In Vitro Phosphorylation in Isolated Horizontal Cells of the Fish Retina: Effects of the State of Light Adaptation

Abstract

Horizontal cells, which are second-order neurons of the vertebrate retina, exhibit synaptic plasticity governed by light and dark adaptation. We have investigated the alterations in the protein phosphorylation patterns of isolated carp (Cyprinus carpio) horizontal cells in relation to their state of light adaptation by using an in vitro phosphorylation assay and compared the resulting data with protein synthesis patterns of the whole retina. Phosphoproteins and [35S]methionine-labelled proteins were analysed by one- and two-dimensional gel electrophoresis followed by autoradiography. The state of light adaptation significantly affected the in vitro phosphorylation of horizontal cell proteins with molecular weights of 68, 56/58, 47, 28 and 15 kDa, but had no effect on the protein synthesis of retinal proteins. In the light the most prominent increase of 32P incorporation was observed in the 47 kDa protein. The biochemical properties of this protein closely resembled those of the growth-associated GAP-48, found in the fish retina. In addition, the phosphorylation of horizontal cell homogenates in the presence of protein kinase activators such as cyclic AMP, calcium, calmodulin and phospholipids revealed that horizontal cells of the fish retina contain cyclic AMP-, calcium/calmodulin- and calcium/phospholipid-dependent protein kinase activity resulting in the phosphorylation of several horizontal cell proteins, including the phosphoproteins which were affected by the state of light adaptation.

Retinoic acid has light-adaptive effects on horizontal cells in the retina

“…However, because at-RA is a much stronger signal with a faster time course than dopamine, a direct effect of at-RA upon protein kinases appears more likely (41,42). We have shown previously that protein biosynthesis is not involved in spinule formation during the first 45 min and that protein phosphorylation is the major mechanism (19).…”

“…The underlying signal cascade of this structural synaptic plasticity is only partially known. It has been shown that activation of protein kinase C by phorbol esters induces the formation of spinules and that, among several other proteins, the growth-associated protein GAP 43 is a likely candidate for phosphorylation (19,20). The involvement of cAMP in the process is still controversial.…”

Molecular Cloning and Functional Expression of zfCx52.6

“…The genomic clone was sequenced, and RACE extension, using retina RNA as the template, was performed to identify expressed sequences from the zfCx52.6 genomic locus. The molecular procedures used have been described in detail previously (18,41,42). The zfCx52.6 gene sequence was deposited in GenBank TM (GI:21311548).…”

Selective synaptic distribution of AMPA and kainate receptor subunits in the outer plexiform layer of the carp retina

“…The latter was used as a positive control. Horizontal cells were isolated from a cell suspension of the carp retina according to a previously described protocol (Janssen-Bienhold et al, 1993). Rat brain P2-fraction and carp retinal membranes and horizontal cells were homogenized in a buffer containing 20 mM Tris/HCl, pH 7.4, 10 mM MgCl 2 , 0.6 mM CaCl 2 , 0.5 mM EGTA, 0.005% Triton X-100 (TX-100), 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 g/ml of leupeptin, and 5 g/ml of aprotinin.…”