Radiation inactivation studies of renal brush border water and urea transport

1985 ◽  
Vol 249 (6) ◽  
pp. F806-F812
Author(s):  
A. S. Verkman ◽  
J. A. Dix ◽  
J. L. Seifter ◽  
K. L. Skorecki ◽  
C. Y. Jung ◽  
...  
Keyword(s):  
Radiation Dose ◽  
Water Transport ◽  
Brush Border ◽  
Membrane Vesicles ◽  
Target Size ◽  
Freezing Process ◽  
Urea Transport ◽  
Transport Pathways ◽  
Radiation Inactivation ◽  
Osmotic Water

Radiation inactivation was used to determine the nature and molecular weight of water and urea transport pathways in brush border membrane vesicles (BBMV) isolated from rabbit renal cortex. BBMV were frozen to -50 degrees C, irradiated with 1.5 MeV electrons, thawed, and assayed for transport or enzyme activity. The freezing process had no effect on enzyme or transport kinetics. BBMV alkaline phosphatase activity gave linear ln(activity) vs. radiation dose plots with a target size of 68 +/- 3 kDa, similar to previously reported values. Water and solute transport were measured using the stopped-flow light-scattering technique. The rates of acetamide and osmotic water transport did not depend on radiation dose (0-7 Mrad), suggesting that transport of these substances does not require a protein carrier. In contrast, urea and thiourea transport gave linear ln(activity) vs. dose curves with a target size of 125-150 kDa; 400 mM urea inhibited thiourea flux by -50% at 0 and 4.7 Mrad, showing that radiation does not affect inhibitor binding to surviving transporters. These studies suggest that BBMV urea transport requires a membrane protein, whereas osmotic water transport does not.

Ca2+ transport pathways in brush-border membrane vesicles of crustacean antennal glands

1993 ◽  
Vol 264 (6) ◽  
pp. R1206-R1213 ◽  
Author(s):  
G. A. Ahearn ◽  
P. Franco
Keyword(s):  
Membrane Potential ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Carrier System ◽  
Transport Pathways ◽  
45Ca Uptake ◽  
Negative Membrane Potential ◽  
Antennal Glands

Calcium uptake by brush-border membrane vesicles of Atlantic lobster (Homarus americanus) kidneys (antennal glands) in independent experiments was stimulated by outwardly directed Na or H gradients. In the absence of external amiloride, 45Ca uptake was strongly stimulated by an outwardly directed Na gradient, and this stimulation was enhanced by the addition of an inside-negative membrane potential. External amiloride (2 mM) reduced 45Ca uptake sixfold and lowered sensitivity to membrane potential. 45Ca influx kinetics (2.5-s uptake) in the presence of an outwardly directed H gradient and inside-negative membrane potential were composed of three components: 1) an amiloride-sensitive carrier system, 2) an amiloride-insensitive carrier system, and 3) a verapamil- and membrane potential-sensitive process that may represent diffusional transfer through a calcium channel. It was concluded that 45Ca entry by the amiloride-sensitive process occurred by a previously described electrogenic 2 Na-1 H antiport mechanism [Ahearn, G., and L. Clay. Am. J. Physiol. 257 (Regulatory Integrative Comp. Physiol. 26): R484-R493, 1989; Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F758-F767, 1990; Ahearn, G., P. Franco, and L. Clay. J. Membr. Biol. 116: 215-226, 1990]. 45Ca influx by the amiloride-insensitive mechanism occurred by an apparent electroneutral 1 Ca-2 Na exchange. Transport stoichiometry of the latter mechanism was tentatively established by experiments determining intravesicular Na binding properties and by its apparent lack of response to a membrane potential. At physiological Na, Ca, and H concentrations in the antennal gland lumen and epithelial cytosol, these three calcium transport pathways individually may make significant contributions to net calcium reabsorption to the blood.

Facilitated diffusion of urate in avian brush-border membrane vesicles

2002 ◽  
Vol 283 (4) ◽  
pp. C1155-C1162 ◽  
Author(s):  
Steven M. Grassl
Keyword(s):  
Membrane Potential ◽  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Facilitated Diffusion ◽  
Transport Pathways ◽  
Proximal Tubule Cells ◽  
Avian Kidney

Membrane transport pathways mediating transcellular secretion of urate across the proximal tubule were investigated in brush-border membrane vesicles (BBMV) isolated from avian kidney. An inside-positive K diffusion potential induced a conductive uptake of urate to levels exceeding equilibrium. Protonophore-induced dissipation of membrane potential significantly reduced voltage-driven urate uptake. Conductive uptake of urate was inhibitor sensitive, substrate specific, and a saturable function of urate concentration. Urate uptake was trans-stimulated by urate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate. Urate uptake was unaffected by an outward α-ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in the presence and absence of an imposed inside-alkaline pH gradient or an outward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux across the brush border membrane of urate-secreting proximal tubule cells.

