G. A. Ahearn

D-glucose transport in decapod crustacean hepatopancreas.

Physiological mechanisms of gastrointestinal absorption of organic solutes among crustaceans remain severely underinvestigated, in spite of the considerable relevance of characterizing the routes of nutrient absorption for both nutritional purposes and formulation of balanced diets in aquaculture. Several lines of evidence attribute a primary absorptive role to the digestive gland (hepatopancreas) and a secondary role to the midgut (intestine). Among absorbed organic solutes, the importance of D-glucose in crustacean metabolism is paramount. Its plasma levels are finely tuned by hormones (crustacean hyperglycemic hormone, insulin-like peptides and insulin-like growth factors) and the function of certain organs (i.e. brain and muscle) largely depends on a balanced D-glucose supply. In the last few decades, D-glucose absorptive processes of the gastrointestinal tract of crustaceans have been described and transport mechanisms investigated, but not fully disclosed. We briefly review our present knowledge of D-glucose transport processes in the crustacean hepatopancreas. A discussion of previous results from experiments with hepatopancreatic epithelial brush-border membrane vesicles is presented. In addition, recent advances in our understandings of hepatopancreatic D-glucose transport are shown, as obtained (1) after isolation of purified R-, F-, B- and E-cell suspensions from the whole organ by centrifugal elutriation, and (2) by protein expression in hepatopancreatic mRNA-injected Xenopus laevis oocytes. In a perspective, the applicability of these novel methods to the study of hepatopancreatic absorptive function will certainly improve our knowledge of this structurally complex organ.

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.

Electrogenic 2 Na+/1 H+ exchange in crustanceans

SummaryHepatopancreatic brush border membrane vesicles of the freshwater prawn,Macrobrachium rosenbergii and the marine lobster,Homarus americanus exhibited22Na uptake which was Cl-independent, amiloride sensitive, and stimulated by a transmembrane H gradient (Hi>Ho). Sodium influx by vesicles of both species were sigmoidal functions of [Na]o, yielding Hill coefficients that were not significantly different (P>0.5) than 2.0. Estimations of half-saturation constants (KNa) were 82.2mm (prawn) and 280.1mm (lobster), suggesting a possible adaptation of this transporter to environmental salinity.Trans-stimulation andcis-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). Similarcis inhibition studies using amiloride as a competitive inhibitor of Na uptake supported the occurrence of dual external sites (prawn,Ki 50 and 1520 μm; lobsterKi 9 and 340 μm). 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.

Intestinal glucose transport in carnivorous and herbivorous marine fishes

SummaryThe influx and transepithelial movements of glucose and their effects on the electrophysiology and Na transport in upper and lower intestines of the herbivorous surgeonfish,Acanthurus mata, and carnivorous eel,Gymnothorax undulatus, were measured. TheKtG andJmaxG of glucose influx into the tissues were higher in the surgeonfish upper intestine than in the surgeonfish lower intestine or in both segments of the eel intestine. A prominent diffusion-like transport component was also measured in all four segments during influx experiments. Net transepithelial glucose fluxes (0.05 mM) were greater in eel intestine than in those of the surgeonfish largely due to an apparent lower apical membrane permeability of the former coincident with reduced backflux of glucose from epithelium to lumen. All four stripped intestinal segments exhibited non-significant (from zero;P>0.05) or small, serosa-negative transepithelial potential differences (−0.1 to −2.2 mV), and low transepithelial resistances (40–88 Ω cm−2). Each tissue displayed significant (P<0.05) serosa negative short-circuit currents and only the upper intestines in both fish showed net transepithelial fluxes of Na. Glucose addition to the mucosa did not significantly (P>0.05) change the transepithelial resistance, but did induce a significant (P<0.05), but slow reduction in serosa negative short-circuit current. Although 3 of the 4 intestinal segments exhibited increasedJnetNa with added luminal glucose, these increased net cation fluxes were not quite significant (P>0.05). It is concluded that coupled Na-glucose transport occurs in these tissues, but that metabolic enhancement of unrelated current-generating mechanisms also takes place and may modify depolarizing effects of organic solute transfer.

NUTRIENT TRANSPORT BY THE CRUSTACEAN GASTROINTESTINAL TRACT: RECENT ADVANCES WITH VESICLE TECHNIQUES

1. Techniques are described for producing purified brush‐border membrane vesicles (BBMV) of crustacean hepatopancreas which can be used to examine the characteristics of solute transport at the apical pole of hepatopancreatic epithelial cells.

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

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. Sodium-dependent glucose uptake by BBMV was electrogenic and was stimulated at low pH by reducing sugar transport Ki, without affecting JM. 3. Glutamate was largely transported in zwitterionic form at pH 4.0 by an electrically silent cotransport mechanism with both Na and Cl. 4. Increased H+ concentration tripled the apparent membrane permeability to glutamate as well as the amino acid transport JM. 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. A model is proposed for hepatopancreatic BBMV at acidic pH which employs ionic chemical gradients and membrane potential as nutrient transport driving forces.

