J.C. Fenwick

Interrelationships between gill chloride cell morphology and calcium uptake in freshwater teleosts

The involvement of the freshwater fish gill chloride cells (CCs) in trans-branchial calcium uptake (JinCa2+) was investigated. This was accomplished by assessing the interspecific relationships between the apical surface area of CCs exposed to the external environment and JinCa2+. Three species of freshwater teleosts, the rainbow trout (Oncorhynchus mykiss), the American eel (Anguilla rostrata) and the brown bullhead catfish (Ictalurus nebulosus), were used. Chronic (ten-day) treatment with cortisol in each species was used as a tool to evoke variations in both JinCa2+ and gill CC morphology in order to assess intraspecific relationships between CC surface area and JinCa2+. The results of quantitative morphometry, based on analysis of scanning electron micrographs, demonstrated that catfish possessed the lowest fractional area of exposed CC (CCFA) on the gill filament epithelium (12,744 ± 2248 μm2/mm2) and was followed, in increasing order, by American eel (21,355 ± 981 μm2/mm2) and rainbow trout (149,928 ± 26,545 μm2/mm2). With the exception of catfish, chronic treatment with cortisol caused significant increases in CCFA owing to proliferation of CCs and/or enlargement of individual CCs (eel only). The rates of JinCa2+ closely reflected the CC fractional area in each species. The results of correlation analysis revealed significant correlations between CC fractional area and JinCa2+ in trout and eel. Owing to the absence of an effect of cortisol treatment, there was no significant correlation in catfish because of insufficient variation in CC fractional area in this species. CC fractional area was significantly correlated with JinCa2+ among the three species examined. These results suggest that CC is involved in calcium uptake in freshwater teleosts and that both intra- and interspecific differences in the rates of calcium uptake can be accounted for by variability in the surface area of exposed CCs on the gill epithelia.

The effects of soft-water acclimation on gill structure in the rainbow trout Oncorhynchus mykiss

Abstract.Rainbow trout (Oncorhynchus mykiss) were exposed to ion-poor (soft) water to test the hypothesis that naturally induced proliferation of branchial chloride cells causes a thickening of the blood-to-water diffusion barrier. This was achieved by using a combination of scanning and transmission electron-microscopic techniques. Fish were exposed to soft-water conditions ([Na+]= 0.055 mmol l-1, [Cl–]≈0.029 mmol 1–1, [Ca2+]≈ 0.059 mmol 1–1, and [K+]≈0.007 mmol 1–1) for 1, 2, and 4 weeks. Marked chloride cell proliferation was evident at all sampling times with an approximate doubling of the gill epithelial surface area covered by chloride cells exposed to the water (”chloride cell fractional area”). The increases in chloride cell fractional area resulted from both increased numbers of cells and expanded apical surfaces of exposed individual cells. As a result of chloride cell proliferation, soft-water exposure was associated with a doubling of the lamellar blood-to-water diffusion distance from 3.26±0.08 μm to 6.58±0.43 μm as determined from transmission electron micrographs. These data demonstrated a positive correlation between chloride cell fractional area and blood-to-water diffusion distance. We conclude that, in trout, chloride cell proliferation during soft-water exposure, while presumably benefiting ionic regulation, may impair gas transfer owing to the associated thickening of the blood-to-water diffusion barrier.

Ca2+-dependent phosphatase and ATPase activities in eel gill plasma membranes - I. Identification of Ca2+ activated ATPase activities with non-specific phosphatase-activities

The characteristics of Ca2+-activated ATPase activities previously often postulated as components for the calcium transporting system in fish gills do not fulfil the requirements of a transport Ca2+-ATPase. The chelation of Ca2+- or Mg2+-ions is a prerequisite for the adenosinephosphate esters to serve as substrate for gill plasma membrane phosphatases. Ca2+-activated ATP hydrolysis results from the activity of a heterogeneous pool of phosphatases located in the plasma membranes of the branchial epithelium, as is concluded from substrate specificity tests and the effects of various inhibitors on these hydrolytic activities. In the present study only non-specific phosphatases could be shown.

In vivo and in vitro effects of Stannius corpuscle extract on the branchial uptake of 45Ca in stanniectomized North American eels (Anguilla rostrata).