Betaine transport in rabbit renal brush-border membrane vesicles

1993 ◽  
Vol 264 (6) ◽  
pp. F948-F955 ◽  
Author(s):  
T. M. Wunz ◽  
S. H. Wright
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Renal Cortex ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Maximal Rate ◽  
Transport Pathways ◽  
Organic Osmolyte ◽  
Renal Brush Border Membrane ◽  
Renal Brush Border

Transport of the organic osmolyte betaine was characterized in brush-border membrane vesicles (BBMV) isolated from rabbit renal cortex. Inwardly directed gradients of either Na+ or H+ supported concentrative uptake in a manner consistent with the presence of parallel Na(+)-betaine and H(+)-betaine cotransport processes. Concentrative uptake occurred in the presence of membrane potential alone, indicating that betaine transport is electrogenic. Accumulation of betaine was not dependent on chloride in the medium. Whereas L-proline inhibited both the H(+)- and Na(+)-sensitive components of betaine transport, glycine blocked the H(+)-sensitive pathway and had little effect on Na(+)-sensitive betaine transport. Both pathways were adequately described by Michaelis-Menten kinetics. Under Na(+)-gradient conditions (pH equilibrium), the maximal rate of total betaine transport (Jmax) = 50.8 +/- 13.3 nmol.mg-1.min-1 and the concentration of total betaine producing half-maximal uptake (Kt) = 4.1 +/- 0.5 mM. Under H(+)-gradient conditions (Na+ free), Jmax = 102.5 +/- 10.5 nmol.mg-1.min-1 and Kt = 2.8 +/- 0.3 mM. Imposition of both Na+ and H+ gradients increased Jmax (142 +/- 25.5 nmol.mg-1.min-1) to a level significantly greater than that noted in the presence of a Na+ gradient alone. We conclude that betaine transport in renal BBMV involves two distinct transport pathways that are differentiated on the basis of sensitivity to either Na+ or H+ and by their specificity to proline and glycine.

Inorganic anion transport in kidney and intestinal brush border and basolateral membranes

1980 ◽  
Vol 238 (6) ◽  
pp. F452-F460 ◽  
Author(s):  
S. Grinstein ◽  
R. J. Turner ◽  
M. Silverman ◽  
A. Rothstein
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Anion Transport ◽  
Significant Inhibition ◽  
Kidney Cortex ◽  
Basolateral Membrane Vesicles ◽  
Inorganic Anion ◽  
Transport Pathways ◽  
Basolateral Membranes

The efflux of inorganic anions from purified brush border and basolateral membrane vesicles from dog kidney cortex was measured under equilibrium exchange conditions. Marked differences in temperature sensitivity and effects of inhibitors were found between the Cl and SO4 transport pathways and between the two types of membranes. SO4 transport in both brush border and basolateral membranes was markedly reduced by cooling, but significant inhibition by 4,4'–diisothiocyano-2,2'–disulfonic stilbene (DIDS) was only observed in basolateral vesicles. In contrast, Cl efflux from both types of vesicles was neither substantially inhibited by DIDS nor by lowering the temperature to 0 degrees C. Phosphate efflux from basolateral membrane vesicles was found to be only partially sensitive to DIDS. Attempts to label the stilbene-sensitive SO4 pathway in basolateral vesicles using [3H2]DIDS as a marker were unsuccessful due to the nonspecific labeling of many membrane components. The asymmetry in inorganic anion transport behavior exhibited by brush border and basolateral membrane vesicles from dog renal proximal tubule was also observed in equivalent vesicles prepared from rat small intestine.

scholarly journals Radiation-inactivation analysis of the Na+/bile acid co-transport system from rabbit ileum

1995 ◽  
Vol 306 (1) ◽  
pp. 241-246 ◽  
Author(s):  
W Kramer ◽  
F Girbig ◽  
U Gutjahr ◽  
S Kowalewski
Keyword(s):  
Bile Acid ◽  
Transport System ◽  
Brush Border ◽  
Binding Proteins ◽  
Brush Border Membrane ◽  
High Energy ◽  
Target Size ◽  
Rabbit Ileum ◽  
Radiation Inactivation ◽  
Bile Acid Binding

The functional-unit molecular size of the Na+/bile acid cotransport system and the apparent target size of the bile-acid-binding proteins in brush-border membrane vesicles from rabbit ileum were determined by radiation inactivation with high-energy electrons. The size of the functional transporting unit for Na(+)-dependent taurocholate uptake was determined to 451 +/- 35 kDa, whereas an apparent molecular mass of 434 +/- 39 kDa was measured for the Na(+)-dependent D-glucose transport system. Proteins of 93 kDa and 14 kDa were identified as putative protein components of the ileal Na+/bile acid cotransporter in the rabbit ileum, whereas a protein of 87 kDa may be involved in passive intestinal bile acid uptake. Photoaffinity labelling with 3- and 7-azi-derivatives of taurocholate revealed a target size of 229 +/- 10 kDa for the 93 kDa protein, and 132 +/- 23 kDa for the 14 kDa protein. These findings indicate that the ileal Na+/bile acid co-transport system is in its functional state a protein complex composed of several subunits. The functional molecular sizes for Na(+)-dependent transport activity and the bile-acid-binding proteins suggest that the Na+/bile acid co-transporter from rabbit ileum is a homotetramer (AB)4 composed of four AB subunits, where A represents the integral 93 kDa and B the peripheral 14 kDa brush-border membrane protein.

