Melatonin Synthesis and Calcium Responses in the Pineal Gland of the Trout

Abstract

The present in vitro study correlates melatonin production of explanted trout pineal organs with intracellular calcium concentrations of isolated and immunocytochemically identified pinealocytes. Melatonin production of pineal organs maintained in flow-through organ culture is closely related to the incident light, showing high levels in the scotopic and mesopic range of illumination and low levels in the photopic range. Diminishing extracellular calcium (Ca2+) and enhancing magnesium (Mg2+) as well as application of Co2+ (3 mM) reduces melatonin release in the light- and dark-adapted state and causes a reversible breakdown of spontaneous oscillations in the intracellular concentration of free calcium ions [Ca2+]i that are observed in approximately 20% of the isolated S-antigen-immunoreactive pinealocytes. Intracellular calcium oscillations are also reversibly blocked by application of 10 μM nifedipine. Depolarization of pinealocytes by treatment with 60 mM KCl elevates [Ca2+]i in 90% of the oscillating and 50% of the non-oscillating cells. The effect of KCl is blocked by 50 μM nifedipine and 3 mM CoCl2. Furthermore, application of CoCl2 reduces the basal [Ca2+]i in non-oscillating pinealocytes. These results suggest that voltagegated L-type calcium channels play a major role in the regulation of [Ca2+]i in trout pinealocytes. It appears that Co2+ and low calcium/high Mg2+ buffer reduces melatonin release through an action on the intracellular calcium concentration of trout pinealocytes. All these data show that the trout pineal organ synthesizes and releases melatonin in relation to the irradiance of the incident light and depends on the presence of extracellular calcium which enters the cell via voltage-gated calcium channels. Treatment of cultured pineal organs with norepinephrine (1 μM) or the β-adrenergic agonist isoproterenol has no significant effect on the light-dependent melatonin release. Furthermore, application of norepinephrine in varying concentrations (100 pM - 50 μM) that were previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes has no effect on [Ca2+]i of oscillating or non-oscillating trout pinealocytes. These data suggest that in the trout pineal organ a norepinephrine sensitive pinealocyte of the mammalian type is not involved in the transduction of light signals into a melatonin response.