489
J. Physiol. (1977), 271, pp. 489-504 With 7 text-ftguree Printed in Great Britain
SODIUM CHLORIDE TRANSPORT ACROSS THE CHICKEN COPRODEUM. BASIC CHARACTERISTICS AND DEPENDENCE ON SODIUM CHLORIDE INTAKE
BY I. CHOSHNIAK*, B. G. MUNCK AND E. SKADHAUGE From the Institute of Medical Physiolcgy, Dept. A, University of Copenhagen, the Panum Institute, DK-2200 Copenhagen N, Denmark (Received 24 January 1977) SUMMARY
1. The transport characteristics of the chicken coprodeum have been examined in vitro using the isolated mucosa. The short-circuit current (I,), the transepithelial electrical potential difference (p.d.), the unidirectional transmural fluxes (J., J.) of sodium and chloride measured in the short-circuited state, and the unidirectional influx of sodium and chloride across the brush border membrane measured under open-circuit conditions have been studied. The effect of the sodium chloride contents of the diet on these parameters have been investigated. 2. The isolated mucosa depends functionally on the presence of glucose in the incubation media. This dependence reflects the need of glucose as a fuel. There is no indication of coupling between transport of -sugars and sodium across the brush border membrane. For preparations from chickens on a low sodium diet a very high and stable ISc can quantitatively be accounted for by the net transport of sodium. Influx of sodium across the brush border membrane is not significantly different from the net flux of sodium. By feeding the chickens a high sodium diet the I,, is reduced by more than 95 %, the net transport of sodium is abolished, and the transepithelial electrical conductance is reduced by more than 50 %. 3. Both unidirectional transepithelial fluxes of chloride, and the serosa to mucosa flux of sodium appear to proceed through a paracellular shunt. 4. Under the conditions of the low sodium diet the paracellular pathway appears to be anion selective. Whereas, under the conditions of the high sodium regimen the paracellular route appears to be cation selective. After adaptation to a high sodium diet the influx of sodium across the brush border membrane is only moderately reduced. Consequently the decisive event in the adaptation must be localized elsewhere. *
Present address: Department of Zoology, University of Tel Aviv, Israel.
490
I. CHOSHNIAK, B. 0. MUNCK AND E. SKADHAUGE INTRODUCTION
Studies in vivo of electrolyte and water transport by the lower chicken intestine (colon and coprodeum) have demonstrated that these transport processes adapt to the dietary load of sodium, and that the effects of a low sodium diet can be partly mimicked by intramuscular administration of aldosterone (Thomas, Skadhauge & Read, 1975). In birds the coprodeum is the final stage for the conservation of sodium and water (Skadhauge, 1973). By the method described by Nellans, Frizzell & Schultz (1974) the coprodeum of the chicken can be freed from the very thick muscular-connective tissue layer, leaving only the mu8cularie8 mucosa with the mucosal tissues. We have employed this preparation in a study of the electrical parameters and the characteristics of sodium and chloride transport across the chicken coprodeum. METHODS
Material&. Adult white Plymouth Rock hens (2-4 kg) were used in all experiments. Before use the birds were kept on one of four diets: (1) a mixture of equal parts of wheat and barley and tap water, (2) commercial chicken pellets and tap water, (3) the wheat and barley mixture, 10 ml. per kg body weight of 9 % sodium chloride (w/v) by crop tube solutionlonce daily, for 1 week, and tap water, (4) the commercial pellets and sodium chloride as in (3) and tap water. The electrolyte intake on these diets is given in Table 1. Chickens were kept on the first diet for at least 9 days, but for no longer than 30 days before use. The third diet was only introduced after at least 9 days on the wheat and barley diet. The chickens were killed by decapitation. The lower bowel including colon, coprodeum, and urodeum (Fig. 1) was removed, opened lengthwise and briefly rinsed in saline. Finally, the mucosal layer of the coprodeum was isolated by dissection (Nellans et al. 1974) and mounted in the appropriate