Dopamine Release via Protein Kinase C Activation in the Fish Retina

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Abstract
Calcium‐dependent phospholipid‐sensitive protein kinase [protein kinase C (PKC)] was partially purified from the carp (Cyprinus carpio) retina through DE 52 ion exchange and Cellulofine gel filtration chromatography. The phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) activated PKC in the nanomolar range. A major 38‐kDa protein in the retinal supernatants (105,000 g) was phosphorylated in vitro by PKC during a short period (3 min). Other phosphoproteins also appeared during a further prolonged period (>15 min). Rod‐bipolar and dopamine (DA) interplexiform cells in the fish retina were immunoreactive to a monoclonal antibody to PKC (α/β‐subtype). The PKC antibody recognized a 78‐kDa native PKC enzyme by means of an immunoblotting method. Subsequently, the effects of two kinds of PKC activators were investigated on [3H]DA release from retinal cell fractions containing DA cells that had been preloaded with [3H]DA. A phorbol ester (TPA) induced a calcium‐ and dose‐dependent [3H]DA release during a short period (2 min), with the minimal effective dose being 1 nM. Other phorbols having no tumor‐promoting activity, such as 4β‐phorbol and 4α‐phorbol 12, 13‐didecanoate, were ineffective on [3H]DA release. A synthetic diacylglycerol [1‐oleoyl‐2‐acetylglycerol (OAG)], which is an endogenous PKC activator, was also able to induce a significant release of [3H]DA. Furthermore, TPA was found to release endogenous DA from isolated fish retina by a highly sensitive HPLC with electrochemical detection method. The OAG‐ or TPA‐induced [3H]DA or DA release was completely blocked by inhibitors of PKC, such as 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7) and staurosporine. In contrast, release of [3H]glycine, γ‐[3H]aminobutyric acid, and D‐[3H]aspartic acid was never induced by TPA. Taken together, the present data indicate that TPA or OAG triggers a release of DA synaptically and that the induction is due to a PKC activation specifically in the membrane of DA cells in the fish retina. The functional role of PKC in rod‐bipolar cells is unknown at present.