Lysine and methionine transport by bovine jejunal and ileal brush border membrane vesicles

Lysine (LYS) and methionine (MET) transport were studied using brush border membrane vesicles from bovine jejunal and ileal tissues. Total transport of LYS and MET was divided into mediated and diffusion components. Mediated uptake was further divided into sodium (Na)-dependent and Na-independent systems. Total LYS and MET uptake by ileal brush border (BB) vesicles tended to be higher than that by jejunal BB vesicles at all concentrations evaluated but differences were significant (P less than .05) at 2.5 and 7.5 mM for LYS and 5, 12.5 and 15 mM for MET. The greater capacity of ileal BB vesicles appeared to be due to the Na-dependent component of LYS uptake and the diffusion component of MET uptake. Transporters had less affinity but higher capacity than for LYS transport in both ileal and jejunal tissue. Methionine transport was greater (P less than .05) than LYS transport in both ileal and jejunal BB vesicles when the initial amino acid concentration was 7.5 mM. But when the initial amino acid concentration was 1.25 mM, MET uptake was greater (P less than .13) than LYS uptake in jejunal, but not in ileal, BB vesicles. The relative contribution of mediated and diffusion uptake systems to total MET and LYS uptake was found to be dependent on substrate concentration. Both intestinal site and substrate concentration influenced the contributions of Na-dependent, Na-independent and diffusion systems to total methionine and lysine uptake.     

L-canavanine inhibits L-arginine uptake by broiler chicken intestinal brush border membrane vesicles

1. Intestinal brush border membrane vesicles (BBMV) were prepared from 3-week-old broiler chickens. 2. Electron microscopy of the BBMV fraction showed single membrane vesicles of different sizes with no electron dense material inside. No other organelles were observed. The sucrase and maltase activities were enriched by factors of 16 and 18, respectively, in the BBMV fraction in comparison with the homogenate. On the other hand, the Na+/K+-ATPase sensitivity to ouabain was increased by a factor of 0.8. 3. The BBMV showed a maximum L-[14C]-arginine uptake (944.9 +/- 22.9 pmoles/mg protein) at 45 s and thereafter it declined slowly. In the presence of 0.5 mM L-canavanine, the L-[14C]-arginine uptake by BBMV was reduced by 43.6% at 45 s. 4. It is concluded that L-canavanine inhibits L-arginine Na+-dependent transport across the enterocyte apical membrane in a highly purified intestinal BBMV from broiler chickens.     

Simultaneous isolation and characterization of brush border and basolateral membrane vesicles from bovine small intestine

Purified brush border and basolateral membranes were isolated from homogenized intestinal enterocytes of Holstein steers by divalent cation precipitation followed by differential and sucrose density gradient centrifugation. Alkaline phosphatase and Na/K adenosine triphosphatase served as marker enzymes for the brush border and basolateral membranes, respectively. The brush border and basolateral membrane fractions were enriched 5.1- and 10.1-fold, respectively, over the cellular homogenate. Electron micrographs, obtained with transmission electron microscopy, confirmed the vesicular nature of the membranes and revealed that basolateral membrane vesicles generally were smaller and more irregular in shape than brush border membrane vesicles. The vesicular nature of isolated membrane preparations was confirmed with osmotic activity experiments. Enrichment of brush border and basolateral membrane fractions compared to the initial homogenate and the vesicular configuration of both preparations indicate that the isolated brush border and basolateral membrane preparations were suitable models for evaluating nutrient transport properties of bovine small intestine. The number of transport experiments possible per animal using the membrane vesicle technique is many times more efficient than some other in vitro techniques (i.e., intestinal rings or everted sacs).     

Na(+)-dependent transport of D-xylose by bovine intestinal brush border membrane vesicles (BBMV) is inhibited by various pentoses and hexoses

To detect whether pentoses and hexoses occurring in rumen bacteria or in hemicellulose ingested with feed and partly released in the small intestine have an affinity for the Na(+)-dependent glucose transporter of the bovine intestinal brush border membrane (BBM), we investigated whether these monosaccharides inhibit Na(+)-dependent transport of 14C-labelled D-xylose across the BBM using brush border membrane vesicles (BBMV) isolated from the mid-jejunum of cows. We used D-xylose as the transport substrate, because it has a low affinity for the Na(+)-dependent glucose transporter and thus its uptake into BBMV is more efficiently competitively inhibited by other sugars than that of D-glucose. D-Ribose, D-mannose and L-rhamnose occurring in rumen bacteria significantly inhibited Na(+)-dependent uptake of D-xylose into BBMV, but their inhibitory effect was less than that of D-glucose, D-xylose and phlorizin. This also applied to L-arabinose (and D-arabinose), which is, like D-xylose and D-galactose, a constituent of hemicellulose, and to 2-deoxy-D-glucose. Of all monosaccharides tested, only D-fructose did not affect Na(+)-dependent D-xylose transport. It is concluded that some pentoses and hexoses occurring in rumen bacteria (D-ribose, D-mannose and L-rhamnose) or hemicellulose (L-arabinose and D-xylose) have a low affinity for the Na(+)-dependent glucose transporter of the bovine BBM and may therefore be absorbed from the jejunum when released in the small intestine.     

