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IN FOCUS
Laboratory of Pharmacology and Chemistry, National Institutes of Health/National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
IN HIS RECENT CANNON LECTURE, Cowley (3) pointed out the parallels between Cannon's time early in the last century and our own, i.e., the challenge facing us now, as then, is that of "systems integration" and understanding how organisms actually achieve homeostasis. We have begun to generate an incredible "array" of detail at the genomic and molecular levels (e.g., Refs. 3, 23), but, as noted by Cowley, that is just the "parts list." The key is to integrate them into functional systems. This is precisely the focus of the American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. Furthermore, as illustrated so effectively by Somero (20), comparative models provide a particularly apt means of teasing out the critical components in such a systems approach. The sections below provide several examples drawn from recent issues of this journal that highlight both the comparative approach and the roles of membrane transporters in homeostasis.
Osmotic, Ionic, and pH Regulation
Fish are surrounded by their aquatic environment, and changes in ionic and osmotic composition of that water must be met on a moment-to-moment basis. Very often the gill, in particular the chloride cell of the gill, plays a critical role in adaptation to both acute and chronic change (2, 13, 16, 18). These responses are complex, involving multiple transport proteins and signaling pathways. Indeed, many of the same transporters play important roles in both osmotic and pH regulation. Not surprisingly, sorting out their specific contributions has proven challenging. As illustrated below, application of molecular and cell culture techniques are beginning to identify the key components in these important regulatory processes at the molecular level and to provide a clearer picture of their integrated response to change.
pH Regulation
As noted by Claiborne et al. (2) in their recent review, fish must maintain a high ventilation rate to meet their oxygen demands and thus cannot significantly alter ventilation in response to changing pH. Consequently, they depend on cellular mechanisms to maintain pH homeostasis locally and on gill transport mechanisms to sustain overall balance. The current model for gill pH regulation (2) depends on carbonic anhydrase to supply protons and bicarbonate and a number of apical and basolateral transporters to move protons and/or bicarbonate to or from the body as needed. Many of the participating enzyme or transport functions have been known or hypothesized for years and some components have been cloned from fish (1, 6, 11, 12, 21). Although most fish live in a narrow range of pH near neutrality, some species do survive in both highly alkaline (pH 10) and acidic (pH 3.5) waters. These fish must adapt to meet their stressful environment, and thus provide models through which to focus on those critical components responsible for pH regulation. Hirata et al. (7) recently applied molecular techniques to one such species, the acid-tolerant Osorezan dace, creating a subtraction library enriched in acid-inducible mRNA. On transfer to these extreme conditions, most fish lose sodium and die. The Osorezan dace shows only a modest fall in plasma sodium (
10-15%) over the first few hours after transfer and recovers toward normal by 12-24 h. This response has two components, a general upregulation of metabolic pathways leading to increased bicarbonate and ammonium production, and a specific set of changes in protein expression in the mitochondria-rich chloride cells of the gill. Na+-K+-ATPase mRNA was markedly increased in the gill, but not in kidney, during this adaptation period consistent with the major contribution of the gill in this process. Likewise, messages for carbonic anhydrase II (CA-II), sodium proton exchanger 3 (NHE3), sodium bicarbonate cotransporter 1 (NBC1), and aquaporin 3 (AQP3) were all elevated in the gills of acid-adapted fish. In contrast, the V-type H+-ATPase was changed very little under these conditions. Immunohistochemistry confirmed not only the presence of these proteins, but also demonstrated their subcellular distribution—apical for NHE3 and basolateral for Na+-K+-ATPase, NBC1, and AQP3—and showed a unique follicular arrangement of the chloride cells in the acid-adapted fish. Together with functional studies of the expressed dace proteins, these observations led the authors to suggest that the primary apical step in proton elimination and sodium conservation was mediated by NHE3, whereas basal Na/HCO3- cotransport and Na+-K+-ATPase were responsible for serosal sodium and bicarbonate extrusion into the plasma, with CA-II playing its usual role in supplying both protons and bicarbonate. What sets this work apart is not that this picture is unique. Indeed, it differs only in modest ways from the working model shown by Claiborne et al. (2) in their review, but Hirata et al. (7) were able to identify specific transporter isoforms and place them in a model based on cloning, expression, and localization data obtained in a species living in a highly acidic environment that requires amplification of pH regulatory processes. Of course, there remain unanswered questions concerning the means through which these changes in expression and transport are achieved. Likewise, proof is needed to show that the mechanisms used by the Osorezan dace under extreme conditions are identical, or similar, to those mediating pH regulation under the less stressful conditions faced by most fish on a day-to-day basis. Nevertheless, this study illustrates an important approach and provides a valuable model with which to address such questions.
A recent study by Piermarini et al. (17) indicates that another transporter, the recently discovered Cl-/HCO3- exchanger pendrin, a member of the SLC26 family of anion exchangers, may also play a role in both acid-base regulation and osmotic balance. They showed that the Atlantic stingray, a euryhaline elasmobranch, expresses a pendrinlike protein at the apical membranes of certain gill epithelial cells. These cells also express high levels of basolateral V-H-ATPase, but not Na+-K+-ATPase. Moreover, this exchanger is expressed more intensely and in more cells over a wider area in the gills (both lamellae and interlamellar regions) in freshwater rays than in seawater-adapted animals. Pendrin expression also appears to be, at least in part, subapical in the seawater animals. These results suggest that pendrin is poised to mediate both base elimination and chloride uptake in the freshwater animal and that its lower expression at the apical membrane of the gill of the seawater-adapted ray is consistent with its need to reduce chloride uptake. Again a number of interesting issues remain to be addressed, in particular, a direct demonstration that stingray pendrin is an anion exchanger like its mammalian relatives and determination of the role, if any, of this transporter in teleost gill. Nevertheless, this provocative observation suggests that pendrin, and perhaps other SLC26A transporters, may contribute to ion and acid-base regulation by the gill.
