RESUMO
While studies clearly point to a role for cortisol signaling in seawater adaptation, very little is known about salinity impact on glucocorticoid receptor (GR) expression in fish. To this end, we investigated the temporal GR expression in the gill and liver of rainbow trout (Oncorhynchus mykiss) to salinity exposure. Trout were subjected to gradual salinity increases (11 ppt for 1 d, 17 ppt for 2 d and 23 ppt for 2 d) over a five day period. Gill Na(+), K(+)-ATPase alpha-subunit mRNA showed a transient elevation with salinity exposure, while gill cystic fibrosis transmembrane conductance regulator mRNA was not significantly affected by salinity. Liver PEPCK transcript levels showed a transient increase at day 1, but not at day 3 or day 5 of salinity exposure, while the activity of this enzyme was significantly depressed at all time points. Liver glycogen content was also significantly reduced by salinity exposure compared to the freshwater group. Gill GR transcript levels were 3-fold greater upon salinity exposure and this level was maintained over the 5 day period, while gill GR protein content remained unchanged except for a significant drop at day 1 of salinity exposure. Liver GR transcript levels showed no significant change with salinity exposure, while GR protein content was transiently elevated at day 3, but not at day 1 or day 5 of salinity exposure. The tissue-specific GR transcript response in the gill leads us to hypothesize a role for osmosensory signal transduction pathway in the regulation of GR expression in fish. Collectively, salinity exposure modulates GR expression and glucocorticoid signaling in rainbow trout.
Assuntos
Perfilação da Expressão Gênica , Oncorhynchus mykiss/genética , Receptores de Glucocorticoides/genética , Cloreto de Sódio/farmacologia , Aclimatação/efeitos dos fármacos , Aclimatação/genética , Aclimatação/fisiologia , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulação da Expressão Gênica/genética , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Oncorhynchus mykiss/fisiologia , Fosfoenolpiruvato Carboxilase/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de TempoRESUMO
We used primary cultures of trout hepatocytes and a physiological dose of cortisol (100 ng/ml), mimicking stressed levels in salmonid fish, to address the impact of glucocorticoid stimulation on glucocorticoid receptor (GR) mRNA abundance and protein content. Cortisol significantly elevated GR mRNA content over a 24-h period; this increase was abolished by actinomycin D, suggesting transcriptional control of GR. However, cortisol significantly decreased GR protein content, leading us to hypothesize that lower GR protein content may be regulating GR mRNA abundance. Indeed, treatment of hepatocytes with MG-132, a proteasomal inhibitor shown to prevent GR degradation by cortisol, abolished cortisol-mediated GR mRNA upregulation. Also, geldanamycin, a heat shock protein 90-specific inhibitor, abolished the GR mRNA increase evident with cortisol but did not modify cortisol-induced increases in abundance of mRNA for phosphoenolpyruvate carboxykinase, a glucocorticoid-responsive gene, or hepatocyte glucose release. Together, our results suggest a negative feedback loop for GR gene regulation by cortisol in trout hepatocytes. The autoregulation of GR may be a crucial step in the physiological stress response process, especially in modulating energy-dependent processes that are glucocorticoid dependent, including gluconeogenesis.
Assuntos
Regulação da Expressão Gênica , Hepatócitos/metabolismo , Hidrocortisona/metabolismo , Oncorhynchus mykiss/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Benzoquinonas , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Lactamas Macrocíclicas , Proteínas Serina-Treonina Quinases/metabolismo , Quinonas/farmacologia , Receptores de Glucocorticoides/genéticaRESUMO
We investigated whether longer-term cortisol exposure modified hepatic glucocorticoid receptor (GR) status and tissue responsiveness to cortisol stimulation in rainbow trout. Fish were given intraperitoneal implants of cortisol (50mg/kg body mass) and this led to elevated plasma cortisol levels mimicking chronically stressed salmonids. There was significantly higher hepatic GR mRNA abundance, despite a drop in GR protein content in the liver of cortisol-treated fish. The tissue responsiveness to cortisol stimulation was apparent from the higher plasma glucose concentration and liver glycogen content. Also, the higher phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance, a key glucocorticoid-responsive gene, by cortisol suggests activation of the GR signalling pathway. There was no significant effect of cortisol treatment on liver PEPCK, alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities compared to the sham fish. The higher heat shock protein (hsp) 90 mRNA abundance and a corresponding elevation in this protein and constitutive hsp70 (hsc70) protein content in the cortisol-treated fish reflects a role for glucocorticoids in the hepatic stress response process. Taken together, the molecular and biochemical responses evident in the liver of trout imply changes favouring tissue responsiveness to glucocorticoids and may be a mechanism to offset GR protein downregulation evident with chronic cortisol stimulation in rainbow trout.