Exposure of freshwater trout (Salmo gairdneri) to waterborne Cd2+ results in accumulation of the metal in the branchial epithelial cells and its appearance in the blood. Cd2+ apparently enters the cells via Ca2+ channels in the apical membrane. Transfer of Cd2+ through the basolateral membrane is probably by diffusion. Inhibition by Cd2+ of transepithelial Ca2+ influx is time-and Cd2+-concentration-dependent. The inhibition of transepithelial Ca2+ influx is accompanied by blockage of apical Ca2+ channels. In line with the assumption that cytosolic Cd2+ inhibits Ca2+ uptake by inhibiting the basolateral Ca2+ pump, we hypothesize that the blockage of Ca2+ channels is an indirect effect of Cd2+ and results from a rise in cytosolic Ca2+ level caused by inhibition of the basolateral membrane Ca2+ pump.
Ca2+ uptake in freshwater fish mainly occurs via the gills (Flik et al. 1985). Ca2+ transport across the gills, a tight ion-transporting epithelium, follows a transcellular route (Perry & Flik, 1988). Micromolar concentrations of cadmium (Cd) in the water inhibit branchial Ca2+ uptake (Verbost et al. 1987a, Reid & McDonald, 1988) and induce hypocalcaemia (Giles, 1984; Pratap et al. 1989). We have advanced circumstantial evidence that the inhibition of Ca2+ uptake by waterborne Cd2+ may result from a competitive inhibition by cytosolic Cd2+ of the Ca2+ pump in the basolateral membrane of the Ca2+-transporting cells in the gills (Verbost et al. 1988).
Cd2+ uptake from the water in freshwater fish mainly occurs via the gills (Williams & Giesy, 1978). Like Ca2+, Cd2+ enters the fish predominantly via a transcellular route because of the tight character of the branchial epithelium (Pärt, 1983). Interference of Cd2+ with the Ca2+ influx route may occur at at least three sites: the apical membrane, where Ca2+ enters the cell via Ca2+ channels (Perry & Flik, 1988), the intracellular Ca2+ buffering systems (Flik et al. 1985) and the basolateral membrane, where Ca2+ is translocated to the blood by a high-affinity Ca2+ pump (Flik et al. 1985). Levels of Cd2+ in fresh water that cause hypocalcaemia (0·1 μmol l−1 Cd in water containing 0·7 mmol l−1 Ca) do not instantly inhibit Ca2+ influx (Verbost et al. 1987a), indicating that no significant competition between waterborne Cd2+ and Ca2+ occurs for the cell entrance step (the concentration ratio Cd/Ca being 1·4×10−4 under these conditions). The same conclusion was reached by Part et al. (1985), who showed that changes in water Ca2+ concentration (0–10 mmol l−1) had no effect on 109Cd accumulation in the gills (the concentration ratio Cd/Ca ranged from 0·7×10−4 to 7·0×10−4). Intracellular Ca2+ interacts with a series of more-or-less Ca2+-specific ligands, such as calmodulin, Ca2+-binding proteins (CaBPs) and Ca2+-ATPase. Calmodulin is a Ca2+-dependent regulator protein with a high Ca2+ affinity and is present in all eukaryotic cells. The affinity of calmodulin for Cd2+ is comparable to that for Ca2+ (Chao et al. 1984; Flik et al. 1987). The affinities of the CaBPs in fish gills for Cd2+ and Ca2+ have not been determined so far, but the vitamin-D-dependent CaBPs from rat kidney and pig duodenal mucosa share with calmodulin the property of having the same high affinity for Cd2+ and Ca2+ (Richardt et al. 1986). To affect the Ca2+ buffering capacity of these proteins in the cell, the intracellular concentration of Cd2+ must reach that of the cytosolic Ca2+ concentration. In comparison with calmodulin or CaBPs, the Ca2+ site of the plasma membrane Ca2+-ATPase in fish gills has an affinity for Cd2+ at least 100 times higher than that for Ca2+ (Verbost et al. 1988). It follows that the Ca2+ extrusion pump is a very sensitive target of Cd2+ in fish gills. Although an inhibition of the basolateral Ca2+ pumps by Cd2+ could explain the diminished branchial Ca2+ uptake following exposure to Cd2+, the possibility that cytosolic Cd2+ also impedes the movement of Ca2+ through apical membrane Ca2+ channels cannot be excluded.
