Hepatopancreatic Brush-border Membrane Vesicles Research Articles

Functional characterization of a novel disaccharide transporter in lobster hepatopancreas.

In animals, the accepted model of carbohydrate digestion and absorption involves reduction of disaccharides into the monosaccharides glucose, fructose, and galactose followed by their individual transmembrane transport into cells. In 2011, a gene for a distinct disaccharide sucrose transporter (SCRT) was found in Drosophila melanogaster and characterized in a yeast expression system. The purpose of the present investigation was to functionally identify and characterize a putative disaccharide transporter analog in the hepatopancreas of the American lobster, Homarus americanus. Purified hepatopancreatic brush-border membrane vesicles (BBMV) were used in transport experiments using 14C-sucrose and a Millipore filter isolation technique. In the absence of sodium, an external pH of 4 significantly stimulated the uptake of 14C-sucrose compared to that occurring at pH 5, 6, or 7. At pH 7, increasing external concentrations of sodium increased 14C-sucrose uptake by BBMV in a hyperbolic fashion and this stimulation was significantly reduced when the pH was changed to 4, suggesting that both protons and sodium ions were each capable of driving the uptake of the sugar. In experiments with a variety of monosaccharides, disaccharides, and trisaccharides, used as potential inhibitors of 14C-sucrose uptake, only maltose and trehalose inhibited carrier-mediated 14C-sucrose transport. An additional experiment showed that 20mM maltose was a competitive inhibitor of 14C-sucrose uptake. The use of a putative lobster SCRT by both maltose and trehalose is nutritionally appropriate for lobsters as they commonly digest glycogen and chitin, polymers of maltose and trehalose, respectively. These findings suggest there is a brush-border proton- or sodium-dependent, hepatopancreatic carrier process, shared by sucrose, maltose, and trehalose, that may function to absorb disaccharides that are produced from digestion of naturally occurring dietary constituents.

Comparative cation dependency of sugar transport by crustacean hepatopancreas and intestine.

ABSTRACTGlucose is transported in crustacean hepatopancreas and intestine by Na+-dependent co-transport, while Na+-dependent D-fructose influx has only been described for the hepatopancreas. It is still unclear if the two sugars are independently transported by two distinct cation-dependent co-transporter carrier systems. In this study, lobster (Homarus americanus) hepatopancreas brush border membrane vesicles (BBMV) were used to characterize, in detail, the cation-dependency of both D-[3H]-glucose and D-[3H]-fructose influxes, while in vitro perfused intestines were employed to determine the nature of cation-dependent sugar transport across this organ. Over the sodium concentration range of 0–100 mM, both [3H]-glucose and [3H]-fructose influxes (0.1 mM; 1 min uptakes) by hepatopancreatic BBMV were hyperbolic functions of [Na+]. [3H]-glucose and [3H]-fructose influxes by hepatopancreatic BBMV over a potassium concentration range of 15–100 mM were hyperbolic functions of [K+]. Both sugars displayed significant (p<0.01) Na+/K+-dependent and cation-independent uptake processes. Transepithelial 25 µM [3H]-glucose and [3H]-fructose fluxes across lobster intestine over luminal sodium and potassium concentration ranges of 0–50 mM and 5–100 mM, respectively, were hyperbolic functions of luminal [Na+] and [K+]. As with hepatopancreatic sugar transport, transepithelial intestinal sugar transport exhibited both significant (p<0.01) Na+/K+-dependent and cation-independent processes. Results suggest that both D-glucose and D-fructose are transported by a single SGLT-type carrier in each organ with sodium being the “preferred”, high affinity, cation for both sugars in the hepatopancreas, and potassium being the “preferred”, high affinity, cation for both sugars in the intestine.

