3H-L-leucine transport by the promiscuous crustacean dipeptide-like cotransporter.

The crustacean intestine and hepatopancreas display a variety of solute transport mechanisms for transmembrane transfer of dietary contents from lumen to epithelial cytosol. An in vitro intestinal perfusion apparatus was used to characterize mucosal to serosoal (MS) and serosal to mucosal (SM) Zn(2+) -dependent (3)H-L-leucine transport by the intestine of the American lobster, Homarus americanus. Transmural 20 µM MS (3)H-L-leucine fluxes across lobster intestine were a hyperbolic function of luminal zinc concentration (1-50 µM) following Michaelis-Menten kinetics (K(m) = 2.67 ± 0.74 µM; J(max) = 19.56 ± 2.22 pmol/cm(2) ×min). Transmural 20 µM SM (3)H-L-leucine fluxes were not affected by serosal zinc, resulting in a highly significant stimulation of net amino acid transfer to the blood by luminal metal. MS fluxes of 20 µM (3)H-L-leucine were also hyperbolic functions of luminal [Cu(2+)], [Mn(2+)], [Na(+)], and [H(+)]. MS flux of (3)H-L-leucine was a sigmoidal function of luminal [L-leucine] and was stimulated by the addition of 20 µM luminal zinc at both pH 7.0 and 5.5. A greater enhanced amino acid transport occurred at the lower pH 5.5. MS flux of 20 µM (3)H-L-leucine in the presence of 20 µM zinc was significantly inhibited by addition of 100 µM luminal glycylsarcosine, and MS flux of 20 µM (3)H-glycylsarcosine was inhibited by 100 µM L-leucine in the presence of 20 µM zinc. Results suggest that (3)H-L-leucine and metals form a complex (e.g., Leu-Zn-Leu] that may functionally mimic dipeptides and use a dipeptide-like transporter during MS fluxes as suggested for fish and mammals.

Dual control of cytosolic metals by lysosomal transporters in lobster hepatopancreas.

This study describes the membrane transport mechanisms used by lobster (Homarus americanus) hepatopancreatic epithelial lysosomes to accumulate and sequester heavy metals from the cytosol, and thereby aid in the regulation of these ions entering the animal from dietary constituents. The present investigation extends previous work describing lysosomal metal uptake by cation exchange with protons and suggests that a second, parallel, lysosomal transport process involving metal-thiol conjugates may work in conjunction with the cation antiporter to control cytoplasmic metal concentrations. Transport of (65)Zn(2+) by lysosomal membrane vesicles (LMV) incubated in 1 mmol l(-1) glutathione (GSH) was not significantly different from metal transport in the absence of the tripeptide. However, preloading LMV with 1 mmol l(-1) alpha-ketoglutarate (AKG), and then incubating in a medium containing 1 mmol l(-1) GSH, more than doubled metal uptake, compared with vesicles equilibrated with chloride or possessing an outwardly directed chloride gradient. Kinetic analysis of lysosomal (65)Zn(2+) influx as a function of zinc concentration, in vesicles containing 1 mmol l(-1) AKG and incubated in 1 mmol l(-1) GSH, revealed the presence of a sigmoidal, low affinity, high capacity carrier process transporting the metal into the organelle. These data indicated the possible presence of an organic anion exchanger in lobster lysosomal membranes. Western blot analysis of LMV with a rabbit anti-rat OAT1 antibody showed the presence of an orthologous OAT1-like protein (approximate molecular mass of 80 kDa) signal from these membranes. These results, and those published previously, suggest the occurrence of two metal transporters on hepatopancreatic membranes, a high affinity, low capacity cation antiporter and a low affinity, high capacity organic anion exchanger. Together these two systems have the potential to regulate cytoplasmic metals over a wide concentration range.

65Zn2+ transport by isolated gill epithelial cells of the American lobster, Homarus americanus.

Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in 65Zn2+ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 muM verapamil, nifedipine, and the calcium ionophore A23187. 65Zn2+ influxes were hyperbolic functions of zinc concentration (1-1,000 microM) and followed Michaelis-Menten kinetics. Calcium reduced both apparent zinc binding affinity (K (m)) and maximal transport velocity (J (max)) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. 65Zn2+ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented.

