Sodium-dependent nucleoside transport in the human intestinal brush-border membrane

The objective of the study was to determine the identity and kinetic characteristics of nucleoside transporters present in the brush-border membrane of the human jejunum. With use of brush-border membrane vesicles, uptake of [3H]uridine was stimulated two- to threefold by an inwardly directed Na+ gradient and was inhibited by both 100 microM thymidine and 100 microM guanosine nucleosides, which serve as model substrates for purine (N1, cif) and pyrimidine (N2, cit) transporters, respectively. [3H]thymidine and [3H]guanosine transport exhibited an overshoot phenomenon only in the presence of a Na+ gradient. Na(+)-thymidine uptake was inhibited by 100 microM cytidine or thymidine but not by guanosine, inosine, formycin B, or hypoxanthine. [3H]guanosine uptake was inhibited by 100 microM inosine, guanosine, or formycin B but not by thymidine or cytidine. Both adenosine and uridine inhibited uptake of [3H]thymidine and [3H]guanosine to a similar extent, indicating that both N1, cif and N2, cit Na(+)-nucleoside transporters are expressed in human jejunum. Enhanced uptake of Na(+)-thymidine by an inside-negative potential difference generated by K+ and valinomycin provides evidence that nucleoside transport is rheogenic, involving net transfer of a positive charge. The Hill coefficient was unity for all three substrates, indicating a Na(+)-nucleoside coupling stoichiometry of 1:1. At saturating Na+ concentration (150 mM) the kinetic parameters (n = 3-4) Michaelis-Menten constant and maximum velocity for uridine, thymidine, and guanosine uptake were 4.15 +/- 1.79, 2.74 +/- 0.58, 12.02 +/- 1.34 microM and 25.93 +/- 7.38, 16.10 +/- 3.64, 63.92 +/- 10.23 pmol.mg-1.10 s-1, respectively. These results suggest that, in contrast to the human kidney that expresses the N4 nucleoside transporter, the human jejunum expresses both N1 and N2 Na(+)-nucleoside transporters.

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Na-D-glucose cotransport in renal brush-border membrane vesicles of an early teleost (Oncorhynchus mykiss)

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1995.269.3.r592 ◽

1995 ◽

Vol 269 (3) ◽

pp. R592-R602 ◽

Cited By ~ 2

Author(s):

C. A. Freire ◽

E. Kinne-Saffran ◽

K. W. Beyenbach ◽

R. K. Kinne

Keyword(s):

Glucose Uptake ◽

Brush Border ◽

Brush Border Membrane ◽

Maximum Velocity ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Hill Coefficient ◽

Gamma Glutamyl Transpeptidase ◽

Renal Brush Border Membrane ◽

Voltage Dependent

Brush-border membrane vesicles (BBMV) enriched with alkaline phosphatase (8.1-fold) and gamma-glutamyl transpeptidase (11.5-fold) were prepared from the rainbow trout kidney. D-[3H]glucose uptake was stimulated by inward Na gradients but not by K, choline, Li, N-methyl-D-glucamine, or mannitol gradients. Na-dependent glucose uptake displayed overshoot in voltage-polarized vesicles (VPV; negative inside) but not in short-circuited vesicles (SCV). Recognition of carbons 2 and 3 of the glucopyranose ring was essential for glucose uptake. Phlorizin inhibited Na-dependent D-glucose uptake with an inhibition constant of 11.4 microM. The Michaelis-Menten constant of glucose was 0.58 mM in VPV and increased to 1.49 mM in SCV, whereas that for sodium was 193 mM in VPV and similar in SCV. Maximum velocity of Na was reduced in SCV. The Hill coefficient was 1 for both Na and glucose in VPV and SCV. Our studies indicate a single Na-D-glucose cotransporter that transports Na and glucose with a 1:1 stoichiometry and voltage-dependent kinetics. The transporter shares functional properties with both mammalian transporters SGLT1 and SGLT2.

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Multiple sodium-dependent nucleoside transport systems in bovine renal brush-border membrane vesicles

Biochemical Journal ◽

10.1042/bj2740027 ◽

1991 ◽

Vol 274 (1) ◽

pp. 27-33 ◽

Cited By ~ 66

Author(s):

T C Williams ◽

S M Jarvis

Keyword(s):

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Nucleoside Transport ◽

Transport Systems ◽

Facilitated Diffusion ◽

Nucleoside Transporter ◽

Renal Brush Border Membrane ◽

Renal Brush Border

Na(+)-dependent nucleoside transport was examined in bovine renal brush-border membrane vesicles. Two separate Na+/nucleoside cotransporters were shown to be present: (1) a system specific for purine nucleosides and uridine, designated as the N1 carrier, and (2) an Na(+)-dependent nucleoside transporter that accepts pyrimidine nucleosides, adenosine and analogues of adenosine, designated as the N2 system. Both systems exhibit a high affinity for nucleosides (apparent Km values approximately 10 microM), are insensitive to inhibition by facilitated-diffusion nucleoside transport inhibitors, are rheogenic and exhibit a high specificity for Na+. Na+ increases the affinity of the influx of guanosine and thymidine, nucleosides that serve as model permeants for the N1 and N2 nucleoside transporters respectively. The Na+/nucleoside coupling stoichiometry is consistent with 1:1 for both carriers.