L-leucine, l-methionine, and l-phenylalanine share a Na+/K+-dependent amino acid transporter in shrimp hepatopancreas

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of (3)H-L-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. (3)H-L-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na(+)- and K(+)-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. (3)H-L-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, L-methionine, at 20mM significantly (p<0.0001) inhibited the 2-min uptakes of 1mM (3)H-L-leucine in both Na(+)- and K(+)-containing incubation media. The residual (3)H-L-leucine uptake in the two media were significantly greater than zero (p<0.001), but not significantly different from each other (p>0.05) and may represent an L-methionine- and cation-independent transport system. (3)H-L-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [L-leucine], following the carrier-mediated Michaelis-Menten equation. In NaCl, (3)H-L-leucine influx displayed a low apparent K M (high affinity) and low apparent J max, while in KCl the transport exhibited a high apparent K M (low affinity) and high apparent J max. L-methionine or L-phenylalanine (7 and 20mM) were competitive inhibitors of (3)H-L-leucine influxes in both NaCl and KCl media, producing a significant (p<0.01) increase in (3)H-L-leucine influx K M, but no significant response in (3)H-L-leucine influx J max. Potassium was a competitive inhibitor of sodium co-transport with (3)H-L-leucine, significantly (p<0.01) increasing (3)H-L-leucine influx K M in the presence of sodium, but having negligible effect on (3)H-L-leucine influx J max in the same medium. These results suggest that shrimp BBMV transport (3)H-L-leucine by a single L-methionine- and L-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na(+) or K(+) acting as co-transport drivers binding to shared activator sites.

K+-dependent 3H-d-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp

The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-D-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-D-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-D-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J(max) and lowered K(m) in both salts. ³H-D-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis-Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-D-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (D-galactose, α-methyl-D-gluco-pyranoside, unlabeled D-glucose, D-fructose, and D-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-D-glucose influx. Only unlabeled D-glucose, D-fructose, and D-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of D-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-D-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-D-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, D-fructose, and D-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter, suggesting that an invertebrate analogue of this protein may occur in shrimp.

Comparative sugar transport by crustacean hepatopancreas and intestine

Glucose is transported in crustacean hepatopancreas and intestine by Na+‐dependent cotransport, while Na+‐dependent D‐fructose influx has only been described for the hepatopancreas. In this study lobster (Homarus americanus) hepatopancreas brush border membrane vesicles (BBMV) were used to characterize the cation‐dependency of both 3H‐D‐glucose and 3H‐D‐fructose influxes, while in vitro perfused intestines were employed to determine the nature of cation‐dependent sugar transport in this organ. Over the sodium concentration range of 0–100 mM, both 3H‐D‐glucose and 3H‐D‐fructose influxes (0.1 mM; 1 min uptakes) by hepatopancreatic BBMV were hyperbolic functions of [Na+], exhibiting Km values of 2.30 +/− 0.59 mM (glucose) and 3.79 +/− 1.02 mM (fructose). 3H‐D‐fructose influx by hepatopancreatic BBMV over a potassium concentration range of 15–100 mM was a hyperbolic function of [K+], exhibiting Km value of 12.5 +/− 0.6 mM. Transepithelial 25 μM 3H‐D‐glucose and 3H‐D‐fructose fluxes across lobster intestine over a luminal sodium concentration range of 1 – 50 mM were hyperbolic functions of luminal [Na+], displaying Km values of 6.05 +/− 0.99 mM (glucose) and 10.58 +/− 1.65 mM (fructose). Results suggest that there may be a single carrier process in both organs with a higher affinity for NaCl than for KCl and displays a substrate specificity considerably different than that demonstrated by mammalian SGLT1.

3H‐D‐glucose transport is both Na+‐ and K+‐dependent in the marine shrimp, Litopenaeus setiferus

This study reports the occurrence of a brush border 3H‐D‐glucose transporter in the digestive tract of a marine shrimp that is similarly stimulated by both Na+ and K+. Hepatopancreatic brush border membrane vesicles loaded with 300 mM mannitol exhibited 0.1 mM 3H‐glucose uptake overshoots, that were membrane‐potential‐ sensitive, when incubated in media containing either 150 mM NaCl or KCl. Sugar influxes (15 sec uptakes) in both NaCl or KCl were significantly (p &lt; 0.05) inhibited by 5 mM phloridzin. 3H‐glucose (0.1 mM) influxes in media containing concentrations of NaCl or KCl from 0 to 400 mM were hyperbolic functions of the salt concentrations with both Km‐ and Jmax –values generally higher for KCl than for NaCl. Significant increases (p &lt; 0.05) in Km‐ and Jmax‐values occurred in both media in the presence of a membrane potential difference (inside negative). 3H‐glucose influx was a hyperbolic function of [glucose] from 0.1 to 5.0 mM in the presence of either 150 mM NaCl or KCl. Influx kinetic constants (Km and Jmax) were higher in NaCl than in KCl, and imposed inside‐negative membrane potentials decreased the kinetic constants in both instances. Results suggest that sugar absorption in shrimp may be largely K+‐dependent during juvenile herbivory and Na+‐dependent in adult omnivory/carnivory. Supported by USDA agriculture and food initiative competitive grant no. 2010‐65206‐20617.

