INTRODUCTION

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SEVERAL STUDIES on Na⁺/H⁺ exchange in a wide variety of vertebrate epithelial and nonepithelial cells have demonstrated that this transport system is almost ubiquitous and that it is involved in intracellular pH (pH_i) and cell volume regulation and in transcellular Na⁺ transport (13). Na⁺/H⁺ stoichiometric ratio, specificity, interaction with other cations, and kinetic parameters have been extensively investigated and reviewed (7, 9, 13, 14). These studies showed that in all tissues examined Na⁺/H⁺ antiporter activity was amiloride sensitive, electroneutral, and exhibited an apparent stoichiometry of 1Na⁺ for 1H⁺.

Recently, several experiments have demonstrated that some invertebrate epithelial cell types possess a Na⁺/H⁺ exchange mechanism that is electrogenic and exhibits a stoichiometric ratio of 2Na⁺:1H⁺ (for recent reviews, see Refs. 1 and 24). In crustaceans, such a different type of antiporter activity has been found in brush-border membrane vesicles (BBMV) prepared from both freshwater prawn (Macrobrachium rosembergii) and marine lobster (Homarus americanus) hepatopancreas (2, 4), from lobster antennal gland epithelium (3), and in plasma membranes isolated from crab (Carcinus maenas) gills (23). Interestingly, in crustaceans an electroneutral Na⁺/H⁺ exchange mechanism has also been characterized on the basolateral domain of lobster hepatopancreatic epithelial cells (12), suggesting that two different types of Na⁺/H⁺ antiporters, i.e., the 1Na⁺:1H⁺ and the 2Na⁺:1H⁺, coexist in crustacean plasma membranes from the same epithelial tissue.

In this article, we examine the properties of a Na⁺/H⁺ exchange mechanism found in BBMV isolated from hepatopancreatic epithelial cells of the Kuruma prawn Penaeus japonicus. Results indicate that this Na⁺/H⁺ exchange activity is different from the electrogenic 2Na⁺:1H⁺ antiporter, extensively characterized in hepatopancreatic plasma membranes of several invertebrate species, and resembles the electroneutral type(s) found in vertebrate cells (13) and crustacean hepatopancreatic basolateral cell membranes (12).

	MATERIALS AND METHODS

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Materials

Preparation of BBMV

Enzyme Assays

Transport Experiments

Fluorescence quenching of the pH-sensitive dye AO, which depends on intravesicular acidification, was evaluated as previously described (26). Briefly, excitation and emission wavelengths were set to 498 and 530 nm, respectively, and both slit widths were set to 5 nm. Ten microliters of a 0.6 mM AO solution (in water), 10 µl of a 1 mM valinomycin solution (in ethanol), and 1,960 µl of a buffer, giving the final composition indicated in the legends to Figs. 1 and 4, were mixed in a glass cuvette. Then, the fluorescence value of this mixture was set to 90 arbitrary fluorescence units, and the reaction was started by injection of 20 µl of a vesicle suspension (160 µg protein) into the cuvette.

Kinetic analysis. To obtain information about kinetic parameters of Kuruma prawn hepatopancreatic BBM Na⁺/H⁺ exchanger, experiments were carried out according to the pH jump technique (27). BBMV prepared in a buffer at pH 5.5 were injected in a cuvette buffer at pH 8.5. This experimental maneuver first provoked a rapid AO fluorescence decay, due to rapid AO accumulation inside the vesicles depending on intravesicular acidic pH, and then a slower fluorescence quenching recovery, due to the dissipation of the preset H⁺ gradient (in > out). Ten seconds after vesicle addition to the cuvette buffer, when the recovery phase was still linear, 50 µl of a NaCl stock solution was added to the cuvette to obtain increasing extravesicular Na⁺ concentrations (ranging from 0 to 200 mM), and the rate of AO fluorescence quenching recovery was measured (for experimental details, see Figs. 2, 3, 5). Initial dissipation rates (F/s) were calculated by interpolating the experimental points within the first 5 s from the beginning of the dissipation, and data obtained were plotted according to Woolf-Augustinsson-Hofstee (22). Kinetic parameters were then calculated by linear regression analysis.

