scholarly journals Effect of Inhibitors on Alanine Transport in Isolated Rabbit Ileum

1967 ◽  
Vol 50 (10) ◽  
pp. 2357-2375 ◽  
Author(s):  
Ronald A. Chez ◽  
Richard R. Palmer ◽  
Stanley G. Schultz ◽  
Peter F. Curran
Keyword(s):  
Intestinal Epithelium ◽  
Brush Border ◽  
Rate Coefficient ◽  
Metabolic Energy ◽  
Metabolic Inhibitors ◽  
Direct Link ◽  
Rabbit Ileum ◽  
Alanine Transport ◽  
Effect Of Inhibitors

The effects of metabolic inhibitors and ouabain on alanine transport across rabbit ileum, in vitro, have been investigated. Net transport of alanine and Na across short-circuited segments of ileum is virtually abolished by cyanide, 2,4-dinitrophenol, iodoacetate, and ouabain. However, these inhibitors do not markedly depress alanine influx from the mucosal solution, across the brush border, into the intestinal epithelium, and they do not significantly affect the Na dependence of this entry process. The results of this investigation indicate that: (a) the Na dependence of alanine influx does not reflect a mechanism in which the sole function of Na is to link metabolic energy directly to the influx process; and (b) the inhibition of net alanine transport across intestine is, in part, the result of an increased rate coefficient for alanine efflux out of the cell across the brush border. Although these findings do not exclude a direct link between metabolic energy and alanine efflux, the increased efflux may be the result of the increased intracellular Na concentration in the presence of these inhibitors. The results of these studies are qualitatively consistent with a model for alanine transport across the brush border which does not include a direct link to metabolic energy.

scholarly journals Studies on the Electrical Potential Profile across Rabbit Ileum

1971 ◽  
Vol 57 (6) ◽  
pp. 639-663 ◽  
Author(s):  
Richard C. Rose ◽  
Stanley G. Schultz
Keyword(s):  
Amino Acids ◽  
Brush Border ◽  
Electrical Potential ◽  
Metabolic Inhibitors ◽  
Electrical Potential Difference ◽  
Intercellular Spaces ◽  
Cell Interior ◽  
Rabbit Ileum ◽  
Lateral Intercellular Spaces ◽  
Rapid Replacement

When isolated strips of mucosal rabbit ileum are bathed by physiological electrolyte solution the electrical potential difference (PD) across the brush border (ψmc) averages 36 mv, cell interior negative. Rapid replacement of Na in the mucosal solution with less permeant cations, Tris or choline, results in an immediate hyperpolarization of ψmc. Conversely, replacement of choline in the mucosal solution with Na results in an abrupt depolarization of ψmc. These findings indicate that Na contributes to the conductance across the brush border. The presence of actively transported sugars or amino acids in the mucosal solution brings about a marked depolarization of ψmc and a smaller increase in the transmural PD (Δψms). It appears that the Na influx that is coupled to the influxes of amino acids and sugars is electrogenic and responsible for the depolarization of ψmc. Under control conditions Δψms can be attributed to the depolarization of ψmc together with the presence of a low resistance transepithelial shunt, possibly the lateral intercellular spaces. However, quantitatively similar effects of amino acids on ψmc are also seen in tissues poisoned with metabolic inhibitors or ouabain. Under these conditions Δψmc is much smaller than under control conditions. Thus, the depolarization of ψmc might not account for the entire Δψms, observed in nonpoisoned tissue. An additional electromotive force which is directly coupled to metabolic processes might contribute to the normal Δψms.

scholarly journals Lysine Transport across Isolated Rabbit Ileum

1969 ◽  
Vol 53 (2) ◽  
pp. 157-182 ◽  
Author(s):  
B. G. Munck ◽  
Stanley G. Schultz
Keyword(s):  
Brush Border ◽  
Maximal Velocity ◽  
Transport Capacity ◽  
Rabbit Ileum ◽  
Mucosal Membrane ◽  
Intestinal Brush Border

Lysine transport by in vitro distal rabbit ileum has been investigated by determining (a) transmural fluxes across short-circuited segments of the tissue; (b) accumulation by mucosal strips; and (c) influx from the mucosal solution across the brush border into the epithelium. Net transmural flux of lysine is considerably smaller than that of alanine. However, lysine influx across the brush border and lysine accumulation by mucosal strips are quantitatively comparable to alanine influx and accumulation. Evidence is presented that the "low transport capacity" of rabbit ileum for lysine is due to: (a) a carrier-mediated process responsible for efflux of lysine out of the cell across the serosal and/or lateral membranes that is characterized by a low maximal velocity; and (b) a high "backflux" of lysine out of the cell across the mucosal membrane. A possible explanation for the latter observation is discussed with reference to the relatively low Na dependence of lysine transport across the intestinal brush border.

