Characterization of a Basolateral Electroneutral Na⁺/H⁺ Antiporter in Atlantic Lobster (Homarus Americanus) Hepatopancreatic Epithelial Vesicles

Na⁺/H⁺ antiport has been documented and studied extensively in nearly every cell type examined, particularly in vertebrates. Both prokaryotic and eukaryotic cells generally possess a Na⁺/H⁺ antiporter within the plasma membrane that is responsible for the net uptake of one sodium ion in exchange for the net extrusion of one hydrogen ion (Grinstein, 1988). This 1:1 electroneutral process displays a sensitivity to amiloride and its 5-amino-substituted derivatives (Aronson, 1985), as well as an affinity for Li⁺ (Dudeja et al. 1989). In eukaryotic cells, the Na⁺/H⁺ exchanger performs a variety of essential cell functions such as intracellular pH and volume regulation, transcellular Na⁺ transport (Grinstein, 1988) and, in addition, is activated in response to mitogenic stimuli associated with cell growth and proliferation (Grinstein et al. 1989).

Studies of a number of vertebrate nonepithelial and epithelial cell types have revealed the kinetic properties of the Na⁺/H⁺ antiporter to be relatively similar in teleosts (Vilella et al. 1991), turtles (Post and Dawson, 1992), chickens (Musch et al. 1992) and mammals (Murer et al. 1976). Kinetic analyses of cultured porcine kidney cells, LLC-PK₁ (Haggerty et al. 1988), and cultured opossum kidney cells (Montrose and Murer, 1990) also indicate that in these polarized epithelia two pharmacologically distinct Na⁺/H⁺ antiporters (isoforms) co-exist within the same cell localized to either the apical or basolateral membrane (see review by Clark and Limbird, 1991). In addition to pharmacological data, immunological techniques verified the presence of two distinct Na⁺/H⁺ exchanger isoforms in individual epithelial cells that were restricted to either the apical or basolateral poles (Biemesderfer et al. 1993).

Molecular genetic methods have enabled certain laboratories to clone the gene encoding the Na⁺/H⁺ antiporter. The first successful clone, referred to as NHE1 (Sardet et al. 1989), is a ubiquitous housekeeping protein found within the basolateral membrane of nearly every polarized epithelial cell examined (Clark and Limbird, 1991). It is likely that, in polarized epithelia, the apical and basolateral Na⁺/H⁺ antiporters are present as isoforms on their respective membranes, with a specialized functional role fulfilled by the apical isoforms. Indeed, genes for three apically localized NHE isoforms have been cloned, corroborating the previous pharmacological and immunological evidence: NHE2 (Tse et al. 1991), NHE3 and NHE4 (Orlowski et al. 1992).

A novel class of electrogenic Na⁺/H⁺ antiporter was described by two independent laboratories working with invertebrate organisms (see reviews by Clark and Limbird, 1991; Towle, 1993). Amiloride-sensitive electrogenic Na⁺/H⁺ exchange was documented in lobster and prawn hepatopancreas (Ahearn and Clay, 1989; Ahearn et al. 1990), crab gill (Shetlar and Towle, 1989) and starfish pyloric cecae (Ahearn and Franco, 1990). Kinetic analysis utilizing brush-border membrane vesicles (BBMVs) isolated from Atlantic lobster (Homarus americanus) hepatopancreas indicated a transport stoichiometry of 2Na⁺:1H⁺ (Ahearn and Clay, 1989). Ahearn and Franco (1990) went on to document Ca²⁺/H⁺ exchange occurring on the 2Na⁺/1H⁺ exchanger in lobster antennal gland. Ca²⁺ transport via the electrogenic 2Na⁺/1H⁺ antiporter has also been described for lobster hepatopancreas BBMVs (Ahearn and Zhuang, 1995), and in starfish pyloric cecae both Ca²⁺ and Zn²⁺ can be transported on the established electrogenic system (Zhuang et al. 1995). Therefore, the electrogenic 2Na⁺/1H⁺ exchanger is unlike any previously described apical Na⁺/H⁺ antiporter and may be represented by a separate gene family.

