Sulfate/oxalate exchange by lobster hepatopancreatic basolateral membrane vesicles

1995 ◽  
Vol 269 (3) ◽  
pp. R572-R577 ◽  
Author(s):  
G. A. Gerencser ◽  
M. A. Cattey ◽  
G. A. Ahearn
Keyword(s):  
Gradient Centrifugation ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Basolateral Membrane Vesicles ◽  
Sulfate Uptake ◽  
Discontinuous Sucrose Gradient ◽  
Carboxylic Anions

Purified basolateral membrane vesicles (BLMV) were prepared from lobster hepatopancreas by osmotic disruption and discontinuous sucrose gradient centrifugation. Radiolabeled sulfate uptake was stimulated by 10 mM intravesicular oxalate compared with gluconate-loaded vesicles. Sulfate/oxalate exchange was not affected by transmembrane valinomycin-induced potassium diffusion potentials (inside negative or inside positive), suggesting electroneutral anion transport. Sulfate uptake was not stimulated by the similar carboxylic anions formate, succinate, oxaloacetate, or ketoglutarate. Sulfate influx occurred by at least one saturable Michaelis-Menten carrier system [apparent Km = 6.0 +/- 1.7 mM; maximum flux (Jmax) = 382.3 +/- 37.0 pmol.mg protein-1 x 7 s-1]. Sulfate/oxalate exchange was significantly reduced by the anion antiport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but was not affected by bumetanide or furosemide. The possible physiological role of this exchange mechanism in anion/sulfate transport across the crustacean hepatopancreas is discussed.

Proton-stimulated Cl-HCO3 antiport by basolateral membrane vesicles of lobster hepatopancreas

1987 ◽  
Vol 252 (5) ◽  
pp. R859-R870 ◽  
Author(s):  
G. A. Ahearn ◽  
M. L. Grover ◽  
R. T. Tsuji ◽  
L. P. Clay
Keyword(s):  
Gradient Centrifugation ◽  
Exchange Process ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Proton Gradient ◽  
Disulfonic Acid ◽  
Tissue Homogenate ◽  
Hyperbolic Function ◽  
Basolateral Membrane Vesicles ◽  
Apparent Diffusion

Purified epithelial basolateral membrane vesicles were prepared from lobster hepatopancreas by sorbitol gradient centrifugation. Na+-K+-adenosinetriphosphatase, alkaline phosphatase, and cytochrome-c oxidase enzyme activities in the final membrane preparation were enriched 9.6-, 1.4-, and 0.4-fold, respectively, compared with their activities in the original tissue homogenate. Vesicle osmotic reactivity was demonstrated using 60-min equilibrium 36Cl uptake experiments at a variety of transmembrane osmotic gradients. 36Cl uptake into vesicles preloaded with HCO3 was significantly greater than into vesicles lacking HCO3. This exchange process was stimulated by a transmembrane proton gradient (internal pH greater than external pH). Proton-gradient-dependent Cl-HCO3 exchange was potential sensitive and stimulated by an electrically negative vesicle interior. 36Cl influx (4-s exposures) into HCO3-loaded vesicles occurred by the combination of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid sensitive, carrier-mediated transfer and "apparent diffusion." 36Cl influx was a hyperbolic function of both internal [HCO3] and internal [Cl]. The two internal anions displayed a 100-fold difference in apparent affinity constants with HCO3 being strongly preferred. 36Cl influx was stimulated more by preloaded monovalent than by divalent anions. Na was an inhibitor of proton-dependent anion antiport, whereas K had no effect. A model for HCl-HCO3 antiport is suggested that employs combined transmembrane concentration gradients of Cl and HCO3 to power anion exchange and transfer protons against a concentration gradient.

Influence of vascular and luminal hexoses on rat intestinal basolateral glucose transport

1994 ◽  
Vol 72 (4) ◽  
pp. 317-326 ◽  
Author(s):  
Raymond Tsang ◽  
Ziliang Ao ◽  
Chris Cheeseman
Keyword(s):  
Glucose Transport ◽  
Plasma Glucose ◽  
Intestinal Adaptation ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Intestinal Transport ◽  
Membrane Vesicles ◽  
Luminal Perfusion

