Receptor-Stimulated Phospholipase A2 Liberates Arachidonic Acid and Regulates Neuronal Excitability Through Protein Kinase C

Abstract

Type B photoreceptors in Hermissendaexhibit increased excitability (e.g., elevated membrane resistance and lowered spike thresholds) consequent to the temporal coincidence of a light-induced intracellular Ca2+ increase and the release of GABA from presynaptic vestibular hair cells. Convergence of these pre- and postsynaptically stimulated biochemical cascades culminates in 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 conventional PKC isoforms, raising questions as to the unique role of synaptic stimulation in the activation of PKC. GABA receptors on the B cell are coupled to G proteins that stimulate phospholipase A2 (PLA2), which is thought to regulate the liberation of arachidonic acid (AA), an “atypical” activator of PKC. Here, we directly assess whether GABA binding or PLA2 stimulation liberates AA in these cells and whether free AA potentiates the stimulation 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 μM GABA, a fast and persistent increase in ADIFAB emission was observed, and this increase was blocked by the PLA2 inhibitor arachidonyltrifluoromethyl ketone (50 μM). Furthermore, direct stimulation of PLA2 by melittin (10 μM) increased ADIFAB emission in a manner that was kinetically analogous to GABA. In response to simultaneous exposure to the stable AA analogue oleic acid (OA, 20 μM) and light (to elevate intracellular Ca2+), B photoreceptors exhibited a sustained (>45 min) increase in excitability (membrane resistance and evoked spike rate). The excitability increase was blocked by the PKC inhibitor chelerythrine (20 μM) and was not induced by exposure of the cells to light alone. The increase in excitability in the B cell that followed exposure to light and OA persisted for ≥90 min when the pairing was conducted in the presence of the protein synthesis inhibitor anisomycin (1 μm), 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 PLA2 signaling cascade that liberates AA, and that this free AA interacts with postsynaptic Ca2+ to synergistically stimulate PKC and enhance neuronal excitability. In this manner, the interaction of postsynaptic metabotropic receptors and intracellular Ca2+ may serve as the catalyst for some forms of associative neuronal/synaptic plasticity.