Long‐Term Dynamic in vitro System for Investigating Rat Pineal Melatonin Secretion

Abstract

Abstract Several details regarding the control of melatonin (MT) secretion from the pinealocytes are still to be clarified. To obtain more data on the mechanism and kinetics of MT secretion and on the interactions between bioactive materials affecting MT production, a perifusion system has been developed in our laboratory. In this dynamic in vitro system the surviving pinealocytes maintain their full responsiveness for at least 5 days. In order to determine the MT contents of large numbers of samples collected in the perifusion system, a sensitive MT radioimmunoassay was set up utilizing our specific MT antibody. In our perifusion system the basal MT release does not change significantly during the 5-day experiments. Norepinephrine (NE) was used at 1 muM concentration for 30 min as a marker of the responsiveness of pinealocytes, given at the beginning and at the end of the same experiments. No significant difference was found in the MT responses to NE stimulation over 5 days. The kinetics of MT response and the dose-response relationship were investigated after NE exposure at various concentrations (100 nM to 10 mM). NE at 100 nM was found to be ineffective. Between 1 muM and 1 mM concentrations NE increased the MT release in a dose-dependent manner. No significant difference was found between the responses above 1 mM concentration. NE seems to be a specific stimulator of pineal MT production. The MT production reached the maximum value after a relatively long lag (2 to 3 h) when NE application had been stopped, and returned to basal values after 5 to 6 h. This prolonged time-course of MT secretion, in contrast with the fast responses of pituitary cells to releasing hormones, suggests that NE stimulated the synthesis of MT rather than the release of stored hormone. The modulatory effects of light-dark cycle on basal and stimulated MT release of perifused pineals was also investigated: Neither basal nor NE-stimulated (100 nM to 10muM) MT release was influenced significantly by light-dark conditions showing that the light-dark cycle does not have a direct modulatory effect on MT secretion under in vitro circumstances. Based on our observations, this perifusion system should be a useful tool for investigating: 1) hormone interactions on the regulation of pineal MT release, and 2) accurate kinetics of MT response.