Differential physiological expression of the invertebrate 2Na+/1H+ antiporter in single epithelial cell type suspensions of lobster hepatopancreas.

Lobster (Homarus americanus) hepatopancreas is a complex, heterogeneous tissue composed of four epithelial cell types that individually contribute to the overall functional properties of digestion, absorption, secretion, and detoxification. Previous studies, using purified hepatopancreatic brush border membrane vesicles, have described the properties of an electrogenic, 2Na+/1H+ antiporter in this tissue that regulates the absorption and secretion of these cations. These studies were not able to localize this cation exchange phenomenon to specific epithelial cell types. In the present study, sodium/proton exchange by purified, single cell, suspensions of lobster (Homarus americanus) hepatopancreatic epithelium was investigated using a centrifugal elutriation method to cleanly separate the four individual cell types for subsequent physiological characterization. Results indicate that all four hepatopancreatic epithelial cell types possessed the 2Na+/1H+ antiporter as a result of its unique sigmoidal influx properties. Hill Coefficients, measures of transport sigmodicity obtained from kinetic analyses of 22Na+ influx by single cell type suspensions, varied from 1.56 +/- 0.30 (R-cell suspensions) to 2.79 +/- 0.41 (F-cell suspensions), suggesting that different numbers of sodium ions may be accommodated by each cell type. Both calcium and zinc were competitive inhibitors of 22Na+ influx in E-cells (calcium Ki = 105.1+/-5.2 microM; zinc Ki = 46.2 +/- 7.8 microM), but the extent to which these divalent cations inhibited monovalent cation transport by each cell type varied. It is concluded that different isoforms of the electrogenic 2Na+/1H+ antiporter may be present in each hepatopancreatic cell type and thereby contribute in differing degrees to the cation regulatory functions performed by the overall organ.

Electroneutral Na+/H+ exchange in brush-border membrane vesicles from Penaeus japonicus hepatopancreas.

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 measuring 22Na+ uptake. Kinetic parameters measured under short-circuited conditions were the Na+ concentration that yielded one-half of the maximal dissipation rate (Fmax) of the preset transmembrane delta pH (KNa) = 15 +/- 2 mM and Fmax = 3,626 +/- 197 delta F.min-1.mg protein-1, with a Hill coefficient for Na+ of approximately 1. In addition, the inhibitory constant for dimethylamiloride was found to be approximately 1 microM. 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-dependent 22Na+ uptake was observed in lobster hepatopancreatic BBMV. Substitution of chloride with gluconate resulted in increasing KNa and decreasing delta Fmax, 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.

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

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 &micro;mol l-1 for amiloride and 940 &micro;mol 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.

Dipeptide transport by crustacean hepatopancreatic brush-border membrane vesicles

Epithelial brush-border membrane vesicles (BBMVs) of lobster (Homarus americanus) hepatopancreas were formed by a Mg2+ precipitation technique. In these BBMVs, [14C]glycylsarcosine ([14C]Gly-Sar) uptake was stimulated by a transmembrane proton gradient. transmembrane K+ diffusion potential (inside negative) stimulated [14C]Gly-Sar uptake above that observed with short-circuited vesicles, while an inwardly directed Na+ gradient had no stimulatory effect on peptide uptake. [14C]Gly-Sar influx (over 10 s) occurred by a low-affinity, saturable, proton-gradient-dependent carrier system (Kt=5.90&plusmn;0.13 mmol l-1, Jmax=4662&plusmn;487 pmol mg-1 protein 10 s-1; mean &plusmn; s.e.m., N=3). This carrier exhibited a high-affinity proton binding site (KH=235&plusmn;25 nmol l-1; pK=6.6) and an apparent 1H+:1Gly-Sar transport stoichiometry. Influx of 0.1 mmol l-1 [14C]Gly-Sar into lobster hepatopancreatic BBMVs was significantly (P<0.01) cis-inhibited by 10 mmol l-1 diethylpyrocarbonate and by a variety of other dipeptides (10 mmol l-1), suggesting a broad transport specificity. These observations strongly suggest that transport of peptides into crustacean hepatopancreas is proton-gradient-dependent and electrogenic, qualitatively resembling the peptide transport paradigm proposed for fish and mammals.

