In some freshwater fish species, the control of gill Na, Cl cotransporter (Ncc2b) by prolactin appears to be instrumental to ionic homeostasis. This study was carried out to examine the signaling pathways involved in prolactin-mediated salt retention using gill explants from Japanese medaka (Oryzias latipes). Ovine prolactin induced a concentration-dependent stimulation of ncc2b with significant effects of 10, 100 and 1000 ng of hormone per mL media (2-6 fold). To understand the molecular mechanisms mediating prolactin control of gill function, we analyzed effects on signaling pathways known to be involved in the hormones action in other systems, namely Stat5, Akt and Erk1/2. Their activation was examined in a time course and concentration response experiment. Prolactin (1 μg mL-1) induced a rapid phosphorylation (stimulation) of Stat5 (10 min) that reached a plateau after 30 min and was maintained for at least 120 min. The effect of prolactin on Stat5 phosphorylation was concentration-dependent (4-12 fold). No activation of Akt or Erk1/2 was observed in either experiment. The Stat5 activation was further investigated in localization studies that demonstrated strong nuclear expression of phosphorylated Stat5 in prolactin-treated gill ionocytes. Using specific inhibitors, we analyzed the signalling pathways mediating prolactin induction of gill ncc2b. Co-incubation experiments showed that Stat5 inhibition blocked prolactin's stimulation of ncc2b expression, while PI3K-Akt and Mek1/2-Erk1/2 pathway inhibitors had no effect. These findings show that ncc2b expression is dependent on prolactin's downstream activation of Stat5 and its subsequent nuclear translocation within branchial ionocytes.
Keywords: Epithelia; Medaka; Osmoregulation; Salt retention; Signaling pathways.
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