Cloning of sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) from Caribbean spiny lobster Panulirus argus.

We have previously reported on calcium transport mechanisms in American lobster, Homarus americanus, using (45)Ca(2+) coupled with vesicle preparations of hepatopancreatic endoplasmic reticulum. The active transport of calcium across membranes bordering calcium-sequestering stores such as sarcoplasmic or endoplasmic reticulum is catalyzed by membrane-spanning proteins, the sarco-endoplasmic Ca(2+)-ATPases (SERCAs). In the study described here we used advanced bioinformatics and molecular techniques to clone SERCA from the economically important Caribbean spiny lobster, Panulirus argus. We report the complete cloning of a full-length SERCA from P. argus antenna cDNA (GenBank accession number AY702617). This cDNA has a 1020-amino acid residue open reading frame which is 90% identical to published sequences of other crustacean SERCA proteins. Our data support the hypothesis that one crustacean and three vertebrate genes controlling calcium transport were derived from a common ancestral gene.

Transepithelial transport of zinc and L-histidine across perfused intestine of American lobster, Homarus americanus.

The intestine of the American lobster, Homarus americanus, was isolated and perfused in vitro with a physiological saline, based on the ion composition of the blood, to characterize the mechanisms responsible for transmural transport of zinc and how the amino acid, L-histidine, affects the net movement of the metal across the tissue. Previous studies with this preparation, focusing on the characteristics of unidirectional mucosa to serosa (M to S) fluxes of (65)Zn(2+) and (3)H-L-histidine, indicated the presence of a brush border co-transport process responsible for simultaneously transferring the metal and amino acid across this tissue as an apparent bis-complex (Zn-[His](2)) using a PEPT-1-like dipeptide carrier mechanism. In addition, both zinc and L-histidine were also transferred toward the blood by separate transporters that were independent of the other substrate. The focus of the present study was to characterize the serosa to mucosa (S to M) flux of (65)Zn(2+) under a variety of conditions, and use these values in conjunction with those from the previous study, to assess the direction and magnitude of net metal movement across the tissue. Transmural S to M transport of (65)Zn(2+) was markedly reduced with the addition of the serosal inhibitors ouabain (32%), excess K(+) (25%), excess Ca(2+) (30%), Cu(2+) (38%), nifedipine (21%), and vanadate (53%). In contrast, this flux was markedly stimulated with the serosal addition of ATP (24%) and excess Na(+) (28%). These results suggest that S to M fluxes of zinc occurred by the combination of the basolateral Na/Ca exchanger (NCX), where zinc replaced calcium, and a basolateral nifedipine-sensitive calcium channel. Transmural M to S (65)Zn(2+) fluxes (5-100 microM) were threefold greater than S to M metal transport, and the addition of luminal L-histidine doubled the net M to S zinc flux over its rate in the absence of the amino acid. The results of this paper and those in its predecessor indicate that zinc transport by the lobster intestine is absorptive and significantly enhanced by luminal amino acids.

L-proline transport by purified cell types of lobster hepatopancreas.

The hepatopancreas of the American lobster, Homarus americanus, has four epithelial cell types that are anatomically distinguishable and can be separated for in vitro investigation of their individual biological roles in the intact organ using centrifugal elutriation. Previous studies employing this separation method have produced hepatopancreatic cell suspensions that have been used to examine the nature of copper transport, 2 Na+/1 H+ exchange, and D-glucose absorption by each cell type in isolation from the other cells comprising the tubular epithelium. The present investigation used this method to study amino acid transport by E-, F-, R-, and B-cells of the lobster hepatopancreas in order to characterize the absorption processes for protein digestion products by this organ and to identify which cell type was most likely the responsible agent for net transcellular transfer of these organic molecules from lumen to blood. Results indicated that heptopancreatic E- and F-cell types were the only cells exhibiting Na+-dependent 3H-L-proline transport. Further examination of 3H-L-proline influx by F-cell suspensions indicated that this cell type possessed plasma membrane Na+-dependent IMINO-like and B0-like transport mechanisms and Na+-independent L-like transport mechanisms. Using selective inhibitors of these separate transport systems (e.g., L-pipecolate, L-alanine, and L-leucine), the IMINO-like transporter appeared to predominate in L-proline influx into F-cells, while lesser amounts of amino acid transport took place by the B0-like and L-like systems. The results of this study suggest that the hepatopancreatic F-cell is the epithelial cell type responsible for the bulk of amino acid absorption by this organ and that the IMINO-like transporter is responsible for most of the L-proline transfer through this agent. It is further suggested that as digestion and absorption proceeds in the hepatopancreas and concentrations of luminal amino acids and sodium fall, Na+-dependent transport systems, like the IMINO-like and B0-like, increase their binding affinities for their substrates to maximize nutrient transfer across the epithelium.

