© 2001 by The Society for Integrative and Comparative Biology
Osmoregulation by Gills of Euryhaline Crabs: Molecular Analysis of Transporters1
1 Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672 and Department of Biology, Lake Forest College, Lake Forest, Illinois 60045
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The physiological mechanisms by which aquatic animals regulate the osmoconcentration of their body fluids remain unclear despite many excellent studies of tissue and cell function. This review summarizes the current status of an ongoing molecular biological approach to investigating transporters and transport-related enzymes in ion-transporting gills of osmoregulating crustaceans. We have identified cDNAs coding for six candidate proteins in gills of the blue crab Callinectes sapidus and the green shore crab Carcinus maenas, including a Na+ + K+-ATPase
-subunit, a V-type H+-ATPase B-subunit, a Na+/H+ exchanger, a Na+/K+/2Cl– cotransporter, two isoforms of carbonic anhydrase, and arginine kinase. Although our account is far from complete, examination of mRNA abundance by quantitative reverse transcription/polymerase chain reaction (RT/PCR) has identified candidates that are preferentially expressed in gill epithelium, including the Na+ + K+-ATPase -subunit and Na+/H+ exchanger. The osmoregulatory response to salinity reduction includes enhanced mRNA expression of at least one form of carbonic anhydrase. |
INTRODUCTION |
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Many of the papers in the symposium Osmoregulation: An Integrated Approach are focused at the cellular level, asking questions about mechanisms by which cells regulate their intracellular osmotic condition. At the organismal level, we can ask similar questions about regulation of extracellular osmolytes, focusing on tissues that are specialized for osmolyte uptake, retention, or excretion. This review addresses such an organismal level of osmoregulation, with an emphasis on applications of molecular biology to studies of osmolyte transporters and transport-related enzymes in gills of euryhaline crustaceans.
Among the brachyuran crabs, some species (e.g., the blue crab Callinectes sapidus) are capable of osmoregulating and thriving in freshwater (Mangum and Amende, 1972
; Cameron, 1978). Others (e.g., the shore crab Carcinus maenas) are less tolerant of freshwater but nevertheless maintain high extracellular osmolyte concentrations well into brackish water (Siebers et al., 1982). Still others (e.g., the lesser blue crab Callinectes similis) appear less capable of osmoregulation over their entire range (Piller et al., 1995; Guerin and Stickle, 1997) (Fig. 1). The range of osmoregulatory capacities expressed in aquatic crustaceans thus affords the possibility of a comparative physiological approach to the identification of the specific genetic capabilities that contribute to osmoregulatory capacity.
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) (Mantel and Farmer, 1983
) (Piller et al., 1995
) (Siebers et al., 1982Which organ is most involved in organismal osmoregulation in aquatic crustaceans? The antennal glands of brachyuran crabs are incapable of producing urine that is anisosmotic to the hemolymph (Cameron and Batterton, 1978
) and thus cannot be considered as important organs in osmoregulation. Rather, the loss of osmolytes via the urine presents a stressful condition in dilute salinities that must be met by active ion uptake. The crustacean hepatopancreas expresses a variety of transporters (Ahearn et al., 1992), but little attention has been paid to their possible involvement in osmoregulation by euryhaline species. Although the crustacean intestine is known to transport ions (Mantel and Farmer, 1983) and appears to be the major site of water uptake at molt (Neufeld and Cameron, 1994), its overall contribution to osmoregulatory ion uptake is believed to be minimal (Chu, 1987).
The majority of experimental evidence suggests that it is the gills of euryhaline crustaceans that serve as the primary site of osmoregulatory ion transport. Although varying widely in morphology, gills and associated structures of osmoregulators typically contain cell types that are reflective of their transporting capabilities (Taylor and Taylor, 1992). Typical characteristics of these epithelial cells include a greatly elaborated membrane surface, particularly in the basolateral region facing the body fluid. Numerous mitochondria fill such cells, providing the ATP and related phosphagens that power transport processes (Fig. 2). Gill epithelial cells that are believed to function principally in gas exchange are much thinner, with limited membrane elaboration and few mitochondria (Copeland and Fitzjarrell, 1968; Goodman and Cavey, 1990).
