scholarly journals Glycyl-l-proline transport in rabbit enterocyte basolateral-membrane vesicles

1990 ◽  
Vol 269 (3) ◽  
pp. 565-571 ◽  
Author(s):  
J Dyer ◽  
R B Beechey ◽  
J P Gorvel ◽  
R T Smith ◽  
R Wootton ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicle ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Peptide Transport ◽  
Marker Enzyme ◽  
Basolateral Membrane Vesicles ◽  
Protein Markers ◽  
Proximal Intestine

The properties of a peptide-transport system in rabbit enterocyte basolateral membrane were examined with glycyl-L-proline as the substrate. Basolateral-membrane vesicles prepared from rabbit proximal intestine were characterized in terms of both purity and orientation. Marker-enzyme assays show that the basolateral-membrane marker, ouabain-sensitive K(+)-activated phosphatase, is enriched 17-fold with respect to the initial homogenate. The activities of enzymes used as markers for other membranes and organelles are low, and contamination of the final membrane fraction with these is minimal. The use of immunoblotting techniques further confirms the absence of brush-border-membrane contamination. Proteins in the basolateral-membrane vesicle preparation gave no cross-reaction with antibodies against the 140 kDa antigen and the Na+/glucose-symport protein, markers specific to the brush-border membrane of the enterocyte. Conversely, antibodies raised against the classical basolateral-membrane marker, the RLA class I histocompatibility complex, reacted strongly with a 43 kDa basolateral-membrane protein. The orientation of the basolateral-membrane vesicles was shown to be predominantly inside-out on determination by two independent criteria. The uptake of [1-14C]glycyl-L-proline by these vesicles is stimulated by the presence of an inwardly directed pH gradient, and this stimulation can be abolished by the proton ionophores carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP) and tetrachlorotrifluoromethylbenzimidazole (TTFB). Transport is also inhibited by HgCl2, thimerosal, Na+ and other glycyl dipeptides.

Is intestinal peptide transport energized by a proton gradient?

1985 ◽  
Vol 249 (2) ◽  
pp. G153-G160 ◽  
Author(s):  
Vadivel Ganapathy ◽  
Frederick H. Leibach
Keyword(s):  
Small Intestine ◽  
Membrane Potential ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Proton Gradient ◽  
Peptide Transport ◽  
Direct Role ◽  
Mucosal Cells

Transport of intact peptides, followed by intracellular hydrolysis in the intestinal mucosal cells, plays an important role in the absorption of protein digestion products in the mammalian small intestine. Even though earlier studies on peptide absorption in intact-tissue preparations have indicated that peptides are transported by an active Na+-dependent mechanism, recent studies with purified brush-border membrane vesicles have unequivocally demonstrated that Na+ does not play a direct role in the translocation of peptides across the membrane. Like most amino acids, peptides are also transported as zwitterions. However, peptide transport causes depolarization of the brushborder membrane in intact mucosal cells as well as in purified membrane vesicles, and the depolarization is the result of a net transfer of positive charge across the membrane during peptide transport. This electrogenic nature of peptide transport is observed even in the absence of Na+. Peptide transport is enhanced by an interior-negative membrane potential and inhibited by an interior-positive membrane potential. An inward proton gradient stimulates peptide transport, and this stimulation is reduced when the proton gradient is subjected to rapid dissipation by the presence of a proton ionophore. These observations strongly suggest that peptides are cotransported with protons in the intestine. There is substantial evidence for the existence of an inward proton gradient in the mammalian small intestine, and therefore it is very likely that this proton gradient is the in vivo energy source for the uphill transport of peptides. The Na+-H+ exchanger in the brush-border membrane, in conjunction with Na+-K+-ATPase at the basolateral membrane, is probably responsible for the generation and maintenance of the proton gradient and may thus be involved indirectly in the intestinal absorption of peptides.

Calcium uptake by brush-border and basolateral membrane vesicles in chick duodenum

1990 ◽  
Vol 258 (1) ◽  
pp. G16-G23 ◽  
Author(s):  
J. Takito ◽  
T. Shinki ◽  
T. Sasaki ◽  
T. Suda
Keyword(s):  
Brush Border ◽  
Calcium Uptake ◽  
Brush Border Membrane ◽  
Calcium Absorption ◽  
Basolateral Membrane ◽  
Control Level ◽  
Membrane Vesicles ◽  
Basolateral Membrane Vesicles ◽  
Dihydroxyvitamin D3 ◽  
Calbindin D28k

