Anion effects on fluid absorption from rat jejunum perfused in vivo

1983 ◽  
Vol 244 (1) ◽  
pp. G33-G39
Author(s):  
M. H. Humphreys ◽  
L. Y. Chou
Keyword(s):  
Atpase Activity ◽  
Water Absorption ◽  
Brush Border ◽  
Intestinal Transport ◽  
Ringer Solution ◽  
Rat Jejunum ◽  
Perfusion Fluid ◽  
Potent Effect

Perfusion of rat jejunal segments in vivo with an isotonic, HCO3-free SO4-Ringer solution resulted in low rates of net sodium (JNanet) and water absorption. When the perfusion fluid was changed to one containing 25 mM Na2SO3, JNanet increased from 4.7 +/- 1.2 to 11.6 +/- 1.5 (SE) mumol X cm-1 X h-1 (P less than 0.001). This increased absorption was accompanied by comparable increases in chloride and water absorption, occurred without a detectable change in potential difference across the perfused segment, and was readily reversed on reinstitution of perfusion with SO4-Ringer. Perfusion with SO3-Ringer had no effect on electrolyte absorption from terminal segments of rat ileum. Addition of L-phenylalanine stimulated absorption from SO4-Ringer perfusate but not from SO3-Ringer perfusate. Addition of 25 mM NaHCO3 to SO4-Ringer perfusate caused parallel increases in JNanet and JHCO3net; when 25 mM NaHCO3 was added to SO4-Ringer perfusate that also contained 25 mM NaSCN, the same increase in JHCO3net occurred but was not associated with any increase in JNanet. These results indicate a potent effect of SO2-3 and HCO-3 to stimulate JNanet from rat jejunum but not from ileum. These anion effects on intestinal transport in vivo resemble their effects on ATPase activity of brush-border fractions from small intestine in vitro and raise the possibility that these effects on ion transport could be mediated through the changes in brush-border ATPase activity, which are brought about by exposure to these anions, although other explanations are also possible.

Intestinal Transport of a Tetrapeptide, l-Leucylglycylglycylglycine, in Rat Small Intestine in Vivo

1979 ◽  
Vol 57 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Y. C. Chung ◽  
D. B. A. Silk ◽  
Y. S. Kim
Keyword(s):  
Amino Acid ◽  
Brush Border ◽  
Transport Mechanism ◽  
Intestinal Transport ◽  
General Mechanism ◽  
Aminopeptidase Activity ◽  
Amino Acid Mixtures ◽  
Jejunal Perfusion

1. The intestinal transport mechanism for the tetrapeptide l-leucylglycylglycylglycine, Leu-Gly-Gly-Gly, and its relation to the transport of free Leu, Leu-Gly and Leu-Gly-Gly were investigated in vivo by means of jejunal perfusion in rats. 2. The rates of net Leu absorption from peptides (Leu-Gly and Leu-Gly-Gly-Gly) were significantly greater than those from the free amino acid mixtures when the test solutions were perfused at a concentration of 15 mmol/l. 3. Net Leu absorption rates from Leu-Gly (10 μmol/ml) and Leu-Gly-Gly (10 μmol/ml) were extensively inhibited (84% and 68% respectively) by Gly-Pro at 100 mmol/l, whereas Gly-Pro had no effect on Leu absorption from Leu-Gly-Gly-Gly. l-Alanine (Ala, 100 μmol/ml), on the other hand, which completely inhibited Leu absorption during perfusion of free Leu, inhibited Leu uptake from Leu-Gly-Gly-Gly only about 50% at all concentrations studied. Ala had no effect on Leu absorption from Leu-Gly and Leu-Gly-Gly (10 μmol/ml). 4. Neither Ala at 100 μmol/ml nor Gly-Pro at 100 μmol/ml had any effect on brush-border aminopeptidase activity in vitro, suggesting that the hydrolytic capacity of the intestinal mucosal brush border was unaltered when Ala or Gly-Pro was included in the perfusion mixture. l-Alanyl-β-naphthylamide (20 μmol/ml), which inhibited brush-border aminopeptidase activity by 85% in vitro, failed to block substantially net Leu absorption from Leu-Gly and Leu-Gly-Gly-Gly. 5. The data presented suggest that, although some of the Leu from the tetrapeptide, Leu-Gly-Gly-Gly, may be hydrolysed before transport, nearly 50% of the tetrapeptide appears to be transported intact. Although Leu-Gly, Leu-Gly-Gly and Gly-Pro seem to share a common transport mechanism, the system used for intact Leu-Gly-Gly-Gly absorption seems to be distinct. However, the present study does not exclude the possibility that binding of the tetrapeptide to the brush-border aminopeptidase alters the affinity of Leu for the amino acid carrier, and therefore further studies are necessary before firm conclusions can be made on the general mechanism of tetrapeptide transport.