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Studies of intact animals, isolated gills, gill lamellae, and membrane vesicles have produced a variety of models of osmoregulatory ion transport in euryhaline crustaceans (Lucu, 1990
; Towle, 1990; Taylor and Taylor, 1992; Pequeux, 1995; Onken and Riestenpatt, 1998). Nearly universal, however, is the assumption that the major driving force for ion transport across gills is provided by the sodium pump or Na+ + K+-dependent ATPase (Towle, 1990). In isolated perfused gills of the blue crab, for example, the Na+ + K+-ATPase inhibitor ouabain blocks more than 50% of the Na+ uptake from external medium to the hemolymph side (Burnett and Towle, 1990) (Fig. 3). Ouabain also blocks ammonia excretion across isolated gills, supporting the idea that a functional sodium pump is essential for this important process as well (Lucu et al., 1989; Weihrauch et al., 1998).
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A second ATP-utilizing pump, the vacuolar-type H+-ATPase, is considered to play an important role in ion uptake by a variety of animal species that successfully tolerate freshwater (Wieczorek et al., 1999
). The V-type ATPase is a highly conserved multisubunit protein similar to the F-type ATP synthase of mitochondria and chloroplasts. Found in eukaryotes, eubacteria, and archaea, the V-type ATPase transports H+ ions against an electrochemical gradient across vacuolar, vesicular, or plasma membranes (reviewed by Nelson et al., 2000). A plasma membrane form of the V-type ATPase has been implicated in energizing NaCl uptake by frog skin, freshwater teleost gill, and gill of the freshwater-tolerant Chinese crab Eriocheir sinensis (Lin and Randall, 1995; Onken and Putzenlechner, 1995; Ehrenfeld and Klein, 1997), where generation of a membrane potential may link its action to current flow through apical Na+ channels or basolateral Cl– channels. The significance of the V-type ATPase in brackish-water-tolerant species is less clear.
Although these ATPases likely provide the electrochemical gradients that drive osmoregulatory ion transport, they must be accompanied by other transporters to complete the process. The Na+ + K+-ATPase is exclusively located in the basolateral membrane of gill epithelial cells (Towle and Kays, 1986), where it likely pumps three Na+ ions outward toward the hemolymph and two K+ (or NH4+) ions inward for each ATP hydrolyzed (Towle and Hølleland, 1987). Thus the net movement of Na+ mediated by Na+ + K+-ATPase is from cytosol to hemolymph, resulting in osmoregulatory accumulation of Na+ (and Cl–, by other mechanisms). Other transporters must mediate movement of Na+ ions from the environment across the apical membrane to the cytosol.
Transporters that may be involved in the apical uptake of Na+ and other ions from the environment include several likely candidates (Fig. 4). One of these is the Na+/H+ exchanger, a transmembrane protein found in many animal cells, where it moves Na+ across the plasma membrane into the cytosol in exchange for H+, depending on a chemical gradient of one of the ions (reviewed by Wakabayashi et al., 1997). Six isoforms of the Na+/H+ exchanger have been described in mammalian species, varying in their tissue and cellular locations and responses to regulatory factors. In gills of teleosts, Na+/H+ exchangers appear to be similar to those of other vertebrates, namely exchanging one Na+ for one H+ (Claiborne et al., 1999). In crustacean gills, however, a Na+/H+ exchanger has been identified that is electrogenic, apparently exchanging two Na+ for one H+ (Shetlar and Towle, 1989). A similar electrogenic exchanger has been described in lobster and prawn hepatopancreas (Ahearn and Clay, 1989; Ahearn et al., 1990). Amiloride, an inhibitor of the Na+/H+ exchanger and epithelial Na+ channels, is known to reduce Na+ uptake across isolated crab gills (Burnett and Towle, 1990) (Fig. 3). Thus the Na+/H+ exchanger (or epithelial Na+ channel) represents one candidate possibly participating in the transfer of Na+ ions across the apical membrane. Because no evidence exists for epithelial Na+ channels in crustacean gills, we have focused our attention on the exchanger.
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A second candidate for an apical mediator of Na+ uptake is the Na+/K+/2Cl– cotransporter, a membrane protein that has been identified in basolateral membranes of salt-excreting epithelia and in apical membranes of salt-absorbing epithelia (Haas, 1994
; Russell, 2000). This electroneutral transporter moves Na+, K+, and 2Cl– ions from extracellular fluid to the cytosol, depending usually on an electrochemical gradient for Na+. Experiments with gills isolated from the shore crab Carcinus maenas have indicated that a portion of Na+ uptake from the medium may be coupled to Cl– uptake, although little effect of the Na+/K+/2Cl– cotransporter inhibitor furosemide could be demonstrated, possibly due to inhibitor interference by the cuticle (Riestenpatt et al., 1996). Although it is unlikely that the electrochemical Na+ gradient would be sufficient to drive ion uptake from fresh water via the cotransporter, the cotransporter may play a role in brackish environments inhabited by species like the shore crab.
