated by the method of least mean squares) mm ma-1; X2 =.1. 8

5 8 SCC AND GT MEASUREMENTS ON RAT ILEUM GI 1 3 l GI 3.9 I Total conductance 1 mmhos cm2 GI 5.3 GI 6i6 Phl Phl Phl 25 3*7 l l Phl 5* l 213 E 6 E 1-4.) C -, 4 U.._ c 2 (n F _- I Time (min) Fig. 3. Effects of glucose (GI) and phlorizin (Phl). The upper outline of the plot represents the short circuit current, and the distance between the upper and the lower outline gives the total conductance. Concentrations of glucose (mm) and phlorizin (#M) are given at the top I I 1 1 I I1 (n E E E -C3 U C a, ~ C 1 F 9 I 9 I I I Short circuit current (pa/cm2) Fig. 4. Effect of 3-O-methylglucose additions on short circuit current and total conductance measurements. Concentrations of 3-O-methylglucose are shown at the top of the Figure. Means and s.e. ofmeans oftwelve experiments. Slope (calculated by the method of least mean squares) m-mhos,za-'. x2 = '969.

6 214 M. GRAQA EMILIO AND C. HENRIQUES DE JESUS In a group of eleven control experiments in which the preparations were kept in glucose-free Krebs solution for the same time as necessary for the glucose additions (ca. 6 min), the values of short circuit current at the start and at the end of this period were, respectively and 38'1 + 3,uA/cm2 (means and S.E.S of means). Similarly, the values of the total conductance were and mmho cm2. These figures are not statistically different. The increase of short circuit current with glucose exhibited kinetics of the Michaelis-Menten type, as shown in Fig. 2. The values calculated for KMn and Vin were, respectively, 2-8 mm and 69,uA/cm2. The initial values of short circuit current could be recovered by washing the preparations with fresh, glucose-free Krebs solution but the value of the total conductance did not change, or decreased only slightly. If the successive additions of glucose were repeated, similar increases in short circuit current were obtained but the change in total conductance was not so noticeable. Similarly, phlorizin (5 x M) reversed the increase in short circuit current caused by glucose but the total conductance remained high, or showed just a small decrease. The plot obtained in one of these experiments is shown in Fig. 3. B. 3--methyl-D-glucose effects The protocol followed in these experiments was strictly similar to the one just described, the same concentrations of 3-O-methyl-D-glucose being used. As in the glucose experiments, addition of successive amounts of this substance were followed by stepwise increases of short circuit current and total conductance, the two variables sharing a linear relationship to one another (Fig. 4). The study of the kinetics of its action on the sodium carrier, shown in Fig. 5, allowed the calculation of a Km value of 7-7 mm and a Vm value of 37,uA/cm2. A good recovery of the initial conditions could be obtained after washing the preparations. C. 2-deoXy-D-glucose effects 2-deoxy-D-glucose was used both in freshly mounted preparations and in preparations which had been subjected to the action of 6-6 mm glucose. Addition of increasing concentrations of that substance ( mm) was followed by successive decreases of the short circuit current, both in the fresh preparations and after glucose treatment; the total conductance remained constant throughout the experiments. The variation of the short circuit current was higher in the cases where glucose had been used previously, the differences between the mean values being statistically significant (P < 1). In both circumstances, the relationship between

7 SeC AND CT MEASUREMENTS ON RAT ILEUM E /s (mm-') Fig. 5. Double reciprocal plot of 3-O-methylglucose concentrations (8) against increase in short circuit current (v). Vm = 37 1A; Km = 7.7 mm. Slope (calculated by the method of least mean squares) mm ma-l; X2 = X67. 2-deoxy-D-glucose concentrations and short circuit current followed the Michaelis-Menten equation (Fig. 6). The Km value was the same in both groups of experiments (47.6 mm). The Vm values were #ta/cm2 for 2-deoxy-D-glucose alone and ,uA/cm2 for 2-deoxy-D-glucose after glucose treatment. Recovery, after washing, was very poor in most cases. The effect of glucose treatment after maximal doses of 2-deoxy-Dglucose was also evaluated in a few experiments; it was seen that the preparations reacted to the usual glucose concentrations ( mm) in the same way as the freshly mounted ones, i.e. with increases of short circuit current and total conductance of the same order of magnitude. D. Isolated epithelium of terminal ileum A group of experiments was carried out in isolated epithelia, after stripping the muscular layers of the intestine by blunt dissection, in order to evaluate the importance of these layers on the total conductance