Effects of taurodeoxycholate on in vivo water and solute transport in rat jejunum in absence and presence of calcium

1986 ◽  
Vol 250 (2) ◽  
pp. G248-G251
Author(s):  
H. V. Ammon ◽  
D. S. Cho ◽  
R. L. Loeffler ◽  
K. L. Reetz
Keyword(s):  
Fatty Acids ◽  
Bile Acids ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Calcium Absorption ◽  
Intestinal Transport ◽  
Fluid Secretion ◽  
Rat Jejunum ◽  
Perfusion Studies

Bile acids and fatty acids enhance the permeability of brush-border membrane vesicles for calcium. It has been postulated that increased influx of calcium into the enterocyte might be responsible for the fluid secretion induced by dihydroxy bile acids and fatty acids. During in vivo perfusion studies of the rat jejunum, 15 mM taurodeoxycholate induced secretion of electrolytes and water (P less than 0.001), reduced glucose absorption (P less than 0.001), and enhanced the absorption of mannitol (P less than 0.0125) and calcium (P less than 0.001). Calcium absorption continued to be enhanced during perfusion of a CaCl2-containing solution following the perfusion with taurodeoxycholate (P less than 0.05). In view of the previously demonstrated enhanced permeability of the apical brush-border membrane in the presence of bile acids, it is very likely that some calcium enters the enterocyte along the steep concentration gradient in the presence of taurodeoxycholate. In spite of enhanced calcium absorption, 15 mM CaCl2 had no effect on control absorption rates or on fluid secretion induced by taurodeoxycholate. The data indicate that the effects of bile acids on intestinal transport are not mediated by an influx of calcium into the enterocyte.

scholarly journals DDEL-12. NANOPARTICLE DELIVERY OF DOXORUBICIN FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii286-iii286
Author(s):  
Caitlin Ung ◽  
Maria Tsoli ◽  
Jie Liu ◽  
Domenico Cassano ◽  
Dannielle Upton ◽  
...  
Keyword(s):  
Treatment Strategies ◽  
Pediatric Brain Tumors ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Confocal Imaging ◽  
Cell Content ◽  
Potent Effect ◽  
Orthotopic Mouse Model ◽  
In Vivo Testing

Abstract DIPGs are the most aggressive pediatric brain tumors. Currently, the only treatment is irradiation but due to its palliative nature patients die within 12 months. Effective delivery of chemotherapy across the blood-brain barrier (BBB) has been a key challenge for the eradication of this disease. We have developed a novel gold nanoparticle functionalised with human serum albumin (Au-NP, 98.8 ±19 nm) for the delivery of doxorubicin. In this study, we evaluated the cytotoxic efficacy of doxorubicin delivered through gold nanoparticles (Au-NP-Dox). We found that DIPG neurospheres were equally sensitive to doxorubicin and Au-NP-Dox (at equimolar concentration) by alamar blue assay. Colony formation assays demonstrated a significantly more potent effect of Au-NP-Dox compared to doxorubicin alone, while the Au-NP had no effect. Furthermore, western blot analysis indicated increased apoptotic markers cleaved Parp, caspase 3/7 and phosphorylated H2AX in Au-NP-Dox treated DIPG neurospheres. Live cell content and confocal imaging demonstrated significantly higher uptake of Au-NP-Dox compared to doxorubicin alone. Treatment of a DIPG orthotopic mouse model with Au-NP-Dox showed no signs of toxicity with stable weights being maintained during treatment. However, in contrast to the above in vitro findings the in vivo study showed no anti-tumor effect possibly due to poor penetration of Au-NP-Dox into the brain. We are currently evaluating whether efficacy can be improved using measures to open the BBB transiently. This study highlights the need for rigorous in vivo testing of new treatment strategies before clinical translation to reduce the risk of administration of ineffective treatments.