Phospholipid fatty acid composition of hepatopancreatic brush-border membrane vesicles from the prawn Penaeus japonicus

Brush-border membrane vesicles (BBMV) were isolated from prawn hepatopancreas as we previously described (Muriana et al (1993) J Biochem 113, 625–629). The characterization of hepatopancreatic BBMV (hBBMV) by monitoring the activity of marker enzymes indicated a relatively pure apical membrane preparation reduced in basolateral contamination. Phospholipid composition of hBBMV was examined by the Iatroscan TLC/FID technique, whereas the fatty acid profile of phospholipids was examined by capillary gas chromatography. Phosphatidylcholine and phosphatidylethanolamine are the principal phospholipids of these membranes. The major fatty acids of phospholipids are palmitic (16:0), palmitoleic (16:ln-7), stearic (18:0), oleic (18:ln-9), eicosapentaenoic (20:5n-3) and docosapentaenoic (22:5n-3) acids. Individual phospholipids are characterized by distinct fatty acid composition, but display a similar ratio of unsaturated-to-saturated fatty acids and a similar unsaturation index.

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

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.

Lipid and Fatty Acid Composition of Hepatopancreatic Brush-Border Mambrane Vesicles from the Prawn Penaeus japonicus1

Brush-border membrane vesicles (BBMV) were isolated from prawn hepatopancreas by a procedure involving Mg2+ preparation. The lipid composition of hepatopancreatic BBMV (hBBMV) was examined by the Iatroscan TLC/FID technique, and the fatty acid profile of total lipids was determined by capillary gas chromatography. hBBMV were characterized by a high content of phospholipids and cholesterol. However, the cholesterol/phospholipid molar ratio is lower in prawn than in vertebrate BBMV, which agrees well with the membrane fluidity of BBMV from different sources. The fatty acid composition of hBBMV was similar to that in marine crustacean oils. The major fatty acids present were palmitic (16:0), palmitoleic (16:1n-7), stearic (18:0), oleic (18:1n-9), and eicosapentaenoic (20:5n-3) acids. The large amount of long-chain polyunsaturated fatty acids seems to be related to a high delta 5-desaturase activity in hBBMV.

Electrogenic 2 Na/1 H antiport in crustacean epithelium is inhibited by a monoclonal antibody.

We have previously published evidence that suggests that Na/H exchange in crustacean and echinoderm epithelia occurs by an electrogenic antiporter protein with two external cation binding sites that accommodate Na, amiloride, or Ca and display a 2:1 monovalent cation antiport stoichiometry. The present study is an initial investigation into the molecular biology of this invertebrate electrogenic exchanger to ascertain its structural similarity to the analogous vertebrate electroneutral antiport system. A panel of monoclonal antibodies was prepared against components of lobster hepatopancreatic epithelial brush-border membranes and assayed immunohistochemically and by Western blotting. The antibodies were tested further in functional assays for their ability to interfere with electrogenic 2 Na/1 H antiport in isolated hepatopancreatic brush-border membrane vesicles. One cell line was identified producing an antibody that significantly inhibited the electrogenic exchange of cations by these membrane preparations and recognized a single protein band on Western blots of hepatopancreas, antennal gland, and gill epithelia corresponding to a molecular mass of 185 kDa. The existence of such an antibody probe may facilitate the purification of the electrogenic antiporter under denaturing conditions, in in vitro expression systems, or in prokaryotic expression libraries.

Electrogenic H(+)-regulated sulfate-chloride exchange in lobster hepatopancreatic brush-border membrane vesicles

