RESUMEN

The mechanism of active, osmoregulatory ion uptake was investigated in the pleopods of the marine isopod Idotea baltica (Pallas). Using isolated split half-podites of isopods acclimated to brackish water (20 salinity) mounted in a micro-Ussing chamber and symmetrically superfused with identical haemolymph-like salines, a mean short-circuit current I(sc) of -445 microA cm(-)(2) was measured in endopodites 3-5, corresponding to an inwardly directed transcellular movement of negative charge. Application of ouabain (5 mmol l(-)(1)) to the basolateral superfusate resulted in the almost total abolition of the I(sc) (reduced from -531 to -47 microA cm(-)(2)), suggesting that the Na(+)/K(+)-ATPase is the driving force for active, electrogenic uptake of NaCl. In contrast, mean I(sc) values close to zero were found in preparations of all exopodites and in endopodites 1 and 2. The specific activities of Na(+)/K(+)-ATPase corresponded with these results. Specific activities were highest in posterior endopodites 3-5 and depended on ambient salinity. In all other rami, the activities were much lower and independent of ambient salinity. Activities in posterior endopodites 3-5 were lowest in isopods acclimated to 30 salinity (2-4 micromol P(i )mg(-)(1 )protein h(-)(1)), increased in individuals kept in 20 salinity (8.4 micromol P(i )mg(-)(1 )protein h(-)(1)) and were highest in isopods acclimated to 15 salinity (18.2 micromol P(i )mg(-)(1 )protein h(-)(1)). When specimens were transferred from 30 to 40 salinity, Na(+)/K(+)-ATPase activity increased in the posterior endopodites. The electrophysiological and Na(+)/K(+)-ATPase activity measurements show that active electrogenic ion transport in this species occurs almost exclusively in posterior endopodites 3-5. The endopodite of the fifth pleopod of I. baltica exhibited a microscopic structure remarkably similar to that described for the lamellae of the phyllobranchiae of brachyurans. It is composed of two opposed epithelial monolayers of ionocytes, each covered by cuticle. Bundles of pillar cells are located within the ionocyte layers, which are separated by a fenestrated lamellar septum of connective tissue. The results obtained in this study indicate that endopodites 3-5 play the main role in osmoregulatory ion uptake of the isopod I. baltica. Moreover, the Na(+)/K(+)-ATPase is the only driving force behind active electrogenic ion uptake across the epithelial cells.

Asunto(s)

Crustáceos/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Electrofisiología , Transporte Iónico , Cloruro de Sodio/metabolismo , Equilibrio Hidroelectrolítico

RESUMEN

Mechanisms of active NaCl uptake across the posterior gills of the shore crab Carcinus maenas were examined using radiochemical and electrophysiological techniques. In order to measure short-circuit current (Isc), transepithelial conductance (Gte) and area-related unidirectional fluxes of Na+ and Cl-, single split gill lamellae (epithelium plus cuticle) of hyperregulating shore crabs were mounted in a modified Ussing chamber. The negative short-circuit current measured with haemolymph-like NaCl saline on both sides of the epithelium could be inhibited by application of basolateral ouabain (ouabain inhibitor constant KOua=56±10 µmol l-1), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; KNPPB=7.5±2.5 mmol l-1) or Cs+ (10 mmol l-1). From the apical side, Isc was nearly completely blocked by Cs+ (10 mmol l-1) or Ba2+ (15 µmol l-1), whereas apical addition of furosemide (1 mmol l-1) resulted in only a small current decrease. Cl- influxes were linearly related to negative Isc. The ratio between net influxes of Cl- and Na+ was found to be approximately 2:1. With a single membrane preparation, achieved by permeabilizing the basolateral membrane with amphotericin B, Cl- influxes which were driven by a concentration gradient were shown to depend on the presence of apical Na+ and K+. On the basis of these observations, we propose that active and electrogenic absorption of NaCl across the gill epithelium of hyperregulating shore crabs proceeds as in the thick ascending limb of Henle's loop in the mammalian nephron. Accordingly, branchial NaCl transport is mediated by apical K+ channels in cooperation with apical Na+/K+/2Cl- cotransporters and by the basolateral Na+/K+-ATPase and basolateral Cl- channels.

RESUMEN

Split gill lamellae (epithelium plus cuticle) of hyperregulating Chinese crabs acclimated to fresh water were mounted in a modified Ussing chamber. Active and electrogenic absorption of sodium and chloride were measured as positive amiloride-sensitive and negative Cl--dependent short-circuit currents (INa, ICl), respectively. Both currents were characterized before and after treatment of the tissue with theophylline or dibutyryl cyclic AMP. Both drugs increased INa and ICl. A simple circuit analysis showed that INa stimulation reflected a marked increase in the transcellular Na+ conductance, whereas the respective electromotive force was unchanged. The Michaelis constant (KNa) for Na+ current saturation was decreased after INa stimulation, indicating an increased affinity of the transport mechanism for its substrate. Consequently, the affinity for the Na+ channel blocker amiloride decreased as expected for a competitive interaction between substrate and inhibitor. Analysis of the amiloride-induced current-noise revealed a marked increase in the number of apical Na+ channels after INa stimulation with theophylline, whereas there was little change in the single-channel current. Stimulation of Cl- absorption was accompanied by a substantial increase in both transcellular conductance and electromotive force, indicating an activation of the apical H+ pump that provides the driving force for active Cl- uptake via apical Cl-/HCO3- exchange and basolateral Cl- channels.