García Lab

Research in the García lab has historically been oriented toward understanding the mechanisms through which light- and dark-adaptation are accomplished in the retina of fishes. Of particular interest are those processes involved in regulating pigment granule movements in the retinal pigment epithelium (RPE). Much of our effort has been oriented toward discovering how acetylcholine activates light-adaptive pigment granule dispersion in retinal pigment epithelium, both in terms of the cell surface receptors involved and the downstream signaling processes. Our research using RPE isolated from green sunfish and bluegill supports a model in which acetylcholine activates a muscarinic receptor, activating a G-protein, which in turn activates phospholipase C, leading to release of calcium from intracellular stores and consequent pigment granule dispersion. Furthermore, using immunohistochemistry we have demonstrated that bluegill RPE express m3 and m5 muscarinic receptors, consistent with our pharmacological results. It remains to be determined whether acetylcholine is a physiologically relevant signal for light adaptive responses in the fish. Our work in zebrafish suggests that muscarinic receptors (m1a, m1b, m3a, m3b, m5a and m5b) are expressed in the eye early in development. Based on others’ work reporting that rudimentary light- and dark-adaptive movements are initiated by 5 days, but not fully developed until adulthood, our work indicates that muscarinic receptors may be present by the time pigment granule movements are initiated. Interestingly, muscarinic receptors (m2 and m5) show diurnal changes in their expression levels at the mRNA level while m3 receptors show changes at the protein level.

We are also interested in dark-adaptive processes, specifically exploring the hypothesis that photoreceptors export cyclic nucleotides in the dark using an ATP-binding cassette transporter called Mrp4, and those cyclic nucleotides, acting as both a first and second messenger, are taken up by the RPE and elicit dark-adaptive pigment granule aggregation. Evidence in support of this model comes from studies on green sunfish and bluegill RPE showing that treating isolated RPE with non-derivatized cAMP induces pigment granule aggregation and that aggregation is inhibited by treating cells with inhibitors of organic anion transport. The same treatment blocks uptake of tritiated cAMP, providing direct evidence of transport-mediated uptake of cAMP. Currently, we are probing zebrafish retina to determine the presence, location and function of Mrp4.

Zebrafish express duplicate forms of many of the genes involved in pigmentation and we are currently working to understand both the regulation of duplicate genes as well as the role of their gene products in influencing pigment granule position in both melanophores (pigmented cells of the skin) and RPE. Interestingly, suprisingly few genes are show a diurnal rhythm in their expression in the eye, but among those that appear to are the genes encoding the rate-limiting enzyme in melatonin synthesis (AANAT), melatonin receptors, dopamine receptors, muscarinic receptors (as mentioned above) and arrestin. We are currently testing whether protein levels change commensurately, particularly in the case of melatonin receptors. Such changes could be relevant to normal, diurnal cycles of activity as well as changes in those cycles associated with aging.