Hill coefficient () was estimated by nonlinear regression analysis based on the Marquardt algorithm (20) using the computer program Statgraphics (STSC, Rockville, MD). Experimental data were fitted to the following Hill-type equation

(1)

where F represents the dissipation rate of the preset transmembrane pH [pH_i < extracellular pH (pH_o)], [Na⁺] is the extravesicular Na⁺ concentration, and K_Na is the Na⁺ concentration that yielded one-half F_max.

²²Na uptake. ²²Na uptake across hepatopancreatic BBMV was measured by the rapid filtration technique as previously described (15). ²²Na⁺ uptake was measured at 22°C by mixing 20 µl (80-100 µg protein) of BBMV suspension with 180 µl of incubation medium containing the radiolabeled solute. Incubation medium composition is indicated in the legend to Fig. 6. ²²Na⁺ uptake was stopped by injection of 3 ml of ice-cold stop solution. BBMV were immediately filtered onto a Millipore filter (0.45 µm) and washed twice with another 3 ml of ice-cold stop solution. Filters, containing the vesicles and their associated radiolabeled solute, were placed in Beckman Ready Solv EP scintillation cocktail and counted in a Beckman LS-1801 scintillation counter. All isotope transport values were corrected for a "blank" obtained by addition of the incubation medium and the vesicles directly to the stop solution before filtering.

Statistics

	RESULTS

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Na⁺/H⁺ Exchange Activity in BBMV Isolated From the Hepatopancreas of P. japonicus

View larger version (9K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   Fig. 1.   A: Kuruma prawn hepatopancreatic brush-border membrane (BBM) Na+/H+ exchange activity in the presence of chloride. BBM vesicles (BBMV) were prepared in a buffer containing (in mM) 60 mannitol, 100 NaCl, 100 KCl, 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), adjusted to pH 7.4 with tris(hydroxymethyl)aminomethane (Tris). To start the experiment, 20 µl of vesicle suspension (160 µg protein) were injected in 1,980 µl of a cuvette buffer containing (final concentration, in mM) 60 mannitol, 100 KCl, 20 HEPES as well as 5 µM valinomycin, 0.5% ethanol, 3 µM acridine orange (AO), adjusted to pH 7.4 with Tris, and 100 tetramethylammonium chloride (trace a); 100 NaCl (control, trace b); and 100 tetramethylammonium chloride plus 0.01 dimethylamiloride (DMA) (trace c). To obtain faster reequilibration of the transmembrane pH gradient, 30 mM NaCl () was added to the reaction mixture. B: effect of Li+ and Ca2+ on Na+/H+ exchange mechanism. BBMV, prepared in a buffer containing (in mM) 60 mannitol, 100 NaCl, 100 KCl, 20 HEPES, adjusted to pH 7.4 with Tris were injected in a cuvette buffer containing (final concentration, in mM) 60 mannitol, 100 KCl, 20 HEPES as well as 5 µM valinomycin, 0.5% ethanol, 3 µM AO, adjusted to pH 7.4 with Tris, 100 mM tetramethylammonium chloride, and either 10 mM LiCl (trace d) or 1 mM CaCl2 (trace e).

Li⁺ has been shown to inhibit the electroneutral 1Na⁺/1H⁺ exchanger located both on vertebrate plasma membranes (7, 13, 16) and lobster hepatopancreatic basolateral membranes (12). Results reported in Fig. 1B show that Na⁺/H⁺ exchange activity of Kuruma prawn hepatopancreatic BBMV was inhibited by 10 mM extravesicular Li⁺ (trace d). In contrast, Ca²⁺ (final concentration in cuvette 1 mM), which has been reported to strongly interact with the electrogenic 2Na⁺/1H⁺ exchange mechanism in crustaceans and echinoderms (1, 6), but not with the electroneutral 1Na⁺/1H⁺ exchanger located on lobster hepatopancreatic basolateral membranes (12), had no effect on Na⁺-dependent proton uptake (trace e). These findings suggest that a specific electroneutral Na⁺/H⁺ exchanger is present in BBMs of Kuruma prawn hepatopancreatic cells.