The Mechanism of Human Placental Urea Transport: A Study Using Placental Brush Border (Microvillous) Membrane Vesicles

2010 ◽  
Vol 17 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Hiroyasu Hisanaga ◽  
Hideaki Iioka ◽  
Ikuko Moriyama ◽  
Kaoru Nabuchi ◽  
Keiko Morimoto ◽  
...  
Keyword(s):  
Brush Border ◽  
Membrane Vesicles ◽  
Urea Transport ◽  
Microvillous Membrane Vesicles

scholarly journals Radiation-inactivation studies on brush-border-membrane vesicles. General considerations, and application to the glucose and phosphate carriers

1988 ◽  
Vol 252 (3) ◽  
pp. 807-813 ◽  
Author(s):  
R Béliveau ◽  
M Demeule ◽  
H Ibnoul-Khatib ◽  
M Bergeron ◽  
G Beauregard ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Dose Range ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Vesicles ◽  
Molecular Size ◽  
Brush Border Membrane Vesicles ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Radiation Inactivation

Radiation-inactivation studies were performed on brush-border-membrane vesicles purified from rat kidney cortex. No alteration of the structural integrity of the vesicles was apparent in electron micrographs of irradiated and unirradiated vesicles. The size distributions of the vesicles were also similar for both populations. The molecular sizes of two-brush-border-membrane enzymes, alkaline phosphatase and 5′-nucleotidase, estimated by the radiation-inactivation technique, were 104800 +/- 3500 and 89,400 +/- 1800 Da respectively. Polyacrylamide-gel-electrophoresis patterns of membrane proteins remained unaltered by the radiation treatment, except in the region of higher-molecular-mass proteins, where destruction of the proteins was visible. The molecular size of two of these proteins was estimated from their mobilities in polyacrylamide gels and was similar to the target size, estimated from densitometric scanning of the gel. Intravesicular volume, estimated by the uptake of D-glucose at equilibrium, was unaffected by irradiation. Uptake of Na+, D-glucose and phosphate were measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Na+-independent D-glucose and phosphate uptakes were totally unaffected in the dose range used (0-9 Mrad). The Na+-dependent uptake of D-glucose was studied in irradiated vesicles, and the molecular size of the transporter was found to be 288,000 Da. The size of the Na+-dependent phosphate carrier was also estimated, and a value of 234,000 Da was obtained.

Effects of membrane fluidizing agents on renal brush border proton permeability

1985 ◽  
Vol 249 (6) ◽  
pp. F933-F940 ◽  
Author(s):  
H. E. Ives ◽  
A. S. Verkman
Keyword(s):  
Water Transport ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
Dependent Manner ◽  
Transport Pathway ◽  
Ph Gradients ◽  
Orange Fluorescence ◽  
Dose Dependent

H+ permeability (PH) of brush border membrane vesicles isolated from rabbit renal cortex was measured from the rate of collapse of preformed pH gradients using acridine orange fluorescence quenching. n-Alkanols increased PH from 0.005 to 0.1 cm/s in a dose-dependent manner. At 25 degrees C, PH increased to 0.01 cm/s at [n-alkanol] = 90 mM (butanol), 30 mM (pentanol), 7 mM (hexanol), and 1.8 mM (heptanol). Activation energy (Ea) of PH was 21.6 kcal/mol (5-50 degrees C), which decreased to 18.5 kcal/mol in the presence of either 200 mM butanol or 12 mM hexanol. Membrane fluidity was estimated from diphenylhexatriene anisotropy (r). n-Alkanols decreased r from 0.25 to 0.18 in a dose-dependent manner. At 25 degrees C, r = 0.22 at [n-alkanol] = 200 mM (butanol), 27 mM (pentanol), 9.5 mM (hexanol), and 2 mM (heptanol). The effects of n-alkanols on PH and r correlated well with known n-alkanol lipid-water partition coefficients. Similar increases in PH and decreases in r were observed for nonalkanol lipid anesthetics. The effects of n-alkanols on the Na+-H+ antiporter and on osmotically driven water transport were also studied. At concentrations of n-alkanol that resulted in a 10-fold increase in PH, there was no significant effect on either Na+-H+ exchange or water transport. These results suggest a lipid pathway for brush border H+ diffusion that is distinct from both the Na+-H+ antiporter and the water transport pathway.

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