Phorbol ester binding sites in the fish retina: correlation with stimulation of endogenous phosphorylation and protein kinase C activation.

Abstract

The injection of phorbol esters into the eyes of dark-adapted teleost fish can mimic light effects in the retina and induces corresponding synaptic plasticity of horizontal cells (HCs). It is therefore very likely that protein kinase C (PKC) mediates light-induced synaptic plasticity. In the present study, we investigated the distribution of PKC, the phorbol ester receptor, in isolated HCs and in the whole retina by using tritated phorbol 12,13-dibutyrate ([3H]PDBu). The binding characteristics analyzed for HC homogenates and retinal homogenates revealed that [3H]PDBu binding is time dependent, specific, saturable, and reversible. Binding sites in HCs displayed a dissociation constant of 11.5 nM and a total number of 2.8 pmol/mg of protein. Autoradiography revealed that [3H]PDBu labeling is present in all retinal layers, including HCs, where it is associated with the somata. Furthermore, the treatment with PDBu strongly affected the endogenous phosphorylation of several membrane, cytosolic, and HC proteins and led to PKC activation as measured by H1 histone phosphorylation. In HCs, the treatment with PDBu in particular affected the amount of 32P incorporated into a group of phosphoproteins (68, 56/58, 47, 28, and 15 kDa) that were recently shown to be affected by light adaptation. These proteins might therefore be considered as important components of the observed morphological and physiological synaptic plasticity of HCs in the course of light adaptation.