Osmotic Regulation
As in the studies of pH regulation presented above, much of the work on osmotic and ionic regulation has capitalized on imposed salinity changes to amplify critical transporter proteins in ionic and osmotic homeostasis.
Saltwater. As reviewed by Marshall (13), the broad features of the processes and proteins involved in adaptation to a hyperosmotic environment are now quite well understood. Sodium and chloride are actively secreted by the gill epithelium to compensate for diffusive water loss and the resulting necessity to drink hypersaline water. The process is driven by basolateral Na+-K+-ATPase. Chloride is transported (a secondary active process) across the basolateral membrane into the chloride cell and exits passively through an apical channel. Sodium follows down its electrochemical gradient through a paracellular route between chloride cells and adjacent accessory cells. Many aspects of the development of this model may be followed through the pages of American Journal of Physiology-Regulatory, Integrative and Comparative Physiology from the ultrastructural work of Karnaky et al. (8) to the primary cultures of McCormick (14) that allowed hormonal control of salinity adaptation to be studied in vitro. More recent studies have focused on the cellular and molecular aspects of adaptation to high salinity. Increasing evidence indicates that there are multiple mitochondria-rich cell subtypes that vary with environmental salinity (5, 22) and that expression of specific Na+-K+-ATPase isoforms also change on adaptation to high salinity (4, 10). In both cases, the functional significance of these observations remains to be established. Fitting more easily into the above model are the studies of Pelis et al. (15) who used immunological techniques to show that an Na+-K+-2Cl- cotransporter was upregulated on acute seawater adaptation and that it colocalized with Na+-K+-ATPase in the chloride cells. Furthermore, during the natural parr-smolt transformation process, Na+-K+-2Cl- cotransporter expression increased through the smolt stage and decreased in the postsmolt animals that did not reach salt water. Thus it appears that the gill Na+-K+-2Cl- cotransporter taps the out-in sodium gradient to drive basolateral chloride entry and raise intracellular chloride above its electrochemical equilibrium. With this driving force, a simple channel should be adequate to mediate the apical step in chloride secretion. In fact, Singer et al. (19) were able to clone a homolog of a cystic fibrosis transmembrane conductance regulator from killifish gill and demonstrate that like the sodium pump and the Na+-K+-2Cl- cotransporter, its expression was upregulated after transfer of the fish to seawater.
Freshwater. The broad outlines of osmoregulation in the face of a hyposmotic environment are generally accepted, but many of the details are uncertain (13, 16, 18). Apical Na uptake takes place via a channel (possibly ENaC) down the electrochemical gradient created by an apical proton pump and the basolateral Na+-K+-ATPase. Chloride entry is thought to be mediated by apical exchange for bicarbonate. A good portion of the uncertainty regarding freshwater ion regulation arises from the sometimes conflicting evidence regarding not only the specific transporters involved, but even the cell type responsible. As noted above there are multiple cell types, apparently including subclasses of both pavement cells and chloride cells (5, 22). Furthermore, there is considerable disagreement as to whether freshwater ion uptake is the exclusive domain of the chloride cells or if the pavement cells also contribute (9, 13, 16, 24). Wood and colleagues (9, 24) have begun to address this issue directly by devising culture conditions under which to study the function of each cell type individually and in coculture. These studies suggest that both cell types do indeed play a role in ion uptake and in slowing diffusive ion loss, particularly in the presence of cortisol, or cortisol and prolactin. Under the conditions used thus far, absorptive fluxes are far lower than those seen in vivo. Nevertheless, for the reasons discussed above, this approach is interesting and, particularly, if applied in concert with molecular tools, holds a great deal of promise for unraveling some of the mysteries still associated with adaptation to freshwater.
FOOTNOTES
Address for reprint requests and other correspondence: J. B. Pritchard, Laboratory of Pharmacology and Chemistry, 110 Alexander Dr., MD F1-03, Research Triangle Park, NC 27709 (E-mail: pritchard{at}niehs.nih.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
-mRNA and -protein levels. Am J Physiol Regul Integr Comp Physiol 278: R101-R110, 2000.-subunit in gills of the teleost Oreochromis mossambicus. Am J Physiol Regul Integr Comp Physiol 275: R926-R932, 1998.This article has been cited by other articles:
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  B. E. Shmukler, C. E. Kurschat, G. E. Ackermann, L. Jiang, Y. Zhou, B. Barut, A. K. Stuart-Tilley, J. Zhao, L. I. Zon, I. A. Drummond, et al. Zebrafish slc4a2/ae2 anion exchanger: cDNA cloning, mapping, functional characterization, and localization Am J Physiol Renal Physiol, October 1, 2005; 289(4): F835 - F849. [Abstract] [Full Text] [PDF]
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