Na+-independent transport of bipolar and cationic amino acids across the luminal membrane of the small intestine

1997 ◽  
Vol 272 (4) ◽  
pp. R1060-R1068 ◽  
Author(s):  
B. G. Munck ◽  
L. K. Munck
Keyword(s):  
Amino Acids ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Short Circuit ◽  
Present System ◽  
Rabbit Ileum ◽  
Beta Alanine ◽  
Brown Adipose ◽  
Cationic Amino Acids

The role of sodium in transport of bipolar and cationic amino acids and their interactions were examined in vitro by measuring unidirectional influx across the brush-border membrane of intact rat jejunal and rabbit ileal epithelia. The chloride-dependent and beta-alanine inhibitable B(0,+) present in rabbit ileum was blocked by combining inhibition by beta-alanine with Na(+)- or Cl(-)-free conditions. Under these conditions, lysine influx across the brush-border membrane is Na+ independent. All Na+-independent influx of cationic and bipolar amino acids is by a system b(0,+) equivalent in the brush-border membrane of both species, where a system y+ is not present. System b(0,+) is shown to be a potent exchanger of intracellular leucine for extracellular lysine and of intracellular lysine for extracellular leucine. The model used to explain leucine stimulation of mucosa to serosa lysine transport can explain Na+ dependence of net lysine absorption. On the assumption that b(0,+) in situ, like the transporter induced by retroperitoneal brown adipose tissue in Xenopus laevi oocytes, acts as an obligatory exchanger, this model can also explain the effects of lysine on short-circuit current and net transport of sodium and the effect on transport capacity by preincubation at Na+-free conditions.

scholarly journals The interaction ofTrypanosoma congolensewith endothelial cells

Parasitology ◽  
1994 ◽  
Vol 109 (5) ◽  
pp. 631-641 ◽  
Author(s):  
A. Hemphill ◽  
I. Frame ◽  
C. A. Ross
Keyword(s):  
Endothelial Cells ◽  
Binding Assay ◽  
Light And Electron Microscopy ◽  
Trypanosoma Congolense ◽  
Metabolic Energy ◽  
Metabolic Inhibitors ◽  
Feeder Cells ◽  
Cell Plasma Membrane ◽  
Cell Plasma

Factors which affect adhesion of culturedTrypanosoma congolensebloodstream forms to mammalian feeder cells have been examined. Using anin vitrobinding assay, the initial events following interaction of trypanosomes with bovine aorta endothelial (BAE) cells were monitored by both light- and electron microscopy. Metabolic inhibitors and other biochemicals were incubated with either cells or parasites, to test whether any inhibited the process. Our findings suggest that adhesion of the parasites is an active process requiring metabolic energy from the trypanosomes, but not from endothelial cells. We also provide data suggesting thatT. congolensebloodstream forms possess a lectin-like domain, localized at distinct sites on their flagellar surface, which interacts with specific carbohydrate receptors, most likely sialic acid residues, on the endothelial cell plasma membrane. We also suggest that the cytoskeletal protein actin is probably involved in this interaction.

scholarly journals Evaluation of human primary intestinal monolayers for drug metabolizing capabilities

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Jennifer E. Speer ◽  
Yuli Wang ◽  
John K. Fallon ◽  
Philip C. Smith ◽  
Nancy L. Allbritton
Keyword(s):  
Phase I ◽  
Intestinal Epithelium ◽  
Brush Border ◽  
Functional Group ◽  
Drug Metabolizing Enzymes ◽  
Metabolizing Enzymes ◽  
Brush Border Enzymes ◽  
In Cells