Kinetics of P. japonicus Hepatopancreatic BBM Na⁺/H⁺ Exchange Mechanism

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View larger version (11K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   Fig. 2.   Kinetics of Kuruma prawn hepatopancreatic BBM Na+/H+ exchange activity. A: BBMV, loaded with a buffer containing (in mM) 60 mannitol, 100 KCl, 20 2-(N-morpholino)ethanesulfonic acid (MES), adjusted to pH 5.5 with Tris, were injected in a cuvette buffer containing (in mM) 60 mannitol and 20 HEPES, adjusted to pH 8.5 with Tris, 5 µM valinomycin, 3 µM AO, 0.5% ethanol, and 100 KCl (short-circuited condition). Ten seconds after vesicle addition to the cuvette buffer, when the recovery phase was still linear, 50 µl of NaCl stock solutions were added to the cuvette buffer () to obtain increasing extravesicular Na+ concentrations ranging from 0 to 200 mM. B: Woolf-Augustinsson-Hofstee plot transformation of AO fluorescence quenching recovery rates under short-circuited conditions. Data obtained as reported in A were plotted according to Woolf-Augustinsson-Hofstee. C: Woolf-Augustinsson-Hofstee plot transformation of AO fluorescence quenching recovery rates under inside-negative membrane potential conditions. BBMV, loaded with a buffer containing (in mM) 60 mannitol, 100 KCl, 20 MES, adjusted to pH 5.5 with Tris, were injected in a cuvette buffer containing (in mM) 60 mannitol, 20 HEPES, adjusted to pH 8.5 with Tris, 5 µM valinomycin, 3 µM AO, 0.5% ethanol, and 100 mM choline chloride. Ten seconds after vesicle addition, 50 µl of NaCl stock solutions were added to the cuvette buffer to obtain extravesicular Na+ concentrations ranging from 0 to 200 mM. Then experimental data were plotted according to Woolf-Augustinsson-Hofstee. F, dissipation rate of the preset transmembrane pH.

Effect of DMA on Na⁺/H⁺ Exchange Activity of P. japonicus Hepatopancreatic BBMV

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Effect of different concentrations of dimethylamiloride ([DMA]) on Kuruma prawn hepatopancreatic Na+/H+ exchange activity. BBMV were prepared in a buffer containing (in mM) 60 mannitol, 100 KCl, 20 MES, adjusted to pH 5.5 with Tris. Twenty microliters of vesicle suspensions (160 µg protein) were injected in 1,980 µl of a cuvette buffer containing (final concentration, in mM) 60 mannitol, 100 KCl, 20 HEPES, adjusted to pH 8.5, 5 µM valinomycin, 3 µM AO, 0.5% ethanol, 10 mM NaCl () or 40 mM NaCl (), and 50, 100, 500, or 1,000 nM DMA, and the recovery rates were measured. Results are presented in a Dixon plot fashion, where v indicates the recovery rate and is expressed as F · min1 · mg protein1.

Characteristics of Na⁺/H⁺ Exchange Activity in the Absence of Chloride

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View larger version (9K): [in this window] [in a new window]   Fig. 4.   Kuruma prawn hepatopancreatic BBM Na+/H+ exchange mechanism measured in the presence of gluconate. Experimental conditions were the same as in Fig. 1A, but with gluconate isosmotically replacing chloride. To obtain faster reequilibration of the transmembrane pH gradient, 30 mM sodium gluconate () was added to the reaction mixture.