Transport of [35S]sulfate by brush-border membrane vesicles (BBMV) of lobster (Homarus americanus) hepatopancreas was stimulated by an outwardly directed chloride gradient. In contrast, sulfate uptake was not enhanced by inwardly directed Na+ or K+ transmembrane gradients. An inside-positive membrane potential (valinomycin and K+) stimulated SO4(2-)-Cl- exchange, whereas an inside-negative membrane potential was inhibitory. Sulfate-sulfate exchange was not affected by alterations of transmembrane potential. An inwardly directed proton gradient, or the presence of low bilateral pH, enhanced SO4(2-)-Cl- exchange, but the H+ gradient alone did not stimulate sulfate uptake in chloride-equilibrated BBMV or in vesicles lacking internal Cl-. The stilbenes 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) strongly inhibited SO4(2-)-Cl- exchange. Sulfate influx occurred by a combination of carrier-mediated transfer, exhibiting Michaelis-Menten kinetics, and nonsaturable "apparent diffusion." 36Cl- influx into sulfate-loaded BBMV was stimulated by an inside-negative transmembrane potential compared with short-circuited vesicles. These results suggest that sulfate-chloride exchange in hepatopancreatic BBMV occurred by an electrogenic carrier mechanism exhibiting a 1:1 flux ratio that was modulated by internal and external H(+)-sensitive regulatory sites. The role of this antiport process in anion secretion is discussed.

Both Na + and Cl − gradients energize NaCl/ l-glutamate cotransport in lobster heptopancreatic brush border membrane vesicles

Previous work with l[ 3H]glutamate transport by lobster (Homarus americanus) hepatopancreatic brush border membrane vesicles (BBMV) indicated that the transport of this amino acid was stimulated by the presence of both Na + and Cl − ions in the external medium, however, the specific catalytic or energetic role of each monovalent ion in amino acid transfer was not established (Ahearn and Clay (1987) J. Exp. Biol. 130, 175–191). The present study employs a variety of experimental treatments with this membrane preparation to clarify the nature of the ion dependency in the cotransport process. A zero-trans time course experiment using inwardly-directed transmembrane Na + or Cl − gradients led to similar transient accumulations of the amino acid above equilibrium values in the presence of equilibrated concentrations of the respective counterions. The uptake overshoots observed in the presence of single ion gradients were significantly increased when gradients of both Na + and Cl − were used simultaneously. When vesicles were pre-equilibrated with l-[ 3H]glutamate and either of the monovalent ions, an inwardly-directed gradient of each counterion led to the transient accumulation of additional labelled amino acid above its equilibrium concentration, indicating that either ion gradient was capable of energizing the net flow of l-glutamate. A cotransport stoichiometry of 1 Na +/1 Cl −/1 l-glutamate was established using the Static Head analysis where a balance of ion and amino acid driving forces were attained with a 7:1 Na + or Cl − gradient (0 >i) against a 7:1 l-glutamate gradient (i> 0).

Electrogenic 2 Na+/1 H+ exchange in crustanceans

Hepatopancreatic brush border membrane vesicles of the freshwater prawn, Macrobrachium rosenbergii and the marine lobster, Homarus americanus exhibited 22Na uptake which was Cl-independent, amiloride sensitive, and stimulated by a transmembrane H gradient (Hi greater than Ho). Sodium influx by vesicles of both species were sigmoidal functions of [Na]o, yielding Hill coefficients that were not significantly different (P greater than 0.5) than 2.0. Estimations of half-saturation constants (KNa) were 82.2 mM (prawn) and 280.1 mM (lobster), suggesting a possible adaptation of this transporter to environmental salinity. Trans-stimulation and cis-inhibition experiments involving variable [H] suggested that the exchangers in both species possessed single internal cation binding sites (pK 6.5-6.7) and two external cation binding sites (prawn, pK 4.0 and 5.7; lobster pK 3.5 and 6.1). Similar cis inhibition studies using amiloride as a competitive inhibitor of Na uptake supported the occurrence of dual external sites (prawn, Ki50 and 1520 microM; lobster Ki9 and 340 microM). Electrogenic Na/H exchange by vesicles from both crustaceans was demonstrated using equilibrium shift experiments where a transmembrane potential was used as the only driving force for the transport event. Transport stoichiometries of the antiporters were determined using Static Head analysis where driving forces for cation transfer were balanced using a 10:1 Na gradient, a 100:1 H gradient, and a stoichiometry of 2.0. These electrogenic 2 Na/1 H exchangers appear thermodynamically capable of generating sufficient gastric acidification for organismic digestive activities.