Abstract Stanniectomized eels became hypercalcemic and developed a greater rate of branchial 45 Ca influx than did control animals. Both of these disturbances could be prevented by the postoperative injection of previously acidified or unacidified extracts of Stannius corpuscles. The increased rate of branchial 45 Ca influx into the isolated perfused poststanniectomized eel gills could also be depressed by the addition of Stannius corpuscle extract to the perfusion fluid. We concluded that Stannius corpuscles contain an acid-stable hypocalcemic factor which exerts its effect on calcium homeostasis by directly attenuating the rate of branchial calcium influx.

Effect of Various Vitamin D Analogs on Plasma Calcium and Phosphorus and Intestinal Calcium Absorption in Fed and Unfed American Eels, Anguilla rostrata

Injection of about 1 ng/g body wt per day of either vitamin D3 or 1,25-(OH)2-vitamin D3 for 7 days induces hypercalcemia and hyperphosphatemia in fed American eels, Anguilla rostrata, but only hyperphosphatemia in unfed eels. These same analogs also stimulated the uptake of 45Ca from intestinal sacs in situ. The vitamin D3 appeared to be relatively more effective than the 1,25-(OH)2D3 metabolite and chlorpromazine inhibited the effect of vitamin D3 on intestinal calcium uptake. 7-Dehydrocholesterol, vitamin D2, and 24,25-(OH)2D3 did not stimulate hypercalcemia, hyperphosphatemia, or intestinal calcium uptake.

Comparative biochemistry and physiology of teleocalcin from sockeye and coho salmon

This is a comparative study of the glycoprotein hormone, teleocalcin, from the corpuscles of Stannius of sockeye (Oncorhynchus nerka) and coho (O. kisutch) salmon. Coho teleocalcin was purified by the same procedures used previously to obtain sockeye teleocalcin and was obtained in a comparable yield. Both salmon teleocalcins had the same molecular weight as estimated by sodium dodecyl sulfate-electrophoresis and both appeared to have the structure of disulfide-linked oligomers. The two hormones were similar on the basis of amino acid and carbohydrate composition and shared 95% homology in the first 40 residues on the N-terminal. The salmon teleocalcins also shared 80% homology with the predicted 1-40 N-terminal sequence from Australian eels (Anguilla australis). Both teleocalcins had potent inhibitory effects on gill calcium uptake in intact rainbow trout (Salmo gairdneri). However, these effects were observed only at the peak in the calcium uptake cycle that is displayed by this species. In North American eels (A. rostrata), the acute administration of both teleocalcins caused significant inhibition of gill calcium uptake without any concomitant changes in plasma calcium levels or other plasma electrolytes. In 4- and 7-day stanniectomy (STX) eels, the acute administration of coho teleocalcin significantly reduced or completely abolished the accelerated gill calcium transport that occurs postoperatively, with no concomitant changes in plasma electrolytes or post-STX hypercalcemia. These experiments provide further evidence that teleocalcin is a regulator of gill calcium transport and has no acute hypocalcemic effects in fish.

The corpuscles of Stannius and calcium regulation in the North American eel (Anguilla rostrata LeSueur)

Abstract The removal of the corpuscles of Stannius elicited hypercalcemia and hypercalciuria in the eel Anguilla rostrata , but the development of these symptoms was dependent upon the presence of exogenous calcium. The hypercalciuria did not develop until after frank hypercalcemiaand was associated with a decrease in the percentage of tubular reabsorption of water and an increase in relative calcium clearance ( C ea C in ). Intact, sham-operated and stanniectomized eels held in acalcemic water displayed hypercalciuria relative to the same groups held in water containing dissolved calcium. In these animals, stanniectomy did not effect a further increase in urine calcium excretion. These findings suggested that eels have a mobile internal store or pool of calcium but that stanniectomy does not affect its mobilization or activity. It is suggested that the corpuscles of Stannius may attennate the overall calcium flux between the animal and its environment.

