Effects of heavy metals on osmoregulation in aquatic organisms have recently been reviewed by Bouquegneau & Gilles (1979). Whereas most of the data in the literature deal with the toxic effects of mercurials, only a few reports can be found on the toxicity of essential trace elements such as Cu and Zn. Rainbow trout exposed to lethal (Skidmore, 1970) and sublethal (Watson & Beamish, 1980) concentrations of Zn maintain a relatively constant internal ionic environment. On the other hand Lewis & Lewis (1971) showed, in the channel catfish, a decreased osmolarity of blood serum after treatment with either Cu or Zn, and Katz (1979) reported an increased Na efflux in freshwater teleosts exposed to heavy metals. An increased gill Na, K-ATPase activity was described in the Zn-treated rainbow trout (Watson & Beamish, 1980). Shephard & Simkiss (1978) showed, in the same species, an increase in the gill protein content of fish exposed to Cu and Zn. These data agree with reports from Cu-treated winter flounder (Baker, 1969) and from Zn-treated dogfish (Crespo, Soriano, Sam-pera & Balasch, 1981; Crespo, 1982) each of which shows an increase in the number of the gill chloride cells.

The opercular epithelium of the killifish, Fundulus heteroclitus, is a flat epithelium with a high density of chloride cells (Karnaky & Kinter, 1977) identical in ultrastructure to that of the gill chloride cells (Karnaky, Kinter, Kinter & Stirling, 1976). This preparation has been studied under short-circuit current conditions in a lucite chamber and proposed as an in vitro model for gill osmoregulatory function (Karnaky, Degnan & Zadunaisky, 1977; Karnaky, 1980). The short-circuit current (Isc) across the epithelium is attributed uniquely to the active transport of chloride ions from the blood to the seawater side of the epithelium (Karnaky et al. 1977 ; Degnan, Karnaky & Zadunaisky, 1977). There is a direct correlation between the number of chloride cells and the Isc, demonstrating that these cells are responsible for chloride secretion (Karnaky et al. 1979).

To investigate the effects of Cu and Zn on chloride transport across the opercular epithelium of 100% seawater-adapted Fundulus heteroclitus, electrical parameters were recorded following the addition of small aliquots (20–200 μl), of Cu (CUSO4. 5H2O) or Zn (ZnSO4. 7H2O) in Ringer’s solution either to both mucosal and serosal sides of the preparation (M + S) or to one side only. Procedures for the dissecting and mounting of the operculum, and descriptions of the Lucite chambers and Ringer’s solution are presented in detail elsewhere (Degnan et al. 1977). The epithelia used for the present study reached a steady-state within 30 min and thereafter exhibited an average spontaneous decay of 11 %/30min.

Table 1 shows that the addition of either Cu or Zn (4 × 10−5 M) to both sides of the preparation caused a statistically significant decrease in both short-circuit current (Isc) and transepithelial potential difference (P.d.). Transmural resistance (R), however, remained unaltered following exposure to either heavy metal. Concentrations as low as 10−5 M (M + S) caused a significant inhibition of the Isc(P< 0·05, n = 3). Initial Isc values were recovered after washing (×3) with Ringer’s solution. When heavy metals were added to the mucosal side only, no inhibition of K was detected, even at higher doses (5 × 10−4 M ; n = 6). After addition of 4 × 10−5 M-CU or Zn to the serosal side only, the pattern for Isc inhibition was the same as the one recorded for M + S exposure (Fig. 1). Zn caused a greater inhibition of the Isc (P<0·02) and P.d. (P <0·01) than did Cu (Table 1).

Table 1.

The effects of Cu and Zn on the electrical properties of the opercular epithelium of seawater-adapted Fundulus heteroclitus

The effects of Cu and Zn on the electrical properties of the opercular epithelium of seawater-adapted Fundulus heteroclitus
The effects of Cu and Zn on the electrical properties of the opercular epithelium of seawater-adapted Fundulus heteroclitus
Fig. 1.

The effect of Cu on the electrical properties of the short-circuited opercular epithelium of seawater-adapted Fundulus heteroclitus. A 4 × 10−5M addition to the serosal side only (epithelium 1) results in marked inhibitions of the Isc (continuous-line) and the P.d. (dots), both of which recover to control values after washing (×3) with Ringer’s solution. This inhibition is absent from mucosal only additions (epithelium 2). The inhibition by Zn (not shown) is also restricted to the serosal side, but is greater than that from equivalent concentrations of Cu.

Fig. 1.

The effect of Cu on the electrical properties of the short-circuited opercular epithelium of seawater-adapted Fundulus heteroclitus. A 4 × 10−5M addition to the serosal side only (epithelium 1) results in marked inhibitions of the Isc (continuous-line) and the P.d. (dots), both of which recover to control values after washing (×3) with Ringer’s solution. This inhibition is absent from mucosal only additions (epithelium 2). The inhibition by Zn (not shown) is also restricted to the serosal side, but is greater than that from equivalent concentrations of Cu.