Enzymes of isolated brush border membrane of Cotugnia digonopora, and their insensitivity to anthelmintics in vitro

Purified brush border membrane of Cotugnia digonopora showed the presence of a number of phosphohydrolases. Among these, alkaline phosphatase was extremely active. Other enzymes such as glucose-6-phosphatase, fructose-1,6-diphosphatase, cAMP-phosphodiesterase, 5'-nucleotidase and adenosine-triphosphatase were also active. Observations were made on the activities of various ATPases; whereas the enzyme was activated by Ca++ and Mg++ in an additive manner, its sensitivity to ouabain was negligible. Furthermore, in the presence of EDTA the enzyme activity was quite significant. The treatment of isolated brush border membrane with mebendazole, niclosamide and praziquantel in vitro did not alter the activity of these enzymes. However, treatment of intact worms drastically affected the integrity of the membrane.     

Two specific sites for binding of K88ab Escherichia coli fimbriae to porcine intestinal brush border membranes

We have studied the characteristics of the binding of the K88ab Escherichia coli fimbrial antigen to porcine brush border membranes by solid phase binding assay. Binding of biotinylated K88ab to brush border membranes followed a sigmoidal dependence and was saturable, apparent saturation occurring with 0.8 ng of fimbriae (approx. 7 ng of fimbriae per microg of brush border protein) irrespective of incubation temperature in the range of room temperature to 4 degrees C. A Hill plot of log [(fimbriae bound)/(maximal binding-fimbriae bound)] vs. log free fimbriae gave a maximal slope of about 2, indicating the existence of two binding sites. From an analysis of an Scatchard plot, apparent binding constants (1)K(2) and (2)K(2) of 7.1 x 10(8) and 17.1 x 10(8)M(-1) were obtained at room temperature. Nor did temperature have any effect on the rate of binding or on receptor affinity (S(0.5)).     

Effects of B-trichothecenes on luminal glucose transport across the isolated jejunal epithelium of broiler chickens

Trichothecenes are closely-related sesquiterpenoids (ring structure) with a 12, 13 epoxy ring and a variable number of hydroxyl, acetyl or other substituents. In chickens, D-glucose and amino acid absorption occurs via carrier-mediated transport. Recently, it has been observed that deoxynivalenol (DON) alters the gut function and impairs glucose and amino acid transport in chickens. The purpose of this work was to determine the effects of different B-trichothecenes [DON, Nivalenol (NIV), 15-Ac-DON and Fusarenon X (FUS X)] on intestinal carrier-mediated sodium co-transport of D-glucose in the small intestine of broiler chickens. Intestinal transport was determined by changes in the short-circuit current (Isc), proportional to ion transmembrane flux, in the middle segment of the jejunum of broilers with the Ussing chamber technique. D-glucose produced an increase of the Isc, and this effect was reverted by different B-trichothecene mycotoxins, indicating that the glucose induced Isc was altered by B-trichothecenes. The addition of glucose after pre-incubation of the tissues with B-trichothecenes had no effect (p > 0.05) on the Isc, suggesting that B-trichothecenes afflicted the Na(+)-D-glucose co-transport. However, FUX had no obvious effect on the measured parameters. It could be concluded from the present study that the glucose co-transporter activity appears to be more sensitive to DON, NIV and 15-Ac-DON suppression than by FUS X in the jejunum of broilers.     

Endogenous prostanoids control ion transport across neonatal porcine ileum in vitro

In contrast to the net absorption of Na and Cl ions observed in vivo, porcine small intestine had a net secretion of these ions in vitro. These discrepancies between in vivo and in vitro results have led to difficulties in interpretation of studies investigating mechanisms of intestinal secretion and diarrhea in this species. To examine the influence of endogenous prostanoids on ion transport in neonatal porcine ileum in vitro, tissues were prepared and studied in indomethacin. Net absorption of Na, reversal of net Cl secretion to net absorption, and decreased short circuit current were observed. Conversely, addition of prostaglandins to indomethacin-treated tissues reversed these effects and reestablished conditions similar to those observed in control tissues. Control tissue was essentially refractory to the effects of exogenous prostaglandins. Results indicate that under in vitro conditions, ion transport in neonatal porcine ileum is tightly regulated by endogenous prostanoids that abolish the neutral NaCl absorptive mechanism and elicit electrogenic Cl secretion. However, concentrations of these prostanoids may have been artificially high as a result of tissue preparation for in vitro study.     

