scholarly journals Energized Ca2+ transport by hepatopancreatic basolateral plasma membranes of Homarus americanus.

1998 ◽  
Vol 201 (2) ◽  
pp. 211-220 ◽  
Author(s):  
Z Zhuang ◽  
G A Ahearn
Keyword(s):  
Transport System ◽  
Carrier Transport ◽  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Homarus Americanus ◽  
Molt Cycle ◽  
Apparent Affinity ◽  
Carrier Mechanism ◽  
P Type

Ca2+ transport by hepatopancreatic basolateral membrane vesicles of Atlantic lobster (Homarus americanus) occurred by at least two independent processes: (1) an ATP-dependent carrier transport system, and (2) a Na+-gradient-dependent carrier mechanism. The sensitivity of ATP-dependent Ca2+ transport to vanadate indicated that it was probably due to a P-type ATPase. This system exhibited an extremely high apparent affinity for Ca2+ (Kt=65.28+/-14.39 nmol l-1; Jmax=1. 07+/-0.06 pmol microg-1 protein 8 s-1). The Na+-gradient-dependent carrier transport system exhibited the properties of a Ca2+/Na+ antiporter capable of exchanging external Ca2+ with intravesicular Na+ or Li+. Kinetic analysis of the Na+-dependence of the antiport indicated that at least three Na+ were exchanged with each Ca2+ (n=2. 91+/-0.22). When Li+ replaced Na+ in exchange for 45Ca2+, the apparent affinity for Ca2+ influx was not significantly affected (with Na+, Kt=14.57+/-5.02 micromol l-1; with Li+, Kt=20.17+/-6.99 micromol l-1), but the maximal Ca2+ transport velocity was reduced by a factor of three (with Na+, Jmax=2.72+/-0.23 pmol microg-1 protein 8 s-1; with Li+, Jmax=1.03+/-0.10 pmol microg-1 protein 8 s-1). It is concluded that Ca2+ leaves hepatopancreatic epithelial cells across the basolateral membrane by way of a high-affinity, vanadate-sensitive Ca2+-ATPase and by way of a low-affinity Ca2+/Na+ antiporter with an apparent 3:1 exchange stoichiometry. The roles of these transporters in Ca2+ balance during the molt cycle are discussed.

Inositol Transport by Hepatopancreatic Brush-Border Membrane Vesicles of the Lobster Homarus Americanus

1988 ◽  
Vol 140 (1) ◽  
pp. 107-121
Author(s):  
TEVA SIU ◽  
GREGORY A. AHEARN
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Homarus Americanus ◽  
Brush Border Membrane Vesicles ◽  
Carrier Mechanism ◽  
Apparent Diffusion ◽  
Noncompetitive Inhibitor ◽  
Inositol Uptake ◽  
Glucose Carrier

The mechanism of [3H]myo-inositol transport by the lobster hepatopancreas was examined using purified brush-border membrane vesicles. Transport was stimulated by a 100 mmoll−1 inward Na+ gradient, but other cation gradients were ineffective, suggesting a Na+-dependent transfer mechanism. The transport system was most efficient at pH7.0 (both sides), rather than in the presence of a pH gradient (pHin = 7.0; pHout = 5.5) or at bilaterally low pH (pHin = pHout = 5.5). The system was shown to be electrogenic in two different ways. First, myo-inositol uptake was stimulated by anions of increasing permeability (SCN− > Cl− > gluconate). Second, an outwardly directed, valinomycin-induced K+ diffusion potential (inside negative) enhanced uptake in comparison with vesicles lacking the ionophore. Myo-inositol was transported by a carrier mechanism with an apparent Kt of 0.79mmoll−1, a Jmax of 6.3pmolmg protein−1 s−1, and by apparent diffusion with a permeability coefficient of 5.92 pmolmg protein−1s−1 (mmolT1)−1. D-Glucose was a noncompetitive inhibitor of myo-inositol uptake, but myo-inositol did not significantly reduce the transport of D-[3H]glucose. Vesicles preloaded with myo-inositol trans-stimulated [3H]myo-inositol uptake, whereas those preloaded with D-glucose did not, suggesting that the inositol carrier did not transport D-glucose. It is proposed that myo-inositol does not share the glucose carrier, and that D-glucose may modulateinositol influx by binding to a ‘regulator’ site on the inositol carrier.