65Zn2+ transport by lobster hepato-pancreatic baso-lateral membrane vesicles.

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether (65)Zn(2+) transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10-15 s and approached equilibrium by 120 s. In the absence of sodium, (65)Zn(2+) influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 microM ATP (increase in K(m) and J(max)) and inhibited by the simultaneous presence of 150 micromol l(-1) ATP+250 micromol l(-1) vanadate (decrease in both K(m) and J(max)). In the absence of ATP, (65)Zn(2+) influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l(-1)) and exhibited a Hill Coefficient of 4.03+/-1.14, consistent with the exchange of 3 Na(+)/1Zn(2+). Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle (65)Zn(2+) influx by both the ATP-dependent (K(i)=205 nmol l(-1) Ca(2+)) and sodium-dependent (K(i)=2.47 micromol l(-1) Ca(2+)) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.

Cellular localization of calcium, heavy metals, and metallothionein in lobster (Homarus americanus) hepatopancreas.

This investigation combines confocal microscopy with the cation-specific fluorescent dyes Fluo-3 and BTC-5N to localize calcium and heavy metals along the length of intact lobster (Homarus americanus) hepatopancreatic tubules and isolated cells. A metallothionein-specific antibody, developed in mollusks with cross-reactivity in crustaceans, showed the tissue-specific occurrence of this metal-binding protein in several organ systems in lobster and in single cell types isolated from lobster hepatopancreas. Individual lobster hepatopancreatic epithelial cell types were separated into pure single cell type suspensions for confocal and antibody experiments. Intact hepatopancreatic tubules showed high concentrations of both calcium and heavy metals at the distal tips of tubules where mitotic stem cells (E-cells) are localized. In addition, a concentrated distribution of calcium signal within isolated single premolt E-cells in solution was disclosed that might suggest an endoplasmic reticulum compartmentation of this cation within these stem cells. Both E- and R-cells showed significantly (P < 0.05) greater intracellular calcium concentrations in premolt than intermolt, suggesting the accumulation of this cation in these cells prior to the molt. Antibody studies with lobster tissues indicated that the hepatopancreas possessed 5-10 times the metallothionein concentration as other lobster organ systems and that isolated E-cells from the hepatopancreas displayed more than twice the binding protein concentrations of other cells of this organ or those of blood cells. These results suggest that crustacean hepatopancreatic stem cells (E-cells) and R-cells play significant roles in calcium and heavy metal homeostasis in this tissue. Interactions between the four hepatopancreatic cell types in this regulatory activity remain to be elucidated.

Biology of the 2Na+/1H+ antiporter in invertebrates.