Abstract Background The intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). The enterocytes are supported by a porous extracellular matrix (ECM) that enables proper cell adhesion and function of brush border enzymes, such as alkaline phosphatase (ALP) and alanyl aminopeptidase (AAP), phase I DMEs that convert a parent drug to a more polar metabolite by introducing or unmasking a functional group, and phase II DMEs that form a covalent conjugate between a functional group on the parent compound or sequential metabolism of phase I metabolite. In our effort to develop an in vitro intestinal epithelium model, we investigate the impact of two previously described simple and customizable scaffolding systems, a gradient cross-linked scaffold and a conventional scaffold, on the ability of intestinal epithelial cells to produce drug metabolizing proteins as well as to metabolize exogenously added compounds. While the scaffolding systems possess a range of differences, they are most distinguished by their stiffness with the gradient cross-linked scaffold possessing a stiffness similar to that found in the in vivo intestine, while the conventional scaffold possesses a stiffness several orders of magnitude greater than that found in vivo. Results The monolayers on the gradient cross-linked scaffold expressed CYP3A4, UGTs 2B17, 1A1 and 1A10, and CES2 proteins at a level similar to that in fresh crypts/villi. The monolayers on the conventional scaffold expressed similar levels of CYP3A4 and UGTs 1A1 and 1A10 DMEs to that found in fresh crypts/villi but significantly decreased expression of UGT2B17 and CES2 proteins. The activity of CYP3A4 and UGTs 1A1 and 1A10 was inducible in cells on the gradient cross-linked scaffold when the cells were treated with known inducers, whereas the CYP3A4 and UGT activities were not inducible in cells grown on the conventional scaffold. Both monolayers demonstrate esterase activity but the activity measured in cells on the conventional scaffold could not be inhibited with a known CES2 inhibitor. Both monolayer culture systems displayed similar ALP and AAP brush border enzyme activity. When cells on the conventional scaffold were incubated with a yes-associated protein (YAP) inhibitor, CYP3A4 activity was greatly enhanced suggesting that mechano-transduction signaling can modulate drug metabolizing enzymes. Conclusions The use of a cross-linked hydrogel scaffold for expansion and differentiation of primary human intestinal stem cells dramatically impacts the induction of CYP3A4 and maintenance of UGT and CES drug metabolizing enzymes in vitro making this a superior substrate for enterocyte culture in DME studies. This work highlights the influence of mechanical properties of the culture substrate on protein expression and the activity of drug metabolizing enzymes as a critical factor in developing accurate assay protocols for pharmacokinetic studies using primary intestinal cells. Graphical abstract

Transport of beta-lactoglobulin across rabbit ileum in vitro

1989 ◽  
Vol 256 (6) ◽  
pp. G943-G948 ◽  
Author(s):  
D. Marcon-Genty ◽  
D. Tome ◽  
O. Kheroua ◽  
A. M. Dumontier ◽  
M. Heyman ◽  
...  
Keyword(s):  
Milk Protein ◽  
Ussing Chamber ◽  
Microtubule Assembly ◽  
Metabolic Inhibitors ◽  
Transepithelial Transport ◽  
Enzyme Linked Immunosorbent Assay ◽  
Food Protein ◽  
Rabbit Ileum ◽  
Beta Lactoglobulin

Intestinal transepithelial transport constitutes a major limiting step in the transfer of food protein antigens to the blood. This transport was studied in isolated rabbit ileum in Ussing chamber in vitro for the milk protein antigen beta-lactoglobulin (beta-Lg). The transepithelial passage of beta-Lg was measured by enzyme-linked immunosorbent assay (ELISA) and radiolabeled protein transfer and compared with that of the nonmetabolizable marker polyethylene glycol (PEG)-4000. When 1 mg/ml of beta-[14C]Lg or [3H]PEG was added to the mucosal side of the tissue, the total uptake, measured as the transfer of radiolabeled material across the ileum, was significantly higher for beta-Lg than for PEG (5.46 +/- 1.75 vs. 1.43 +/- 0.26 micrograms.h-1.cm-2). Measured by ELISA, 6-9% of the total amount of beta-Lg transported was absorbed in an intact antigenic form. This transport of intact beta-Lg was inhibited by the metabolic inhibitors 50 mM 2-deoxyglucose and 1 mM azide added simultaneously, was reduced by the microtubule assembly inhibitor 0.05 mM colchicine, and was enhanced by 20 mM ammonia, which inhibits lysosomal proteolytic activity. These results indicate that beta-Lg is efficiently absorbed by the intestinal mucosa of adult animals, partly in intact antigenic form and that beta-Lg transport is probably transcellular, as observed for other proteins. The finding that beta-Lg is absorbed in intact antigenic form agrees with other reports implying that beta-Lg is the main factor responsible for milk protein immunoreactivity and intolerance.

Mechanism of inhibition of alanine absorption by Na ricinoleate.