Indirect coupling to Na+ of p-aminohippuric acid uptake into rat renal basolateral membrane vesicles

1987 ◽  
Vol 253 (5) ◽  
pp. F795-F801 ◽  
Author(s):  
H. Shimada ◽  
B. Moewes ◽  
G. Burckhardt
Keyword(s):  
Transport System ◽  
Equilibrium Distribution ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Kidney Cortex ◽  
Acid Uptake ◽  
Basolateral Membrane Vesicles ◽  
Rat Kidney Cortex ◽  
Indirect Coupling

Experiments with basolateral membrane vesicles prepared from rat kidney cortex were performed to study the mechanism by which p-aminohippuric acid (PAH) is taken up across the contraluminal membrane and is concentrated in proximal tubule cells. An inward Na+ gradient failed to stimulate [3H]PAH uptake compared with K+ or Li+ and did not cause intravesicular PAH accumulation above equilibrium distribution. In the absence of Na+, the dicarboxylates glutarate and suberate cis-inhibited and trans-stimulated [3H]PAH uptake, indicating a common transport system. In the presence of Na+, 10 microM glutarate in the incubation medium did not cis-inhibit, but rather stimulated [3H]PAH uptake and caused PAH accumulation above equilibrium distribution ("overshoot"). Li+ diminished this stimulation, but was without effect on [3H]PAH/PAH- and [3H]PAH/glutarate exchange. The data indicate the coexistence of a Na+ -coupled, Li+-sensitive transport system for dicarboxylates and a Li+ -insensitive PAH/dicarboxylate exchanger in the basolateral membrane. We propose that dicarboxylates are cotransported with Na+ into the cell and subsequently exchange for extracellular PAH at the basolateral membrane. PAH uptake is thereby indirectly coupled to Na+ via the Na+/dicarboxylate cotransporter.

Ca2+ transport processes of lobster hepatopancreatic brush-border membrane vesicles

1996 ◽  
Vol 199 (5) ◽  
pp. 1195-1208 ◽  
Author(s):  
Z Zhuang ◽  
G Ahearn
Keyword(s):  
Epithelial Cells ◽  
Transport System ◽  
Divalent Cation ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Homarus Americanus ◽  
Transport Processes ◽  
Brush Border Membrane Vesicles ◽  
Carrier Mediated Transport

45Ca2+ uptake by hepatopancreatic brush-border membrane vesicles of Atlantic lobster (Homarus americanus) occurred by a combination of three independent processes: (1) an amiloride-sensitive carrier-mediated transport system; (2) an amiloride-insensitive carrier-mediated transport system; and (3) a verapamil-inhibited channel process responsive to transmembrane potential. Both carrier-mediated processes were antiporters and capable of exchanging external Ca2+ with intravesicular Na+ or H+. The kinetic parameters of both carrier-mediated processes have been reported previously. External amiloride and Zn2+ were both competitive inhibitors of 45Ca2+ influx, reducing entry of the divalent cation at a single binding site with Ki values of 370 µmol l-1 for amiloride and 940 µmol l-1 for Zn2+. It is concluded that the mechanisms controlling Ca2+ entry into hepatopancreatic epithelial cells include a previously reported electrogenic 2Na+/1H+ antiporter, an electroneutral 2Na+/1Ca2+ antiporter and a verapamil-sensitive Ca2+ channel, which might also be used for the entry of Zn2+ and possibly other heavy metals. Evidence from an equilibrium-shift experiment, based on the thermodynamics of a coupled transport process, suggested that both monovalent (Na+) and divalent (Ca2+ and Zn2+) cations may enter hepatopancreatic epithelial cells through a common carrier-mediated transport protein. This suite of hepatopancreatic brush-border Ca2+ transport processes qualitatively resembles that previously reported for the luminal membrane of lobster antennal glands and suggests that crustacean epithelial cells from different organs may handle this divalent cation by similar means.

Dicarboxylate transport in renal basolateral and brush-border membrane vesicles

1992 ◽  
Vol 70 (1) ◽  
pp. 106-112 ◽  
Author(s):  
Yong Keun Kim ◽  
Jin Sup Jung ◽  
Sang Ho Lee
Keyword(s):  
Transport System ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Rabbit Kidney ◽  
Substrate Affinity ◽  
Dicarboxylate Transport ◽  
Overshoot Phenomenon

