Submitted to the Department of Biology, Lafayette College, May, 1986.The sailfin molly is a euryhaline cyprinodont fish which hyperosmoregulates in concentrated sea water media. In order to test the hypothesis that osmoregulatory ion transport into and out of the fish is powered by gill Na, K-ATPase (sodium pump), salinity-induced changes in gill filament Na, K-ATPase enzyme specific activity (ESA) were measured. The enzyme was characterized for optimal concentrations of K, Mg, ATP and optimal pH. Fish were acclimated for one month in one of five media: fresh water, 17.5%, 30%, 57.5% or 75% sea water (SW). Minimal ESA was found in gills from fish in 30% SW, a medium in which the plasma is approximately isoosmotic to the external medium. Fish from more dilute media and more concentrated media had significantly higher gill ESA than 30% SW fish. These findings support the hypothesis that both inward ion transport in dilute media and outward ion transport in concentrated media are driven by gill epithelium Na, K-ATPase.
Introduction
Living organisms can be described as aqueous solutions contained within a membrane, the body surface (Schmidt-Nielsen, 1983). The composition of these aqueous solutions must be maintained within narrow limits; large deviations from these levels often cannot be tolerated (e.g.,Claude Bernard's principle of homeostasis). Organisms are frequently faced with external environments very different from their own internal environments. In these cases the problem for the organism is maintaining its internal environment in the face of both chemical and electrical gradients which may induce fluxes of various solutes and water into or out of the organism. Further, since the organism is not impermeable, it is sometimes necessary to use energy (active transport) to move these solutes against their electrochemical gradients to maintain homeostasis.
With the exception of the hagfishes and sharks, most fishes maintain an internal concentration approximately equivalent to 30% sea water (Evans, 1979). Therefore, fish in both fresh water and sea water are constantly faced with the problem of ionic and osmotic regulation. In fresh water the fish must deal with osmotic water uptake and loss of solutes through permeable epithelial tissues and via the urine, while in sea water it experiences osmotic water loss and solute uptake through permeable epithelial tissues. Since the teleost kidney is not capable of forming a concentrated urine for solute excretion in sea water and some salts are lost to the environment in the urine in fresh water, another organ must be utilized for solute regulation. This organ is the gill.
The active transport of the major plasma cation, sodium, has been linked to a system utilizing an enzyme known as Na, K-dependent, Mg -requiring, adenosine triphosphatase (Na, K-ATPase). The enzyme is an integral membrane protein containing two alpha and two beta subunits with a total molecular weight for the unit of approximately 250,000 daltons (Kepner and Macey, 1968). The enzyme is a ubiquitous component of animal cell membranes. Na, K-ATPase uses energy from the hydrolysis of ATP to move sodium out across the cell membrane while a counterion (generally K) is transported into the cell (Glynn and Carlish, 1975).
Euryhaline organisms, which can survive in a wide range of salinities, often show an increase in gill Na, K-ATPase activity with variations in the external above or below internal concentrations. Towle et al.(1977) and Jacob and Taylor (1983) showed that the teleost, Fundulus heteroclitus, increased its gill Na, K-ATPase activity when acclimated in sea water concentrations greater or less than its blood osmotic concentration. The ability of the same sodium pump to drive both gill salt excretion in a sea water environment and gill salt uptake in a fresh water environment has been questioned. Butler and Carmichael (1972) suggested that sodium transport across the gills of the eel, Anguilla rostrata, is a reversible process powered by the same transport system. The finding that the Na, K-ATPase molecule exhibits a distinct polarity and is restricted to the basolateral cell membrane (Karnaky et al.,1976; Towle et al.,1983) has provided major insight into the proposed mechanism of ion exchange (Fig. 1).
Note that there are two different mechanisms involved, one for absorption of salts in fresh water and one for secretion of salts in sea water and that both mechanisms depend on the basolateral Na, K-ATPase enzyme to power them. The proposed mechanisms of exchange is based on a series of findings.
First, the Na. K-ATPase enzyme is located in the basolateral membrane, as previously mentioned. Epstein et al.,(1980) and Richards and From (1970) proved this by subjecting intact fish to both internally- and externally-applied ouabain, a specific inhibitor of Na, K-ATPase. Ouabain in the external medium had no inhibitory effect on ion exchange, while intraperitoneal injections of ouabain profoundly inhibited the fluxes of both sodium and chloride, thus documenting that the enzyme must have a basolateral location. Karnaky et al.(1976) also demonstrated the basolateral location of the enzyme through tritiated ouabain autoradiography.