8 216 M. GRAQA EMILIO AND C. HENRIQUES DE JESUS > '15 1/s (mm-1) Fig. 6. Double reciprocal plots of 2-deoxy-D-glucose concentrations (8) against decrease in short circuit current (v). * without glucose: Vm = -384 #sa; Km = 476 mm. Slope 1118±48-8mMnmA1; X2 = '2789. A after glucose: Vm = uA; Km = 47-6 mm. Slope 73± 42-7 mm ma-1;x2 = of the preparations. The effects of glucose and 2-deoxy-D-glucose were studied with the protocol previously described. The results, shown in the graphs of Fig. 7 and Fig. 8, can be compared with those obtained using the non-dissected preparations, with respect to the changes in short circuit current, although the Km values were higher for both sugars in the isolated epithelium. The mean initial value for the total conductance was 1 1 mmhos cm2, higher than the corresponding value for the intact tissue (8.7 mmhos cm2), but the increases of total conductance with increasing glucose concentrations were identical in both cases. Final mean values after 6-6 mm glucose treatment were, respectively, 12X3 and 11*4 mmho cm2. As in the former experiments, the total conductance was not altered by the addition of 2-deoxy-D-glucose. E. Effects of 3-deoxy-3-fluor-D-glucose A small amount of this substance was available, so that its effects on the short circuit current and total conductance of four preparations of

9 SOC AND GT MEASUREMENTS ON RAT ILEUM N I I I ( CO l4 ( n MOD C;) LbCb I I I 217 en E E (A E * C.) C. I- 111F i1-9 I I I Short circuit current (/ia/cm2) Fig. 7. Effect of glucose on short circuit current and total conductance in isolated epithelium of rat ileum. Glucose concentrations are indicated at the top of the graph. Means and s.e. of means of twelve experiments. Slope (calculated by the method of least-mean squares) -61 ± *2 m-mhos 6A-1; %2 = /s (mmm) 1/s (mmm) Fig. 8. Double reciprocal plots of glucose and 2-deoxyglucose concentrations against, respectively, increases or decreases in short circuit current in isolated epithelium of rat ileum. * glucose: Vm = 69 lisa; Km = 6-5 mm. Slope = 73*2 ± 79 mm ma-1; x2 = A 2-deoxy-D-glucose: V. = -55 PA; K. = 8 MM. Slope = 1482_ 25-3 mm ma-1;x2 = 1286.

10 218 M. GRAQA EMILIO AND C. HENRIQUES DE JESUS isolated epithelium were studied by adding increasing concentrations to the mucosal half-chambers. The total conductance also increased, but at least in two preparations most of the rise was seen after the first addition of the drug. The results of these experiments are shown in Fig * N 16 E E M 14-~ ~ ~ ~ 4 14~~~~~~ C o 4~~~~~~~~~ Fig E4 3o4 4o4 1 1, Short circuit current (jia/cm2) Fig. 9. Effect of 3-deoxy-3-fluorglucose on short circuit current and total conductance in 4 preparations of isolated epithelium of rat ileum. The concentrations of 3-deoxy-3-fluorglucose are indicated at each point. TABLE 1. Comparison of short circuit current values in experiments where Eagle's medium was used and experiments with the usual Krebs solution. Short circuit current values are quoted in iua/cm2 Control Change in short circuit current (after 6 6 mm glucose) Eagle's S.D n x Krebs S.D n t test P < 1 P > 5

11 SCC AND GT MEASUREMENTS ON RAT ILEUM 219 F. Use of Eagle's minimum essential medium as nutrient solution in the serosal half-chambers In the group of experiments where this solution was utilized, the protocol followed the same steps as described under 'effects of glucose', the difference being that the serosal bath was substituted by Eagle's medium before starting additions of glucose. The control values of short circuit current before additions of glucose were higher than the corresponding values obtained using Krebs solution (Table 1) but the effects of glucose were not statistically different, so that the use of the nutrient medium was discontinued. DISCUSSION It has been shown that a good agreement exists between the net flux of sodium across the rat ileum and the short circuit current (Curran, 196; Clarkson & Toole, 1964; Taylor, Wright, Schultz & Curran, 1968). Thus, in the present study, the short circuit current is considered as a measure of net sodium transport from mucosa to serosa. The results obtained in this preparation with successive additions of glucose confirm previous findings in the new-born pig and rabbit ileum with respect to the increase in conductance accompanying the increase in short circuit current. In the case of rat ileum, the linear relationship between the two parameters could be well defined, as in most preparations it was possible to discriminate between the effects of 5 glucose concentrations. The same phenomenon was reproduced with another hexose that is 'actively' transported but not metabolized, 3-O-methyiglucose, and also with a sugar molecule which is considered to be the closest to glucose in its affinity to the sodium sugar carrier, 3-deoxy-3-fluorglucose (Barnett, Ralph & Munday, 1969), and which is not metabolized as well. It seems worthwhile to stress the increase in conductance obtained using our experimental conditions, as this effect was not considered in the original work of Schultz & Zalusky (1964a) on rabbit ileum; nor was it considered in the studies of Barry, Smyth & Wright (1965) in rat jejunum, and it has been neglected since then. Presumably, the fact that we have been measuring short term effects of the sugar additions facilitates the evaluation of small variations of the conductance. In long term experiments, like the ones involving flux measurements, these changes may be easily disregarded as part of the large individual scattering of conductance values shown by the preparations. Another important point resulting from the conductance determinations is that the same order of variation is found in stripped epithelia as in the intact tissue, although the 'control' values are slightly higher in the