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Nucleoside transport in brush border membrane vesicles from human kidney

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(92)90156-g ◽

1992 ◽

Vol 1105 (1) ◽

pp. 1-9 ◽

Cited By ~ 47

Author(s):

Marcelo M. Gutierrez ◽

Claire M. Brett ◽

Ronda J. Ott ◽

Andrew C. Hui ◽

Kathleen M. Giacomini

Keyword(s):

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Human Kidney ◽

Brush Border Membrane Vesicles ◽

Nucleoside Transport

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Substrate selectivity, potential sensitivity and stoichiometry of Na+-nucleoside transport in brush border membrane vesicles from human kidney

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(93)90202-b ◽

1993 ◽

Vol 1149 (2) ◽

pp. 202-208 ◽

Cited By ~ 49

Author(s):

Marcelo M. Gutierrez ◽

Kathleen M. Giacomini

Keyword(s):

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Human Kidney ◽

Brush Border Membrane Vesicles ◽

Nucleoside Transport ◽

Substrate Selectivity ◽

Potential Sensitivity

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Electroneutral Na+/H+exchange in brush-border membrane vesicles fromPenaeus japonicus hepatopancreas

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1998.274.2.r486 ◽

1998 ◽

Vol 274 (2) ◽

pp. R486-R493 ◽

Cited By ~ 1

Author(s):

Sebastiano Vilella ◽

Vincenzo Zonno ◽

Laura Ingrosso ◽

Tiziano Verri ◽

Carlo Storelli

Keyword(s):

Kinetic Parameters ◽

Brush Border ◽

Brush Border Membrane ◽

Electrical Potential ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Hill Coefficient ◽

Uptake Kinetic ◽

Ph Dependent ◽

Transmembrane Electrical Potential

An electroneutral Na+/H+exchange mechanism (dimethylamiloride inhibitable, Li+ sensitive, and Ca2+ insensitive) was identified in brush-border membrane vesicles (BBMV) from Kuruma prawn hepatopancreas by monitoring Na+-dependent H+ fluxes with the pH-sensitive dye acridine orange and measuring22Na+uptake. Kinetic parameters measured under short-circuited conditions were the Na+ concentration that yielded one-half of the maximal dissipation rate ( F max) of the preset transmembrane ΔpH ( K Na) = 15 ± 2 mM and F max = 3,626 ± 197 Δ F ⋅ min−1 ⋅ mg protein−1, with a Hill coefficient for Na+ of ∼1. In addition, the inhibitory constant for dimethylamiloride was found to be ∼1 μM. The electroneutral nature of the antiporter was assessed in that an inside-negative transmembrane electrical potential neither affected kinetic parameters nor stimulated pH-dependent (intracellular pH > extracellular pH)22Na+uptake. In contrast, electrogenic pH-dependent22Na+uptake was observed in lobster hepatopancreatic BBMV. Substitution of chloride with gluconate resulted in increasing K Na and decreasing Δ F max, which suggests a possible role of chloride in the operational mechanism of the antiporter. These results indicate that a Na+/H+exchanger, resembling the electroneutral Na+/H+antiporter model, is present in hepatopancreatic BBMV from the Kuruma prawn Penaeus japonicus.

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Lysine transport by brush-border membrane vesicles of eel intestine: interaction with neutral amino acids

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1990.259.6.r1181 ◽

1990 ◽

Vol 259 (6) ◽

pp. R1181-R1188 ◽

Cited By ~ 2

Author(s):

S. Vilella ◽

G. A. Ahearn ◽

G. Cassano ◽

M. Maffia ◽

C. Storelli

Keyword(s):