scholarly journals Dissection of Swa2p/Auxilin Domain Requirements for Cochaperoning Hsp70 Clathrin-uncoating Activity In Vivo

2006 ◽  
Vol 17 (7) ◽  
pp. 3281-3290 ◽  
Author(s):  
Jing Xiao ◽  
Leslie S. Kim ◽  
Todd R. Graham
Keyword(s):  
Atpase Activity ◽  
Point Mutation ◽  
Mutational Analysis ◽  
Weak Interactions ◽  
Tetratricopeptide Repeat ◽  
Uba Domain ◽  
J Domain ◽  
Tpr Domain

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.

Metabolic importance of Na+/K+-ATPase activity during sea urchin development.

1997 ◽  
Vol 200 (22) ◽  
pp. 2881-2892 ◽  
Author(s):  
P Leong ◽  
D Manahan
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Sea Urchin ◽  
Sodium Pump ◽  
Sea Water ◽  
Strongylocentrotus Purpuratus ◽  
Lytechinus Pictus ◽  
Stimulation Of

Early stages of animal development have high mass-specific rates of metabolism. The biochemical processes that establish metabolic rate and how these processes change during development are not understood. In this study, changes in Na+/K+-ATPase activity (the sodium pump) and rate of oxygen consumption were measured during embryonic and early larval development for two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus. Total (in vitro) Na+/K+-ATPase activity increased during development and could potentially account for up to 77 % of larval oxygen consumption in Strongylocentrotus purpuratus (pluteus stage) and 80 % in Lytechinus pictus (prism stage). The critical issue was addressed of what percentage of total enzyme activity is physiologically active in living embryos and larvae and thus what percentage of metabolism is established by the activity of the sodium pump during development. Early developmental stages of sea urchins are ideal for understanding the in vivo metabolic importance of Na+/K+-ATPase because of their small size and high permeability to radioactive tracers (86Rb+) added to sea water. A comparison of total and in vivo Na+/K+-ATPase activities revealed that approximately half of the total activity was utilized in vivo. The remainder represented a functionally active reserve that was subject to regulation, as verified by stimulation of in vivo Na+/K+-ATPase activity in the presence of the ionophore monensin. In the presence of monensin, in vivo Na+/K+-ATPase activities in embryos of S. purpuratus increased to 94 % of the maximum enzyme activity measured in vitro. Stimulation of in vivo Na+/K+-ATPase activity was also observed in the presence of dissolved alanine, presumably due to the requirement to remove the additional intracellular Na+ that was cotransported with alanine from sea water. The metabolic cost of maintaining the ionic balance was found to be high, with this process alone accounting for 40 % of the metabolic rate of sea urchin larvae (based on the measured fraction of total Na+/K+-ATPase that is physiologically active in larvae of S. purpuratus). Ontogenetic changes in pump activity and environmentally induced regulation of reserve Na+/K+-ATPase activity are important factors that determine a major proportion of the metabolic costs of sea urchin development.