This report utilizes a recently developed Percoll density gradient technique to isolate pure basolateral membrane vesicles (BLMVs) from Atlantic lobster hepatopancreas to investigate whether the basolateral membrane of hepatopancreatic epithelial cells possesses Na⁺/H⁺ exchange activity. This investigation delineates a basolateral-membrane-associated Na⁺/H⁺ antiporter that functionally resembles a vertebrate NHE, differing substantially from the electrogenic system proposed for both the brush-border membrane of hepatopancreatic epithelia and other membranes of crustacean and echinoderm tissues.

Live intermolt Atlantic lobsters, Homarus americanus (H. Milne Edwards), were obtained from commercial dealers in Hawaii and maintained unfed for up to 7 days in a continuous-flow, refrigerated (10 ˚C) filtered seawater aquarium.

Hepatopancreatic basolateral membrane vesicles (BLMVs) were prepared from fresh organs of individual (0.5 kg) lobsters. BLMVs were generated fresh for each experiment utilizing a self-orienting Percoll gradient centrifugation technique adapted from a procedure developed for fish (Davies et al. 1987).

Hepatopancreatic tissue was quickly excised and placed into a hypotonic buffered sucrose medium [MSS buffer: 250 mmol l⁻¹ sucrose, 20 μmol l⁻¹ Tris/HCl, 300 μmol l⁻¹ phenylmethylsulfonylfluoride (PMSF), pH 7.4] and homogenized with a Kinematica GmbH Polytron. An initial centrifugation step (2500 g) removed large cellular debris. A crude separation of basolateral membranes was obtained from a second centrifugation at 20 400 g. The crude basolateral membrane pellet was resuspended in MSS buffer and combined with a premixed dilution of Percoll and centrifuged at 47 800 g for 1 h. The resultant Percoll-gradient bands were enzymatically assayed for basolateral marker enzyme (Na⁺/K⁺-ATPase) and brush-border membrane marker enzyme (alkaline phosphatase) enrichment. High levels of enrichment for Na⁺/K⁺-ATPase (15.4-fold) and minimal alkaline phosphatase enrichment (1.2-fold) indicated a band containing a high concentration of BLM with minimal BBM contamination (Table 1). Enzyme assays were based on protocols reported by Berner and Kinne (1976) and performed at 22 ˚C. The basolateral membrane fraction was homogenized with preloading buffer (buffer contents varied with experiment, see Results for details) and centrifuged for 40 min at 47 800 g. The resulting pellet was resuspended and washed in fresh preloading buffer and centrifuged a final time for 40 min at 47 800 g.

Isolated BLMV uptake studies were conducted at 15 ˚C and were started by diluting the vesicle suspension into a medium containing trace amounts of ²²Na and unlabeled NaCl or sodium gluconate. The compositions of the final resuspension solution and incubation media are described separately for each experiment. Na⁺ uptake was terminated by placing the vesicles into 2 ml of ice-cold stop solution utilizing the Millipore filtration technique developed by Hopfer et al. (1973). Filters were then dissolved in liquid scintillation cocktail (Ecolume) and the radioactivity counted in a Beckman LS 8100 scintillation counter. Na⁺ uptake is expressed as nmol mg⁻¹ protein filter⁻¹ (protein levels were assayed using the BioRad assay). Na⁺ uptake into BLMVs was corrected for non-specific binding by subtracting ‘blank’ values where vesicles and radiolabeled incubation medium were simultaneously injected into ice-cold stop medium without prior mixing. Each experiment was repeated two or three times using membranes from different animals. Each time point is presented in a figure as the mean of 3–5 replicates and the associated standard error. Statistical comparisons were accomplished using Student’s t-tests. A value of P<0.05 was considered significant.

²²NaCl was obtained from DuPont, Inc, USA. Amiloride, dimethylamiloride, valinomycin, phenylmethylsulfonylfluoride (PMSF), tetramethylammonium hydroxide (TMA-OH), Percoll and other reagent grade chemicals were obtained from Sigma Chemical Co, St Louis, MO, USA.