12 22 M. GRAQA EMILIO AND C. HENRIQUES DE JESUS former cases. This suggests that the contribution of the resistance of the muscular layer to the total resistance is not so important, in this case, as in rabbit ileum (Field, Fromm & McColl, 1971) and that the variation in conductance associated with sugar additions is not dependent on the presence of the muscular layer. The interaction between short circuit current and glucose or 3-- methylglucose concentrations followed the Michaelis-Menten kinetics, as happens in rabbit ileum (Schultz & Zalusky, 1964b). The Km values determined from our experiments are somewhat smaller than the values found by those authors (for glucose, 2-8 mm in rat and 4 mm in rabbit ileum; for 3--methylglucose, 7-7 mm in rat and 17 mm in rabbit). The effect of 2-deoxy-D-glucose on this preparation was a decrease in short circuit current without any noticeable change in total conductance, either in the presence or in the absence of glucose. This result is different from the findings in rabbit ileum (Henriques de Jesus, 1977), where the additions of 2-deoxy-D-glucose caused a decrease in short circuit current and an increase in total conductance, these being linearly correlated. This was interpreted as meaning that the change in the electrical parameters was associated with an alteration of a resistive element in the electrical model. In the present case, the fact that the only detected change is in the short circuit current suggests that the effect is metabolic. In fact, 2-deoxy-D-glucose is known as an inhibitor of glucose metabolism, although different effects have been observed in different preparations. Its effect on isolated frog gastric mucosa as inhibitor of acid secretion has been attributed to the formation of a phosphate that cannot be metabolised, resulting in effective cytoglupenia (Sachs, Shoemaker & Hirschowitz, 1965). Owen, Kaplan, Roy & Essig (1975) found that 2-deoxy-Dglucose caused a decrease in the short circuit current in frog skin, dependent on the concentration of glucose in the bathing medium, and suggested a mechanism of competitive inhibition between the two sugars. In our experiments, the effects of 2-deoxy-D-glucose and glucose seem to be independent, the Km of 2-deoxy-D-glucose being the same as in freshly mounted preparations. The Vm value is higher after treatment with glucose, probably because there is a higher metabolic inhibition affecting the sodium pump. Studies on intestinal epithelial cells isolated from the rat (Bihler & Cybulsky, 1973) or the chick (Kimmich & Randles, 1976) and on rabbit intestine (Goldner, Hajjar & Curran, 1969) showed that 2-deoxy-D-glucose enters the cells via a saturable pathway, non sodiumdependent, and does not accumulate against a gradient, unlike glucose. These facts also suggest that this molecule does not act in the same way as other hexoses transported by a carrier-type of process. Phlorizin has long been known to inhibit the transport of glucose by

