Intestinal Salt Absorption in the Stenothermic Antarctic Fish, Notothenia Rossii

Osmoregulation in marine teleosts involves replenishment of branchial water loss by intestinal absorption of salt and water with subsequent Na⁺ excretion by the gills (reviewed by Ellory & Gibson, 1983). It is, therefore, of interest to determine the rate of salt transport in the anterior intestine (as a major site of osmoregulation) of an Antarctic fish species, Notothenia rossii, at 4°C and compare it with that of temperate marine species at their ambient temperature. Transport across fish intestine follows the classical Prechtian pattern of temperature adaptation (Precht, 1958; Smith, 1976; Gibson, Ellory & Cossins, 1985), making this comparison with temperate species a test of the energy-saving theory.

It is also pertinent to ask if Antarctic fish intestine is a Cl⁻-transporting epithelium in the Na⁺,K⁺,Cl⁻-coupled pattern present in other marine teleosts such as plaice and flounder (Field et al. 1978; Ramos & Ellory, 1981; Gibson, 1985). Early work by House & Green (1965) on Cottus, a Northern hemisphere species which could be regarded as closer to Antarctic fish than the flat fishes, indicated possible differences in the mechanism of epithelial transport with small positive values for transepithelial potential difference (PD).

In the present paper, therefore, we have measured electrical parameters and transepithelial Cl⁻ fluxes in the anterior intestine of Notothenia rossii. Cl⁻ absorption was followed in preference to Na⁺ absorption because of its more central role in osmoregulation in other teleosts (e.g. Hirano, 1974; Lau, 1982). The effects of mucosal application of bumetanide, to inhibit the apical cell membrane Na⁺,K⁺,Cl⁻ cotransporter (Field et al. 1978; Ramos & Ellory, 1981), and amphotericin B, to reveal electrogenic basolateral cell membrane Na⁺ pumps mediated through its effect on increasing mucosal Na⁺ permeability (Reuss, 1981; Ellory, Lahlou & Ramos, 1981), were also tested.

Antarctic fish, Notothenia rossii, collected in South Georgia, were obtained on the day of use from British Antarctic Survey, Madingley Road, Cambridge, where they were kept in aerated recirculating sea water at 4°C and fed daily on shrimps. Pieces of stripped intestine were mounted in conventional Ussing chambers (Ramos & Ellory, 1981) 0·7 cm² in area under voltage-clamp conditions. Construction of electrodes and circuits has been described previously (Ramos & Ellory, 1981), except that calomel electrodes were found to give better stability if made and kept at 4°C.

Saline for Antarctic fish was modified from that described by O’Grady et al. (1983) and had the following composition (in mmoll⁻¹): NaCl, 250; KCl, 5; MgSO₄, 3; NaHCO₃, 2; CaC1₂, 2·5; L-alanine, 2·5; D-glucose, 5; buffered with Mops, 5; and Tris, 10; and adjusted with Tris base to give a pH of 7·8 at 4 °C. It was bubbled with O₂ during the experiments. Bumetanide (Leo Pharmaceutical Products, Aylesbury, Bucks) was used at 40μmoll⁻¹ final concentration in saline. Amphotericin B (Fungizone: E. R. Squibb & Sons Inc., Princeton, NJ) was used as a saturated suspension at a nominal concentration of 80μgml⁻¹.

Bidirectional Cl⁻ fluxes were measured using ⁷⁷Br⁻ (Medical Research Council Cyclotron Unit, Hammersmith Hospital, London) and ³⁶C1⁻ (Amersham International, Amersham, Bucks), see Ramos & Ellory (1981). Preliminary experiments showed that ⁷⁷Br⁻ behaved as a Cl^- tracer in nototheniids, as observed in other teleosts (Ramos & Ellory, 1981; Gibson, 1985).

Samples of anterior intestine from N. rossii gave consistent results for electrical parameters with negative short-circuit currents (SCCs) and PDs of —19·5 ± 1·9μAcm⁻² (±S.E.M., N = 28) and — l·8±0·2mV (N = 28), respectively, with a transepithelial resistance of 94 ± 10 Ωcm² (N = 28). The tissues proved to be very stable maintaining these values over at least 2h following the initial equilibration period, during which SCC and PD usually drifted to more negative values by up to 30%.

From the tracer experiments, there was a net chloride absorption of 2·91 ± 0·51μmol cm⁻² h⁻¹(N = 16) with J_ms and J_5m being 6·57±0·62 (N= 16) and 3·66 ±0·48 (N = 16), respectively.