View larger version (10K): [in this window] [in a new window]   View larger version (9K): [in this window] [in a new window]   Fig. 5.   Effect of gluconate on the kinetic parameters of the Na+/H+ antiporter. A: BBMV, loaded with a buffer containing (in mM) 60 mannitol, 100 potassium gluconate, 20 MES, adjusted to pH 5.5 with Tris, were injected into a cuvette buffer containing (in mM) 60 mannitol, 20 HEPES, adjusted to pH 8.5 with Tris, 5 µM valinomycin, 3 µM AO, 0.5% ethanol, and 100 mM potassium gluconate. Ten seconds after vesicle addition to the cuvette buffer, 50 µl of sodium gluconate stock solutions were added to the cuvette to obtain increasing extravesicular Na+ concentrations (ranging from 0 and 75 mM), and the rate of AO fluorescence quenching recovery was measured. B: the experimental conditions used in this experiment are the same as detailed for A but with chloride isosmotically replacing gluconate.

To further demonstrate that the Na⁺/H⁺ exchange activity described in hepatopancreatic BBMV of Kuruma prawn was not transporting any net electrical charge and that the use of gluconate as a Na⁺-accompanying anion in the incubation medium did not play any role in the mode of the antiporter, we measured the time course of ²²Na⁺ across the hepatopancreatic BBMV of Kuruma prawn in the presence of an inwardly directed pH gradient (pH_o = 8.5; pH_i = 5.5) as a driving force either in the presence or in the absence of an artificially imposed transmembrane electrical potential (Fig. 6A). As a control, the time course of ²²Na⁺ was measured in the absence of both pH and electrical membrane gradient. Data reported in Fig. 6A clearly show that the ²²Na⁺ uptake was strongly stimulated by the presence of a transmembrane pH gradient (pH_o = 8.5; pH_i = 5.5) and that the additional presence of an (inside negative) transmembrane electrical potential difference did not further increase its uptake values. Na⁺ uptake exhibited an "overshoot" phenomenon both in the absence and in the presence of membrane potential, reaching a maximum value (after 15 s) 2.5-3 times higher than the equilibrium value. In contrast, a much slower time course with no overshoot phenomenon was observed when equal pH values were present in both intravesicular and extravesicular media (pH_i = pH_o = 5.5). Since the electroneutral Na⁺/H⁺ exchanger found in Kuruma prawn hepatopancreatic BBMV departs from the electrogenic paradigm fully assessed in lobster hepatopancreatic BBMV, we also performed in parallel a control experiment using a BBMV preparation from Atlantic lobster hepatopancreas. As shown in Fig. 6B, under the same experimental conditions as in Fig. 6A we were able to observe a Na⁺/H⁺ exchange activity that was strongly dependent on the application of an inside-negative membrane potential. These results suggest that the Na⁺/H⁺ antiporter identified in Kuruma prawn hepatopancreatic BBMV is different from those previously described using other invertebrate BBM preparations.

View larger version (19K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   Fig. 6.   Time course of 22Na+ uptake driven by an outwardly directed proton gradient measured both in the presence and in the absence (short-circuited condition) of an artificially imposed (inside-negative) electrical membrane potential. A: uptake in Kuruma prawn BBMV. BBMV, preloaded with 60 mM mannitol, 200 mM potassium gluconate, 50 µM valinomycin, and 20 mM MES, buffered at pH 5.5 with Tris, were mixed with an incubation buffer containing (in mM) either 60 mannitol, 200 potassium gluconate, 0.1 22Na+, 20 HEPES, buffered at pH 8.5 with Tris () or 60 mannitol, 200 tetramethylammonium gluconate, 0.1 22Na+, 20 HEPES, buffered to pH 8.5 with Tris () or 60 mannitol, 200 potassium gluconate, 0.1 22Na+, 20 MES, buffered to pH 5.5 with Tris (). B: uptake in Atlantic lobster BBMV. 22Na+ uptake was performed under the same experimental conditions as reported in A.