Characteristics of succinate transport were determined in basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated in parallel from rabbit renal cortex. The uptake of succinate was markedly stimulated by the imposition of an inwardly directed Na+ gradient, showing an "overshoot" phenomenon in both membrane preparations. The stimulation of succinate uptake by an inwardly directed Na+ gradient was not significantly affected by pH clamp or inhibition of Na+–H+ exchange. The Na+-dependent and -independent succinate uptakes were not stimulated by an outwardly directed pH gradient. The Na dependence of succinate uptake exhibited sigmoidal kinetics, with Hill coefficients of 2.17 and 2.38 in BLMV and BBMV, respectively. The Na+-dependent succinate uptake by BLMV and BBMV was stimulated by a valinomycin-induced inside-negative potential. The Na+-dependent succinate uptake by BLMV and BBMV followed a simple Michaelis–Menten kinetics, with an apparent Km of 22.20 ± 4.08 and 71.52 ± 0.14 μM and a Vmax of 39.0 ± 3.72 and 70.20 ± 0.96 nmol/(mg·min), respectively. The substrate specificity and the inhibitor sensitivity of the succinate transport system appeared to be very similar in both membranes. These results indicate that both the renal brush-border and basolateral membranes possess the Na+-dependent dicarboxylate transport system with very similar properties but with different substrate affinity and transport capacity.Key words: dicarboxylate transport, brush border membrane, basolateral membrane, inhibitors, rabbit kidney.

Citrate uptake by basolateral and luminal membrane vesicles from rabbit kidney cortex

1983 ◽  
Vol 244 (6) ◽  
pp. F686-F695 ◽  
Author(s):  
K. E. Jorgensen ◽  
U. Kragh-Hansen ◽  
H. Roigaard-Petersen ◽  
M. I. Sheikh
Keyword(s):  
Transport System ◽  
Membrane Vesicle ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Basolateral Membrane Vesicles ◽  
Experimental Conditions ◽  
Luminal Membrane ◽  
Dependent Transport

The mechanisms of tubular transport of citrate in renal basolateral and luminal membrane vesicles were studied under various experimental conditions. Both membrane preparations take up citrate by a Na+-dependent transport system, although with different characteristics. The uptake of citrate by basolateral membrane vesicles was insensitive to changes in membrane potential, which is indicative of electroneutral transport of the anion. The Na+-dependent uptake of citrate by luminal membrane vesicles was influenced by the presence of Na+salt anions of different permeabilities in the order: chloride greater than sulfate greater than gluconate. Furthermore, addition of citrate to membrane vesicle-potential-sensitive dye suspensions resulted in optical changes of the dye, indicative of electrogenic transfer of this compound. The apparent affinity of the citrate transport system located in luminal membrane vesicles, in contrast to basolateral membrane vesicles, was sensitive to changes in medium pH and was higher than that of basolateral membrane vesicles in the pH range studied. On the basis of these results a model for the transport of citrate by rabbit kidney proximal tubule is proposed.

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.

Urate transport in Homarus americanus hepatopancreas: studies on membrane vesicles and R cells

1995 ◽  
Vol 269 (2) ◽  
pp. R339-R349
Author(s):  
A. T. Nies ◽  
R. K. Kinne ◽  
E. Kinne-Saffran ◽  
M. K. Grieshaber
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Homarus Americanus ◽  
Acid Uptake ◽  
Maximal Velocity ◽  
Active System ◽  
Purine Analogues ◽  
Urate Transport ◽  
Vesicular Membrane

[2-14C]urate uptake was studied in hepatopancreatic basolateral membrane vesicles and in R cell suspensions of the American lobster by Millipore filtration techniques. Unspecific binding of urate to the vesicular membrane was 25.5 +/- 3.0% of equilibrium. Vesicular uptake showed a diffusional and a saturable component (Km) 0.37 +/- 0.04 mM and maximal velocity (Vmax) 16.5 +/- 1.2 pmol urate.mg protein-1.s-1). [2-14C]urate uptake was significantly trans-stimulated by urate. Purine analogues, probenecid, p-aminohippuric acid, pyrazinoic, and oxonic acid cis-inhibited urate transport. Urate uptake was not affected by Na+ or K+ transmembrane gradients but stimulated by 1 mM 2-oxoglutarate at the cis-side in Na(+)-containing media. Cellular urate uptake was inhibited by pyrazinoic acid. Uptake was saturable (Km 0.53 +/- 0.11 mM and Vmax 3.7 +/- 0.4 pmol urate.mg protein-1.s-1) and Na(+)-independent. However, 2-oxoglutarate stimulated uptake in Na(+)-containing media. These results suggest that urate uptake across the basolateral membrane occurs via a specific, Na(+)-independent transport system that may operate in the exchange mode accepting 2-oxoglutarate as countertransported substrate. In vivo, urate uptake thereby would be a tertiary active system driven by a 2-oxoglutarate gradient established across the cell membrane by the operation of a Na(+)-2-oxoglutarate cotransport system.