Freshly isolated BLMVs preloaded with 200 mmol l⁻¹ mannitol and 25 mmol l⁻¹ Hepes at pH 7.0 were exposed to medium containing 0, 50, 100, 300, 600 or 900 mmol l⁻¹ sucrose, 25 mmol l⁻¹ Hepes and 1 mmol l^{−1 22}NaCl at pH 7.0 in order to ascertain whether sealed, functional vesicles were obtained. Fig. 1 illustrates the linear dependence of Na⁺ uptake on the osmolality of the outside medium, thereby confirming that intravesicular volume is dependent on a transmembrane osmotic gradient. The slope statistically intersects (r²=0.94; P<0.05) the y-axis at the origin, which indicates a negligible amount of non-specific Na⁺ binding that is not accounted for by subtracting blank values. The observed osmolality of the outside medium at 0 mmol l⁻¹ sucrose (60 mmol kg⁻¹) is attributable to the Hepes/Tris buffer and NaCl.

Na⁺/H⁺ exchange was demonstrated in short-circuited lobster hepatopancreas BLMVs in the presence of an outwardly directed H⁺ gradient as shown by a 2.5-fold ²²Na uptake overshoot at 1 min during a 60 min experiment (Fig. 2). This overshoot was unaffected by 10 mmol l⁻¹ external Ca²⁺, but the addition of 3 mmol l⁻¹ amiloride or the absence of a pH gradient abolished the uptake overshoot, with Na⁺ entry into the vesicles occurring at a greatly attenuated rate.

As previously mentioned, the apical membrane of lobster hepatopancreatic epithelium features an electrogenic 2Na⁺/1H⁺ antiporter. The following experiment was designed to examine the possibility that the BLM porter may be a similar protein. Na⁺/H⁺ exchange is enhanced in the presence of a membrane potential compared with short-circuited conditions (Fig. 3). Amiloride does not completely inhibit Na⁺/H⁺ exchange relative to conditions in which there is no pH gradient. This is in contrast to the effect of amiloride on Na⁺/H⁺ exchange under electroneutral experimental conditions, suggesting an amiloride-insensitive, electrogenic component to Na⁺ uptake in BLMVs. External Ca²⁺ (10 mmol l⁻¹) only partially inhibited Na⁺ uptake, but did not eliminate the attenuated overshoot at 5 min.

In order to clarify the sensitivity of voltage-dependent Na⁺ uptake to external Ca²⁺ and amiloride, an experiment was conducted in which Na⁺ uptake into BLMVs was a function of a transmembrane potential only (inside negative). Relative to control uptake values, 3 mmol l⁻¹ amiloride had no inhibitory activity, yet 10 mmol l⁻¹ Ca²⁺ did reduce Na⁺ uptake into BLMVs during the first 20 min (Fig. 4). Taken together, the time course data clearly indicate the presence of a basolateral Na⁺/H⁺ exchanger that may operate as an electroneutral (1Na⁺:1H⁺⁾ antiporter, unlike that described for apical membranes from the same tissue.

A time course of ²²Na⁺ uptake using three different Na⁺concentrations (5, 50 and 100 mmol l⁻¹) for periods of 1, 2, 3, 4, 6, 8, 10 and 12 s was examined in order to determine a range over which Na⁺ entry remained linear for each Na⁺ concentration (not shown). The maximum time point at which all three cation concentrations illustrated a linear uptake was 2.5 s. Therefore, Na⁺ uptake recorded at 2.5 s would yield accurate initial rates of Na⁺ entry.

The kinetics of Na⁺ uptake at 2.5 s as a function of external Na⁺ concentration was examined for Na⁺ concentrations ranging from 0 to 100 mmol l⁻¹ in BLMVs. BLMVs were preloaded with 300 mmol l⁻¹ mannitol, 50 mmol l⁻¹ potassium gluconate, 25 mmol l⁻¹ Mes/Tris and 50 μmol l⁻¹ valinomycin at pH 5.5, and incubated in medium containing 50 mmol l⁻¹ potassium gluconate, 25 mmol l⁻¹ Hepes/Tris and variable sodium gluconate and tetramethylammonium gluconate concentrations at pH 7.5. Na⁺ uptake into BLMVs was terminated at 2.5 s and corrected for non-specific binding by subtracting ‘blank’ values. The final net uptake values for each Na⁺ concentration are shown in Fig. 5.