	DISCUSSION

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The present study shows evidence for the existence of a Li⁺-sensitive, Ca²⁺-insensitive, electroneutral Na⁺/H⁺ exchanger on a BBMV preparation of Kuruma prawn (P. japonicus) hepatopancreatic epithelial cells.

It is known that Li⁺ is transported by the electroneutral 1Na⁺/1H⁺ antiporter (13), and it has been shown to competitively inhibit the electroneutral 1Na⁺/1H⁺ exchanger on both vertebrate plasma membranes (7, 13, 16) and lobster hepatopancreatic basolateral membranes (12). In contrast, Li⁺ does not affect apical 2Na⁺/1H⁺ antiporter in lobster hepatopancreatic membrane vesicles (12; Z. Zhuang, unpublished observations). Our data on Kuruma prawn show that the DMA-inhibitable intravesicular acidification induced by the (outwardly directed) Na⁺ gradient is strongly inhibited by extravesicular Li⁺ (Fig. 1B). On the other side, Ca²⁺, which is transported by the electrogenic 2Na⁺/1H⁺ exchange mechanism found in crustaceans and echinoderms (1, 6, 28), does not affect Na⁺-dependent proton uptake in Kuruma prawn hepatopancreatic BBMV. Interestingly, Ca²⁺ was also found to be ineffective on the electroneutral 1Na⁺/1H⁺ exchanger located on lobster hepatopancreatic basolateral membranes (12). Therefore, the lack of any effect by Ca²⁺ and the inhibition shown by Li⁺ suggest that the carrier mechanism described in Kuruma prawn hepatopancreatic BBMV is different from the electrogenic one previously found in the apical membranes of invertebrate epithelial cells (12).

Na⁺/H⁺ exchange activity in Kuruma prawn hepatopancreatic BBMV is a function of Na⁺ concentration (see Fig. 2), with a K_Na ranging between 11 and 23 mM (with V = 0). This value is in agreement with those found in vertebrate epithelia for which K_Na values between 10 and 30 mM were reported (8, 18, 21) and is different from those found for H. americanus and M. rosenbergii hepatopancreatic BBMV for which K_Na values are between 80 and 280 mM (4). Interestingly, a K_Na value of 28 mM has been recently reported for the basolateral electroneutral Na⁺/H⁺ exchanger of lobster hepatopancreatic epithelial cells.

Data reported so far indicate that in crustacean hepatopancreatic apical membranes an electrogenic (2Na⁺/1H⁺) antiporter is present (2-4, 12, 23). In contrast, all data collected in our experiments support the idea that an electroneutral (1Na⁺/1H⁺) exchange system is operating on Kuruma prawn hepatopancreatic BBMV. In fact, from data reported in Fig. 2, obtained both in the presence and in the absence of a transmembrane electrical membrane potential difference, similar kinetic parameters could be calculated (K_Na = 18 ± 3 mM and F_max = 3,451 ± 247 F · min¹ · mg protein¹, with V < 0; K_Na = 15 ± 2 mM and F_max = 3,626 ± 197 F · min¹ · mg protein¹, with V = 0). When fitted to a Hill-type equation, experimental data reported in the same Fig. 2 showed a Hill coefficient proximal to 1 for Na⁺, which suggests an apparent stoichiometry of 1Na⁺/1H⁺. This value was not affected by the presence of an (inside-negative) transmembrane electrical potential ( = 0.86 ± 0.21, with V = 0, and  = 0.85 ± 0.16, with V < 0). It is notable that in a study on the Na⁺/H⁺ exchange activity in membrane vesicles isolated from crab gills under similar experimental conditions Shetlar and Towle (23) have reported a Hill coefficient for Na⁺ of ~2. The absence of any net electrical charge transport during the exchange activity results also from data reported in Fig. 6A, which demonstrate that pH-dependent ²²Na⁺ uptake is insensitive to the application of an outwardly directed (inside-negative) electrical membrane potential. Taken together, these results suggest the conclusion that an electroneutral (1Na⁺/1H⁺ ratio) exchanger, with characteristics similar to vertebrate Na⁺/H⁺ antiporters (13) and to the lobster hepatopancreatic BLMV (12), is present on the apical membrane of Kuruma prawn hepatopancreatic epithelial cells.