The functional expression of membrane transport proteins that are responsible for exchanging sodium and protons is a ubiquitous phenomenon. Among vertebrates the Na+/H+ antiporter occurs in plasma membranes of polarized epithelial cells and non-polarized cells such as red blood cells, muscle cells, and neurons, and in each cell type the transporter exchanges one sodium for one hydrogen ion, is inhibited by amiloride, and regulates intracellular pH and sodium concentration within tight limitations. In polarized epithelial cells this transporter occurs in two isoforms, each of which is restricted to either the brush border or basolateral cell membrane, and perform somewhat different tasks in the two locations. In prokaryotic cells, sodium/proton exchange occurs by an electrogenic 1Na+/2H+ antiporter that is coupled to a primary active proton pump and together these two proteins are capable of tightly regulating the intracellular concentrations of these cations in cells that may occur in environments of 4 M NaCl or pH 10-12. Invertebrate epithelial cells from the gills, gut, and kidney also exhibit electrogenic sodium/proton exchange, but in this instance the transport stoichiometry is 2Na+/1H+. As with vertebrate electroneutral Na+/H+ exchange, the invertebrate transporter is inhibited by amiloride, but because of the occurrence of two external monovalent cation binding sites, divalent cations are able to replace external sodium and also be transported by this system. As a result, both calcium and divalent heavy metals, such as zinc and cadmium, are transported across epithelial brush border membranes in these animals and subsequently undergo a variety of biological activities once accumulated within these cells. Absorbed epithelial calcium in the crustacean hepatopancreas may participate in organismic calcium balance during the molt cycle and accumulated heavy metals may undergo complexation reactions with intracellular anions as a detoxification mechanism. Therefore, while the basic process of sodium/proton exchange may occur in invertebrate cells, the presence of the electrogenic 2Na+/1H+ antiporter in these cells allows them to perform a wide array of functions without the need to develop and express additional specialized transport proteins. J. Exp. Zool. 289:232-244, 2001.

Copper transport by lobster hepatopancreatic epithelial cells separated by centrifugal elutriation: measurements with the fluorescent dye Phen Green.

The hepatopancreas of the American lobster (Homarus americanus) possesses four types of epithelial cells arranged along blind-ended tubules. At the distal tips of these tubules, stem cells termed E-cells differentiate into three other cell types, R-cells, F-cells and B-cells, each of which have different absorptive and secretory roles in the biology of the overall organ. This investigation uses centrifugal elutriation to separate the individual hepatopancreatic epithelial cell types of Homarus americanus and to investigate their plasma membrane copper transport properties using the copper-sensitive fluorescent dye Phen Green. Results show highly dissimilar endogenous concentrations of copper in each cell type and within the vacuoles (vesicles) released from these cells during the centrifugation process ([copper] in vacuoles>E-cells>R-cells>F-cells approximately B-cells). All four cell types were able to absorb copper from external concentrations ranging from 0.01 to 8 micromol l(-1), but considerable differences in transport rates occurred between the cell types. External calcium (0--10 mmol l(-1)) stimulated the uptake of external copper in a saturable fashion, suggesting the occurrence of carrier-mediated metal uptake. Addition of the Ca(2+) channel blocker verapamil (30 micromol l(-1)) to the external medium reduced the uptake rate of copper by all four cell types, but to different extents in each type of cell. External zinc (0--1000 nmol l(-1)) was a competitive inhibitor of copper influx in E- and R-cells, suggesting that the two metals shared the same binding and transport mechanism. A model is proposed which suggests that copper may enter all hepatopancreatic epithelial cell types by a divalent cation antiport process that exchanges intracellular Ca(2+) (or other cations) with either external copper or zinc. Verapamil-sensitive Ca(2+) channels may allow access of external calcium to cytoplasmic exchange sites on the antiporter or to activator sites on the same transport protein. The results suggest that elutriation is an excellent technique for the separation of complex invertebrate organ systems into their separate cell types and for analyzing the physiological properties of each cell type in isolation.

Chloride transport by lobster hepatopancreas is facilitated by several anion antiport mechanisms.

Three anion antiporters have previously been demonstrated in lobster hepatopancreatic basolateral membrane vesicles (BLMV) to perform vital physiological functions in the crustacean. Cl(-) was shown to be transported by all three of the documented antiporters. The stilbene, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, also known as SITS, strongly inhibited Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-) and Cl(-)/HCO(3)(-) exchange. It was concluded that Cl(-) could be transported by different modes of the documented existing anion antiporters in the lobster hepatopancreatic BLMV.

The oxalate/sulfate antiporter in lobster hepatopancreas: internal and external binding constants.

Utilizing a purified basolateral plasma membrane vesicle (BLMV) preparation containing a sulfate/oxalate antiporter, it was demonstrated that sulfate exhibited similar binding characteristics to the transporter whether bound internally or externally. It was also demonstrated that oxalate had similar binding characteristics to the antiporter whether it was bound internally or externally. Oxalate had a greater affinity to the transporter than did sulfate. Several organic anions affected binding and, therefore, overall transport by the antiporter. Most notably, sulfate was the only anion that stimulated oxalate uptake into BLMVs, which suggests a conservative binding specificity for the antiporter. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and/or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited the transport rate, confirming the existence of oxalate/sulfate exchange by the transporter. These results suggest that oxalate, not sulfate, regulates the transport rate because of its greater affinity to the transporter.

