Effects of enzyme and anion transport inhibitors on in vitro incorporation of inorganic carbon and calcium into endolymph and otoliths in salmon Oncorhynchus masou.

The transepithelial transport of inorganic carbon to endolymph and its subsequent deposition on otoliths were pharmacologically examined by incubating the sacculus containing an otolith with NaH(14)CO(3). Calcium incorporation was also studied. Carbon incorporation into endolymph and otoliths was saturated with increased concentrations of bicarbonate ions in the incubation medium and was followed by the Michaelis-Menten equation with a K(m) of 26.3 mM and 0.4 mM, respectively. Carbon incorporation decreased with an increase in chloride concentrations in the medium. Calcium incorporation was not affected by chloride and bicarbonate ions up to 10 mM. Higher concentrations of bicarbonate ions reduced calcium incorporation into both fractions. Carbon incorporation into endolymph and otoliths was inhibited by acetazolamide, disulfonate stilbenes (DIDS and SITS), thiocyanate, and ouabain. Calcium incorporation was not affected by these inhibitors. Amiloride inhibited carbon incorporation into otoliths alone. These results suggest that HCO(3)(-)-ATPase and Cl(-)/HCO(3)(-)-exchangers are involved in the transepithelial transport of bicarbonate ions to the endolymph. Carbonic anhydrase was also suggested to play a role in carbonate production for otolith calcification.

Aluminium and cadmium inhibit vitellogenin and its mRNA induction by estradiol-17 beta in the primary culture of hepatocytes in the rainbow trout Oncorhynchus mykiss.

Effects of Al and Cd on vitellogenin (VTG) and VTG mRNA induction by estradiol-17 beta (E(2)) were examined in primary hepatocyte cultures of rainbow trout. Hepatocytes were precultured for 2 days and then E(2) (2 x 10(-6) M) and Al (10(-6)-10(-4) M) or Cd (10(-9)-10(-6) M) were simultaneously added to the incubation medium. Hepatocytes were cultured for 5 more days. Media and hepatocytes were then analyzed by SDS-PAGE and Northern blotting for VTG and VTG mRNA, respectively. These metals had no appreciable effect on the viability of hepatocytes in culture. However, Al and Cd interfered with VTG production and VTG mRNA expression. Al reduced VTG production in a concentration-dependent way and a significant reduction occurred at Al concentrations greater than 5 x 10(-5) M. VTG mRNA expression also decreased with a negative correlation with Al concentrations (r = -0.98). The inhibition of VTG production by Cd was not concentration-dependent. This metal markedly inhibited VTG production and VTG mRNA expression at 10(-6) M. The Al-induced inhibition of VTG production was restored 7 days after Al removal, but the Cd-induced inhibition was not restored. These results suggest that Al and Cd inhibit VTG production at the transcriptional level to reduce VTG mRNA expression by different mechanisms.

Exposure of Goldfish (Carassius auratus) to Bluegills (Lepomis macrochirus) Enhances Expression of Stress Protein 70 mRNA in the Brains and Increases Plasma Cortisol Levels.

The effect of psychological stress on HSP70 mRNA in the brains and plasma cortisol levels in goldfish was examined. Stress was induced by exposure to a predator (bluegills). HSP70 mRNA and cortisol were determined by Northern blotting and ELISA, respectively. Goldfish exposed to four predators in the same tank without a partition showed marked increases in HSP70 mRNA and cortisol levels 6 hr and 12 hr after commencement of exposure. When goldfish were separated from bluegills with a net partition, HSP70 mRNA expression was enhanced after 6 hr, and returned to the control level after 12 hr. Plasma cortisol levels increased after 2 hr, and returned to the control level after 6 hr. When goldfish were placed in a transparent tank around which bluegills were swimming, HSP70 mRNA expression and cortisol levels increased after 6 hr and 12 hr. Goldfish exposed to water circulating through a tank with bluegills showed no sign of changes in HSP70 mRNA expression or cortisol levels. These results suggest that psychological stress enhanced HSP70 mRNA expression in the brains and increased plasma cortisol levels via visual perception.

Inhibitory effects of aluminium on vitellogenin induction by estradiol-17 beta in the primary culture of hepatocytes in the rainbow trout Oncorhynchus mykiss.

