RESUMO
We previously found that when goldfish were exposed to a potential predator, bluegills, the goldfish experienced an increase in HSP70 mRNA expression in the brains and increased plasma cortisol levels. In the present study, we examined the potential causative relationship between HSP70 mRNA expression and plasma cortisol levels. Cortisol agonists (corticotropin releasing factor and cortisol) and antagonists (metyrapone and betamethasone) were used to modulate plasma cortisol levels. HSP70 mRNA expression and plasma cortisol levels were analyzed by Northern blotting and ELISA, respectively. Goldfish treated with the cortisol agonists showed marked increases in plasma cortisol levels and also in brain HSP70 mRNA expression. When goldfish were exposed to bluegills, plasma cortisol levels increased and HSP70 mRNA expression was enhanced after 6 hr. However, pre-treatment with the cortisol antagonists 24 hr prior to the exposure inhibited the enhancement as well as the increase in plasma cortisol levels. These results suggest that plasma cortisol plays a key role in the enhancement of brain HSP70 mRNA expression in goldfish stressed by exposure to bluegills.
Assuntos
Regulação da Expressão Gênica , Carpa Dourada/sangue , Carpa Dourada/genética , Proteínas de Choque Térmico HSP70/genética , Hidrocortisona/sangue , Perciformes/fisiologia , Comportamento Predatório , Animais , Comportamento Animal/fisiologia , Betametasona/farmacologia , Northern Blotting , Química Encefálica , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Glucocorticoides/farmacologia , Hidrocortisona/agonistas , Hidrocortisona/antagonistas & inibidores , Metirapona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/sangue , Estresse Fisiológico/genética , Fatores de TempoRESUMO
Carbonic anhydrase (CA) in the inner ear sacculus was examined by activity assay, Western blotting and immunohistochemistry to determine its role in otolith calcification. An immunoreactive protein with a molecular mass of approximately 28 kDa was detected by Western blotting. The CO2 hydration activity in the cytosol fraction of the sacculus was 5.4 units/mg protein, while little or no activity was detected in the nuclear and mitochondrial fractions. The enzyme activity was highly inhibited by acetazolamide. The concentration of 50% inhibition was 8.16 nM and the inhibition constant of the activity was 8.25 nM. Transitional and squamous epithelial cells of the sacculus were immunopositive with an anti-CA II antibody, but sensory epithelial cells and mitochondria-rich cells in the transitional epithelium were not. These results suggest that transitional epithelial cells other than mitochondria-rich cells and squamous epithelial cells play an important role in otolith calcification by supplying bicarbonate to otoliths and/or by eliminating protons from endolymph.