It has been reported that, in different mammalian cell types, amiloride appears to inhibit the Na⁺/H⁺ exchanger at a single site with K_i values ranging between 7 and 99 µM (7). Ethylisopropylamiloride (EIPA) and DMA are more effective inhibitors of the Na⁺/H⁺ exchange activity than amiloride. Mean effective concentration (EC₅₀) values of (in µM) 30 for amiloride, 1 for EIPA, and 1 for DMA are considered cutoff values to pharmacologically classify Na⁺/H⁺ systems as "sensitive" or "insensitive" (11). With a K_i value for DMA of ~1 µM (see Fig. 3) and a calculated EC₅₀ greater than 1 µM (cutoff value for DMA), Kuruma prawn hepatopancreatic BBM Na⁺/H⁺ exchanger can therefore be included in the insensitive class of Na⁺/H⁺ exchangers, to which belong (with few exceptions) the apically located electroneutral Na⁺/H⁺ exchangers described in vertebrates (11).

From our data, it appears that chloride plays an important role in the operational mechanism of Kuruma prawn hepatopancreatic BBM Na⁺/H⁺ exchanger (see Figs. 1A, 4, and 6A), since substitution of chloride with gluconate results in a decrease in F_max and Na⁺ affinity. Under our experimental conditions, a possible involvement of a proton-dependent chloride ion transport (i.e., a Cl/OH antiporter) in the alteration of Na⁺-dependent H⁺ uptake can be ruled out, since no chloride ion-dependent H⁺ accumulation could be detected (data not shown). To our knowledge, this dependence on chloride has not been previously evaluated for the electrogenic Na⁺/H⁺ exchanger(s) in H. americanus hepatopancreas and antennal gland and M. rosenbergii hepatopancreas (2-4). Further studies need to be performed to fully evaluate the role of chloride in the operational mechanism of the exchanger.

All together, results reported in this article show evidence of the existence of an electroneutral Na⁺/H⁺ antiporter in the hepatopancreatic BBMV of Kuruma prawn, in contrast to the apical membranes of other crustacean epithelia, where electrogenic Na⁺/H⁺ exchanger(s) seem to play the relevant role in Na⁺ and H⁺ translocation across BBMV of hepatopancreatic cells (2, 4; Fig. 6B). Why so striking a difference? Recently, it has been observed that the electrogenic 2Na⁺/1H⁺ antiporter is specifically associated with the BBMs of only one of the cell types present in crustacean hepatopancreatic epithelium, i.e., the R cells (17), which represent a high percentage of the epithelial cells in H. americanus hepatopancreas (J. Duerr and G. A. Ahearn, personal communication). Interestingly, preliminary observations of histology sections of Kuruma prawn hepatopancreas have shown a low number of R cells in this tissue (Vilella, unpublished observations). This finding would correlate the lack of electrogenic Na⁺/H⁺ exchange activity in Kuruma prawn BBMV to the low amount of R cells, thus explaining the striking difference in Na⁺/H⁺ exchange stoichiometric ratio in Kuruma prawn hepatopancreatic BBMV preparation with respect to H. americanus or M. rosembergii. Whether the presence of a low amount of R cells is either a typical feature of Kuruma prawn hepatopancreatic epithelium or is due to physiological, nutritional, seasonal and/or other environmental (i.e., temperature) changes remains a question to be answered.

Perspectives

In conclusion, a better understanding of crustacean Na⁺/H⁺ exchange system(s) would greatly improve the basic knowledge of phylogenetic relationships among crustaceans and elucidate their still unknown involvement in specific physiological activities.