The experiments described here were designed to test the hypothesis that Cd2+ enters the Ca2+-transporting cell via Ca2+ channels, and that cytosolic Cd2+ may affect apical Ca2+ channels. Two types of experiments were performed, (i) Shortterm accumulation of 45 Ca and 109Cd from the water into the branchial epithelium was determined to evaluate the movements of Ca2+ and Cd2+ through the apical membranes into the epithelium, (ii) Branchial influx of Ca2+ and Cd2+ was measured to evaluate the movement of these ions through both apical and basolateral plasma membranes. In these two types of experiments, apical membrane permeability for Ca2+ was manipulated by adding exogenous La3+ and by injections of the hormone hypocalcin. Recently, it was concluded that this hormone, produced in the Stannius corpuscles of fish (Wendelaar Bonga & Pang, 1986), controls apical membrane permeability to Ca2+ (Lafeber, 1988).
Materials and methods
Freshwater rainbow trout (Salmo gairdneri) ranging in mass from 20 to 40 g were kept indoors and acclimated to city of Nijmegen tapwater ([Ca] = 0·70 ± 0·02 mmol l−1, N = 20) under the conditions described previously (Verbost et al. 1987a). In experiments with La3+, carbonate-free artificial tapwater was used to prevent precipitation of La2(CO3)3. The composition of the artificial tapwater was (in mmol l−1): CaCl2, 0·7; MgCl2, 0·2; NaCl, 3·8; and KC1, 0·06 in demineralized water (pH 7·6). In experiments with Co2+, CoCl2 was added to normal tapwater. The ionic content of tapwater was (in mmol l−1): Ca2+, 0·7; Mg2+, 0·38; Na+, 0·61; K+, 0·05; Cl−, 0-66; SO42−, 0·32; and HCO3−, 315 (pH 7·6).
The total calcium content of water was determined with a calcium kit (Sigma Chemical). Water total cadmium content was determined by atomic absorption spectrophotometry. Protein was measured with a reagent kit (BioRad) using bovine serum albumin as reference. Tracer content of water samples and tissue digests was determined by liquid scintillation analysis. Aqueous samples (0·5 ml) were mixed with 4·5 ml of Aqualuma scintillation fluid.
Fish were pre-exposed to Cd2+ (nominal 1·0 or 0·1 μmol l−1) up to 16 h before experimentation. Water Cd2+ [added as Cd(NO3)2] concentrations were carefully monitored, and a maximum 10% deviation from the calculated concentrations was accepted. Pre-exposure was followed by a 1 h flux period in which the Cd2+ concentration in the water was kept constant (see radio tracer techniques).
Purified hypocalcin and crude extract of Stannius corpuscles were prepared as described by Lafeber et al. (1988b). The hypocalcin content of extracts of Stannius corpuscles, determined by ELISA (Kaneko et al. 1988) was 120–150 μg mg−1 protein. The dose of hypocalcin injected (intraperitoneally) was 2·2–3·0 nmol hypocalcin 100 g−1 fish. Hormone or extract was injected l h before tracer exposure. Injection of saline, used as vehicle, served as control.
Gill tracer accumulation
Fish were transferred to 3·01 of aerated recirculating water containing l·0MBql−1·45CaCl2, 0·9 MBql−1·109CdCl2 or 0·2 MBql−1·22 NaCl in tapwater. After 30min (22Na) or l h (45Ca, 109Cd) of tracer exposure the fish were quickly (2 min) anaesthetized in bicarbonate-buffered methane sulphonate salt (MS 222, 0·5 l−1, pH 7·4) and injected intraperitoneally with 5000 i.u. of sodium heparin per 100 g of fish. The gills were rapidly cleared of blood by perfusion with saline (10 ml 0·9% NaCl) via the ventral aorta. The perfusate was collected by suction after opening the atrium to determine its tracer content. Next, the gill arches were excised, rinsed for 5 s in demineralized water and blotted on wet tissue paper. Gill epithelium was carefully scraped off onto a glass plate with a microscope slide (approx. 0·4 g wet mass, weighed to ± 0·001 g) and dissolved overnight at 60°C in tissue dissolver (NCS, Amersham). The digested tissue was neutralized with glacial acetic acid, scintillation fluid (9 vols) was added, and the radioactivity determined. The remainder of the fish was processed for the determination of total body radioactivity (see below).
Influx was determined on the basis of total body radioactivity. Fish bodies and scraped gill arches were microwave-cooked (2 min) and homogenized in a blender with distilled water (volume: 65% of body mass). Triplicate samples of the homogenate (approx. 0·4 g weighed to ± 0·001 g) were processed for determination of radioactivity, as described above for the gill scrapings. Total body tracer content included the combined activities of homogenate, gill scrapings and perfusate.
