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 tritiated phorbol 12,13dibutyrate ([ 3 H]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 [ 3 H]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 . Key Words: Phorbol ester binding-Protein kinase C-Protein phosphorylation-Retina-Horizontal cells.