1979 ◽  
Vol 236 (5) ◽  
pp. E534
Author(s):  
J J Hajjar ◽  
D M Murphy ◽  
R L Scheig
Keyword(s):  
Fatty Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Intestinal Cell ◽  
Rabbit Ileum ◽  
Ileal Mucosa ◽  
Mechanism Of Inhibition ◽  
Rabbit Intestine

The mechanism of inhibition of alanine absorption by Na ricinoleate has been examined in the rabbit intestine. This fatty acid in a concentration of 2--5 mM inhibits alanine absorption in vivo and in vitro. The inhibition is more evident in the jejunum than in the ileum. Strips of ileal mucosa treated with Na ricinoleate gain Na. Sodium ricinoleate inhibits alanine influx across rabbit ileum, even in the presence of a sodium gradient across these cells. The results suggest that the main action of Na ricinoleate is on the alanine-transport system at the brush-border membrane. The fatty acid may also inhibit amino acid absorption by increasing intestinal cell Na concentration, which results in a decreased Na gradient across the brush-border membrane.

scholarly journals Myosin 1a Regulates Osteoblast Differentiation Independent of Intestinal Calcium Transport

2019 ◽  
Vol 3 (11) ◽  
pp. 1993-2011
Author(s):  
Scott Munson ◽  
Yongmei Wang ◽  
Wenhan Chang ◽  
Daniel D Bikle
Keyword(s):  
Intestinal Epithelium ◽  
Brush Border ◽  
Calcium Transport ◽  
Osteoblast Differentiation ◽  
Epithelial Cell Line ◽  
Null Mouse ◽  
Normal Numbers ◽  
Calmodulin Binding ◽  
Intestinal Calcium Transport

Abstract Myosin 1A (Myo1a) is a mechanoenzyme previously thought to be located exclusively in the intestinal epithelium. It is the principle calmodulin-binding protein of the brush border. Based on earlier studies in chickens, we hypothesized that Myo1a facilitates calcium transport across the brush border membrane of the intestinal epithelium, perhaps in association with the calcium channel Trpv6. Working with C2Bbe1 cells, a human intestinal epithelial cell line, we observed that overexpression of Myo1a increased, whereas the antisense construct blocked calcium transport. To further test this hypothesis, we examined mice in which either or both Myo1a and Trpv6 had been deleted. Although the Trpv6-null mice had decreased intestinal calcium transport, the Myo1a-null mouse did not, disproving our original hypothesis, at least in mice. Expecting that a reduction in intestinal calcium transport would result in decreased bone, we examined the skeletons of these mice. To our surprise, we found no decrease in bone in the Trpv6-null mouse, but a substantial decrease in the Myo1a-null mouse. Double deletions were comparable to the Myo1a null. Moreover, Myo1a but not Trpv6 was expressed in osteoblasts. In vitro, the bone marrow stromal cells from the Myo1a-null mice showed normal numbers of colony-forming units but marked decrements in the formation of alkaline phosphatase–positive colonies and mineralized nodules. We conclude that Myo1a regulates osteoblast differentiation independent of its role, if any, in intestinal calcium transport, whereas Trpv6 functions primarily to promote intestinal calcium transport with little influence in osteoblast function.

scholarly journals Effects of temperature, metabolic inhibitors and some other factors on fluid-phase and adsorptive pinocytosis by rat peritoneal macrophages

1979 ◽  
Vol 180 (3) ◽  
pp. 567-571 ◽  
Author(s):  
M K Pratten ◽  
J B Lloyd
Keyword(s):  
Low Temperature ◽  
Energy Supply ◽  
Cytochalasin B ◽  
Fluid Phase ◽  
Peritoneal Macrophages ◽  
Metabolic Energy ◽  
Metabolic Inhibitors ◽  
Dibutyryl Cyclic Amp ◽  
Effects Of Temperature

Low temperature, NaF and 2,4-dinitrophenol could each abolish the pinocytic uptake of 125I-labelled poly(vinylpyrrolidone) or colloidal [198Au]gold by rat peritoneal macrophages cultured in vitro. Cytochalasin B caused only partial inhibition, even at 10 microgram/ml, and colchicine (10 or 25 microgram/ml) inhibited uptake of colloidal [198Au]gold much more than that of 125I-labelled poly(vinylpyrrolidone). Dibutyryl cyclic AMP and ouabain were without effect on uptake of 125I-labelled poly(vinylpyrrolidone), and slight stimulation was seen with ATP and theophylline. Uptake of 125I-labelled poly(vinylpyrrolidone) was abolished by EGTA (5mM), but restored by adding CaCl2 (5mM). The results appear not to support the conventional criteria for the division of pinocytic phenomena into macropinocytosis, requiring a metabolic energy supply and cytoskeletal components, and micropinocytosis, requiring neither.

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