To check whether the tracer associated with the gill scrapings reflected accumulation into the epithelium, and not adsorption to the external side of the epithelium, six trout were exposed to 109Cd and six to 45 Ca for 1 h and the gills perfused and excised as described above. Then, the left gill arches were rinsed in 1 mmol l−1 EDTAand those from the right side in demineralized water (control). No difference in radioactivity was found between samples from either side for either isotope, indicating that the tracers were associated with the internal face of the epithelial cells.
Influx of Cd2+ or Ca2+ was calculated from the total body radioactivity after 1 h of exposure to 109Cd or 45Ca, respectively, and the respective mean tracer specific activities of the water. Fluxes were normalized to fish mass by linear extrapolation and expressed in nanomoles (Cd2+) or micromoles (Ca2+) per hour per 100 grams of fish (Lafeber et al. 1988b).
Na+ influx was calculated from the plasma 22Na activity (blood samples were taken directly after anaesthetizing the fish), the apparent radiospace at 30min for Na+ (approx. 125 ml kg−1) and the specific activity of the water (Payan & Maetz, 1973; Bath & Eddy, 1979).
The amount of Cd2+ accumulated in gill soft tissue was calculated from the tissue tracer content (activity per gram wet mass, <qg′) and the mean specific activity of 109Cd in the water (SAW) using the equation: Cd2+ accumulated in l h = qg′/SAW (in nanomoles Cd2+ per gram wet mass). For this calculation we have assumed that during the experiment no significant transflux or backflux from blood to cells or from cells to water occurred. The Ca2+ accumulation in the cellular compartment could not be calculated from the tracer accumulation because of the multicompartment behaviour of cells when exposed to the Ca2+ tracer (Borle, 1981). Therefore, to compare the accumulation of 109Cd and 45Ca in gill tissue (Fig. 1A), relative values are presented (qg′/qw′; where qw′ is the water activity per millilitre). Values found for control fish were designated as 100%.
Results are presented as means ± S.E. For statistical evaluation the Mann-Whitney U-test was used; significance was accepted for P ⩽ 0·05.
Manipulation of apical membrane
Fig. 1A summarizes the effects of several treatments that affect apical Ca2+ permeability on 45Ca and 109Cd accumulation in the gills. La3+ (1 μmol l−1) and Co2+ (100 μmol l−1) in the water reduced the Ca2+ accumulation in gill soft tissue by 71% and 33%, respectively, compared with controls. Because of the limited availability of purified hypocalcin we used both Stannius corpuscle extract and purified hypocalcin. Injection of Stannius extract or hypocalcin reduced 45Ca accumulation in gills by 63 % and 37 %, respectively.
To evaluate the effects of the aforementioned treatments on Cd2+ accumulation in the gills, a water Cd concentration had to be established that did not influence the apical permeability to Cd2+ during the 1 h 109Cd accumulation measurement. Since even a 4h exposure to 0·1 μmol l−1 Cd had no effect on the Cd2+ (or Ca2+) accumulation (see Figs 2, 3), this concentration was used. The results showed that treatments that decreased 45Ca accumulation had a similar, and proportional, effect on 109Cd accumulation in the gills (Fig. 1A).
Fig. IB summarizes the effects of the treatments on Ca2+ and Cd2+ influx. Ca2+ influx decreased by 77% when LaCl3 was added to the water. Injections of Stannius extracts and of hypocalcin reduced Ca2+ influx by 55 % and 43 %, respectively. Comparable effects were also found on the Cd2+ influx (Fig. IB). These results show that for both Ca2+ and Cd2+ a reduction in tracer accumulation in the gills is accompanied by a reduction in whole-body influx of the ions.
Effects of Cd2+ exposure on Ca2+ influx
Fig. 2A shows the effects of time of exposure to exogenous Cd2+ on the 45Ca accumulation rate in gill epithelium. A time-related inhibition of 45Ca accumulation was found, with a significant 25% inhibition after 6h of exposure to 0·1 μmol l−1 Cd, and with 60% and 70% inhibition after 9 and 17 h, respectively, of exposure to 0·1 μmol l−1 Cd (exposure time to Cd2+ included pre-exposure and the l h flux period). When fish were exposed to 1·0 μmol l−1 Cd, however, 45Ca accumulation was inhibited by 25 % within 1 h (without pre-exposure), whereas a maximum inhibition of 60% was observed after 3–4 h of exposure. The same maximum inhibition of 45Ca accumulation was reached for 0·1 and 1·0 μmol l−1 Cd. Exposure of trout for 16·5 h to 1·0 μmol l−1 Cd did not affect 22Na accumulation in branchial tissue.
