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