高脂饲料对细鳞鱼稚鱼刷状缘膜水解酶发育的影响

试验评价了蛋白质含量为48%时,脂肪含量分别为15%、20%、25%、30%的饲料对开口后20～60 d的细鳞鱼稚鱼肠道水解酶-γ-谷氨酰肽转移酶、碱性磷酸酶活性发育的影响。结果表明,适度的饲料脂肪含量可以提高水解酶活性,并强于生物饵料,促进了肠道对营养物质的消化。饲料脂肪为20%～30%的水平时,水解酶的活性相对较高。     

Decreased activity of brush border membrane-bound digestive enzymes in small intestines from pigs experimentally infected with porcine epidemic diarrhea virus

Brush border membrane-bound digestive enzymes such as disaccharidases (lactase, sucrase, and maltase), leucine aminopeptidase N, and alkaline phosphatase were measured in jejunum from pigs experimentally infected with porcine epidemic diarrhea virus (PEDV). Three piglets from the infected and control groups were euthanized by electrocution and subjected to necropsy at 24, 36, 48, 60, and 72 hours post-inoculation (hpi). The infection of PEDV to jejunum resulted in significant decreases in brush border membrane-bound digestive enzymes such as disaccharidases (lactase, sucrase, and maltase), leucine aminopeptidase N, and alkaline phosphatase. PEDV replication results in massive destruction of villous enterocytes leading to a marked reduction of intestinal epithelial surface and brush border membrane-bound digestive enzyme activity. Reduced enzymatic activity and villous atrophy in the small intestine is thought to result in a maldigestive and malabsorptive diarrhea.     

Effect of age on activation of porcine intestinal guanylate cyclase and binding of Escherichia coli heat-stable enterotoxin (STa) to porcine intestinal cells and brush border membranes.

Development of age-dependent resistance to enterotoxigenic Escherichia coli was studied, using isolated enterocytes and brush border membranes (BBM) from 7-day-old and 7-week-old pigs. Binding of 125I-labeled heat-stable (125I-STa) enterotoxin to enterocytes and BBM was specific, temperature- and time-dependent, saturable, and partially reversible. Scatchard analysis revealed a single class of receptors. Mean +/- SD avidity of binding (apparent affinity constant, Ka) of 125I-STa to enterocytes from 7-day-old and 7-week-old pigs was 2.14 +/- 0.29 x 10(8) and 2.72 +/- 0.25 x 10(8) L/mol, respectively. Numbers of STa receptors were calculated to be 64,903 +/- 2,900/enterocyte for 7-day-old pigs and 53,029 +/- 3,117/enterocyte for 7-week-old pigs. Numbers of STa receptors expressed per milligram of BBM protein from 7-day-old pigs were 2.66 x 10(11), compared with 2.29 x 10(11) for BBM from 7-week-old pigs. By 5 minutes after addition of STa to reaction mixtures, intracellular cyclic guanosine monophosphate concentration increased 13.9-fold in enterocytes from 7-day-old pigs and 8.7-fold in enterocytes from 7-week-old pigs. The particulate guanylate cyclase activity associated with BBM from 7-week-old pigs was slightly more sensitive to low amounts of STa, compared with BBM from 7-day-old pigs; however, differences were not observed at intermediate and high amounts. These data indicate that lack of a secretory response to STa by older pigs is not attributable either to decreased numbers of STa receptors or to decreased signal response between the STa receptor and membrane-bound guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of glycated ovocystatin to rat renal brush border membranes

Glycated proteins are considered as one of the factors involved in the pathogenesis of diabetic complications, including nephropathy. These proteins are formed endogenously under conditions of hyperglycemia, as well as being provided with food containing sugars, which was subjected to high temperature. Examples are egg products. One of the proteins found in eggs in a relatively high concentration is chicken cystatin (ovocystatin). It is now believed that some proteins can passage the intestinal epithelium by transcytosis directly into the bloodstream. Thus, glycated protein present in food can be an additional source of glycotoxins. The aim of this study was to compare the affinity of native and glycated cystatin to the brush border membranes of rat kidney. Kinetic analysis was performed with surface plasmon resonance technique using sensor chip L1. Dissociation constants for native and glycated cystatin (K_d) were 2.76 μmol/L and 3.82 μmol/L, respectively. The results of our study indicate that glycation only slightly affects binding of cystatin to brush border membranes. This suggests that glycated cystatin and other glycated proteins may also be efficiently taken up in the kidney proximal tubule. The observation may be important for understanding the mechanisms involved in the development of diabetic nephropathy.     

Chick small intestine brush border contains lipase and phospholipase activity.