Fig. 2 indicated that 3 mmol l⁻¹ effectively abolished Na⁺ influx into hepatopancreatic BLMVs. In order to define more accurately the inhibitory kinetics of amiloride on the BLMV Na⁺/H⁺ exchange mechanism, an experiment was conducted in which vesicles were preloaded with 100 mmol l⁻¹ mannitol, 50 mmol l⁻¹ potassium gluconate and 25 mmol l⁻¹ Mes at pH 5.5. Vesicles were incubated in medium consisting of 100 mmol l⁻¹ mannitol, 50 mmol l⁻¹ potassium gluconate and 25 mmol l⁻¹ Hepes at pH 7.5 and variable amiloride concentrations (0–3 mmol l⁻¹) with either 1 or 5 mmol l⁻¹ NaCl present. Fig. 6 shows a Dixon plot of the effects of increasing amiloride concentrations on uptake over a 5 s period (time increased to accommodate binding of amiloride) at both 1 mmol l⁻¹ and 5 mmol l⁻¹ Na⁺. Single slopes for each Na⁺ concentration suggest a single binding site for amiloride on the Na⁺/H⁺ antiporter. The intersections of the plots at 1 mmol l⁻¹ and 5 mmol l⁻¹ Na⁺ are above the horizontal axis, which may suggest a competitive inhibitory effect of amiloride, and yield an apparent K_i of 39 μmol l⁻¹. However, the intersection is not significantly (P>0.05) above the horizontal axis and therefore does not permit a definitive statement about the nature of the inhibition to be made.

The kinetics of uptake of Na⁺ into BLMVs as a function of Na⁺ or amiloride concentration is consistent with the vertebrate paradigm of an electroneutral Na⁺/H⁺ exchange and departs from the electrogenic model described for the apical membrane of the same cell. To investigate further the similarity of BLMV Na⁺/H⁺ exchange to the process described in vertebrate cells, the effect of externally applied Li⁺ was assessed. Fig. 7 is a Dixon plot of the inhibitory effects of variable Li⁺ concentrations on ²²Na influx. Vesicles were preloaded with 100 mmol l⁻¹ mannitol, 50 mmol l⁻¹ potassium gluconate and 25 mmol l⁻¹ Mes at pH 5.5 and were incubated for 5 s in 100 mmol l⁻¹ mannitol, 50 mmol l⁻¹ potassium gluconate, 25 mmol l⁻¹ Hepes at pH 7.5 with either 1 or 5 mmol l⁻¹ NaCl and different concentrations of LiCl (0–6 mmol l⁻¹). One slope for each Na⁺ concentration suggests a single binding site for Li⁺, and the intersection of these two slopes above the horizontal axis suggests a competitive inhibition with an apparent K_i of 493 μmol l⁻¹.

Fig. 8 illustrates the relationship between amiloride-sensitive Na⁺ uptake and the external Na⁺ concentration as a percentage of the preloaded vesicle isotopic contents. Static head conditions (no net flux) were met at an external Na⁺ concentration of 0.5 mmol l⁻¹. Equation 3 predicts a value of n approximately equal to 1.0, and it may be subsequently predicted that the transport stoichiometry of Na⁺/H⁺ exchange is likely to be approximately 1.0.

To rule out the possibility of a basolateral membrane-potential-sensitive carrier, an experiment was conducted in which vesicles were preloaded with 100 mmol l⁻¹ mannitol, 100 mmol l⁻¹ potassium gluconate and 25 mmol l⁻¹ Hepes at pH 8.0. The vesicles were then incubated in 100 mmol l⁻¹ mannitol, 100 mmol l⁻¹ tetramethylammonium gluconate and 25 mmol l⁻¹ Hepes at pH 8.0 in order to produce an inside-negative potential (inside-positive potentials were generated by reversing the preloading and incubation media). External Na⁺ concentration was varied from 50 to 250 mmol l⁻¹ using sodium gluconate and was osmotically balanced by increasing the mannitol concentration in the preloading medium. Fig. 9A illustrates a direct linear relationship (r²=0.95, inside negative; r²=0.97, inside positive; P<0.05 for both lines) between Na⁺ concentration and influx rate, supporting the possible presence of a functional channel. This linear uptake is significantly reduced (P<0.05) when the inside of the vesicle is positive relative to the external medium, supporting the electrogenic component of Na⁺ influx.

