Melatonin, probably best known for its influence on circadian and seasonal rhythms within vertebrates, is actually involved in many other physiological processes. Derived from the neurochemical serotonin, melatonin is a small molecule that can penetrate all tissues easily. For this reason, it would be expected that melatonin would be present in many different organs at concentrations similar to those in plasma, potentially with the same diurnal rhythm. However, if melatonin is found in tissues at concentrations higher than this, it may suggest an active uptake and/or local synthesis of melatonin and a potential role for the molecule within this organ. Physiological effects of melatonin are mediated via high-affinity G-protein coupled receptors, which can be identified in vitro by autoradiography and conventional binding assays using 2-[125I]Iodomelatonin, a radiolabelled marker for melatonin. In the present study, Ewa Kulczykowska,Richard Balment and their group of researchers decided to examine for the first time the potential involvement of melatonin in fish osmoregulatory tissues. They set out to determine the concentration of melatonin as well as the distribution of the specific binding of 2-[125I]Iodomelatonin,which would indicate the location of melatonin receptors, in the kidney, gill and intestine of rainbow trout, flounder and seabream.
The fish were held under a controlled 12 h light:12 h dark illumination regime for at least a period of three days before experimentation. After this acclimation period, plasma melatonin in the trout was measured by high performance liquid chromatography (HPLC), whereas plasma melatonin in seabream and flounder, as well as tissue melatonin levels in all three fish, were assayed using a total melatonin radioimmunoassay. Consistent with findings throughout the animal kingdom, the plasma melatonin levels in trout, flounder and seabream were significantly higher at night than during the day. However,melatonin levels remained unchanged in almost all of the tissues in the three species over a 24 hour cycle. They did not show the diurnal variation that had been found in the fish's plasma, suggesting that levels within these tissues are regulated independently of plasma concentrations. The team also demonstrated 2-[125I]Iodomelatonin binding in the kidney, gill and small intestine and that iodomelatonin binding did not fluctuate during the day, suggesting that the action of melatonin in osmoregulatory tissues is not photoperiod-dependent. Furthermore, GTPγS, a compound specific for G-protein receptors, added to the incubation medium significantly reduced the binding of 2-[125I]Iodomelatonin in gill and intestine of trout and flounder by 30–50%, strongly suggesting that the 2-[125I]Iodomelatonin binding sites are associated with a G-protein in these tissues.
This study is the first to provide evidence for the presence of melatonin binding in fish osmoregulatory tissues. Up until now, reports of melatonin binding in fish have been limited to the brain and the heart. Interestingly,in all three species, the concentration of melatonin in the intestine was significantly higher than those in the plasma, with the greatest difference occurring during the day when plasma levels were at their lowest. This suggests that in addition to the pineal gland and the retina, the intestine may be an important source of melatonin, as seen in other vertebrates. Its high melatonin content together with its G-protein mediated iodomelatonin binding suggests that melatonin in the intestine in particular may play an important physiological role in fish, more so than in the gill and kidney. What exactly that role is, has yet to be determined.