chambers. Each chicken provided tissue enough for four Ussing chambers or for six to eight measurements of influx across the brush border membrane. ['H]polyethyleneglycol (molecular weight: 4000) was purchased from New England Nuclear Co. 22Na and MCl were obtained from The Radiochemical Centre, Amersham, England. All chemicals used were of the highest purity commercially available. Methods. In all experiments the tissues were incubated in a Krebs phosphate buffer, the composition of which was Na 140 mM; K 8 mM; Ca 2-6 mM; Mg 1 mu; Cl 140 mM; P 8 mM; S04 1 mM; pH 74 D-glucose 15 mu. All experiments were performed at 37 'C during aeration with pure oxygen. Unidirectional tran8mural fluxe8 were measured by the Ussing-Zerahn (1951) technique used essentially as described previously (Munck & Rasmussen, 1977). Influx across the brush border membrane (J,,,,) was measured by the method which Schultz, Curran, Chez & Fuisz (1967) described for rabbit ileum. The tissue segments were mounted on a lucite plate with the mucosal side facing upwards. On top of the plate a lucite block was clamped in which four wells were drilled exposing four mucosal areas of 0-62 cm2. On one side of the block two drillings were made into each well, one for injection and withdrawal of the test solution, and one for injection of ice-cold isotonic mannitol solution to terminate the incubation. On the other side of the lucite block a drilling was made through which a large flow of oxygen can be injected to oxygenate the epithelium and to provide vigorous stirring of the solution.
NaCi TRANSPORT ACROSS COPRODEUM
491
TABLE 1. Estimate of the sodium load received by a 3 kg chicken assuming a food consumption of 50 g per kg body weight m-equivjbird 24 hr
Wheat and barley Commercial food Wheat and barley and NaCl load Commercial food and NaCl load
Na 04
Cl 2, 5 12 49 58
18 46 64
K 18 34 18 34
J. Physiol. (1977), 271, pp. 489-504 With 7 text-ftguree Printed in Great Britain
SODIUM CHLORIDE TRANSPORT ACROSS THE CHICKEN COPRODEUM. BASIC CHARACTERISTICS AND DEPENDENCE ON SODIUM CHLORIDE INTAKE
BY I. CHOSHNIAK*, B. G. MUNCK AND E. SKADHAUGE From the Institute of Medical Physiolcgy, Dept. A, University of Copenhagen, the Panum Institute, DK-2200 Copenhagen N, Denmark (Received 24 January 1977) SUMMARY
1. The transport characteristics of the chicken coprodeum have been examined in vitro using the isolated mucosa. The short-circuit current (I,), the transepithelial electrical potential difference (p.d.), the unidirectional transmural fluxes (J., J.) of sodium and chloride measured in the short-circuited state, and the unidirectional influx of sodium and chloride across the brush border membrane measured under open-circuit conditions have been studied. The effect of the sodium chloride contents of the diet on these parameters have been investigated. 2. The isolated mucosa depends functionally on the presence of glucose in the incubation media. This dependence reflects the need of glucose as a fuel. There is no indication of coupling between transport of -sugars and sodium across the brush border membrane. For preparations from chickens on a low sodium diet a very high and stable ISc can quantitatively be accounted for by the net transport of sodium. Influx of sodium across the brush border membrane is not significantly different from the net flux of sodium. By feeding the chickens a high sodium diet the I,, is reduced by more than 95 %, the net transport of sodium is abolished, and the transepithelial electrical conductance is reduced by more than 50 %. 3. Both unidirectional transepithelial fluxes of chloride, and the serosa to mucosa flux of sodium appear to proceed through a paracellular shunt. 4. Under the conditions of the low sodium diet the paracellular pathway appears to be anion selective. Whereas, under the conditions of the high sodium regimen the paracellular route appears to be cation selective. After adaptation to a high sodium diet the influx of sodium across the brush border membrane is only moderately reduced. Consequently the decisive event in the adaptation must be localized elsewhere. *
Present address: Department of Zoology, University of Tel Aviv, Israel.
