Research in Veterinary Science 1992, 53, 19-24
Effects of nicarbazin on sugar intestinal absorption in rabbits V. SORRIBAS, M. P. ARRUEBO, H. NAVARRO, A. I. ALCALDE, Departamento de Fisiologia y Farmacologia, Universidad de Zaragoza, Facultad de Veterinaria, Miguel Servet, 177, 50013-Zaragoza, Spain
experiments have shown that nicarbazin diminished growth and feed efficiency at several concentrations (McDougald and McQuistion 1980, Stephenson et al 1985, Ferrato et al 1988, Bartov 1989a). These effects have been explained as an increase of catabolism (Farny 1956, McDougald and McQuistion 1980, Bartov 1989b) that should be responsible for the lower resistance of birds to heat and their higher mortality. On the other hand, various reports have demonstrated that nicarbazin at recommended levels (125 ppm), did not depress the growth and feed efficiency which only appeared at higher doses (McDougald and Mathis 1984, Welch et al 1988). Furthermore, a recent study of heat stress has shown that nicarbazin increases body temperature and causes great deviations in blood acid-base balance, blood lactate concentration and heart rate (McDougald and Mathis 1984, Beers et al 1989). The anticoccidial action of nicarbazin is located in the small intestine, and one of the most important roles of the small intestine is nutrient absorption. This intestinal absorption is in part mediated by carriers which transport selectively the different nutrient molecules (sugars, amino acids, etc). The aim of the present work was to study whether nicarbazin affects intestinal sugar transport.
Nicarbazin is an anticoccidial drug, used mainly in birds, which can also be used in rabbits. It has been shown to produce several effects, such as inhibition of growth and feed efficiency in poultry. The aim of the present work was to determine whether nicarbazin alters intestinal absorption of sugar. Results obtained show that nicarbazin decreases D-galactose accumulation in the jejunal tissue and increases mucosal to serosal transepithelial fluxes of this sugar, in both cases in a dosedependent way. Furthermore, nicarbazin seems not to modify the sugar diffusion across the intestinal epithelium. The drug also stimulates the sugar uptake in brush border and basolateral membrane vesicles. The results suggest that in rabbits nicarbazin increases sugar intestinal absorption mediated by carriers.
NICARBAZIN, an equimolar complex of 4,4dinitrocarbanilide (DNC) and 2-hydroxy-4,6dimethyl-2-pirimidol (HDP), is an anticoccidial drug used since 1956, mainly in birds, though it may also be used in rabbits (Coudert 1979). The great effectiveness of nicarbazin against various species of coccidia has resulted in its extensive use (Reid and McDougald 1985). The drug is administered systematically with the food, because it is not very soluble in water, usually at 125 mg kg-1 (ppm), but occasionally it has been used at up to 200 ppm (McDougald 1986). Apart from its anticoccidial action several other effects have been produced by nicarbazin, depending on the dose. Cuckler et al (1956) reported that nicarbazin at over 200 ppm depressed feed efficiency and growth in poultry. Later studies showed that nicarbazin at 125 ppm diminished energy retention, and at 250 ppm reduced both energy and protein retention in chickens (Stutz and Johnson 1974). More recent
Materials and methods
Animals The handling, equipment and sacrifice of the animals was done according to the European Council Legislation 86/609/EEC for the protection of experimental animals. The work was carried out on male New Zealand rabbits weighing 1-5 to 2.0 kg, which were maintained at a constant temperature of 24°C with free access to water and standard rabbit fodder. 19
20
V. Sorribas, M. P. Arruebo, H. Navarro, A. L Alcalde
Intestinal tissue experiments After killing the animal by a blow on the nape, the proximal jejunum was removed and rinsed free of intestinal contents with ice-cold Ringer's solution. The tissue was then stripped of its serosal and external muscle layers. The Ringer's solution contained in mmol litre-l: 140 NaC1, I0 KCO3, 0-4 KH2PO4, 2-4 K2HPO4, 1.2 CaC12 and 1.2 MgC12, and was continuously aerated with 95 per cent 02/5 per cent CO2. Cell water determinations. Pieces of jejunum were incubated in Ringer's solution at 37°C containing 0.02 gCi ml ~ ~4C-labelled polyethylene glycol (PEG), for 20 and 30 minutes. The pieces of mucosa were then gently blotted on humid filter paper and