Effects of Na+, H+, and Cl− on alanine transport by lobster hepatopancreatic brush border membrane vesicles

SummaryEpithelial brush border membrane vesicles (BBMV) of lobster hepatopancreas were formed by a magnesium precipitation technique previously described (Ahearn et al. 1985).3H-l-alanine transport by these vesicles was sodium and potassium insensitive, in contrast to a strong Na-dependency exhibited by3H-d-glucose transport. Initial alanine entry rates (15 s uptake) were stimulated and transient alanine uptake overshoots were observed when external pH was acidic (e. g. pH 4.0, 5.0 or 6.0) and a Cl gradient was imposed across the vesicular wall; at pHo=7.4 alanine uptake was reduced in rate and hyperbolic in character. Alanine uptake from an acidic extravesicular environment in the absence of Cl responded to a transmembrane electrical potential difference created by an outwardly-directed, valinomycin-induced, potassium diffusion potential, suggesting that the alanine molecule alone carried sufficient charge under these conditions to respond to the electrical gradient. External 5.0 mMl-lysine andl-serine similarly inhibited the influx and overshoot properties of 0.05 mM3H-l-alanine uptake, whereas 5.0 mMl-leucine had virtually no effect. Trans-stimulation of alanine initial uptake rates and an enhancement of alanine accumulation against a concentration gradient were observed by vesicles preloaded with 1 mMl-lysine, but not by vesicles lacking amino acids or those containing 1 mMl-leucine orl-serine.3H-l-alanine influx from acidic external environments in the presence of a Cl gradient occurred by a combination of carrier-mediated transfer and ‘apparent diffusion’. Decreasing pHo from 6.0 to 4.0 elevated alanineKt from 0.55 to 2.64 mM, while alanineJM increased from 55 to 550 pmol/mg protein· 15 s. ‘Apparent diffusional permeability’ of the membranes to alanine under these conditions increased slightly. These results suggest, but do not conclusively prove, that alanine transport across BBMV of lobster hepatopancreas may occur by way of a classical y+ transprot protein at acidic pH. The extent of this transport is determined by the magnitude of the transmembrane chloride gradient which serves as a powerful driving force for cationic amino acids in this tissue.

CALCIUM TRANSPORT MECHANISMS OF CRUSTACEAN HEPATOPANCREATIC MITOCHONDRIA

Mechanisms of calcium transport across crustacean hepatopancreatic mitochondrial membranes were investigated in the Atlantic lobster, Homarus americanus. Hepatopancreatic mitochondria were purified by combining methods developed for isolation of these organelles from mammalian and crustacean tissues involving the incorporation of differential centrifugation and Percoll-gradient centrifugation. Enrichment of the preparation was assessed using purification of enzyme markers and electron microscopic examination. Pure hepatopancreatic mitochondria displayed 45Ca2+ uptake by an apparent electrogenic, ruthenium red-inhibited (Ki = 1000 ± 137 nM), transport process that was sensitive to cytoplasmic pH and heavy metals such as Zn2+. 45Ca2+ efflux from mitochondria took place by an apparent diltiazem-inhibited, electroneutral, 2Na+/1Ca2+ antiporter and an apparent diltiazem-insensitive 2H+/1Ca2+ antiporter. One or both antiporters were capable of exchanging preloaded 45Ca2+ for external Zn2+. The apparent uptake protein did not display saturation kinetics over the concentration range selected, but was specifically inhibited by ruthenium red, strongly suggesting the occurrence of a mammal-like uniporter protein in these crustacean mitochondria. These apparent uptake and efflux transport systems are discussed with regard to calcium storage during the molt cycle and the role they may play in heavy metal detoxification. J. Exp. Zool. 283:147–159, 1999.

Changes in fluid transport across the perfused midgut of the freshwater prawn, Macrobrachium rosenbergii during the molting cycle

Abstract 1. 1. Net water flux was measured across the perfused midgut of MacrobraMum rosenbergii during different stages of the molting cycle. 2. 2. Compared to fluid transport rates exhibited during intermolt. fluxes were elevated in animals approaching ecdysis. 3. 3. However, fluxes were much reduced in postmolt individuals. 4. 4. The significance of these findings in relation to salt and water balance of the organism is discussed.

SULFATE/BICARBONATE ANTIPORT BY LOBSTER HEPATOPANCREATIC BASOLATERAL MEMBRANE VESICLES

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton or sodium symport or antiport processes involving a variety of anions. It had been previously observed in lobster hepatopancreas that sulfate could be secreted, however, the mechanisms for this potential secretory process had not been fully described. Therefore, the present study was done to delineate one of the potential processes for this transport. Purified basolateral membrane vesicles (BLMV) were prepared from lobster hepatopancreas by osmotic disruption and discontinuous sucrose gradient centrifugation. Transport of (35S) sulfate into BLMV was stimulated by an outwardly directed bicarbonate gradient compared with gluconate-loaded vesicles. An inside-positive membrane potential (valinomycin and K+) stimulated sulfate/bicarbonate exchange; whereas an inside-negative membrane potential was inhibitory. Sulfate/sulfate exchange was not affected by alterations of transmembrane potential difference. External protons stimulated sulfate/bicarbonate exchange although proton gradients had no effect on sulfate/bicarbonate exchange in the BLMV. The stilbenes, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), strongly inhibited sulfate/bicarbonate exchange. These results suggest that sulfate/bicarbonate exchange in hepatopancreatic BLMV occurred by an electrogenic carrier mechanism exhibiting a 1:1 flux ratio. The possible physiological role of this antiport process in sulfate transport across the crustacean hepatopancreas is discussed. J. Exp. Zool. 284:158–167, 1999.