490
I. CHOSHNIAK, B. 0. MUNCK AND E. SKADHAUGE INTRODUCTION
Studies in vivo of electrolyte and water transport by the lower chicken intestine (colon and coprodeum) have demonstrated that these transport processes adapt to the dietary load of sodium, and that the effects of a low sodium diet can be partly mimicked by intramuscular administration of aldosterone (Thomas, Skadhauge & Read, 1975). In birds the coprodeum is the final stage for the conservation of sodium and water (Skadhauge, 1973). By the method described by Nellans, Frizzell & Schultz (1974) the coprodeum of the chicken can be freed from the very thick muscular-connective tissue layer, leaving only the mu8cularie8 mucosa with the mucosal tissues. We have employed this preparation in a study of the electrical parameters and the characteristics of sodium and chloride transport across the chicken coprodeum. METHODS
Material&. Adult white Plymouth Rock hens (2-4 kg) were used in all experiments. Before use the birds were kept on one of four diets: (1) a mixture of equal parts of wheat and barley and tap water, (2) commercial chicken pellets and tap water, (3) the wheat and barley mixture, 10 ml. per kg body weight of 9 % sodium chloride (w/v) by crop tube solutionlonce daily, for 1 week, and tap water, (4) the commercial pellets and sodium chloride as in (3) and tap water. The electrolyte intake on these diets is given in Table 1. Chickens were kept on the first diet for at least 9 days, but for no longer than 30 days before use. The third diet was only introduced after at least 9 days on the wheat and barley diet. The chickens were killed by decapitation. The lower bowel including colon, coprodeum, and urodeum (Fig. 1) was removed, opened lengthwise and briefly rinsed in saline. Finally, the mucosal layer of the coprodeum was isolated by dissection (Nellans et al. 1974) and mounted in the appropriate chambers. Each chicken provided tissue enough for four Ussing chambers or for six to eight measurements of influx across the brush border membrane. ['H]polyethyleneglycol (molecular weight: 4000) was purchased from New England Nuclear Co. 22Na and MCl were obtained from The Radiochemical Centre, Amersham, England. All chemicals used were of the highest purity commercially available. Methods. In all experiments the tissues were incubated in a Krebs phosphate buffer, the composition of which was Na 140 mM; K 8 mM; Ca 2-6 mM; Mg 1 mu; Cl 140 mM; P 8 mM; S04 1 mM; pH 74 D-glucose 15 mu. All experiments were performed at 37 'C during aeration with pure oxygen. Unidirectional tran8mural fluxe8 were measured by the Ussing-Zerahn (1951) technique used essentially as described previously (Munck & Rasmussen, 1977). Influx across the brush border membrane (J,,,,) was measured by the method which Schultz, Curran, Chez & Fuisz (1967) described for rabbit ileum. The tissue segments were mounted on a lucite plate with the mucosal side facing upwards. On top of the plate a lucite block was clamped in which four wells were drilled exposing four mucosal areas of 0-62 cm2. On one side of the block two drillings were made into each well, one for injection and withdrawal of the test solution, and one for injection of ice-cold isotonic mannitol solution to terminate the incubation. On the other side of the lucite block a drilling was made through which a large flow of oxygen can be injected to oxygenate the epithelium and to provide vigorous stirring of the solution.
NaCi TRANSPORT ACROSS COPRODEUM
491
TABLE 1. Estimate of the sodium load received by a 3 kg chicken assuming a food consumption of 50 g per kg body weight m-equivjbird 24 hr
Wheat and barley Commercial food Wheat and barley and NaCl load Commercial food and NaCl load
Na 04
Cl 2, 5 12 49 58
18 46 64
K 18 34 18 34