Electroneutral Na+-2 Cl−-Leucine Cotransport by Lobster Hepatopancreatic Brush-Border Membrane Vesicles

ABSTRACT Uptake of L-[3H]leucine by lobster hepatopancreatic brush-border membrane vesicles was stimulated by a transmembrane NaCl gradient (o&amp;gt;i), but not by identical gradients of NaSCN or other Cl− salts (e.g. K+, Li+, NH4+, Cs+ or choline), suggesting that amino acid transfer depended upon both Na+ and Cl−. In NaCl medium at acidic pH, leucine uptake was largely electroneutral and unresponsive to a transmembrane potential generated by permeable anions; however, in Na+-free medium, amino acid transport was strongly electrogenic under the same conditions. Leucine influx occurred by a combination of two carrier processes at physiologically acidic pH. One exhibited an influx Kt of 0·59 mmol 1−1, a JM of 390pmol mg protein−1 s−1 and a cotransport stoichiometry of 1 Na+ : 2 Cl+: 1 leucine. This process was most strongly cis-inhibited by the nonpolar amino acids phenylalanine, methionine and isoleucine, and most weakly inhibited by the more polar species methylaminoisobutyric acid (MeAlB), hydroxyproline, glutamate and arginine. The second leucine carrier system showed a very low binding affinity and could not be distinguished from diffusion, was Na+-and Cl−-independent, and was cis-inhibited by more polar amino acids such as lysine, hydroxyproline, MeAIB, alanine and glutamate. These results suggest that brush-border leucine transport in lobster hepatopancreas at acidic pH may occur by a combination of a modified L-system, that includes ion cosubstrates, and either by a second undefined Na+-independent process with a broad structural specificity or by multiple Na+-independent processes.

Sodium-coupled sugar and amino acid transport in an acidic microenvironment

1. 1. Nutrient transport mechanisms of lobster hepatopancreatic epithelial brush border membrane vesicles (BBMV) are strongly influenced by the acidic nature of the tubular lumen. 2. 2. Sodium-dependent glucose uptake by BBMV was electrogenic and was stimulated at low pH by reducing sugar transport K 1, without affecting J M . 3. 3. Glutamate was largely transported in zwitterionic form at pH 4.0 by an electrically silent cotransport mechanism with both Na and Cl. 4. 4. Increased H + concentration tripled the apparent membrane permeability to glutamate as well as the amino acid transport J M . 5. 5. At pH 4.0 leucine was transported as a cation by two dissimilar carrier systems: a Na-independent process shared by polar amino acids, and an electroneutral Na-2Cl-dependent mechanism shared with non-polar amino acids. 6. 6. A model is proposed for hepatopancreatic BBMV at acidic pH which employs ionic chemical gradients and membrane potential as nutrient transport driving forces.

Na+-Cl−-Glutamate Cotransport by Lobster Hepatopancreatic Brush Border Membrane Vesicles

ABSTRACT l-[3H]glutamate uptake by lobster hepatopancreatic brush border membrane vesicles, formed by a magnesium precipitation technique, was stimulated by a transmembrane NaCl gradient (o&amp;gt;i), but not by identical gradients of KCl, TMA-Cl, NaSCN or NaNO3-, suggesting that the amino acid transfer depends specifically upon both Na+ and Cl−. In the presence of a NaCl gradient (o&amp;gt;i), glutamate uptake was strongly dependent upon bilateral pH and increased markedly as pH was lowered from 7·0 to 4·0. NaCl-dependent l-[3H]glutamate uptake was not trans-stimulated by preloading vesicles with K+. At pH 5·0, NaCl-dependent glutamate uptake occurred by an electroneutral process, whereas Na+-dependent d-[3H]glucose uptake at this pH was electrogenic. l-[3H]glutamate influx exhibited both carrier-mediated and apparent diffusional transport components. Decreasing pH had no significant effect on glutamate influx (Kt), but tripled both maximal carrier-mediated entry rate (JM) and the apparent diffusional permeability (P) of the membrane to this compound. l-[3H]glutamate influx was strongly trans-stimulated by vesicles preloaded with l-and d-glutamate, l-and d-aspartate, l-cysteate, l-tyrosine and l-asparagine, but not by vesicles preloaded with l-leucine, l-glutamine, l-proline, l-alanine or l-lysine. l-[3H]glutamate influx at pH 4·0 was a hyperbolic function of both external Na+ and Cl− concentrations at fixed concentrations of the respective counterions, suggesting cotransport between the amino acid and the two ions with a stoichiometry of 1 Na+ : 1 Cl− : 1 glutamate.