Branchial mitochondria-rich cells in the dogfish Squalus acanthias

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Abstract

In marine teleost fishes, the gill mitochondria-rich cells (MRCs) are responsible for NaCl elimination; however, in elasmobranch fishes, the specialized rectal gland is considered to be the most important site for salt secretion. The role of the gills in elasmobranch ion regulation, although clearly shown to be secondary, is not well characterized. In the present study, we investigated some morphological properties of the branchial MRCs and the localization, and activity of the important ionoregulatory enzyme Na+/K+-ATPase, under control conditions and following rectal gland removal (1 month) in the spiny dogfish, Squalus acanthias. A clear correlation can be made between MRC numbers and the levels of Na+/K+-ATPase activity in crude gill homogenates (r2=−0.69). Strong Na+/K+-ATPase immunoreactivity is also clearly associated with the basolateral membrane of these MRCs. In addition, the dogfish were able to maintain ionic balance after rectal gland removal. These results all suggest a possible role of the dogfish gill in salt secretion. MRCs were, however, unresponsive to rectal gland removal in terms of changes in number, fine structure and Na+/K+-ATPase activity, as might be expected if they were compensating for the loss of salt secretion by the rectal gland. Thus, the specific role that these MRCs play in ion regulation in the dogfish remains to be determined

Introduction

In marine teleost fishes, the gill contributes significantly to whole animal ion regulation, through active NaCl elimination. Within the branchial epithelium, a population of mitochondria-rich cells (MRCs) or chloride cells has been identified as the cell-type involved in the active Cl efflux (Foskett and Scheffey, 1982). High activities of the basolateral sodium pump (Na+/K+-ATPase), associated with these cells, are important in providing the driving force for Cl entry via a basolateral Na+/K+/2Cl cotransporter, while the accumulating intracellular Cl exits apically via a CFTR-like Cl channel (see Marshall and Bryson, 1998). The transepithelial potential is sufficient to drive Na+ efflux on a paracellular route via leaky tight junctions, between mitochondria-rich chloride and accessory cells (Sardet et al., 1979).

In the cartilaginous fishes, specifically the elasmobranchs, the role of the gills in active ion regulation is quite clearly secondary to the specialized salt secreting rectal gland (Burger and Hess, 1960) and they are actually the site of net NaCl uptake (see Shuttleworth, 1988). The mechanism of NaCl elimination in the rectal gland parenchymal cells is the same as has just been described in the teleost gill MRCs above (see Riordan et al., 1994). Interestingly, MRCs have also been characterized in the gills of elasmobranches, however, with some notable morphological differences (Doyle and Gorecki, 1961, Hughes and Wright, 1970, Wright, 1973, Haywood, 1975, Laurent and Dunel, 1980, Crespo et al., 1981, Crespo, 1982). The amplification of the basolateral membrane domain in teleost fishes is produced by an extensive tubular system, while in elasmobranches the plasma membrane is heavily folded into a basal labyrinth. Also, the elasmobranch gill has a low permeability (Evans, 1979) and Na+ efflux (Bentley et al., 1976, Horowicz and Burger, 1968), which is likely to be associated with an absence of the MR accessory cell type and its associated leaky tight junctions. There is, however, some indirect evidence suggesting that the elasmobranch gill has an active Cl efflux (Bentley et al., 1976).

In experiments in which the rectal gland had been rendered non-functional by ligation or excision, dogfish have been able to maintain plasma electrolytes at normal levels after prolonged periods (1 month) (Burger, 1965, Evans et al., 1982, Evans, 1993). Chan et al. (1967) have shown, in the rectal gland ectomised lip shark (Hemiscyllium plagiosum), that the muscle acts as a buffer for salt loading, but this would have a limited role in the long term. Urine flow rates were found to double in rectal glandless fish, however, Cl levels remained isotonic to plasma and the kidney still could not completely compensate for the loss of rectal gland function (Burger, 1962, Burger, 1965). The gills contribute the remaining component by either increasing efflux or decreasing influx. Presumably, the small population of MRCs in the epithelium performs branchial NaCl elimination and they are able to up-regulate their activity.

In the study by Chan et al. (1967), Na+ ion efflux did not increase in response to a salt load, leading to the conclusion that the gill does not contribute significantly to ion homeostasis in the absence of a functional rectal gland. However, it should be noted that the gills have a much larger Cl efflux (∼5-times; Bentley et al., 1976, Evans et al., 1982) that may be active (Bentley et al., 1976) and, since only Na+ and not Cl fluxes were measured, it remains uncertain as to whether the gills do have a significant role in active ion regulation.

In the present study, the possibility that branchial MRCs are mobilized to help maintain plasma electrolyte levels in the absence of a rectal gland was investigated. In addition to the measurement of plasma osmolytes (Na+, Cl, K+, urea), gill Na+/K+-ATPase activity and branchial MRC numbers [no. MRCs per interlamellar space (ILS)] were measured. The gill epithelium was observed using scanning and transmisson electron microscopy to determine if any subcellular changes were taking place. Na+/K+-ATPase was also immunolocalized in gill tissue using a mouse monoclonal antibody to the α subunit of chicken Na+/K+-ATPase, to determine the tissue distribution.

Section snippets

Animals

Dogfish, Squalus acanthias, were obtained from commercial trawlers by hook and line, and housed in a large circular tank (288 m3) with flowing seawater (10.5 °C, 31‰), at the Bamfield Marine Station (Bamfield, BC, Canada). Mean body mass (mean±S.E.M., n) was 2.86±0.36 kg, n=12.

Surgery

Fish were anaesthetized with MS-222 (1:10000 tricaine methanesulfonate; Syndel Laboratories Ltd., Richmond, BC, Canada) and then placed on an operating table while the gills were irrigated with aerated seawater containing

Results and discussion

This is the first study to demonstrate a clear relationship between branchial mitochondria-rich cells and the important ionoregulatory enzyme, Na+/K+-ATPase, in the dogfish. The elasmobranch gill is capable of unidirectional Cl effluxes ranging from 20–90% of uptake rates (see Shuttleworth, 1988) and the localization of Na+/K+-ATPase to the MRC indicates that they are likely responsible for the elimination of Cl.

The removal of the rectal gland, the otherwise primary organ for NaCl

General discussion

Branchial mitochondria-rich cells in dogfish display high levels of Na+/K+-ATPase immunoreactivity and the activity measured in crude gill homogenates can be correlated with numbers of these cells. We have thus established that there is a close relationship between Na+/K+-ATPase and the MRC type in this cartilaginous fish. This relationship has long been established in teleost fish gills. However, unlike teleost fish MRCs, the branchial MRCs of this cartilaginous fish do not appear responsive

Acknowledgments

This work was supported by an NSERC operating grant to DJR and a university fellowship to JMW. We would also like to thank the late John Boom for help in setting up this study.

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