weighed, then extracted in 1 ml 0.1 tool litre -1 H N O 3 overnight. Samples of the extracts and samples of the bathing solutions were then counted. Following extraction the tissues were dried at 80°C for 24 hours and then reweighed. Tissue water was calculated as the difference between wet and dry weights. Nicarbazin at four concentrations was present in the bathing solution from the start of the incubation: 30 ppm -- 0.07 mM; 60 ppm = 0.141 mM; 125 ppm = 0-293 mM; and 1250 ppm = 2.931 raM. Nicarbazin showed no significant effect either on the extracellular space, tissue water fraction or on cell water fraction at any of these concentrations (data not shown). Sugar uptake measurements. Strips of jejunum weighing about 100 mg were incubated for 30 minutes at 37°C in 10 ml Ringer's solution containing 0.01 gCi m1-1 14C-D-galactose plus unlabelled D-galactose, and nicarbazin at 30, 60, 125 and 1250 ppm. D-galactose was used because it is less metabolised than D-glucose by the intestinal tissue, and the two sugars are transported by the same mechanisms. At the end of the experiment the tissues were washed with two or three gentle shakings in ice-cold Ringer's solution and blotted carefully on both sides to remove excess moisture. The tissue was weighed wet and extracted by shaking for 15 hours in 0-5 ml 0.1 tool litre -1 H N O 3. Samples were taken from the bathing solutions and from the extracts of the tissues for radioactivity counting. All the modifiers (nicarbazin, etc) were added to the incubation solution at the beginning of the incubation
period. The results are expressed as gmol Dgalactose m1-1 cell water, after correction for the extracellular space. ~~Transepithelial flux measurements. The stripped mucosa was mounted as a flat sheet in Ussingtype chambers. The bathing solution on the mucosal and serosal surfaces of the tissue was maintained at 37°C using a circulating water bath. Both solutions contained D-galactose and nicarbazin at the same concentration (4 m M and 125 or 1250 ppm, respectively). Mucosal to serosal sugar fluxes (Jm-s) were measured by adding 0.04 gCi ml-1 14C-labelled D-galactose on the mucosal side, and serosal to mucosal fluxes (Js-m) were measured by adding it on the serosal side. Samples were removed from the side in which labelled substrate was not added, at 20 minute intervals for 80 minutes, and after a 30 minute preincubation period. One sample only was taken for counting from the radioactively labelled side. Samples of the radioactive solution were counted using a liquid scintillation counter. Membrane vesicle experiments Brush border membrane vesicles (BBMV) and basolateral m e m b r a n e vesicles (BLMV) were obtained from rabbit enterocytes through a simultaneous method. BBMVwere prepared according to the MgZ+/EGTAprecipitation method of Hauser et al (1980). The BLMV were purified by using a sucrose gradient which was obtained from two concentrations of this disaccharide (16 and 34 per cent, with densities 1.063 and 1.146 g cm -3, respectively). The two final vesicle preparations were resuspended in a mixture of 500 m M sorbitol, 4 m M lithium azide, and a metal ion-free 10 m M Hepes/7mM n-butylamine/7 m M maleic acid buffer (HMBA), p H 7'4 (Alvarado and Mahmood 1979). Substrate uptake was measured by using a rapid filtration method (Kessler et al 1978) and a short-time incubation apparatus (CNRS/INRA prototype, France) as previously described (Brot-Laroche et al 1986). The incubation medium contained the following: 4 m M lithium azide and 10 m M HMBA;0.1 m M L-leucine plus 14C-labelled L-leucine; N a S C N 100/0 m M (out/in) to obtain an initial metal-ion gradient and an electric potential due to SCN-; enough sorbitol to adjust the osmolarity to 500 mosM litre-1. Nicarbazin was mixed directly with the
Effect of nicarbazin on D-galactose intestinal absorption vesicles to reach a concentration of 125 ppm in the membranes. The experiments were carried out as uptake time-courses of sugar. L-leucine uptakes are expressed as pmol L-leucine mg -1 protein of vesicles. The protein concentration of vesicles was measured with the Bio-Rad assay kit (Bradford 1976). The vesicles were stored in liquid nitrogen until the day of experiment.
Chemicals All reagents were of the highest purity commercially available. D-galactose, D-glucose and phlorizin were obtained from Sigma, PEG and nicarbazin were obtained from Merck, and the labelled products from Amersham International.