Amino Acids ◽

Transport System ◽

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Hill Coefficient ◽

European Eel ◽

Diffusional Permeability ◽

Wide Range

L-[3H]lysine uptake was measured in brush-border membrane vesicles prepared from intestinal mucosa of the European eel Anguilla anguilla. Lysine uptake occurred via 1) a nonsaturable component with an apparent diffusional permeability (P) of 0.58 microliter.mg protein-1.min-1,2) a Na-dependent transport system [half-saturation constant (Kapp) 0.16 mM, maximal transport rate (Jmax) 3.57 nmol.mg protein-1.min-1]; 3) a Na-independent transport system (Kapp 0.17 mM, Jmax 2.77 nmol.mg protein-1.min-1). Both carrier-mediated processes were accelerated by the presence of an intravesicular negative membrane potential. Hill analysis of L-lysine influx, over a wide range of external Na concentrations, resulted in a Hill coefficient (n) of approximately 2, suggesting that two or more Na ions may be associated with amino acid transport. The inhibition of lysine uptake by other amino acids was studied. Na-dependent lysine uptake was competitively inhibited by proline [inhibitory constant (Ki) approximately 2 mM] and may occur by a system specific for cationic amino acids. Na-independent lysine uptake was competitively inhibited by alanine (Ki approximately 1 mM) and may occur by a classic L system.

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Carrier-mediated mechanism for biotin transport in rabbit intestine: studies with brush-border membrane vesicles

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1991.261.1.r94 ◽

1991 ◽

Vol 261 (1) ◽

pp. R94-R97 ◽

Cited By ~ 3

Author(s):

H. M. Said ◽

I. Derweesh

Keyword(s):

Brush Border ◽

Concentration Gradient ◽

Brush Border Membrane ◽

Maximum Velocity ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Experimental Conditions ◽

Simple Diffusion ◽

Rabbit Intestine ◽

High Concentrations

Simple diffusion has been reported as the mechanism of biotin transport in rabbit intestine. In this study, we reevaluated this concept by examining biotin transport in rabbit intestine using optimal experimental conditions and a well-established brush-border membrane vesicles (BBMV) technique. Uptake of biotin by rabbit intestinal BBMV was found by an osmolarity study to be mostly the result of transport of the vitamin into an osmotically sensitive intravesicular space with little binding to membrane surfaces. Biotin transport in rabbit intestinal BBMV was 1) Na+ gradient dependent (out greater than in) with a clear “overshoot” phenomenon, indicating the accumulation of the substrate against a concentration gradient; 2) initial rate of biotin transport by the Na+ gradient-dependent component was saturable as a function of substrate concentration with apparent Km and maximum velocity (Vmax) values of 6.7 microM and 10.7 pmol.mg protein-1 x 10 s-1, respectively; 3) inhibited by high concentrations of unlabeled biotin and its related compounds desthiobiotin and thioctic acid in the presence, but not absence, of a Na+ gradient; and 4) not affected by inducing a relatively positive or negative intravesicular space with the use of valinomycin-induced K+ diffusion potential. These findings indicate that the biotin transport mechanism in rabbit intestine is carrier mediated in nature. Furthermore, this mechanism is Na+ gradient dependent, capable of accumulating the substrate against a concentration gradient and transport the vitamin via an electroneutral process.

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Characterization of a sodium-dependent concentrative nucleobase-transport system in guinea-pig kidney cortex brush-border membrane vesicles

Biochemical Journal ◽

10.1042/bj3030901 ◽

1994 ◽

Vol 303 (3) ◽

pp. 901-905 ◽

Cited By ~ 17

Author(s):

D A Griffith ◽

S M Jarvis

Keyword(s):

Guinea Pig ◽

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Hill Coefficient ◽

Kidney Cortex ◽

Pig Kidney ◽

Na Ions

The characteristics of hypoxanthine transport were examined in purified brush-border membrane vesicles isolated from guinea-pig kidney. Hypoxanthine uptake in the vesicles was specifically stimulated by both Na+ and an inside-negative potential, resulting in a transient accumulation of intravesicular hypoxanthine. Na(+)-dependent hypoxanthine influx was saturable (apparent Km 4.4 +/- 2.1 microM, Vmax. 128 +/- 29 pmol/min per mg of protein at 100 mM NaCl and 22 degrees C). Guanine, thymine, 5-fluorouracil and uracil inhibited hypoxanthine uptake (Ki values 1-30 microM), but adenine and the nucleosides inosine and thymidine were without effect. Guanine competitively inhibited Na(+)-dependent hypoxanthine influx, suggesting that it was a substrate for the active nucleobase transporter in guinea-pig renal membrane vesicles. A sigmoidal dependence between hypoxanthine influx and Na+ concentration was obtained (KNa 13 +/- 2 mM; Hill coefficient, h, 2.13 +/- 0.14), suggesting that at least two Na+ ions are transported per hypoxanthine molecule. This system differs from the Na(+)-nucleobase carrier in cultured LLC-PK1 renal cells, which has a stoichiometric coupling ratio of 1:1. These results represent the first demonstration of an active electrogenic nucleobase carrier in renal apical membrane vesicles.

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