Effects of Al on estradiol-induced vitellogenin (VTG) induction were electrophoretically examined in primary hepatocyte cultures in rainbow trout. Hepatocytes were precultured for 2 days and then estradiol-17 beta (E2, 6 x 10(-6) M) and Al (10(-6)-10(-4) M) were added to the incubation medium. The hepatocytes were cultured for 5 more days. Spent media were analyzed by SDS-PAGE and the relative rate of VTG synthesis was evaluated by a measurement of the integrated optical density of the main VTG band and was expressed as the percentage of VTG to total proteins including the VTG. The addition of Al to the incubation medium had no effect on the viability of hepatocytes in the culture. However, it specifically reduced VTG synthesis by hepatocytes in a concentration-dependent way and there was a significant reduction at Al concentrations greater than 6 x 10(-5) M. VTG synthesis by E2-primed hepatocytes was also reduced by Al concentrations of more than 6 x 10(-5) M 2-6 days after Al addition. Enriched Ca concentrations (1.8 to 2.5 or 5.0 mM) in the incubation medium had no protective effect on the reduction of VTG synthesis by Al. These results suggest that the synthesis of VTG is more susceptible to Al than are other hepatocyte-derived proteins.

Effects of extracellular calcium concentrations and calcium antagonists on vitellogenin induction by estradiol-17 beta in primary hepatocyte culture in the rainbow trout Oncorhynchus mykiss.

The involvement of calcium in vitellogenin (Vg) synthesis in response to estradiol-17 beta (E2, 2 x 10(-6) M) was electrophoretically examined in primary hepatocyte culture in the rainbow trout Oncorhynchus mykiss. The relative rate of Vg synthesis was evaluated by measuring the integrated optical density of the Vg band after SDS-PAGE and expressed as a percentage of Vg to total proteins. The induction of Vg by E2 did not occur at 0 meq Ca/liter, while some proteins other than Vg (main subunit) were weakly synthesized. Vg synthesis increased in a calcium concentration-dependent way and reached the maximum at 5 meq Ca/liter in the incubation medium. The addition of bovine growth hormone (50 ng/ml) and/or ovine prolactin (100 ng/ml) had no effect on stimulating Vg synthesis by E2, regardless of the calcium concentrations in the medium. Lanthanum and verapamil (10(-4) M) markedly inhibited Vg synthesis, while diltiazem (10(-4) M) was insignificant. Reactive blue (10(-4) was also effective in inhibiting Vg synthesis to about 60% of the control. These results suggest that the synthesis of Vg, a calcium-binding protein, is more susceptible to calcium than are other proteins. Receptor-operated and verapamil-sensitive calcium channels have been proposed to be involved in calcium entry into hepatocytes in rainbow trout.

Involvement of growth hormone and prolactin in the induction of vitellogenin synthesis in primary hepatocyte culture in the eel, Anguilla japonica.

Multihormonal effects of bovine growth hormone (GH) and ovine prolactin (PRL) on vitellogenin (Vg) synthesis in response to estradiol-17 beta (E2) were electrophoretically examined in primary hepatocyte culture in the eel, Anguilla japonica. Newly synthesized protein after hormonal treatment was identified as Vg by immunoblot and immunoelectrophoresis. A single injection of E2 into hypophysectomized eels failed in the induction of Vg synthesis. Similarly, E2 alone was insufficient to induce Vg synthesis in the culture. On the other hand, the combination of E2 with GH and/or PRL markedly stimulated Vg synthesis. Neither GH alone nor PRL alone had any effect on the induction of Vg synthesis by cultured hepatocytes. Hepatocytes isolated from E2-primed eels also required GH or PRL for continuation of Vg synthesis. The highest Vg synthesis occurred at doses of 50 ng GH/ml and 1 microgram PRL/ml in the presence of 2 x 10(-6) M E2. These results indicate that the multihormonal stimulation of GH and/or PRL as well as E2 is essential for the active synthesis of Vg in eels.

Short- and long-term effects of ammonium chloride on RNA, DNA and protein concentrations in muscle and serum in the rainbow trout, Oncorhynchus mykiss.

1. Effects of ammonium-induced acidosis on white muscle RNA, DNA and protein concentrations were examined in the rainbow trout, Oncorhynchus mykiss. Serum protein concentrations were also determined. 2. A single administration of ammonium chloride at a dose of sublethal level caused no changes in these parameters after 3 and 6 hr. 3. Multiple administrations at a lesser dose caused significant decreases in RNA and serum protein concentrations 48 and 72 hr after the first administration. 4. DNA and muscle protein concentrations remained unchanged throughout the experimental period. 5. Significant decreases in RNA-DNA ratios and RNA-muscle protein ratios were primarily attributed to decreases in RNA concentrations.

Effects of a dietary deficiency in calcium on growth and calcium uptake from the aquatic environment in the goldfish, Carassius auratus.

1. Young goldfish, Carassius auratus, were fed a calcium-deficient diet for 8 or 56 days and then their net uptake of calcium from the aquatic environment was examined using 45Ca. 2. They showed normal growth and the normal level of plasma calcium in both experiments. 3. In the short-term experiment, no dietary effect was found in the uptake of environmental calcium. In the long-term experiment, however, the rate of uptake was significantly stimulated by 18%. 4. The results indicate the presence of a compensatory action in calcium uptake between dietary and aquatic sources.