Receptor-stimulated phospholipase A(2) liberates arachidonic acid and regulates neuronal excitability through protein kinase C

Type B photoreceptors in Hermissenda exhibit increased excitability (e.g., elevated membrane resistance and lowered spike thresholds) consequent to th e temporal coincidence of a light-induced intracellular Ca2+ increase and t he release of GABA from presynaptic vestibular hair cells. Convergence of t hese pre- and postsynaptically stimulated biochemical cascades culminates i n the activation of protein kinase C (PKC). Paradoxically, exposure of the B cell to light alone generates an inositol triphosphate-regulated rise in diacylglycerol and intracellular Ca2+, co-factors sufficient to stimulate c onventional PKC isoforms, raising questions as to the unique role of synapt ic stimulation in the activation of PKC. GABA receptors on the B cell are c oupled to G proteins that stimulate phospholipase A(2) (PLA(2)), which is t hought to regulate the liberation of arachidonic acid (AA), an "atypical" a ctivator of PKC. Here, we directly assess whether GABA binding or PLA(2) st imulation liberates AA in these cells and whether free AA potentiates the s timulation of PKC. Free fatty-acid was estimated in isolated photoreceptors with the fluorescent indicator acrylodan-derivatized intestinal fatty acid -binding protein (ADIFAB). In response to 5 muM GABA, a fast and persistent increase in ADIFAB emission was observed, and this increase was blocked by the PLA(2) inhibitor arachidonyltrifluoromethyl ketone (50 muM). Furthermo re, direct stimulation of PLA(2) by melittin (10 muM) increased ADIFAB emis sion in a manner that was kinetically analogous to GABA. In response to sim ultaneous exposure to the stable AA analogue oleic acid (OA, 20 muM) and li ght (to elevate intracellular Ca2+), B photoreceptors exhibited a sustained (>45 min) increase in excitability (membrane resistance and evoked spike r ate). The excitability increase was blocked by the PKC inhibitor chelerythr ine (20 muM) and was not induced by exposure of the cells to light alone. T he increase in excitability in the B cell that followed exposure to light a nd OA persisted for greater than or equal to 90 min when the pairing was co nducted in the presence of the protein synthesis inhibitor anisomycin (1 mu m), suggesting that the synergistic influence of these signaling agents on neuronal excitability did not require new protein synthesis. These results indicate that GABA binding to G-protein-coupled receptors on Hermissenda B cells stimulates a PLA(2) signaling cascade that liberates AA, and that thi s free AA interacts with postsynaptic Ca2+ to synergistically stimulate PKC and enhance neuronal excitability. In this manner, the interaction of post synaptic metabotropic receptors and intracellular Ca2+ may serve as the cat alyst for some forms of associative neuronal/synaptic plasticity.