Two intracellular enzymes that may support active ion transport across crustacean gills are carbonic anhydrase and arginine kinase. Carbonic anhydrase is believed to provide the counterions H+ and HCO3– for Na+ and Cl– exchange by catalyzing the hydration of CO2 within gill cells (reviewed by Henry, 1996). The activity of the cytoplasmic form of carbonic anhydrase increases dramatically in gills of euryhaline crabs upon transfer from high to low salinities (Henry, 1988), apparently enhancing the supply of counterions for NaCl uptake.
In the tissues of many invertebrates, arginine kinase catalyzes the hydrolysis of phosphoarginine, phosphorylating adenosine diphosphate to form ATP. ATP-utilizing processes such as active ion transport may depend on the ATP buffering function of arginine kinase or on its mitochondrion-to-cytosol shuttle function (Ellington and Hines, 1991). Following transfer of crabs from high to low salinities, the enzymatic activity of cytosolic arginine kinase approximately doubles in gills of the strong osmoregulator Callinectes sapidus, but does not change in the more limited osmoregulator Carcinus maenas (Kotlyar et al., 2000).
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MOLECULAR ANALYSIS OF TRANSPORTERS |
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Our approach to characterizing candidate transporters and transport-related enzymes depends on the application of the techniques of molecular biology. To qualify for inclusion in a working model of osmoregulatory ion transport, the following questions should be answered:
(1) Is the candidate cDNA identifiable, in our case by reverse transcription and polymerase chain reaction (RT/PCR)? Although this question may seem simplistic in relation to model building, a clear absence of a particular transporter cDNA would lead to eliminating that transporter from the model.
(2) Is the candidate mRNA expressed preferentially in ion-transporting gills? Comparisons with tissues less specialized for osmoregulatory ion transport may identify transporters that are highly expressed in gills.
(3) Is candidate mRNA and/or protein expression sensitive to osmoregulatory challenge? Although transporters may be regulated at a variety of levels, including cell signaling cascades altering phosphorylation levels of the transport protein, an osmoregulatory response at the transcriptional or translational level could help to identify components of a model.
To address the first question, we have focused initially on transporters and transport-related enzymes for which nucleotide or amino acid sequence information exists from other species and tissues. Conserved regions of these sequences are identified and used as the basis for designing degenerate oligonucleotide primers for PCR in attempts to amplify the transporter of interest from cDNA prepared by reverse transcription of gill mRNA. If a related transporter is expressed in the tissue being examined, then a distinct product of the polymerase chain reaction should be amplified and can then be sequenced directly without subcloning. Analysis of the sequence via open reading frame detection and an alignment search of GenBank (Altschul et al., 1997) may identify the transporter of interest. If such experiments fail to reveal the target transporter, either the primers are not effective or the target template does not exist in the tissue being examined. If the target is identified, then we can proceed with further analysis.
Answers to the second and third questions require an analytical tool that measures the relative level of transporter mRNA in the tissue. Northern blots or RNAse protection assays can be used in these applications but suffer from lack of sensitivity. We have chosen to use quantitative RT-PCR for these purposes, demonstrating that the method can detect less than a two-fold difference in the level of a specific mRNA between two tissue samples (Towle et al., 1997; Kotlyar et al., 2000). In this method, the polymerase chain reaction is performed using gene-specific primers and a concentration of template that limits the amount of product formed. To ensure that template is indeed limiting, PCR is carried out for a limited number of amplifications (18–22), requiring product detection that is considerably more sensitive than the conventional ethidium bromide staining. We have found that incorporating biotinylated dUTP into the product (replacing some of the dTTP) provides a tag that can be easily detected (Towle et al., 1997).