In order to strengthen the argument that a non-carrier mechanism is operating over the same concentration range that the electroneutral Na⁺/H⁺ antiporter is functional, BLMVs were preloaded as described above such that they possessed an inside-negative membrane potential. Na⁺ concentration was varied externally from 0 to 50 mmol l⁻¹ with sodium gluconate and osmotically balanced with appropriate amounts of mannitol present in the preloading medium. Na⁺ influx as a function of external Na⁺ concentration was assessed at pH 8.0 and 4.5. Fig. 9B illustrates a linear function (r²=0.94, pH=4.5; r²=0.96, pH=8,0; P<0.05 for both lines) for BLMV Na⁺ influx that is significantly reduced (P<0.05) at pH 4.5, suggesting that a non-saturatable, pH-sensitive process is accommodating Na⁺ entry into BLMVs.

The present study strongly supports the existence of an electroneutral Na⁺/H⁺ antiporter on the basolateral membrane of lobster hepatopancreatic epithelial cells which physiologically resembles members of the NHE family of Na⁺/H⁺ antiporters (Fig. 10). The lobster BLM antiporter differs from the previously documented apical electrogenic Na⁺/H⁺ exchanger reported in lobster and prawn hepatopancreas (Ahearn and Clay, 1989; Ahearn et al. 1990), in starfish pyloric ceca (Ahearn and Franco, 1990) and in blue crab gill (Shetlar and Towle, 1989).

Na⁺ uptake by the basolateral Na⁺/H⁺ antiporter is a hyperbolic function of Na⁺ concentration, as illustrated in Fig. 5, with a calculated K_m of 28±1.7 mmol l⁻¹ and a J_max of 1.74±0.13 μmol mg⁻¹ min⁻¹, values that fall within the range reported for the vertebrate NHE isoforms (Clark and Limbird, 1991).

In order to verify the 1:1 stoichiometric relationship between the simultaneous flux of Na⁺ and H⁺ predicted by the Michealis–Menten kinetic model, the static head method of Turner and Moran (Turner, 1983) was applied to Na⁺/H⁺ antiport in BLMVs. Because a 10-fold transmembrane gradient of Na⁺ balanced a 10-fold gradient of H⁺ and led to static head conditions, the transport stoichiometry of Na⁺/H⁺ exchange was predicted to be 1.0.

In contrast, kinetic analysis of Na⁺ influx on the BBM from the same tissue revealed a sigmoidal curve reflecting the cooperative binding of, probably, 2 Na⁺ externally and 1 H⁺ internally, resulting in an electrogenic antiport process subsequently corroborated by static head analysis (Ahearn and Clay, 1989). It is thus clear that the apical and basolateral membranes of hepatopancreas epithelium possess functionally different Na⁺/H⁺ antiporters. Furthermore, Ca²⁺/H⁺ exchange in BLMVs is undetectable (Z. Zhuang, unpublished data), whereas in BBMVs Ca²⁺ has been shown to be exchanged in lieu of Na⁺ by the apical 2Na⁺/1H⁺ antiporter (Ahearn and Franco, 1990).

Over a broad range of external Na⁺ concentrations, the presence of an inside-negative potential alone produced a linear influx over the entire range examined (Fig. 9A). This figure also indicates the inhibitory effect of an inside-positive potential on solute influx. To eliminate the possibility that, at lower Na⁺ concentrations, an electrogenic carrier may be contributing to the electrogenic influx pathway, Na⁺ uptake by BLMVs was monitored while external Na⁺ concentrations ranged from 0 to 50 mmol l⁻¹. Diffusion across the membrane was observed; it was significantly reduced at pH 4.5 relative to pH 8.0. Ion channels are indeed sensitive to pH (Palmer and Frindt, 1987). According to Hille (1968), at pH 4.5, Na⁺ channel conductivity diminished by approximately 50 % compared with that measured at physiological pH, consistent with the effect observed for Na⁺ influx into lobster BLMVs. The precise nature of this putative basolateral membrane ion channel is unknown.