Statistics All results are expressed as means _+ sE. The comparison between means was evaluated by a two-way (animals and treatments) analysis of variance (ANOVA)procedure as outlined by Steel and Torrie (1980). The Fisher's protected least significant difference test (PLSD) was used as a multiple-comparison method to compare data among groups, and considered statistically significant when P
Effects of nicarbazin on sugar intestinal absorption in rabbits V. SORRIBAS, M. P. ARRUEBO, H. NAVARRO, A. I. ALCALDE, Departamento de Fisiologia y Farmacologia, Universidad de Zaragoza, Facultad de Veterinaria, Miguel Servet, 177, 50013-Zaragoza, Spain
experiments have shown that nicarbazin diminished growth and feed efficiency at several concentrations (McDougald and McQuistion 1980, Stephenson et al 1985, Ferrato et al 1988, Bartov 1989a). These effects have been explained as an increase of catabolism (Farny 1956, McDougald and McQuistion 1980, Bartov 1989b) that should be responsible for the lower resistance of birds to heat and their higher mortality. On the other hand, various reports have demonstrated that nicarbazin at recommended levels (125 ppm), did not depress the growth and feed efficiency which only appeared at higher doses (McDougald and Mathis 1984, Welch et al 1988). Furthermore, a recent study of heat stress has shown that nicarbazin increases body temperature and causes great deviations in blood acid-base balance, blood lactate concentration and heart rate (McDougald and Mathis 1984, Beers et al 1989). The anticoccidial action of nicarbazin is located in the small intestine, and one of the most important roles of the small intestine is nutrient absorption. This intestinal absorption is in part mediated by carriers which transport selectively the different nutrient molecules (sugars, amino acids, etc). The aim of the present work was to study whether nicarbazin affects intestinal sugar transport.
Nicarbazin is an anticoccidial drug, used mainly in birds, which can also be used in rabbits. It has been shown to produce several effects, such as inhibition of growth and feed efficiency in poultry. The aim of the present work was to determine whether nicarbazin alters intestinal absorption of sugar. Results obtained show that nicarbazin decreases D-galactose accumulation in the jejunal tissue and increases mucosal to serosal transepithelial fluxes of this sugar, in both cases in a dosedependent way. Furthermore, nicarbazin seems not to modify the sugar diffusion across the intestinal epithelium. The drug also stimulates the sugar uptake in brush border and basolateral membrane vesicles. The results suggest that in rabbits nicarbazin increases sugar intestinal absorption mediated by carriers.
NICARBAZIN, an equimolar complex of 4,4dinitrocarbanilide (DNC) and 2-hydroxy-4,6dimethyl-2-pirimidol (HDP), is an anticoccidial drug used since 1956, mainly in birds, though it may also be used in rabbits (Coudert 1979). The great effectiveness of nicarbazin against various species of coccidia has resulted in its extensive use (Reid and McDougald 1985). The drug is administered systematically with the food, because it is not very soluble in water, usually at 125 mg kg-1 (ppm), but occasionally it has been used at up to 200 ppm (McDougald 1986). Apart from its anticoccidial action several other effects have been produced by nicarbazin, depending on the dose. Cuckler et al (1956) reported that nicarbazin at over 200 ppm depressed feed efficiency and growth in poultry. Later studies showed that nicarbazin at 125 ppm diminished energy retention, and at 250 ppm reduced both energy and protein retention in chickens (Stutz and Johnson 1974). More recent
Materials and methods
Animals The handling, equipment and sacrifice of the animals was done according to the European Council Legislation 86/609/EEC for the protection of experimental animals. The work was carried out on male New Zealand rabbits weighing 1-5 to 2.0 kg, which were maintained at a constant temperature of 24°C with free access to water and standard rabbit fodder. 19
20
V. Sorribas, M. P. Arruebo, H. Navarro, A. L Alcalde