Na-K-ATPase activity along the rabbit, rat, and mouse nephron

1979 ◽  
Vol 237 (2) ◽  
pp. F114-F120 ◽  
Author(s):  
A. I. Katz ◽  
A. Doucet ◽  
F. Morel
Keyword(s):  
Atpase Activity ◽  
Thick Ascending Limb ◽  
Sensitivity Limit ◽  
Henle's Loop ◽  
Collecting Tubule ◽  
Pars Recta ◽  
Hydrolysis Of ◽  
Total Enzyme

Na-K-ATPase activity along the rabbit, rat, and mouse nephron was determined with a micromethod that measures directly labeled phosphate released by the hydrolysis of [gamma-32P]ATP. Na-K-ATPase activity was highest in the rat, intermediate in the mouse, and lowest in the rabbit nephron. With the exception of rabbit cortical thick ascending limb, the enzyme profile was similar in the three species: Na-K-ATPase activity per millimeter tubule length was highest in the distal convoluted tubule and thick ascending limb of Henle's loop, intermediate in the proximal convoluted tubule, and lowest in the pars recta and collecting tubule. The enzyme was present in the thin limbs of Henle's loop, but its activity was very low and measurements were close to the sensitivity limit of the method. Both the absolute activity and the fraction of the total enzyme represented by Na-K-ATPase were severalfold higher than in kidney homogenates. Finally, the Na-K-ATPase activity measured in certain segments of the rat and rabbit nephron in this study seems sufficient to account in theory for the active component of the net sodium transport found in the corresponding region of the nephron with either in vivo or in vitro single tubule microperfusion techniques.

Anion-stimulated ATPase activity of brush border from rat small intestine.

1979 ◽  
Vol 236 (1) ◽  
pp. E70 ◽  
Author(s):  
M H Humphreys ◽  
L Y Chou
Keyword(s):  
Alkaline Phosphatase ◽  
Atpase Activity ◽  
Brush Border ◽  
Atp Hydrolysis ◽  
Intestinal Transport ◽  
Mitochondrial Enzymes ◽  
Intestinal Alkaline Phosphatase ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Small Intestinal

Differential centrifugation of rat small intestinal homogenates produced a crude brush border (BB) fraction that was enriched 15-fold for the marker enzymes, alkaline phosphatase and sucrase; contamination with mitochondrial enzymes, monoamine oxidase and succinate dehydrogenase, was minimal. ATP hydrolysis by this BB fraction was stimulated by addition of several anions to the incubation medium: HCO3 and Cl were equally effective in this regard, with NO3, NO2, SO4, and acetate being less stimulatory. SCN and CNO inhibited ATPase activity, whereas the divalent anion SO3 was stimulatory at low concentrations (less than 25 mM) but inhibitory at 100 mM. Maximum anion stimulation was observed at a Mg concentration of 0.5 mM, and pH optimum was 8.5. Kinetic analysis showed that HCO3 increased the Vmax without altering the Km for ATP; the Ka for this effect of HCO3 was 35 mM. This enzyme activity was completely inhibited by 20 mM L-phenylalanine, 10 mM L-cysteine, and 3 mM EDTA, compounds that also inhibited intestinal alkaline phosphatase. These results demonstrate the presence of anion-stimulated ATPase activity in rat small intestinal brush border and suggest that this activity may be related to intestinal alkaline phosphatase. The role of this enzyme in intestinal transport is not known, but could relate to the regulation of intestinal absorption and secretion.

Mechanism of maleic acid-induced glucosuria in dog kidney

1976 ◽  
Vol 231 (4) ◽  
pp. 1024-1032 ◽  
Author(s):  
M Silverman ◽  
L Huang
Keyword(s):  
Brush Border ◽  
Maleic Acid ◽  
Brush Border Membrane ◽  
Multiple Indicator Dilution ◽  
Dog Kidney ◽  
Proximal Tubular ◽  
Multiple Indicator ◽  
Prior Administration