The application of bumetanide (40μmoll⁻¹) in the mucosal solution produced an immediate fall in SCC and PD to less than 20% of control values within 60 min and with a half-time of about 20 min (Fig. 1). Transepithelial resistance did not change significantly, the control value was 90 ± 13 Ωcm²(N = 16) and that after bumetanide was 93 ± 20 Ωcm²(N = 15).

Net chloride absorption was also inhibited, falling to 1·65 ±0·31 μmolcm⁻²h⁻¹ after 60min. Inhibition was achieved by a fall in J_ms from 6·57 ± 0·62μmol cm⁻²h⁻¹(N = 16) in the control period to 4·88 ± 0·56μmolcm⁻²h⁻¹ (N = 16) after 60min with bumetanide, without J_sm changing significantly, 3·66 ±0·48 (N= 16) and 3·25 ± 0·37μmol cm⁻²h⁻¹(N = 16), respectively.

In 12 tissues, amphotericin B (80μgml⁻¹) was applied to the mucosal solution, following previous addition of bumetanide. SCC and PD were stimulated to positive values, rising from — 5·9±4·0μA (N = 12) and —0·3±0·2mV (N = 12) following bumetamide to + 12·0 ± 3·1μA cm⁻²(N = 12) and +1·1 ±0·2 mV (N= 12) after 60 min in amphotericin. Again transepithelial resistance was unaffected, with values of 154 ± 30Ωcm²(N = 12) before, and 140 ± 24Ωcm²(N = 12) after, amphotericin B.

Intestinal Cl⁻ transport of Antarctic nototheniids at 4°C would thus appear to be similar to that of temperate marine teleosts measured at room temperature, samples of stripped anterior intestine mounted in Ussing chambers showing a negative SCC and PD with a net Cl⁻ absorption in excess of the current, which indicates a significant net movement of other ions, presumably net Na⁺ absorption.

PD, SCC and Cl⁻ absorption were all inhibited by mucosal application of the loop-diuretic, bumetanide, the effect on Cl⁻ transport being mediated via a reduction in J_ms, without a change in J_sm, as observed in plaice for piretanide (Ramos & Ellory, 1981). The ionophore amphotericin B stimulated SCC and PD to positive values, consistent with the presence of an electrogenic Na⁺ pump in the basolateral cell membrane (Graf & Giebisch, 1979; Reuss, 1981). Furthermore, it is known from isolated intestinal sac experiments that fluid absorption in the Antarctic fish Pagothenia borchgrevinki (Boulenger) is reduced by serosal application of ouabain (O’Grady et al. 1983).

In other marine teleosts (Field et al. 1978; Ramos & Ellory, 1981; Ellory et al. 1981; Gibson, 1985) similar findings have been attributed to the function of a basolateral cell membrane Na⁺,K⁺-ATPase coupled to a bumetanide-sensitive Na⁺,K⁺,C1⁻ cotransporter at the apical cell membrane.

Fig. 2 compares nototheniid transport parameters measured at 4 °C with those of temperate marine teleosts measured at 22°C. The magnitudes of the fluxes, at the respective experimental temperatures, are similar, and since one would expect fluxes in temperate fish to be considerably reduced at 4°C compared with that at room temperature (Smith, 1976; Gibson et al. 1985) or with respect to nototheniids, Antarctic fish must, therefore, possess cellular adaptations to account for the relatively high Cl⁻ transport rates at 4°C. Conventional models of epithelial transport suggest that the main regulatory sites include the permeability of the apical and basolateral cell membranes, the intercellular junctions, and the basolateral cell membrane Na⁺ pumping capacity (e.g. Field et al. 1978). Previous work on temperature acclimation on teleost intestine has ascribed a principal role to the activity of the Na⁺,K⁺-ATPase (Smith & Ellory, 1971 ; Gibson et al. 1985) resulting from changes in turnover rate, membrane fluidity or ATP supply (e.g. Smith & Ellory, 1971; Cossins, 1983; Wodtke, 1981). Metabolic compensations do occur in Antarctic fish (Scholander et al. 1957; Holeton, 1974) but further studies are required to elucidate the adaptive strategy of Antarctic fish intestine.

In conclusion, the intestinal component of osmoregulation in nototheniids would appear to be uncompromised. The present results suggest that intestinal NaCl transport is no different, either qualitatively or quantitatively, from that of other temperate marine teleosts except that it is very well adapted to function at low temperatures. The mechanism of this adaptation deserves further study.

JSG thanks the MRC for support during this work. The Antarctic fish were kindly donated by British Antarctic Survey, Madingley Road, Cambridge.