One major hypothesis rests on the prediction that levels of mRNA coding for osmoregulatory transporters may be higher in the tissues that are specialized for such transport compared to those that are specialized for other functions. Moreover, we predict that osmoregulatory stress may lead to an increase in the transcription rate of the relevant genes, most likely leading to enhanced mRNA levels. Quantitative RT-PCR by itself cannot measure transcription rates or mRNA stability but rather provides a snapshot of mRNA availability for translation. It is clear that post-transcriptional regulatory mechanisms, including translational and cell-signaling controls, may be essential components of the osmoregulatory response, as summarized elsewhere in this symposium. The focus here, however, lies in the initial identification of candidate transporters using primarily molecular approaches.
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CANDIDATE TRANSPORTERS AND TRANSPORT-RELATED ENZYMES |
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Na+ + K+-ATPase
Originally described enzymatically in homogenates of nerves from the shore crab Carcinus maenas (Skou, 1957
), the Na+ + K+-ATPase is known to increase in activity in gills of aquatic animals when they are osmotically stressed. For example, transferring blue crabs (Callinectes sapidus) from 35% salinity to 5% leads to a doubling of Na+ + K+-ATPase activity in the ion-transporting posterior gills (Towle et al., 1976). Similar results are seen in the shore crab Carcinus maenas (Siebers et al., 1982) and many other species. These results imply that the energetic costs of osmoregulation in stressful conditions are met, at least in part, by enhanced activity of the Na+ + K+-ATPase.
It has not been clear whether the salinity-related changes in Na+ + K+-ATPase activity result from enzyme activation or from enzyme (and perhaps mRNA) synthesis. Western blot analysis of Na+ + K+-ATPase -subunit protein in plasma membranes prepared from Carcinus maenas gills indicated that crabs acclimated for 3 wk to low salinity showed twice the amount of -subunit protein compared with crabs acclimated to high salinity or to low salinity for just 4 hr (Lucu and Flik, 1999).
Recent molecular data from our laboratory have revealed a higher abundance of Na+ + K+-ATPase -subunit mRNA as well as protein in posterior gills of Callinectes sapidus compared to anterior gills. However, we observed little effect of salinity change on either -subunit mRNA or -subunit protein (Paulsen et al., 2000; Towle et al., 2001). Thus our evidence suggests that tissue differences in Na+ + K+-ATPase activity in gills of the blue crab are the result of gene regulation, leading to enhanced mRNA and protein levels. Salinity-related changes in Na+ + K+-ATPase activity, perhaps exerted by hormone-sensitive translational or cell signaling processes, may be superimposed upon the likely differences in gene transcription. In conclusion, molecular properties of the Na+ + K+-ATPase fulfill two of our criteria for inclusion in a transport model, suggesting a central role of the Na+ + K+-ATPase in osmoregulatory ion transport.
Vacuolar-type H+-ATPase
cDNAs coding for the B subunit of the V-type ATPase have been identified and at least partially sequenced in gills of five brachyuran crab species (Weihrauch et al., 2000). A detailed study of the expression of B subunit mRNA in gills of Carcinus maenas suggests that the V-type H+-ATPase is not expressed preferentially in posterior ion-transporting gills, nor is its expression enhanced by osmoregulatory challenge (Fig. 5) (Weihrauch et al., 2000). The V-type H+-ATPase thus satisfies the first criterion for inclusion as a candidate transporter in C. maenas, but not the second and third. On the other hand, in the freshwater-tolerant Chinese crab E. sinensis, V-type H+-ATPase B subunit mRNA is preferentially expressed in ion-transporting posterior gills of osmotically challenged animals (Weihrauch et al., 2000), thus meeting all three of the proposed criteria for inclusion in transport models. The shore crab C. maenas may not survive in freshwater because it is incapable of activating membrane-specific V-type ATPase gene transcription.
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salinity. Total RNA (2 µg) was treated with RNAse-free DNAse and reverse transcribed to cDNA using oligo-dT as primer. Five percent of the resulting product was used as template for polymerase chain reaction in the presence of biotinylated-dUTP. Biotin-containing amplification products were separated by gel electrophoresis, blotted, and visualized with streptavidin-conjugated alkaline phosphatase. The number of amplification cycles is indicated. M = biotinylated DNA marker. Exerpted from Na+/H+ Exchanger
A Na+/H+ exchanger cDNA has been amplified and sequenced from gills of two crab species, Carcinus maenas and Callinectes sapidus (Newton et al., 1996
; Towle et al., 1997). Closely related to the vertebrate isoforms previously sequenced, it is not known whether the cloned exchanger is identical with the electrogenic exchanger described in membrane vesicles from crustacean epithelia (Shetlar and Towle, 1989). Nevertheless, the cloned exchanger is expressed very strongly at the mRNA level in gills, showing much lower abundance in all other tissues examined (Fig. 6). Thus the Na+/H+ exchanger meets the first two criteria for retention as a candidate transporter involved in osmoregulation. Whether exchanger mRNA levels respond to osmoregulatory challenge is the subject of current work.
