Ouabain, a potent inhibitor of the enzyme Na+/K+-ATPase, is known to be a steroid hormone in mammals that is involved in blood pressure and volume regulation. Recently, a group from the University of Hawaii examined whether ouabain also acts as a hormone in fish and reported that ouabain is present in the plasma and various tissues of the euryhaline Mozambique tilapia, Oreochromis mossambicus. When the researchers transferred tilapia between fresh and salty water, they discovered that changes in plasma concentrations of ouabain in tilapia were well correlated with changes in plasma osmolality and cortisol, an important hormone during salinity change. Now, this same research group has followed up with an equally intriguing analysis of the functional role of ouabain in fish osmoregulation.
The present study set out to establish whether ouabain interacts with growth hormone and prolactin (known osmoregulatory hormones in fish) in the same manner as cortisol. During seawater acclimation, cortisol stimulates Na+/K+-ATPase activity and the proliferation of chloride cells that are responsible for the active extrusion of Cl-. In addition, cortisol stimulates the release of growth hormone, which is also involved in seawater adaptation, and inhibits the release of prolactin, an important hormone in freshwater osmoregulation, from the pituitary gland. Prolactin release is dependent on calcium entering pituitary gland cells and the synthesis of cAMP, both of which are prevented by cortisol.
To assess the role of ouabain in fish osmoregulation, Kajimura, Grau and the group examined the release of osmoregulatory hormones by tilapia pituitary glands. They isolated whole pituitary glands from tilapia and placed them individually in a 96-well plate containing 200 μl of incubation media. With this in vitro preparation, they measured prolactin and growth hormone release from tilapia pituitary glands in response to different doses of ouabain. To see if ouabain, like cortisol, inhibits prolactin release by preventing calcium uptake and cAMP synthesis, they measured prolactin release in response to calcium (using the calcium ionophore A23187) and to cAMP (using the analogue dbcAMP and the phosphodiesterase inhibitor IBMX) in the presence or absence of ouabain. The group found that at physiological concentrations,ouabain exerted a dose-dependent inhibitory effect on prolactin, whereas supra-physiological doses of ouabain stimulated prolactin release. By contrast, ouabain stimulated growth hormone release at lower concentrations but had an inhibitory effect at higher levels. Both A23187 and dbcAMP + IBMX had stimulatory effects on prolactin release, and these effects were inhibited by ouabain when it was also placed in the incubation media. This suggests that, like cortisol, ouabain regulates the mechanism of prolactin release. Furthermore, using microspectrofluorometry and a calcium-sensitive dye, the team was able to quantify `real-time' changes in calcium levels within pituitary cells. Their data indicated that at physiological concentrations,ouabain rapidly reduces intracellular calcium concentrations, just as cortisol does.
The group has shown convincingly that, similar to the actions of cortisol during salinity change, ouabain plays an important role in fish osmoregulation by inhibiting the release of prolactin via the Ca2+ and cAMP signal transduction pathways. The team believes that ouabain in the hypothalamus of the brain may be involved in the regulation of pituitary prolactin release and hope to prove this hypothesis in the future by using a ouabain-specific antibody and immunocytochemistry. Until then, we will wait for more news about this exciting new fish hormone!