Calcium uptake was compared between duodenal brush-border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from vitamin D-deficient chicks and those injected with 625 ng of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. The uptake by BBMV in the 1 alpha,25-(OH)2D3-treated birds attained a maximum (280% of the control) at 12 h and was maintained at an elevated level (210%) at 24 h after the injection of the vitamin. In contrast, ATP-dependent calcium uptake by BLMV reached a maximum (185% of the control) at 6 h and decreased to the control level at 24 h. The kinetic analysis revealed that 1 alpha,25(OH)2D3 increased Vmax values without any changes in apparent Km values in both BBMV and BLMV. The activity of ATP-dependent calcium uptake was localized exclusively in the basolateral membrane, and the activity was inhibited by vanadate (IC50, 1 microM), but not by oligomycin, theophylline, calmodulin, trifluoperazine, or calbindin D28K. These results indicate that calcium transport through both the brush-border and basolateral membranes is involved in the 1 alpha,25(OH)2D3-dependent intestinal calcium absorption. The initiation of calcium absorption by 1 alpha,25(OH)2D3 appears to be due to an increase in the rate of calcium efflux at the basolateral membrane rather than the rate at the brush-border membrane.

Evidence for carrier-mediated Cl-SO4 exchange in rabbit ileal basolateral membrane vesicles

1987 ◽  
Vol 253 (3) ◽  
pp. G404-G410 ◽  
Author(s):  
C. M. Schron ◽  
R. G. Knickelbein ◽  
P. S. Aronson ◽  
J. W. Dobbins
Keyword(s):  
Anion Exchange ◽  
Brush Border ◽  
Initial Velocity ◽  
Brush Border Membrane ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Basolateral Membrane Vesicles ◽  
Beta Hydroxybutyrate

In rabbit ileal basolateral membrane (BLM) vesicles, an outwardly directed Cl gradient ([Cl] in/out = 60/6 mM) stimulated the initial velocity of SO4 uptake compared with uptake in the absence of Cl. Under Cl gradient conditions, SO4 was transiently accumulated at a concentration twice that found at equilibrium ("overshoot"). Chloride gradient-stimulated SO4 uptake was markedly reduced by inhibitors of anion exchange (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) and was saturable (SO4 Km = 0.302 +/- 0.064 mM; Vmax = 1.59 +/- 0.22 nmol SO4 . mg protein-1 . min-1). SO4 uptake by BLM vesicles was not stimulated by imposition of an inside-positive electrical potential, suggesting that the stimulation by a Cl gradient was not due to an induced electrical potential. Oxalate, nitrate, iodide, and bromide inhibited the initial velocity of Cl gradient-stimulated SO4 uptake, whereas phosphate, beta-hydroxybutyrate, lactate, and p-aminohippurate had no effect. When SO4 uptake by BLM vesicles was compared with that of brush-border membrane vesicles, Cl gradient-stimulated SO4 uptake was found predominantly in the BLM preparation. In conclusion, these findings provide evidence for a carrier on the ileal basolateral membrane that mediates Cl-SO4 exchange.

scholarly journals Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer.

1989 ◽  
Vol 108 (6) ◽  
pp. 2193-2200 ◽  
Author(s):  
J P Gorvel ◽  
Z Mishal ◽  
F Liegey ◽  
A Rigal ◽  
S Maroux
Keyword(s):  
Energy Transfer ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Aminopeptidase N ◽  
Fluorescence Energy Transfer ◽  
Membrane Domains ◽  
Basolateral Membrane Vesicles ◽  
Fluorescence Energy

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.

scholarly journals Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex

1989 ◽  
Vol 264 (1) ◽  
pp. 223-231 ◽  
Author(s):  
T C Williams ◽  
A J Doherty ◽  
D A Griffith ◽  
S M Jarvis
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Facilitated Diffusion ◽  
Basolateral Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Outer Cortex ◽  
Overshoot Phenomenon

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

Urate transport in the proximal tubule: in vivo and vesicle studies

1985 ◽  
Vol 249 (6) ◽  
pp. F789-F798 ◽  
Author(s):  
A. M. Kahn ◽  
E. J. Weinman
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Anion Exchanger ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Transport Processes ◽  
Basolateral Membrane Vesicles ◽  
Urate Transport ◽  
Rat Proximal Tubule