From our results it is apparent that low concentrations of Cu or Zn can affect chloride transport across the opercular epithelium of the seawater-adapted Fundulus heteroclitus. In contrast to our findings for Cu and Zn, Hg inhibits Isc across the killifish opercular epithelium after mucosal as well as serosal exposure (Degnan & Miller, 1980). The absence of effects from the mucosal side argues against the direct action of these heavy metals on the chloride cell apical membrane. Likewise, these data suggest that these heavy metals do not rapidly penetrate the opercular epithelium.

The fact that Cu and Zn are effective only when added to the serosal side suggests that these metals might interact with Na,K-ATPase located on the basolateral membrane of the chloride cell (Karnaky et al. 1976).

To test the effects of Cu and Zn on the Na pump we studied the in vitro inhibition of the specific activity of Na,K-ATPase. 25µl crude gill homogenates (2–3 mg protein/ml) treated with 0·001% Na deoxycholate were initially preincubated for 10 min at 37 °C in a final volume of 0·5 ml [0·4 mM-EGTA (pH 8·1), 10 mM-Na azide, 2·5mM-MgCl2, 104mM-NaCl, 16mM-KCl, 40mM-Tris-HCl (pH 7·2)] with or without heavy metals. The reaction was started by the addition of Mg-Tris-ATP (3 mM-ATP, pH 7·5) and stopped with 2% trichloroacetic acid. The inorganic phosphate liberated by ATP hydrolysis was determined according to the method of Fiske & Subarrow (1925). Na,K-ATPase activity was calculated as the difference between ATPase activity in the presence and absence of 0·3 mM-ouabain. Protein content of the homogenate was determined according to the method of Lowry, Rosebrough, Farr & Randall (1951).

Fig. 2 shows the dose response curves for Zn and Cu. A statistically significant (P<0·02) decrease in Na,K-ATPase specific activity was first detected following exposure to 10−5M-Zn and 10−4M-Cu, which caused inhibitions of 7 % and 20%, respectively. These results suggest that the inhibition of chloride secretion (Isc) across the opercular epithelium of seawater-adapted Fundulus heteroclitus following exposure to 4 × 10−5 M-Cu or Zn is only partially due to an inhibition of the Na pump. Moreover, Cu and Zn dose response curves for the inhibition of enzyme activity do not differ statistically from each other, yet Zn causes a greater inhibition of the Isc and P.d. than does Cu. The striking difference in responses to these heavy metals suggests that the Na pump is not the only mechanism involved in their toxicity.

Fig. 2.

The influence of Cu and Zn on Na,K-ATPase from crude whole gill homogenates of seawater-adapted Fundulus heteroclitus. Specific activity is expressed as mean ± S.E.M. as a percentage of the control

Fig. 2.

The influence of Cu and Zn on Na,K-ATPase from crude whole gill homogenates of seawater-adapted Fundulus heteroclitus. Specific activity is expressed as mean ± S.E.M. as a percentage of the control

According.to recent models of chloride cell function (Silva, Solomon, Spokes & Epstein, 1977 ; Ernst, Dodson & Karnaky, 1980) chloride is transported initially into chloride cells from the blood side by a Na-facilitated, neutral-coupled carrier at the basolateral interface. This ‘secondary’ active transport is driven by the low intracellular Na gradient established by the primary active transport of Na out of the cell by basolateral, plasmalemmal Na,K-ATPase. Clearly, Cu and Zn are potent inhibitors of chloride transport across the opercular epithelium. They inhibit Na,K-ATPase and may interact as well with the coupled NaCl carrier. Since the Isc across the killifish opercular epithelium is inhibited by a-adrenergic agents (Degnan et al. 1977; Mendelsohn, Cherksey & Degnan, 1981) Cu and Zn may also act directly on a-adrenergic receptors. Additionally, heavy metals are thought to alter permeability of the cell membrane (Rothstein, 1959; Kinter & Pritchard, 1977). Further work on this in vitro preparation may help elucidate the underlying mechanisms of Cu and Zn toxicity to electrolyte transport processes.

We would like to thank E. Bell for her technical assistance. We would also like to acknowledge L. Garretson, Dr R. Perez and Dr W. P. Dubinsky for their helpful comments. This work was supported by NIH grant GM 29099 to Dr Karnaky and C.I.R.I.T. grant (Generalitat de Catalunya) to Dr S. Crespo.

