Photochemical reactivity of RPE melanosomes is increased after disruption by pulsed laser exposures

Purpose. We have previously shown that melanin isolated from the retinal pigment epithelium (RPE) is capable of oxidizing various cellular components, such as ascorbic acid and linoleic acid, during visible light exposure. We now show that the reactivity of the RPE melanosomes depends on their structural integrity, and may be markedly increased if the melanosomes are broken apart. Methods. RPE melanosomes were prepared from fresh bovine eyes, mixed with 7 mM NADPH in Tris buffer (pH 7.2), and exposed to the 2^nd-harmonic output of a Q-switched Nd:YAG laser (532 nm, 1800 10-nsec pulses at 10 Hz) in order to induce photodisruption. The photochemical activity of the melanosomes was then "pumped" by exposure to the continuous wave output of an Argon ion laser (488 nm) for five minutes. Oxidation of NADPH was measured by loss of absorbance at 340 nm. Results. Compared to native melanosomes not pre-exposed to the Nd:YAG laser, the photodisrupted melanosomes oxidized up to three times more NADPH during the 5′ pump period, with a quantum yield of >10^-5. The amount of NADPH oxidized by disrupted melanosomes increased in proportion to the energy density delivered by the YAG laser, up to a maximum at about 300 mJ/cm²/pulse. Physical disruption of the melanosomes was confirmed by scanning and transmission electron microscopy. Conclusions. Photodisruption of the melanosomes evidently allows the NADPH access to the interior of the melanosome where it is oxidized by light-induced melanin radicals. This finding suggests that the intact surface of the native melanosome, probably a protein coat, acts to prevent excited melanin from reacting with susceptible cell components. Breakdown of this barrier, for example in age or by injury, may be a mechanism leading to increased photooxidative stress in the eye.