The effects of exogenous Cd2+ on whole-body Ca2+ and Na+ influx are shown in Fig. 2B. Exposure to 0·l μmol l−1 Cd for up to 6h had no significant effect on Ca2+ influx, but after 9 and 17 h Ca2+ influx was inhibited by 67% and 87%, respectively. With 1 μmol l−1 exogenous Cd, Ca2+ influx was significantly (49%) inhibited within 1h of exposure, and after 3–4 h of exposure a maximum 80% inhibition was observed. Exposure of fish to 1 μmol l−1 Cd for 16·5 h had no effect on Na+ influx.
Effects of Cd2+ exposure on Cd2+ influx
The effects of exogenous Cd2+ on the 109Cd accumulation in gill epithelium are shown in Fig. 3A. Exposure to 0·1 μmol l−1 Cd for up to 4 h had no significant effect, but 109Cd accumulation decreased by 64 % after 6 h and by 75 % after 17 h of exposure to Cd2+. With 1·0 μmol l−1 Cd in the water, six times more Cd2+ accumulated in gill soft tissue than with 0·1 μmol l−1 Cd in the water. If we designate Cd2+ accumulation after l h as 100%, an 80% inhibition occurred during the second hour of incubation, which was almost the maximum inhibition observed after longer exposure (up to 17 h).
In Fig. 3B the effects of exogenous Cd2+ on whole-body Cd2+ influx are shown. The Cd2+ influx at 0·1 μmol l−1 external Cd was around 3·4 nmol h−1100 g−1 fish. Exposure to Ol^moll-1 Cd for 4h did not significantly affect Cd2+ influx. However, after 6h or more Cd2+ influx decreased by 55%. In fish exposed to l·0 μmol l−1 Cd for l h, Cd2+ influx was around 60 nmol h−1100 g−1 fish, 18 times higher than in those exposed to 0·1 μmol l−1 Cd. Cd2+ influx during the second hour of exposure to 1 μmol l−1 Cd and thereafter decreased by 80 %.
Two major conclusions are drawn from this study. Cd2+ in the water at a concentration that provokes a specific hypocalcaemia inhibits both Ca2+ and Cd2+ influx, but not Na+ influx. The inhibitory effect of Cd2+ is concentration-and time-dependent. Treatments that inhibit branchial Ca2+ influx (exposure to La3+ in the water or hypocalcin injections) inhibit branchial Cd2+ influx from the water.
Transepithelial Cd2+ influx
The main purpose of our study was to examine how Cd2+ passes across the branchial epithelium. We tested the hypothesis that Cd2+ follows the Ca2+ route through the epithelium. This idea was prompted by the similarity in charge and ionic radius of Ca2+ and Cd2+.
Whole-body Cd2+ influx amounted to 3·4 nmol h−1100 g−1 fish at OTgmoll-1 external Cd and 61·9 nmol h−1100 g−1 fish at 1·0 μmol l−1 Cd. These values for whole-body Cd2+ influxes are of the same order as the values found by Part & Svanberg (1981) for Cd2+ influx in isolated head preparations of trout. These results extend the observations of Williams & Giesy (1978) that the gills are the predominant site of Cd2+ influx. Our finding that inhibition of Cd2+ influx is accompanied by a decrease in Cd2+ accumulation in gill soft tissue establishes that the Cd2+ influx is transcellular.
Cd2+ accumulation in branchial epithelium
Our earlier experiments with the perfused isolated head technique indicated that Cd2+ may be transferred across the apical membrane via La3+-inhibitable Ca2+ channels (Verbost et al.1987a). The present results on intact trout confirm and extend these earlier findings: both La3+ treatment and hypocalcin treatment decrease Ca2+ and Cd2+ accumulation in the gills. These observations indicate an effect of hypocalcin on apical membrane Ca2+ channels, and thus also extend the data of Lafeber et al. (1988a), who showed that hypocalcin inhibits transepithelial Ca2+ influx. Moreover, the observation that both Ca2+ and Cd2+ accumulation are inhibited by these treatments strongly suggests that these ions enter the cells via the same pathway.