Intestinal tissue experiments After killing the animal by a blow on the nape, the proximal jejunum was removed and rinsed free of intestinal contents with ice-cold Ringer's solution. The tissue was then stripped of its serosal and external muscle layers. The Ringer's solution contained in mmol litre-l: 140 NaC1, I0 KCO3, 0-4 KH2PO4, 2-4 K2HPO4, 1.2 CaC12 and 1.2 MgC12, and was continuously aerated with 95 per cent 02/5 per cent CO2. Cell water determinations. Pieces of jejunum were incubated in Ringer's solution at 37°C containing 0.02 gCi ml ~ ~4C-labelled polyethylene glycol (PEG), for 20 and 30 minutes. The pieces of mucosa were then gently blotted on humid filter paper and weighed, then extracted in 1 ml 0.1 tool litre -1 H N O 3 overnight. Samples of the extracts and samples of the bathing solutions were then counted. Following extraction the tissues were dried at 80°C for 24 hours and then reweighed. Tissue water was calculated as the difference between wet and dry weights. Nicarbazin at four concentrations was present in the bathing solution from the start of the incubation: 30 ppm -- 0.07 mM; 60 ppm = 0.141 mM; 125 ppm = 0-293 mM; and 1250 ppm = 2.931 raM. Nicarbazin showed no significant effect either on the extracellular space, tissue water fraction or on cell water fraction at any of these concentrations (data not shown). Sugar uptake measurements. Strips of jejunum weighing about 100 mg were incubated for 30 minutes at 37°C in 10 ml Ringer's solution containing 0.01 gCi m1-1 14C-D-galactose plus unlabelled D-galactose, and nicarbazin at 30, 60, 125 and 1250 ppm. D-galactose was used because it is less metabolised than D-glucose by the intestinal tissue, and the two sugars are transported by the same mechanisms. At the end of the experiment the tissues were washed with two or three gentle shakings in ice-cold Ringer's solution and blotted carefully on both sides to remove excess moisture. The tissue was weighed wet and extracted by shaking for 15 hours in 0-5 ml 0.1 tool litre -1 H N O 3. Samples were taken from the bathing solutions and from the extracts of the tissues for radioactivity counting. All the modifiers (nicarbazin, etc) were added to the incubation solution at the beginning of the incubation
period. The results are expressed as gmol Dgalactose m1-1 cell water, after correction for the extracellular space. ~~Transepithelial flux measurements. The stripped mucosa was mounted as a flat sheet in Ussingtype chambers. The bathing solution on the mucosal and serosal surfaces of the tissue was maintained at 37°C using a circulating water bath. Both solutions contained D-galactose and nicarbazin at the same concentration (4 m M and 125 or 1250 ppm, respectively). Mucosal to serosal sugar fluxes (Jm-s) were measured by adding 0.04 gCi ml-1 14C-labelled D-galactose on the mucosal side, and serosal to mucosal fluxes (Js-m) were measured by adding it on the serosal side. Samples were removed from the side in which labelled substrate was not added, at 20 minute intervals for 80 minutes, and after a 30 minute preincubation period. One sample only was taken for counting from the radioactively labelled side. Samples of the radioactive solution were counted using a liquid scintillation counter. Membrane vesicle experiments Brush border membrane vesicles (BBMV) and basolateral m e m b r a n e vesicles (BLMV) were obtained from rabbit enterocytes through a simultaneous method. BBMVwere prepared according to the MgZ+/EGTAprecipitation method of Hauser et al (1980). The BLMV were purified by using a sucrose gradient which was obtained from two concentrations of this disaccharide (16 and 34 per cent, with densities 1.063 and 1.146 g cm -3, respectively). The two final vesicle preparations were resuspended in a mixture of 500 m M sorbitol, 4 m M lithium azide, and a metal ion-free 10 m M Hepes/7mM n-butylamine/7 m M maleic acid buffer (HMBA), p H 7'4 (Alvarado and Mahmood 1979). Substrate uptake was measured by using a rapid filtration method (Kessler et al 1978) and a short-time incubation apparatus (CNRS/INRA prototype, France) as previously described (Brot-Laroche et al 1986). The incubation medium contained the following: 4 m M lithium azide and 10 m M HMBA;0.1 m M L-leucine plus 14C-labelled L-leucine; N a S C N 100/0 m M (out/in) to obtain an initial metal-ion gradient and an electric potential due to SCN-; enough sorbitol to adjust the osmolarity to 500 mosM litre-1. Nicarbazin was mixed directly with the
Effect of nicarbazin on D-galactose intestinal absorption vesicles to reach a concentration of 125 ppm in the membranes. The experiments were carried out as uptake time-courses of sugar. L-leucine uptakes are expressed as pmol L-leucine mg -1 protein of vesicles. The protein concentration of vesicles was measured with the Bio-Rad assay kit (Bradford 1976). The vesicles were stored in liquid nitrogen until the day of experiment.
Chemicals All reagents were of the highest purity commercially available. D-galactose, D-glucose and phlorizin were obtained from Sigma, PEG and nicarbazin were obtained from Merck, and the labelled products from Amersham International.
Statistics All results are expressed as means _+ sE. The comparison between means was evaluated by a two-way (animals and treatments) analysis of variance (ANOVA)procedure as outlined by Steel and Torrie (1980). The Fisher's protected least significant difference test (PLSD) was used as a multiple-comparison method to compare data among groups, and considered statistically significant when P
