Ca2+ dependent activation of extracellular signal regulated kinases 1 and 2: role of intrasynaptosomal Ca2+ stores

The aim of the thesis was to examine how Ca2+ activates the ERK pathway and the contribution of Ca2+ released from intracellular stores in physiological and pathophysiological conditions using isolated nerve terminals (synaptosomes) in a presynaptic model. The Ca2+-dependent phosphorylation/activation of ERK1 and ERK2 stimulated by depolarisation of the plasma membrane or by Ca2+ influx mediated by the ionophore ionomycin was significantly reduced by the removal of external Ca2+. Intrasynaptosomal Ca2+ contribution to the Ca2+-dependent component of ERK1 and ERK2 phosphorylation/activation was indicated by the depletion of intrasynaptosomal Ca2+ or inhibition of the smooth endoplasmic reticulum Ca2+-ATPase pump. Two main pathways were found to lead to the release of Ca2+ from intrasynaptosomal stores. Firstly, external Ca2+ influx directly activated ryanodine receptors (RyRs) to mediate Ca2+-induced Ca2+ release (CICR). Secondly, Ca2+ influx or activation of GPCRs coupled to Gq/11 activated phospholipase C (PLC) to effect PIP2 metabolism and IP3 production, with consequent activation of IP3-induced Ca2+ release (IPCR). The activation of group I metabotropic glutamate receptor (mGluR1/5) stimulation supported IPCR. Intriguingly, inhibition of Ca2+ influx through voltage-dependent calcium channels (VDCCs) by stimulating GABAB, group III mGluRs, 5-HT1A and A1 receptors was suppressed by prior depletion of the smooth endoplasmic reticulum. Mitochondria and acidic compartments also appear to store Ca2+ intrasynaptosomally, with mitochondrial depolarisation resulting in a transient increase in ERK1 and ERK2 phosphorylation/activation. Finally, a pathophysiological model of nerve terminal ischemia showed that intrasynaptosomal Ca2+ release contributes to the Ca2+-dependent component of phosphorylation/activation of ERK1 and ERK2 occurring when Na+/K+- ATPase is inhibited. In conclusion, extracellular Ca2+ influx and intracellular Ca2+ store release together support Ca2+ mediated stimulation of the ERK pathway in synaptosomes. This has important implications in the cross-talk of signalling pathways to ERK1 and ERK2 phosphorylation/activation and neurotransmitter release from nerve terminals in physiological and pathophysiological conditions