Whole-body Cd2+ and Ca2+ influx
As the decrease in tissue accumulation was proportional to the decrease in transcellular Ca2+ and Cd2+ flux, both whole-body Ca2+ and whole-body Cd2+ influx depend on passage of the ions through the apical membranes of the epithelium. Ca2+ and Cd2+ transfer across the epithelium should not be expected to be the same, because of the differences in tissue status of the ions (at the beginning of an experiment the gill cells contain millimolar concentrations of Ca and no Cd). However, if epithelial cells were unable to discriminate between Ca2+ and Cd2+, 109Cd could be used as Ca2+ tracer (and 45Ca as Cd2+ tracer). The tracer uptake studies show, however, that the epithelial cells do discriminate between Ca2+ and Cd2+ (109Cd being differentially accumulated in the gills compared with 45Ca, using similar amounts of radioactivity), which indicates a difference in cellular buffering and/or a difference in basolateral transfer.
To understand transepithelial Cd2+ uptake it is important to know how Cd2+ passes through the basolateral membrane. Because we obtained no evidence for an ATP-driven translocation from experiments with basolateral membrane vesicles in our laboratory (results not shown), and because the transepithelial Cd2+ influx is too high to be explained by diffusion through a pure lipid bilayer, some mechanism of facilitated diffusion seems likely. A similar conclusion was drawn for Cd2+ transfer through basolateral membranes in rat duodenum (Foulkes, 1986).
Short-term regulation of transepithelial Ca2+ influx takes place at the level of the apical membrane, which is under the control of fast-acting hypocalcin (Lafeber et al. 1988a). Indeed, the transepithelial influx of Ca2+ proved to be proportionally related to Ca2+ accumulation in the gills. The rate of transport of Cd2+via the gills appears to depend on the permeability of the apical membrane to Cd2+, as well as on passage through the cytosol and/or the basolateral membrane of the cell.
In the calculations of Ca2+ accumulation and Cd2+ influx, we have assumed that no backflux from cytosol to water or from blood to cytosol occurred. This assumption seems justified as the influx of Cd2+ is very low (62 nmol h−1 100 g−1 fish in water containing 1·0 μmol l−1 Cd and 0·7 mmol l−1 Ca) and the Cd2+ space is large. Also, backflux of Cd2+ through the apical membrane will be negligible because of the electrical potential difference between cytosol and water (the cytosol being negative; Perry & Flik, 1988).
Transepithelial Na+ influx
Chloride cells are generally considered as the sites of Na+ uptake in the gills (Avella et al. 1987). The Na+/K+-ATPase activity, located in the basolaferal membrane of these cells, is regarded as the most important driving force for transepithelial Na+ uptake in fish gills (deRenzis & Bornacin, 1984). Branchial Na+ influx is not affected by Cd2+ exposure, in contrast to Ca2+ influx, which also depends on ATPase activity (Ca2+-ATPase; Flik et al. 1985) colocalized with Na+/K+-ATPase. This suggests that Na+/K+-ATPase activity is not inhibited by Cd2+ concentrations that inhibit Ca2+-ATPase. This is in agreement with in vitro studies, which indicate a much lower sensitivity of the Na+/K+-ATPase than the Ca2+-ATPase for Cd2+. The concentration of Cd2+ causing 50% inhibition in vitro of Na+/K+-ATPase from various origins, for instance rabbit proximal tubule (Diezi et al. 1988), cultured vascular smooth muscle cells (Tokushige et al. 1984) or rat brain synaptosomes (Lai et al. 1980) is in the micromolar range. We found for trout gills, that the branchial para-nitrophenyl phosphatase (pNPPase) activity, which reflects the K+-dependent dephosphorylation step in the Na+/K+-ATPase reaction cycle, was inhibited by 50 % by 0-25 /zmol I-1 Cd2+ (G. Flik, unpublished results). These data indicate that the concentration of free cytosolic Cd2+ that causes inhibition of Ca2+ influx must be below the micromolar range, as Na+ influx was not affected. Such a low cytosolic Cd2+ concentration also excludes significant binding of Cd2+ to calmodulin, as the Km of calmodulin for Cd2+ is in the micromolar range (Flik et al. 1987). The formation of Cd2+-calmodulin complexes has been proposed as the primary cause of cellular Cd2+ toxicity (e.g. Suzuki et al. 1985). The present results do not support such a calmodulin-mediated toxicity mechanism for Cd2+.