Baker
,
J. T. P.
(
1969
).
Histological and electron microscopical observations on copper poisoning in the Winter Flounder (Pseudopleuronectes americanus)
.
J. Fish Res. Bd Can
.
26
,
2785
2793
.
Bouquegneau
,
J. M.
&
Gilles
,
R.
(
1979
).
Osmoregulation and pollution of the aquatic medium
.
In Mechanisms of osmoregulation in animals
(ed.
R.
Gilles
), pp.
563
580
.
J. Wiley
.
Crespo
,
S.
(
1982
).
Surface morphology of dogfish (Scyliorhinus canicula) gill epithelium, and surface morphological changes following treatment with zinc sulphate: a scanning electron microscope study
.
Mar. Biol
.
67
,
159
166
.
Crespo
,
S.
,
Soriano
,
E.
,
Sampera
,
C.
&
Balasch
,
J.
(
1981
).
Zinc and copper distribution in excretory organs of the dogfish Scyliorhinus canicula and chloride cell response following treatment with zinc sulphate
.
Mar. Biol
.
65
,
117
123
.
Degnan
,
K. J.
,
Karnaky
,
K. J.
, Jr
. &
Zadunaisky
,
J. A.
(
1977
).
Active chloride transport in the in vitro opercular skin of a teleost (Fundulus heteroclitus) a gill-like epithelium rich in chloride cells
.
J. Physiol., Lond
.
271
,
155
191
.
Degnan
,
K. J.
&
Miller
,
D. S.
(
1980
).
Effects of HgCl2 on electrical characteristics of Fundulus heteroclitus opercular skin, a gill model
.
Bull. Mt. Desert Isl. Biol. Lab
.
20
,
114
116
.
Ernst
,
S. A.
,
Dodson
,
W. C.
&
Karnaky
,
K. J.
, Jr
. (
1980
).
Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus)
.
Cell Biol
.
87
,
488
497
.
Fiske
,
C.
&
Subarrow
,
Y.
(
1925
).
The colorimetric determination of phosphorus
.
J. biol. Chem
.
66
,
325
400
.
Karnaky
,
K. J.
, Jr
. (
1980
).
Ion secreting epithelia: chloride cells in the head-region of Fundulus heteroclitus
.
Am. J. Physiol
.
238
,
185
198
.
Karnaky
,
K. J.
, Jr
.,
Degnan
,
K. J.
&
Zadunajsky
,
J. A.
(
1977
).
Chloride transport across isolated opercular epithelium of killifish: A membrane rich in chloride cells
.
Science, N.Y
.
195
,
203
205
.
Karnaky
,
K. J.
, Jr
.,
Degnan
,
K. J.
&
Zadunajsky
,
J. A.
(
1979
).
Correlation of chloride cell number and short-circuit current in chloride-secreting epithelia of Fundulus heteroclitus
.
Bull. Mt. Desert Isl. Biol. Lab
.
19
,
109
111
.
Karnaky
,
K. J.
, Jr
. &
Kinter
,
W. B.
(
1977
).
Killifish opercular skin: a flat epithelium with a high density of chloride cells
.
J. exp. Zool
.
199
,
355
364
.
Karnaky
,
K. J.
, Jr
.,
Kinter
,
L. B.
,
Kinter
,
W. B.
&
Stirling
,
C. E.
(
1976
).
Teleost chloride cell. II. Autoradiographic localization of gill Na,K-ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments
.
J. Cell Biol
.
20
,
157
177
.
Katz
,
B.
(
1979
).
Relationship of the physiology of aquatic organisms to the lethality of toxicants: A broad overview with emphasis on membrane permeability
.
Aquat. Toxicol. (A.S.T.M.) Ref. STP
667
,
62
79
.
Kinter
,
W. B.
&
Pritchard
,
J. B.
(
1977
).
Altered permeability of cell membranes. In Handbook of Physiology. Section 9. Reactions to Environmental Agents
.
Am. Physiol. Soc
., pp.
563
576
.
Lewis
,
S. D.
&
Lewis
,
W. M.
(
1971
).
The effect of zinc and copper on the osmolality of blood serum of the channel catfish, Ictalurus punctatus Rafinesque, and golden shiner, Notomigonus crysoleucas, Mitchill
,
Trans. Am. Fish Soc
.
4
,
639
643
.
Lowry
,
O. H.
,
Rosebrouch
,
N. J.
,
Farr
,
A. L.
&
Randall
,
R. J.
(
1951
).
Protein measurement with the Folin phenol reagent
.
J, biol. Chem
.
193
,
265
275
.
Mendelsohn
,
S. A.
,
Cherksey
,
B. D.
&
Degnan
,
K. J.
(
1981
).
Adrenergic regulation of chloride secretion across the opercular epithelium: the role of cyclic AMP
.
J. comp. Physiol
.
145
,
29
35
.
Rothstein
,
A.
(
1959
).
Cell membrane as site of action of heavy metals
.
Fedn. Proc. Fedn. Am. Socs exp. Biol
.
18
,
1026
1038
.
Shephard
,
K.
&
Simkiss
,
K.
(
1978
).
The effects of heavy metal ions on Ca2+ ATPase extracted from fish gills
.
Comp. Biochem. Physiol
.
61B
,
69
72
.
Silva
,
P.
,
Solomon
,
R.
,
Spokes
,
K.
&
Epstein
,
F. H.
(
1977
).
Ouabain inhibition of gill Na,K-ATPase: relationship to active chloride transport
.
J. exp. Zool
.
199
,
419
426
.
Skidmore
,
J. F.
(
1970
).
Respiration and osmoregulation in rainbow trout with gills damaged by Zn sulphate
.
J. exp. Biol
.
52
,
481
494
.
Watson
,
T. A.
&
Beamish
,
W. H.
(
1980
).
Effects of Zinc on branchial ATPase activity in vivo in rainbow trout, Salmo gairdneri
.
Comp. Biochem. Physiol
.
66C
,
77
82
.