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Na+/K+/2Cl– Cotransporter
A putative Na+/K+/2Cl– cotransporter has been identified and sequenced in gills of blue crab Callinectes sapidus (GenBank Accession Number AF190129 [GenBank] ) (Towle, 1998
). Gills of the shore crab C. maenas also express the Na+/K+/2Cl– cotransporter (Weihrauch and Towle, unpublished), contrary to earlier reports from our lab. Preliminary evidence suggests that the cotransporter is expressed preferentially in posterior gills of C. sapidus (Towle, 1998) (Fig. 7). Current work is examining whether gills of C. maenas express the cotransporter strongly and also whether osmoregulatory challenge enhances mRNA and protein expression.
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Carbonic anhydrase
We have successfully amplified two different isoforms of carbonic anhydrase cDNA from gills of Callinectes sapidus and Carcinus maenas (Gehnrich et al., 2000a, b
). Messenger RNA coding for one of the isoforms is much more abundant than the mRNA coding for the second isoform. Both are more abundant in posterior gills than in anterior gills. In addition, the levels of carbonic anhydrase mRNA increase dramatically when crabs are transferred from high to low salinity (Fig. 8). Our evidence suggests therefore that carbonic anhydrase satisfies all three criteria for inclusion as a transport-related enzyme critical for osmoregulation by crab gill, likely enhancing the availability of counterions for transporter proteins.
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Arginine kinase
In the course of molecular work on transporters, we serendipitously amplified a cDNA coding for arginine kinase, producing a complete cDNA sequence for Carcinus maenas and a complete open reading frame for Callinectes sapidus (Kotlyar et al., 2000
). Although the enzymatic activity of arginine kinase is enhanced following transfer of C. sapidus from high to low salinity, mRNA abundance is not affected by this osmoregulatory challenge. In the weaker osmoregulator C. maenas, both enzyme activity and mRNA abundance were not responsive to salinity reduction (Fig. 5). We can conclude that arginine kinase expression is not tightly coupled to osmoregulatory processes in these two species. The enzyme may, however, mediate buffering of ATP levels that are generally required for active ion transport and other processes. |
CONCLUSION |
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Four of the six candidate transporters and transport-related enzymes so far analyzed meet two of the criteria for inclusion in a working model of NaCl uptake across gills of hyperosmoregulating crabs. These are the Na+ + K+-ATPase
-subunit, the Na+/H+ exchanger, the Na+/K+/2Cl– contransporter, and at least one form of carbonic anhydrase. Our preliminary data indicate that the transcription of carbonic anhydrase is enhanced following salinity reduction. The V-type H+-ATPase B-subunit seems not to be expressed preferentially in ion-transporting gills of the brackish-water-tolerant Carcinus maenas, but it may be important in freshwater adaptation by the Chinese crab Eriocheir sinensis. Much remains to be investigated regarding candidate transporters and transport-related enzymes in gills of osmoregulating crustaceans, particularly with regard to localizing the mRNA and proteins within the epithelial cells. The existence of multiple splicing products and protein isoforms must be investigated more completely. Molecular cloning and complete sequencing of the cDNAs coding for candidate transporters will allow the in vitro expression of each protein, which can then be used to generate antibodies for immunocytochemistry, permitting further exploration of osmoregulatory ion transport mechanisms.
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ACKNOWLEDGMENTS |
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This review is dedicated to the memory of Charlotte P. Mangum, mentor, collaborator, and friend. Her early encouragement and constructive criticism formed the basis of the work summarized here. The author's research is supported by the National Science Foundation (IBN-9807539) and the Foster G. McGaw Foundation. The symposium was supported by the National Science Foundation (IBN-9904521) and by the Divisions of Comparative Physiology and Biochemistry, Invertebrate Zoology, and Comparative Endocrinology of the Society for Integrative and Comparative Biology.
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FOOTNOTES |
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1 From the Symposium Osmoregulation: An Integrated Approach presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2000, at Atlanta, Georgia.
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