Localization of the primary Cd2+ target in the gills
Our data show that Ca2+ and Cd2+ enter the gills via the same route. This implies that Cd2+ is concentrated in the ion-transporting cells of the gills, the chloride cells, since these cells account for the branchial Ca2+ transport (Fenwick, 1989). As a consequence, inhibition of Ca2+ transport will occur as soon as Cd2+ has accumulated to a level sufficient to inhibit the Ca2+-ATPase transport system (Verbost et al. 1988). However, the present results also show a reduction in the rate of accumulation of Ca2+ in gills after prolonged Cd2+ exposure. We suggest that this effect is caused by a decrease in the permeability of the apical membrane to Ca2+, possibly by an indirect blockage of Ca2+ channels by cytosolic Cd2+. This hypothesis is supported by several observations. First, the inhibition by Cd2+ is not acute, in contrast to the inhibition by La3+ (Verbost et al. 1987a). Moreover, short-term exposure to Cd2+ (0 · 1 μ mol l− 1 Cd) has no effect on the rate of accumulation of Ca2+ in the tissue, whereas long-term exposure (17 h) decreases the rate of accumulation of Ca2+. Second, long-term exposure to Cd2+ decreases the rate of accumulation of Cd2+ in the gills, whereas short-term exposure to Cd2+ has no effect. These observations indicate that Ca2+ channels become blocked when Cd2+ accumulates in the epithelial cells, as happens after a prolonged exposure to waterborne Cd2+. These findings also support our conclusion that external Cd2+ cannot cause the inhibition of Ca2+ influx by competition with Ca2+ at mucosal sites, because the inhibition of influx is not instantaneous.
Which mechanism underlies the blockage by Cd2+ of the permeability of the apical membrane to Ca2+? Once Cd2+ has entered the Ca2+-transporting cell it could affect the Ca2+ channels in several ways. The basolateral Ca2+ pump has an extremely high affinity for Cd2+ (I50 = 3 nmol l−1; Verbost et al. 1988) and, therefore, nanomolar intracellular Cd2+ concentrations inhibit Ca2+ extrusion. We suggest that inhibition of the Ca2+ pump leads to an increased intracellular Ca2+ concentration, [Ca2+]i. Possibly, [Ca2+]i rises to levels that close the apical membrane Ca2+ channels. Thus, [Ca2+]i serves as a feedback signal to control Ca2+ entry at the apical membrane. An analogous model was proposed for rat small intestine enterocytes (Van Os, 1987). Both an overcapacity of Ca2+ pumps and buffering of Cd2+ by cytosolic binding proteins could explain the delay in the inhibition of Ca2+ uptake by Cd2+.
To test the hypothesis that Cd2+ closes Ca2+ channels by increasing [Ca2+]i would require examination with Ca2+ fluorochromes to show the predicted rise in [Ca2+]i. However, this was not feasible because Cd2+ quenches Quin2 fluorescence and interferes with Ca2+-dependent fluorescence of Fura-2 and Indo-1 (P. M. Verbost & G. Visser, personal observations). The Ca2+ binding sites of Fura-2 and Indo-1 are EGTA-type sites (Tsien, 1980) with a much higher affinity for Cd2+ than for Ca2+ (Sillen & Martell, 1964). There is, however, indirect evidence for a rise in [Ca2+]i upon exposure to Cd2+ of various other cell types: erythrocytes show an acceleration in age-related changes (Kunimoto et al. 1985), protein phosphorylation in human platelets is increased (Pezzi & Cheung, 1987), water absorption in rat duodenum is reduced (Toraason & Foulkes, 1984) and in rat skeletal muscle cells the degradative enzymes phosphorylase-b kinase, phospholipases and proteases are activated (Toury et al. 1985). All these phenomena are known to be mediated by a rise in [Ca2+]i. In human lymphocytes Cd2+ produces an increase in the accumulation of 45 Ca and in the rate of mitogenesis (Parker, 1974). The latter effect is also dependent on a rise in [Ca2+]i (Parker, 1974).
Although apical membrane Ca2+ channels may also be affected in fish exposed to Cd2+, we conclude that Ca2+ transport in the gills becomes inhibited primarily because the basolateral membrane Ca2+ pump has such an extremely high sensitivity to Cd2+. This conclusion for fish gills may be extended to explain intoxication of mammalian Ca2+-transporting epithelia, such as the kidneys and the intestine, by Cd2+. Recent data on Cd2+ inhibition of Ca2+ pumps from rat kidney and rat intestinal tissues (Verbost et al. 1987b) confirm the applicability of the gill model.
This study was supported by a grant from the Foundation for Fundamental Biological Research (BION), which is subsidized by the Dutch Organisation for Scientific Research (NWO). Mr F. A. T. Spanings is thanked for his excellent fish husbandry.