The influence of luminal and vascular hexoses in rats on glucose transport across the jejunal basolateral membrane (BLM) was measured using isolated membrane vesicles prepared from infused animals. In vivo vascular infusions of glucose produced an increase in glucose transport across BLM vesicles. Sucrose, mannose, galactose, and fructose had no significant effect. Plasma glucose concentrations were unaffected by galactose and sucrose vascular infusions, while mannose and fructose produced a modest rise, and glucose increased plasma glucose to 20 mM. Insulin release was significantly increased by vascular infusion of glucose and fructose, while mannose produced only a small sustained rise. Sucrose and galactose had no effect. Perfusion through the lumen of the rat jejunum in vivo, for up to 4 h, with glucose, fructose, sucrose, or lactate (100 or 25 mM) produced a significant increase in the maximal rate of glucose transport (up to 4- to 5-fold) across BLMs. Galactose and mannose had no effect. Luminal glucose perfusion produced a small nonsignificant increase in glucose inhibitable cytochalasin B binding to BLM vesicles, and no change was seen in the microsomal pool of binding sites. The abundance of GLUT2 in the jejunal BLM, as determined by Western blotting, was unaffected by luminal perfusion of 100 mM glucose for 4 h. Fructose almost completely inhibited the carrier-mediated uptake of glucose in control and upregulated jejunal BLM vesicles. These results are discussed in relation to the physiological role of the upregulation of GLUT2 activity by luminal and vascular hexoses.Key words: intestinal transport, basolateral membrane, glucose transport, intestinal adaptation.

Isolation, visualization, characterization, and osmotic reactivity of crayfish BLMV

1998 ◽  
Vol 274 (3) ◽  
pp. R725-R734
Author(s):  
Michele G. Wheatly ◽  
Jennifer R. Weil ◽  
Phyllis B. Douglas
Keyword(s):  
Procambarus Clarkii ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Freshwater Crayfish ◽  
Basolateral Membrane Vesicles ◽  
Marine Crustaceans ◽  
Antennal Gland ◽  
Discontinuous Sucrose Gradient ◽  
Uptake Studies ◽  
Inside Out

Procedures were developed to isolate basolateral membrane vesicles (BLMV) from gill, hepatopancreas, and antennal gland of intermolt freshwater crayfish, Procambarus clarkii. Individual procedures involved a discontinuous sucrose gradient (gill), a 65% sucrose cushion (hepatopancreas), or differential centrifugation (antennal gland). BLMV were visualized, characterized (37°C), and tested for osmotic reactivity with a view to using them for Ca2+ uptake studies. Mean diameters of BLMV were 159 nm (gill), 363 nm (hepatopancreas), and 226 nm (antennal gland). Enrichments of basolateral membranes and mitochondria in BLMV were, respectively, 18- and 1.7-fold for gill, 9- and 0.4-fold for hepatopancreas, and 10- and 1-fold for antennal gland. Apical contamination was negligible in BLMV. Percentages of resealing of vesicles as inside out, right side out, or leaky/sheets were 17:27:56% (gill), 14:26:60% (hepatopancreas), and 21:39:40% (antennal gland). Vesicles exhibited osmotic reactivity, as indicated by a linear relationship between vesicular45Ca2+uptake and osmolality. Nonspecific45Ca2+binding was 20% in gill, 39% in hepatopancreas, and 31% in antennal gland. Data were compared with published values for marine crustaceans.

Sulfate transport by chick renal tubule brush-border and basolateral membranes

1987 ◽  
Vol 252 (1) ◽  
pp. R85-R93 ◽  
Author(s):  
J. L. Renfro ◽  
N. B. Clark ◽  
R. E. Metts ◽  
M. A. Lynch
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Protein A ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Transport Processes ◽  
Precipitation Method ◽  
Disulfonic Acid ◽  
Sulfate Transport ◽  
Sulfate Uptake

Brush-border and basolateral membrane vesicles (BBMV and BLMV, respectively) were prepared from chick kidney by a calcium precipitation method and by centrifugation on an 8% Percoll self-generating gradient, respectively. In BBMV a 100-mM Na gluconate gradient, out greater than in, caused concentrative sulfate uptake approximately fivefold greater at 1 min than at 60 min (equilibrium) whether or not the membranes were short-circuited with 100 mM K gluconate, in = out, plus 20 micrograms valinomycin/mg protein. A 48-mM HCO3- gradient, in greater than out, stimulated a 2.5-fold higher uptake at 1 min than at 60 min, and short circuiting as above had no effect on the magnitude of this response. Imposition of a H+ gradient (pH 5.4 out vs. pH 7.4 in) caused concentrative uptake fourfold higher at 1 min than at equilibrium. Short circuiting as above or addition of 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly inhibited the pH gradient effect. Creation of an inside positive electrical potential with 100 mM K gluconate, out greater than in, plus valinomycin, also caused concentrative sulfate uptake. The K gradient in the absence of valinomycin had no effect on sulfate uptake (compared with isosmotic mannitol). Based on inhibitor/competitor effects, these are distinct sulfate transport processes. In chick BLMV, imposition of an HCO3- gradient, in greater than out, produced concentrative sulfate uptake; however, neither Na+ nor H+ gradients had significant effects at 15 s. 4-Acetamido-4'-isothiocyanostilbene 2,2–-disulfonic acid disodium at 0.1 mM was an effective inhibitor of BLMV bicarbonate-sulfate exchange; however, neither Cl-, SCN-, nor CCCP inhibited.