scholarly journals IMMUNOLOCALIZATION OF AN ANTIGEN ASSOCIATED WITH THE INVERTEBRATE ELECTROGENIC 2Na+/1H+ ANTIPORTER

1994 ◽  
Vol 189 (1) ◽  
pp. 85-104 ◽  
Author(s):  
C Kimura ◽  
G Ahearn ◽  
L Busquets-Turner ◽  
S Haley ◽  
C Nagao ◽  
...  
Keyword(s):  
Brush Border ◽  
Plasma Membranes ◽  
Cation Exchanger ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Homarus Americanus ◽  
Brush Border Membranes ◽  
Antennal Gland ◽  
Organ Systems ◽  
Vacuolar Membranes

Epithelial plasma membranes from crustacean gut, kidney and gills have been shown recently to display an electrogenic 2Na+/1H+ antiporter that differs considerably in its physiological properties from the vertebrate electroneutral 1Na+/1H+ exchange paradigm. In this study, we describe the histological and cytological localization of an antigen associated with invertebrate electrogenic 2Na+/1H+ antiport in lobster (Homarus americanus) tissues using a monoclonal antibody (MAb 11) raised in mice against purified brush border membranes of the hepatopancreatic epithelium. Previous work showed that MAb 11 inhibited electrogenic 2Na+/1H+ and Ca2+/H+ exchange by hepatopancreatic brush border membrane vesicles, but was without effect on Na+-dependent d-glucose transport, suggesting a restricted inhibitory specificity to the cation exchanger. MAb 11 binding occurred at hepatopancreatic epithelial R-cell brush border membranes, at plasma membranes of the antennal gland and gill podocytes, and at vacuolar membranes of hepatopancreatic B- and R-cells, gill nephrocytes and epithelial cells of the antennal gland labyrinth and gill lamellae, as assessed by FITC-labelled secondary antibodies. Control FITC-labelled antibodies raised in mice against vertebrate keratin proteins displayed only weak non-specific binding to the tissues and cells responding intensely to MAb 11, supporting the specific nature of MAb 11 binding to its cognate antigen. The broad histological and cytological distribution of MAb 11 binding to plasma membranes and vacuolar membranes from several lobster organ systems suggests that the physiological activities regulated by its antigen, possibly an element of the invertebrate electrogenic cation exchanger, may be diverse.

Stoichiometry of organic anion/dicarboxylate exchange in membrane vesicles from rat renal cortex and hOAT1-expressing cells

2003 ◽  
Vol 285 (4) ◽  
pp. F775-F783 ◽  
Author(s):  
Amy Aslamkhan ◽  
Yong-Hae Han ◽  
Ramsey Walden ◽  
Douglas H. Sweet ◽  
John B. Pritchard
Keyword(s):  
Plasma Membranes ◽  
Renal Cortex ◽  
Organic Anion ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Hill Coefficient ◽  
Xenopus Laevis Oocytes ◽  
Coupling Ratio ◽  
Kinetic Measurements ◽  
Static Head

Although membrane vesicle studies have established the driving forces that mediate renal organic anion secretion and the organic anion transporter Oat1 has now been cloned in several species, its stoichiometry has remained uncertain. In this study, we used electrophysiology, kinetic measurements, and static head experiments to determine the coupling ratio for Oat1-mediated organic anion/dicarboxylate exchange. Initial experiments demonstrated that uptake of PAH by voltage-clamped Xenopus laevis oocytes expressing rOat1 led to net entry of positive charge, suggesting that coupling was one-to-one. This conclusion was confirmed by kinetic analysis of PAH and glutarate fluxes in native basolateral membrane vesicles from the rat renal cortex, which showed a Hill coefficient of 1. Similarly, static head experiments on the rat vesicles also showed a 1:1 coupling ratio. To confirm these conclusions in a system expressing a single cloned transporter, Madin-Darby canine kidney cells were stably transfected with the human exchanger hOAT1. The hOAT1-expressing cell line showed extensive PAH transport, which was very similar in all respects to transport expressed by hOAT1 in Xenopus oocytes. Its Km for PAH was 8 μM and glutarate effectively trans-stimulated PAH transport. When stoichiometry was assessed using plasma membranes isolated from the hOAT1-expressing cells, both kinetic and static head data indicated that hOAT1 also demonstrated a 1:1 coupling between organic anion and dicarboxylate.

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