Second, the hypothesis that chloride is exchanged for bicarbonate in the branchial epithelium has been widely confirmed (Maetz and Romeu, 1964; Kerstetter and Kirschner, 1972), while the existence of Na+/NH4+ exchange remains controversial. Maetz and Romeu showed initially that Na+ was exchanged for NH4+ in the gills of the goldfish, Carassius auratus,while Kerstetter et al.(1970) showed that increased sodium concentration in the external medium affected the efflux of acid (H+) but not NH4+, therefore giving rise to the proposed Na+/H+ coupled exchange. Maetz (1973) then suggested that Na+ influx in fresh water goldfish is equivalent to the combined efflux of both NH4+ and H+. Recent work, however, on the perfused, isolated trout head has shown that almost all Na+ influx is specifically linked with NH4+ excretion (Payan, 1978, and Payan, Matty and Maetz, 1975).
Finally, morphological changes in gill epithelium have been found in fish acclimated in different environmental salinities, suggesting that chloride cells may increase their permeability to ions during adaptation to sea water (Hwang and Hirano, 1985). This study revealed that in a high salinity environment chloride cell interdigitations degenerated between adjacent cells, thus producing a "leaky" junction. No such leaky junctions were present in fresh water adapted fish. This morphological evidence suggests that the gill may have an increased permeability to ions in sea water than in fresh water. The authors also noted that the chloride cell in juvenile flounder gills starts developing of degenerating interdigitations (= leaky junctions) within three hours after transfer to a new salinity. Girard and Payan (1980) also noted this increased permeability of gill epithelium, specifically the primary lamellar epithelium, by monitoring influxes of radioactive isotopes (24Na, 36Cl, 14C-mannitol) from the external medium in sea water adapted trout.
The two mechanisms for ion exchange, absorption and secretion, are thought to occur within two different cell types in the teleost gill, the respiratory cell and the chloride cell, respectively (Girard and Payan, 1980). Much evidence has been found to support the function of the chloride cell as the site of active ion transport in sea water fish gills ever since Keys and Wilmer (1932) discovered it. This cell shows common characteristics with other known transport systems in epithelial tissues: increased basolateral cell surface area and numerous mitochondria (Philpott, 1968). As noted previously, chloride cell morphology changes directly with varying salinities (Hwang and Hirano, 1985). Shira and Utida (1970) demonstrated that chloride cells of sea water adapted teleosts are larger and generally more numerous than those of fresh water adapted teleosts, while Karnaky, Ernst and Philpott (1976) showed a four-fold increase in Na, K-ATPase activity, chloride cell hypertrophy and proliferation of the basolateral membrane. Also, studies have shown that the Na, K-ATPase occurs in high concentration within these chloride cells (Langdon and Thorpe, 1984, Karnaky et al., 1976). Chloride cells also contain an extensive, three-dimensional network of branching tubules which are invaginations of the basolateral cell surface (Philpott, 1968). Interstitial fluid found within the tubules is in intimate contact with the cell membrane and tubular cell surfaces, thus increasing the surface area for ion exchange with interstitial fluid. These tubules have been found to increase in number upon adaptation to sea water (Karnaky et al.,1976).
Recent studies reveal that the two cell types (respiratory and chloride cells) are found in two anatomically distinct locations within the gill with very different exposures to the blood. Gill blood circulation has been studied in numerous teleosts and the existence of two blood pathways in each gill filament is well documented (Girard and Payan, 1966; Steen and Kruysse, 1964). The two vascular spaces present in the gill circulation are limited by distinct epithelia (Laurent and Dunel, 1978). Respiratory cells are found strictly in the epithelium of the secondary lamellae which is supplied by arterial blood flow, while chloride cells are typically found in the epithelium bordering the sinus of the primary lamellae and are supplied with venous blood. These positional differences add support to the proposed ion exchanged mechanisms, since respiratory cells (which are involved in gas exchange, nitrogenous waste removal and acid-base balance) are supplied by an arterial blood flow and they exchange sodium and chloride for ammonia and bicarbonate, respectively, in the secondary lamellae. Chloride cells, which deal strictly with salt excretion, are located in the primary gill lamellae, away from the respiratory cells. Thus, the changes associated with adaptation to sea water or fresh water environments are: 1) an increase or decrease in the number an/or size of chloride cells, 2) an increase or decrease in the Na, K-ATPase activity within the chloride cell and 3) the development or degeneration of interdigitations between the chloride cell and adjacent cells. These changes occur specifically in the primary lamellae of the teleost gill and not in the secondary lamellae (Girard and Payan, 1980).
The present study was undertaken to determine the osmoregulatory performance of the black molly, Poecilia latipinna, to examine some of the properties of its gill Na, K-ATPase and to determine changes in enzyme activity in each of the four gills as a function of acclimation to media of different salinities. P. latipinnawas chosen for study because it is known to be a successful osmoregulator (Evans, 1975).
The picture of a male sailfin molly at the top of this page is taken from Aquarium Atlas,by Rudiger Reihl and Hans A. Baensch, MERGUS-Verlag Hans A. Baensch, 1986, Melle, Germany.