1. Uptake of triglycerides and phosphoglycerides was demonstrated in ligated washed chick intestinal segments in situ. Mucosal uptake was observed both in the jejunum and ileum of chicks and was not accompanied by significant luminal hydrolysis of acylglycerides. 2. Brush border membrane vesicles were prepared and lipase and phospholipase activities were demonstrated. These activities were enriched in parallel to the increase in activity of maltase and gamma-glutamyl transpeptidase in all small intestinal areas. 3. In order to determine if the pancreatic and brush border membrane lipolytic activities were similar different properties of these activities were examined. The relative activities of pancreatic and brush border membranes towards triolein and phosphatidylcholine differed. The pH-triolein activity curve and the use of different additives showed some diverging effects between the activities. These differences were not, however, sufficient to conclude that the brush border activity towards triolein is distinct from that of pancreatic lipase. 4. Lipolytic activity in the brush border may play a role in acylglyceride digestion, uptake and transport in the small intestine of the chick.     

Investigation of the effect of black walnut extract on in vitro ion transport and structure of equine colonic mucosa

OBJECTIVE: To examine the secretory response (in the presence and absence of prostaglandin inhibition) in vitro and structural alterations of colonic mucosa in horses after intragastric administration of black walnut extract (BWE). ANIMALS: 14 adult horses. PROCEDURE: Seven horses were administered BWE intragastrically and monitored for 11 hours. Tissue samples were obtained from the right ventral, left ventral, and right dorsal colons (RVC, LVC, and RDC, respectively) of the 7 BWE-treated and 7 control horses. Tissue samples were examined via light microscopy, and the extent of hemorrhage, edema, and granulocytic cellular infiltration (neutrophils and eosinophils) was graded. Colonic mucosal segments were incubated with or without flunixin meglumine (FLM) for 240 minutes; spontaneous electrical potential difference and short-circuit current (Isc) were recorded and used to calculate mucosal resistance. RESULTS: Colonic tissues from BWE-treated horses (with or without FLM exposure) had an overall greater Isc during the 240-minute incubation period, compared with tissues from control horses. The resistance pattern in RVC, LVC, and RDC samples (with or without FLM exposure) from BWE-treated horses was decreased overall, compared with control tissues (with or without FLM exposure). Histologically, colonic mucosal tissues from BWE-treated horses had more severe inflammation (involving primarily eosinophils), edema, and hemorrhage, compared with tissue from control horses. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, BWE administration appears to cause an inflammatory response in colonic mucosal epithelium that results in mucosal barrier compromise as indicated by decreased mucosal resistance with presumed concomitant electrogenic chloride secretory response, which is not associated with prostaglandin mediation.     

Characterization of equine vocalization

Bioacoustics is the study of sound in animals and includes, but is not limited to, animal communication with associated behavior, sound production anatomy and neurophysiology, auditory capacities and auditory mechanisms, and animal welfare. The present research investigates the vocalizations of horses during stressful situations. Stress can be positive or negative. Distress is anything that affects the animal in a negative way, such as in mare and foal separation. Eustress is anything that affects the animal in a positive way, such as morning feeding time in a horse barn. The purpose of the current research is to find spectral differences in the recorded vocalizations of stalled horses that indicate both distress and eustress using the Hidden Markov Model (HMM). Greenwood Function Cepstral Coefficient values suggest that there are spectral differences between vocalizations in a distress and eustress situation. These consistent results indicate that further research to obtain and evaluate vocalizations of horses may provide a productive tool in understanding equine welfare.     

Differential toxic effects of gentamicin on cultured renal epithelial cells (LLC-PK1) on application to the brush border membrane or the basolateral membrane

Aminoglycoside antibiotics are generally accepted to accumulate in renal proximal tubule cells from the luminal surface and show toxic effects on the cells. The binding affinity and membrane permeability of aminoglycoside antibiotics are different at the brush border membrane (BBM) and the basolateral membrane (BLM) of proximal tubule cells. This study was performed, therefore, to investigate the differential effects of the aminoglycoside antibiotic gentamicin (GM) on cultured LLC-PK1 cells, a pig kidney proximal epithelial cell line, after addition to the BBM or the BLM side. LLC-PK1 cells were cultured on microporous membranes until forming confluent monolayers, and then GM was added to either the BBM or the BLM side. GM caused release of enzymes from the organelles, with a higher level of release observed following addition to the BBM side than that to the BLM side. Patterns of [3H]GM uptake by the cells differed in a manner dependent on whether it was added to the BBM or the BLM side. That is, the cellular uptake from the BBM side increased with incubation time, while that from the BLM side showed rapid saturation. These results suggested that aminoglycoside antibiotics show differential effects on cultured proximal epithelial cells and have differential patterns of cellular uptake when added to the BBM or the BLM side.