Na⁺ conductance in epithelial basolateral membranes is not a novel observation. Post and Dawson (1992), in their description of a turtle colon basolateral Na⁺/H⁺ antiporter, described an amiloride-sensitive electroneutral exchanger that was also capable of an uncoupled Na⁺-selective conductance. Garty et al. (1987) described an amiloride-sensitive Na⁺ conductance in the basolateral membrane of toad bladder as an L-type Na⁺ channel (K_i=13 μmol l⁻¹) and did not consider the possibility that the conductance was a property of the Na⁺/H⁺ antiporter. The dual nature of the antiporter proposed by Post and Dawson (1992) was supported in part by the observation that both Na⁺/H⁺ exchange and Na⁺ conductance were blocked by similar concentrations of amiloride. In our studies, Na⁺ conductance was unaffected by externally applied 3 mmol l⁻¹ amiloride or 500 μmol l⁻¹ dimethylamiloride (data not shown), suggesting that the Na⁺/H⁺ antiporter was not responsible for the observed basolateral Na⁺ conductance. However, Palmer and Frindt (1987) reported that at pH 6.5 a marked decrease occurs in Na⁺ channel activity in rat cortical collecting tubules, whereas both Post and Dawson’s (1992) and our own data (not shown) do not reveal diminished Na⁺ conductance at pH 6.5.

In a wide range of mammalian cells, amiloride appears to interact at a single site that blocks transport function in the Na⁺/H⁺ exchanger with K_i values ranging from 7 to 99 μmol l⁻¹ (Aronson, 1985). Lobster BLMV Na⁺/H⁺ exchange is also inhibited by amiloride with a K_i of 39 μmol l⁻¹. In general, amiloride has been shown to be a competitive inhibitor of Na⁺ entry (Aronson, 1985), interacting with at least two amino acids, one of which also binds Na⁺ (Counillon and Pouyssegur, 1993). Our data suggest competitive inhibition by amiloride on lobster BLMV Na⁺/H⁺ exchange; however, the intercept is not significantly above the horizontal axis and therefore inhibition may be non-competitive.

In addition to amiloride, Li⁺ (K_i=493 μmol l⁻¹) also acts as a competitive inhibitor of Na⁺/H⁺ exchange in lobster BLMVs, although with less inhibitory efficacy. Li⁺ is typically transported as an alternative substrate in Na⁺/H⁺ exchange (Aronson, 1985). Mahnensmith and Aronson (1985) present evidence that Li⁺ and amiloride compete for a single site in renal epithelial vesicles, which is consistent with the effects of both amiloride and Li⁺ on Na⁺/H⁺ exchange in lobster BLMVs. An inhibitory effect of Li⁺ is in contrast to the physiology of the apical 2Na⁺/1H⁺ antiporter, which is not affected by Li⁺ (Z. Zhuang, unpublished observation).

This report is the first physiological characterization of an electrically silent Na⁺/H⁺ antiporter in crustacean epithelia. The antiporter resembles the NHE family of Na⁺/H⁺ antiporters described for all vertebrate species studied and is dissimilar to the electrogenic model of crab and lobster brush-border epithelia. It is unknown at present whether this novel electroneutral Na⁺/H⁺ exchanger is a unique or a homologous NHE isoform.

Recent attempts (Towle and Wu, 1994) to clone the gene encoding the electrogenic 2Na⁺/1H⁺ antiporter in crab, using degenerate primers based on NHE sequences in order to screen crab gill cDNA, have produced a gene sequence which shows extensive homology with vertebrate NHE sequences. Hydropathy analysis yielded a pattern of hydrophobic and hydrophilic domains which closely resembled the pattern of the NHE1 isoform. NHE1 is the ubiquitous ‘housekeeping’ antiporter and, in polarized vertebrate epithelia, is generally localized to the basolateral membrane (Biemesderfer et al. 1993). Unfortunately, neither the tissue specificity nor the cellular localization of the cloned crab sequence is known and functional expression of cDNA has not been reported.

It is plausible that the basolateral electroneutral Na⁺/H⁺ antiporter described for the lobster hepatopancreatic epithelium plays a housekeeping role in maintaining intracellular pH and cell volume akin to the role of NHE1. The electrogenic 2Na⁺/1H⁺ antiporter on the brush-border membrane may reflect an adaptive response of Na⁺/H⁺ antiport to specific aspects of Na⁺ and/or Ca²⁺ transport (Ahearn et al. 1994) and may be representative of a novel gene family. Future investigations into the regulatory events and structural components of the lobster BLMV Na⁺/H⁺ antiporter should clarify its precise physiological role and homology to the NHE gene family.

The authors thank Z. Zhuang, M. Klein, D. Tupper and V. Pennington for technical support and helpful discussions. This study was supported by grants from the National Science Foundation (DCB89-0365 and IBN-9317230).