Ultraviolet-B Radiation Effects on Antioxidant Status and Survival in the Zebrafish, Brachydanio rerio¶

Abstract Direct impact of ambient (1.95 W/m2) and subambient doses of UV-B radiation on muscle/skin tissue antioxidant status was assessed in mature zebrafish (Brachydanio rerio). The influence of these doses on hatching success and survival in earlier life stages was also examined. Subambient doses of UV-B radiation in the presence (1.28 W/m2) and absence (1.72 W/m2) of a cellulose acetate filter significantly depressed muscle/skin total glutathione† (TGSH) levels compared with controls (0.15 W/m2) and low (0.19 W/m2) UV-B–treated fish after 6 and 12 h cumulative exposure. Ambient UV-B exposure significantly decreased muscle/skin glutathione peroxidase (GPx) activity after a 6 h exposure; activities of glutathione reductase (GR) were unchanged over this exposure period. Superoxide dismutase (SOD) and catalase activities peaked after 6 and 12 h cumulative exposure, respectively, but fell back to control levels by the end of the exposure period. The changes in tissue antioxidant status suggested UV-B–mediated increases in cytosolic superoxide anion radicals (O2−) and hydrogen peroxide (H2O2). This apparent UV-B–mediated increase in oxidative stress is further supported by a significant increase in muscle/skin thiobarbituric acid reactive substances (TBARS). Hatching success of newly fertilized eggs continuously exposed to ambient UV-B was only 2% of the control value. Even at 30 and 50% of ambient UV-B, hatching success was only 80 and 20%, respectively, of the control. Newly hatched larvae exposed to an ambient dose of UV-B, experienced 100% mortality after a 12 h cumulative exposure period. This study supports a major impact of UV-B on both the mature and embryonic zebrafish.

Sodium dependent ion transporters in trout gills

Biochemical procedures developed to isolate plasma membranes from the branchial epithelium of rainbow trout (Oncorhynchus mykiss) yield membrane fractions that are specifically enriched in the plasma membrane marker enzyme Na+/K+-ATPase. As the bulk of the branchial Na+/K+-ATPase is assumed to be confined to the mitochondria-rich chloride cells, such membrane preparations must contain the essence of the enzymatic machinery of the chloride cells. Basal Na+ activity in branchial (chloride) cells is around 10 millimolar and, accordingly, we find a Km for Na+ of the Na+/K+-ATPase of 13 millimolar, indicating that the enzyme may be regulated by changes in cytosolic sodium. The Na+-gradient across the serosal plasma membrane created by this pump provides energy for 3Na+/Ca2+-exchange and bumetanide-sensitive Na+/K+/2Cl--cotransport. Here we further postulate the presence of a Na+/Cl--cotransporter, indicated by thiazide-sensitive, bumetanide-insensitive transport of Na+ and Cl-; this cotransporter activity awaits the characterization of its kinetics. The Na+/Ca2+-exchanger has kinetic characteristics compatible with a regulatory role of cytosolic Na+ in the activity of this carrier. Both Na+/Ca2+-exchange and Ca2+-ATPase activity may contribute to transport of Ca2+, the former having lower affinity for calcium but a higher capacity than the latter carrier. The Na+/K+/2Cl--cotransporter has kinetics that favor a regulatory role for plasma K+ in the activity of this carrier. Seawater adaptation leads to increased activity of cotransporter molecules in the plasma membrane fractions (the activity increases relative to that of the Na+/K+-ATPase) and this may reflect a function in Cl--extrusion performed by the chloride cells in a seawater environment. A function for the cotransporter in the gills of freshwater fish may be the regulation of cell volume.

Effect of stanniectomy on calcium activated adenosinetriphosphatase activity in the gills of fresh water adapted North American eels, Anguilla rostrata LeSueur.

Abstract Branchial Ca 2+ -ATPase activity calculated on the basis of tissue weight was higher in stanniectomized eels than in sham-operated eels 2 weeks after surgery. But the enzyme showed similar characteristics. In both groups one half of the maximal activity occurred at a calcium concentration of 0.45 m M and maximal activity at 2.0 m M . The optimal pH (7.8) and temperature (40 to 45°) were also the same. However, the total protein concentration of the final extracted enzyme preparation was significantly higher in the stanniectomized group. These data suggest that stanniectomy resulted in the de novo synthesis of more branchial Ca 2+ -ATPase.