The multiple indicator-dilution technique in vivo and isolated brush-border membranes in vitro have been used to explore the mechanism of maleic acid-induced glucosuria in dog kidney. The interaction of D-glucose with the antiluminal membrane from the peritubular fluid surface is unaltered. It is demonstrated that alpha-methyl-D-glucoside (alpha MG) enters and exits from the proximal tubular cell only across the brush-border membrane. Then using alphaMG as a reference indicator, it is shown that maleic acid does not cause complete inhibition of D-glucose interaction with the antiluminal membrane from the cytoplasmic surface. The binding of [3H]phlorizin both in vivo and in vitro is not affected by prior administration of maleic acid, indicating that D-glucose interaction with the outside surface of the brush border is also not affected by maleic acid. The data are therefore consistent with the concept that maleic acid-induced glucosuria is due either to i) partial inhibition of D-glucose movement from cytoplasm across the antiluminal membrane into the blood, ii) stimulated movement back across the brush-border membrane into urine, or iii) a combination of the two effects.

Intestinal filtration-secretion due to increased intraluminal pressure in rabbits

1982 ◽  
Vol 242 (1) ◽  
pp. G65-G75
Author(s):  
E. A. Swabb ◽  
R. A. Hynes ◽  
W. G. Marnane ◽  
J. S. McNeil ◽  
R. A. Decker ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
Surface Area ◽  
Intestinal Transport ◽  
Blood Flow Rate ◽  
Intraluminal Pressure ◽  
Rabbit Ileum ◽  
Venous Dilatation ◽  
Small Intestinal

The mechanism of changes in small intestinal transport due to acutely increased intraluminal hydrostatic pressure (IHP) was investigated in detail using perfused in vivo rabbit intestinal segments. IHP affected passive transport in vivo by increasing effective mucosal surface area in the small intestine (indicated by 3HOH transport and tissue architectural changes) and increasing small intestinal permeability (indicated by a proportionately greater increase in mannitol than erythritol secretory clearance). IHP did not alter ileal blood flow rate measured by radioactive microspheres, despite grossly evident venous dilatation, or active intestinal transport in the ileum as measured by a) in vitro ion transport in the absence of elevated hydrostatic pressure, b) mucosal adenylate cyclase or Na-K-ATPase activities, and c) glucose-stimulated water and electrolyte absorption. Acutely increased IHP appears to influence the hydrodynamics of the mucosal microcirculation in the rabbit ileum to produce a driving force for passive filtration-secretion, which is associated with and possibly augmented by increased tissue permeability and effective surface area.

scholarly journals Mycotoxins: Biotransformation and Bioavailability Assessment Using Caco-2 Cell Monolayer

Toxins ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 628
Author(s):  
Van Nguyen Tran ◽  
Jitka Viktorová ◽  
Tomáš Ruml
Keyword(s):  
Cell Monolayer ◽  
Intestinal Barrier ◽  
Intestinal Transport ◽  
Human Colon ◽  
In Vitro Models ◽  
Biologically Active ◽  
In Vitro Techniques ◽  
In Vivo Cytotoxicity

The determination of mycotoxins content in food is not sufficient for the prediction of their potential in vivo cytotoxicity because it does not reflect their bioavailability and mutual interactions within complex matrices, which may significantly alter the toxic effects. Moreover, many mycotoxins undergo biotransformation and metabolization during the intestinal absorption process. Biotransformation is predominantly the conversion of mycotoxins meditated by cytochrome P450 and other enzymes. This should transform the toxins to nontoxic metabolites but it may possibly result in unexpectedly high toxicity. Therefore, the verification of biotransformation and bioavailability provides valuable information to correctly interpret occurrence data and biomonitoring results. Among all of the methods available, the in vitro models using monolayer formed by epithelial cells from the human colon (Caco-2 cell) have been extensively used for evaluating the permeability, bioavailability, intestinal transport, and metabolism of toxic and biologically active compounds. Here, the strengths and limitations of both in vivo and in vitro techniques used to determine bioavailability are reviewed, along with current detailed data about biotransformation of mycotoxins. Furthermore, the molecular mechanism of mycotoxin effects is also discussed regarding the disorder of intestinal barrier integrity induced by mycotoxins.

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