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

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.

Antiport-driven sulfate secretion in an invertebrate epithelium.

A novel invertebrate gastrointestinal transport mechanism has been shown to couple chloride/sulfate exchange in an electrogenic fashion. In the lobster, Homarus americanus, the hepatopancreas, or digestive gland, exists as an outpocketing of the digestive tract, representing a single cell layer separating the gut lumen and an open circulatory system comprised of hemolymph. Investigations utilizing independently prepared brush-border and basolateral membrane vesicles revealed discrete antiport systems which possess the capacity to bring about a transcellular secretion of sulfate. The luminal antiport system functions as a high affinity, one-to-one chloride-sulfate exchanger that is stimulated by an increase in luminal hydrogen ion concentration. Such a system would take advantage of the high chloride concentration of ingested seawater, as well as the high proton concentrations generated during digestion, which further suggests a potential regulation by resident sodium-proton exchangers. Exchange of one chloride for one divalent sulfate ion provides the driving force for electrogenic vectorial translocation. The basolateral antiport system was found to be electroneutral in nature, responsive to gradients of the dicarboxylic anion oxalate, while lacking in proton stimulation. No evidence of sodium-sulfate cotransport, commonly reported for the brush border of vertebrate renal and intestinal epithelia, was observed in either membrane preparation. The two antiporters together can account for the low hemolymph to seawater sulfate levels previously described in decapod crustaceans. A secretory pathway for sulfate based upon electrogenic chloride-antiport may appear among invertebrates partly in response to digestion taking place in a seawater environment.

Sulfate/oxalate exchange by lobster hepatopancreatic basolateral membrane vesicles.

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.

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

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.

Both Na+ and Cl- gradients energize NaCl/L-glutamate cotransport in lobster hepatopancreatic 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 (o greater than i) against a 7:1 L-glutamate gradient (i greater than o).

Sodium and calcium share the electrogenic 2 Na(+)-1 H+ antiporter in crustacean antennal glands.

Na uptake by short-circuited epithelial brush-border membrane vesicles of Atlantic lobster (Homarus americanus) antennal gland labyrinth was Cl independent, amiloride sensitive, and stimulated by a transmembrane H+ gradient [( H]i greater than [H]o; i is internal, o is external). Na influx (2.5-s uptake) was a sigmoidal function of [Na]o (25-400 mM) when pHi = 5.0 and pHo = 8.0 and followed the Hill equation for binding cooperatively [apparent maximal influx (Jmax) = 271 nmol.mg protein-1.s-1, apparent affinity constant for Na (KNa) = 310 mM Na, and Hill coefficient (n) = 2.41]. Amiloride acted as a competitive inhibitor of Na binding to two external sites with markedly dissimilar apparent amiloride affinities (Ki1 = 14 microM; Ki2 = 1,340 mM). Electrogenic Na-H antiport by these vesicles was demonstrated by equilibrium-shift experiments in which an imposed transmembrane electrical potential difference was the only driving force for exchange. A transport stoichiometry of 2 Na to 1 H was demonstrated with the static-head technique in which a balance of driving forces was attained with 10:1 Na gradient and 100:1 H gradient. External Ca, like amiloride, was a strong competitive inhibitor of Na-H exchange, acting at two sites on the outer vesicular face with markedly different apparent divalent cation affinities (Ki1 = 20 microM; Ki2 = 500 microM). Ca-H exchange by electrogenic Na-H antiporter was demonstrated in complete absence of Na by use of an outward H gradient in presence and absence of amiloride. Both external amiloride (Ki1 = 70 microM; Ki2 = 500 microM) and Na (Ki1 = 12 mM; Ki2 = 380 mM) were competitive inhibitors of Ca-H exchange. These results suggest that the electrogenic 2 Na-1 H exchanger characterized for this crustacean epithelium may also have a role in organismic Ca balance.

Electrogenic 2 Na+/1 H+ exchange in crustaceans.

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.