Pier Lorenzo Marchiafava

Melatonin as an antioxidant in retinal photoreceptors

Abstract: Dark‐adapted, single photoreceptors isolated from the frog retina produce reactive oxygen species (ROS) after about 1 min of illumination with saturating light that we verified by their oxidation of preloaded dihydrorhodamine 123 (DHR) into the fluorescent rhodamine 123 (RHO). In this preparation we tested the antioxidant effects of vitamin E and of melatonin. Melatonin at picomolar and low nanomolar concentrations was determined to be 100 times more potent in inhibiting the light‐induced oxidative processes than was vitamin E. On the contrary, both compounds exerted potent prooxidant effects at micromolar concentrations that is above the physiological levels of melatonin. This provides evidence that physiological concentrations of melatonin in a living cell may exert protective actions against a natural oxidant stimulus (light). This helps to define the functional role of endogenous melatonin in photoreceptors, which by their physiological characteristics, are among the marked producers of ROS in the organism.

MELATONIN INDUCES MEMBRANE CONDUCTANCE CHANGES IN ISOLATED RETINAL ROD RECEPTOR CELLS

Experiments were conducted to verify whether the neurohormone melatonin influences the membrane conductance of photoreceptors isolated from the frog retina. It has been found that 20 microM melatonin decreases membrane conductances both in the linear and non linear ranges by <0.4 nS. These actions are estimated to produce in dark adapted photoreceptors an increase of the response to a dim light induced change of the dark current of about 21%, i.e. from 1.3 to 1.62 mV/pA.

Chapter 1 The electrical responses of the trout pineal photoreceptors to brief and prolonged illumination

Intracellular recordings from 103 photoreceptors in the excised pineal body of adult trouts were obtained by using single electrode current- and voltage-clamp techniques. The photoresponses to brief flashes showed the same polarity but a slower time course than those previously recorded from retinal photoreceptors of lower vertebrates. Pineal photoreceptors showed spectral sensitivity peaks at about 495 and 521 nm and absolute sensitivity comparable to retinal cone cells of the same species. The photoreceptor membrane conductance, measured under voltage clamp during moderate illumination was about 10% lower than in the dark, and the extrapolated reversal potential of the response was at 60 mV above the dark membrane potential. The addition of 3-isobutyl-1-methylxanthine (IBMX) to the perfusate was followed by a receptor depolarization in the dark and by a slow-down of the response kinetic. Pineal receptor cells produce constant amplitude responses during steady illumination, without displaying the delayed slow depolarization typically associated with light adaptation of retinal photoreceptors. Photoresponses to brief flashes superimposed on a steady illumination are decreased in amplitude by an amount directly related to the background intensity. Increase of the background intensity leads to threshold increments without significant changes of the saturation intensity, resulting in a gradual compression of the cell dynamic range. These results were discussed relative to light adaptation in retinal photoreceptors. The conclusion can be drawn that the response properties of pineal photoreceptors during steady illumination are part of an unknown, self-regulating mechanism to lock the rate of metabolism and secretion of indolamines to the absolute level of diurnal light.

Photoresponses and light adaptation of pineal photoreceptors in the trout

The photoresponses of 116 photoreceptors in the excised perfused pineal body of adult trout were intracellularly recorded. Brief flashes produced slower, but otherwise com parable, responses to retinal receptor cells. T he absolute sensitivity, as well as the light-induced membrane conductance changes, were also similar to those o f retinal cone cells in the same species. Steady illumination decreased the cell sensitivity to superim posed flashes, in accordance with the Weber—Fechner relation, indicating the presence o f light adaptation. This conclusion is supported by the non-exponential nature of the amplitude—intensity relation and by the conspicuous shortening of the time-to-peak with increasing flash intensity. The time-dependent response decay typical of retinal photoreceptors was not found, however. The fact that, during constant illumination, the pineal photoresponses maintain the same amplitude set at the initial peak perhaps represents an original mechanism of light adaptation which allows these cells to maintain their voltage-dependent synthesis and secretion of indolamines as a constant relation with daily illumination.

The Photoresponses of the Trout Pineal Cells

The pineal body of the trout is a strongly vascularized rounded structure (about 1 mm diameter), lying upon the dorso- lateral portion of the diencephalon and attached to it by means of a peduncle about 2 mm long. The pineal body is essentially a photoneuroendocrine organ since several lines of evidences indicate its light regulated influence on locomotion, gonadotropic activity as well as on the production of melatonin, an hormon involved in body pigmentation (Oksche, 1986).