13 SCC AND GT MEASUREMENTS ON RAT ILEUM 221 intestinal cells (Newey, Parsons & Smyth, 1959; Crane, 196; Alvarado & Crane, 1964). We utilized it in some experiments with the hope of reversing the effects of glucose on the electrical parameters. In fact, the increase in short circuit current induced by glucose was abolished, but no change was detected in the total conductance. The same effect was observed in the glucose experiments, after washing the preparations with glucose-free Krebs solution. A possible interpretation of this finding is that the increase in total conductance is due to an enlargement of the intercellular spaces caused by the enhanced sodium and water transport. Phlorizin, by inhibiting the sodium glucose carrier, or the withdrawal of glucose, will cause a decrease in short circuit current, but the geometry of the intercellular spaces may be permanently altered, so that the total conductance of the preparations remains high. This problem, as well as other questions evoked by the work done so far, will be the object of further research in the near future with the help of micro-electrode techniques which will allow us to discriminate between changes in resistance in the mucosal and the serosal barriers. Another aspect that seems to deserve further investigation is the detailed study of the transients when changes in short circuit current are imposed on the preparations, since these changes seem to have different time constants. This study will be made possible by the use of a voltage clamp device, specially designed to detect independently rapid changes of the parameters being measured. Finally, we would like to point out the possible interest of the use of nutrient-bathing solutions when long term experiments with this type of preparation is attempted. In the present case, the use of minimum essential medium did improve the control conditions, although the difference was not sufficiently large to justify its use in experiments which never lasted for longer than 2-3 hr. We are grateful to Dr D. Aikman (University of East Anglia) for his help in the determination of statistical parameters. 3-deoxy-3-fluor-D-glucose was obtained through the courtesy of Dr J. E. G. Barnett. We acknowledge with thanks the technical help of Mrs M.. Ferreira. REFERENCES ALVARADO, F. & CRANE, R. K. (1964). Studies on the mechanism of intestinal absorption of sugars. VII. Phenylglycoside transport and its possible relationship to phlorizin inhibition of the active transport of sugars by the small intestine. Biochim. biophy8. Acta 93, BARNETT, J. E. G., RALPH, A. & MUNDAY, K. A. (1969). Structural requirements for active intestinal transport. Spatial and bonding requirements at C.3 of the sugar. Biochem. J. 114,

14 222 M. GRAQA EMILIO AND C. HENRIQUES DE JESUS BARRY, R. J. C., SMYTH, D. H. & WRIGHT, E. M. (1965). Short circuit current and solute transfer by rat jejunum. J. Physiol. 181, BIHLER, I. & CYBULS1KY, R. (1973). Sugar transport at the basal and lateral aspect of the small intestinal cell. Biochim. biophys. Acta 298, CLARKSON, T. W. & TOOLE, S. R. (1964). Measurement of short circuit current and ion transport across the ileum. Am. J. Physiol. 26, CRANE, R. K. (196). Intestinal absorption of sugars. Physiol. Rev. 4, CURRAN, P. F. (196). Na, Cl and water transport by rat ileum in vitro. J. gen. Physiol. 43, EMWLIO, M. G., BALLS, M. & MENANO, H. P. (1975). Use of a tissue culture medium for in vitro studies on the ion transport capacity of amphibian epithelia. Experientia 31, FIELD, M., FROMM, D. & McCoLL, I. (1971). Ion transport in rabbit ileal mucosa. I. Na and Cl fluxes and short circuit current. Am. J. Physiol. 22, GOLDNER, A. M., HAJJAR, J. J. & CURRAN, P. F. (1969). 2-Deoxyglucose transfer in rabbit intestine. Biochim. biophys. Acta 173, HENRIQUES DE JESUS, C. (1974). Ion transport in the ileum of the new-born pig. Ph.D. Thesis, Cambridge University. HENRIQUES DE JESUS, C. (1977). Short circuit current and total conductance measurements in rabbit ileum. J. Physiol. (in the press). HENRIQUES DE JESUS, C. & SMITH, M. (1974). Protein and glucose-induced changes in sodium transport across the pig small intestine. J. Physiol. 243, KIMMICH, E. A. & RANDLES, J. (1976). 2-Deoxyglucose transport by intestinal epithelial cells isolated from the chick. J. Membrane Biol. 27, NEWEY, H., PARSONS, B. J. & SMYTH, D. M. (1959). The site of action of phlorizin in inhibiting intestinal absorption of glucose. J. Physiol. 148, OWEN, A., CAPLAN, RoY S. & ESSIG, A. (1975). The interaction of 2-deoxy-Dglucose and glucose: effects on the short circuit current of frog skin. Biochim. biophys. Acta 389, SACUS, G., SHOEMAKER, R. & HIRSCHOWITZ, B. I. (1965). Action of 2-deoxy-Dglucose on frog gastric mucosa. Am. J. Physiol. 29, SCHULTZ, S. G. & CURRAN, P. F. (197). Coupled transport of sodium and of organic solutes. Physiol. Rev. 5, SCHULTZ, S. G. & ZALUSKY, R. (1964a). Ion transport in isolated rabbit ileum. I. short circuit current and Na fluxes. J. gen. Physiol. 47, SCHULTZ, S. G. & ZALUSKY, R. (1964b). Ion transport in isolated rabbit ileum. II. The interaction between active sodium and active sugar transport. J. gen. Physiol. 47, TAYLOR, A. E., WRIGHT, E. M., SCHULTZ, S. G. & CURRAN, P. F. (1968). Effects of sugars on ion fluxes in intestine. Am. J. Physiol. 214, USSING, H. H. & WINDHAGER, E. E. (1964). The nature of the shunt path and active sodium transport path through frog skin epithelium. Acta physiol. scand. 61,