The transport of urate in the mammalian nephron is largely confined to the proximal tubule. Depending on the species, net reabsorption or net secretion is observed. The rat, like the human and the mongrel dog, demonstrates net reabsorption of urate and has been the most extensively studied species. The unidirectional reabsorption and secretion of urate in the rat proximal tubule occur via a passive and presumably paracellular route and by a mediated transcellular route. The reabsorption of urate, and possibly its secretion, can occur against an electrochemical gradient. A variety of drugs and other compounds affect the reabsorption and secretion of urate. The effects of these agents depend on their site of application (luminal or blood), concentration, and occasionally their participation in transport processes that do not have affinity for urate. Recent studies with renal brush border and basolateral membrane vesicles from the rat and brush border vesicles from the dog have determined the mechanisms for urate transport across the luminal and antiluminal membranes of the proximal tubule cell. Brush border membrane vesicles contain an anion exchanger with affinity for urate, hydroxyl ion, bicarbonate, chloride, lactate, p-aminohippurate (PAH), and a variety of other organic anions. Basolateral membrane vesicles contain an anion exchanger with affinity for urate and chloride but not for PAH. Both membrane vesicle preparations also permit urate translocation by simple diffusion. A model for the transcellular reabsorption and secretion of urate in the rat proximal tubule is proposed. This model is based on the vesicle studies, and it can potentially explain the majority of urate transport data obtained with in vivo techniques.

Folate transport by human intestinal brush-border membrane vesicles

1987 ◽  
Vol 252 (2) ◽  
pp. G229-G236 ◽  
Author(s):  
H. M. Said ◽  
F. K. Ghishan ◽  
R. Redha
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Folate Transport ◽  
Intestinal Brush Border Membrane ◽  
Transport Carrier ◽  
Incubation Buffer

Transport of folic acid (Pte-Glu) across the brush-border membrane of human intestine was studied using brush-border membrane vesicle (BBMV) technique. The transport of Pte-Glu was higher in BBMV prepared from the jejunum than those prepared from the ileum (0.70 +/- 0.05 and 0.14 +/- 0.02 pmol X mg protein-1 X 10 s-1, respectively). The transport of Pte-Glu appeared to be carrier mediated and was pH dependent and increased with decreasing incubation buffer pH; saturable (Kt = 1.69 microM, Vmax = 4.72 pmol X mg protein-1 X 10 s-1); inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate, methotrexate, and 5-formyltetrahydrofolate (Ki = 2.2, 1.4 and 1.4 microM, respectively); not affected by inducing a relatively positive or negative intravesicular compartment; independent of Na+ gradient; and inhibited by 4,4'-diisothiocyanatostlibene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor. The increase in Pte-Glu transport on decreasing incubation buffer pH appeared to be in part mediated through a direct effect of acidic pH on the transport carrier and in part through the pH gradient imposed by activating Pte-Glu-:OH- exchange and/or Pte-Glu-:H+ co-transport mechanisms. The important role of an acidic extravesicular environment in Pte-Glu transport is consistent with a role for the intestinal surface acid microclimate in folate transport. These results demonstrate that Pte-Glu transport in human BBMV occurs by a carrier-mediated system that is similar to that described for rat and rabbit intestinal BBMV.

Na+-independent D-glucose transport in rabbit renal basolateral membranes

1988 ◽  
Vol 254 (5) ◽  
pp. F711-F718 ◽  
Author(s):  
P. T. Cheung ◽  
M. R. Hammerman
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Brush Border ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Rabbit Kidney ◽  
Basolateral Membrane Vesicles ◽  
Proximal Tubular

To define the mechanism by which glucose is transported across the basolateral membrane of the renal proximal tubular cell, we measured D-[14C]glucose uptake in basolateral membrane vesicles from rabbit kidney. Na+-dependent D-glucose transport, demonstrable in brush-border vesicles, could not be demonstrated in basolateral membrane vesicles. In the absence of Na+, the uptake of D-[14C]glucose in basolateral vesicles was more rapid than that of L-[3H]glucose over a concentration range of 1-50 mM. Subtraction of the latter from the former uptakes revealed a saturable process with apparent Km of 9.9 mM and Vmax of 0.80 nmol.mg protein-1.s-1. To characterize the transport component of D-glucose uptake in basolateral vesicles, we measured trans stimulation of 2 mM D-[14C]glucose entry in the absence of Na+. Trans stimulation could be effected by preloading basolateral vesicles with D-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose, but not with L-glucose or alpha-methyl-D-glucoside. Trans-stimulated D-[14C]glucose uptake was inhibited by 0.1 mM phloretin or cytochalasin B but not phlorizin. In contrast, Na+-dependent D-[14C]glucose transport in brush-border vesicles was inhibited by phlorizin but not phloretin or cytochalasin B. Our findings are consistent with the presence of a Na+-independent D-glucose transporter in the proximal tubular basolateral membrane with characteristics similar to those of transporters present in nonepithelial cells.

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