Sensitivity of rat renal luminal and contraluminal sulfate transport systems to DIDS

1986 ◽  
Vol 250 (2) ◽  
pp. F226-F234 ◽  
Author(s):  
C. Bastlein ◽  
G. Burckhardt
Keyword(s):  
Transport System ◽  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Transport Systems ◽  
Basolateral Membrane Vesicles ◽  
Sulfate Transport ◽  
Sulfate Uptake ◽  
Basolateral Membranes ◽  
Irreversible Inhibition

4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was tested as an inhibitor of the sulfate transport systems in rat renal brush border and basolateral membrane vesicles. Na+-driven sulfate uptake into brush border membrane vesicles was half-maximally inhibited at 350 microM DIDS. Proton gradient-driven sulfate uptake into basolateral membrane vesicles was competitively inhibited by DIDS with a Ki of 2.4 microM. The Km for delta pH-driven sulfate uptake was 5.4 microM. The different affinities of the sulfate transport systems for DIDS correlated with different substrate specificities. The luminal transport system accepted a smaller range of anions than the contraluminal system and did not operate as a Na+-independent anion exchanger. After treatment of basolateral membrane vesicles with 50 microM DIDS at pH 8.4 for 30 min, an irreversible inhibition of sulfate uptake was observed. With brush border membranes, only a small irreversible inhibition was obtained. Lack of inhibition after treatment of basolateral membranes with DIDS at pH 6.4 indicated that DIDS reacted with deprotonated amino groups of the transport protein. Sulfate was protected from the irreversible inhibition by DIDS. Sodium-driven uptake of L-glutamate and methylsuccinate into basolateral membrane vesicles was not irreversibly inhibited by DIDS, indicating a specific action of DIDS on the contraluminal sulfate transport system. Irreversible and substrate-protectable inhibition of sulfate transport render DIDS suitable for future affinity labeling studies on the sulfate transport system in basolateral membranes.

Na+-H+ antiporter of rat colonic basolateral membrane vesicles

1989 ◽  
Vol 257 (4) ◽  
pp. G624-G632 ◽  
Author(s):  
P. K. Dudeja ◽  
E. S. Foster ◽  
T. A. Brasitus
Keyword(s):  
Exchange Process ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Basolateral Membrane Vesicles ◽  
Proton Efflux ◽  
Sodium Gradient ◽  
Transport Inhibitors ◽  
22Na Uptake ◽  
Na Uptake

The present experiments were conducted, using acridine orange and 22Na uptake techniques, to demonstrate the presence of an electroneutral Na+-H+ exchange process in rat colonic basolateral membrane vesicles. Results consistent with the existence of a distinct Na+-H+ antiporter in these vesicles include the following: 1) an outwardly directed Na+ gradient stimulated proton influx (Na+in, 100 mM; pHin 7.5/pHout 7.5) and an inwardly directed sodium gradient (Na+out, 5-50 mM; pHin 6.0/pHout 7.5) stimulated proton efflux; 2) sodium-stimulated proton influx was minimally decreased (approximately 10-25%) under voltage clamp conditions (addition of valinomycin in the presence of K+ on both sides of vesicles), indicating that Na+ for H+ exchange in these vesicles could not be explained solely on the basis of a membrane potential; 3) an outwardly directed proton gradient (pHin 5.5/pHout 7.5) stimulated 22Na uptake into these vesicles and a threefold "over-shoot" was observed; 4) 22Na uptake and sodium-stimulated proton efflux were saturable with a Km for Na+ of 5.8 +/- 0.9 and 7.0 +/- 0.3 mM, respectively; 5) amiloride (1 mM) significantly inhibited both sodium-stimulated proton efflux (approximately 69%) and 22Na uptake (approximately 89%), but other transport inhibitors (acetazolamide, 4-acetamido-4'isothiocyanostilbene-2,2'-disulfonic acid, and bumetanide) had no effect on 22Na uptake; 6) N-methylglucamine+ (a nonpermeant cation) did not affect pH gradient-stimulated 22Na uptake, whereas Li+ inhibited Na+ uptake; 7) an inwardly directed Li+ gradient stimulated proton efflux and the Km for Li+ was 12.5 +/- 1.5 mM. These findings establish the existence of an electroneutral Na+-H+ exchange mechanism in rat colonic basolateral membrane vesicles.

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