Stimulated increase in free cytosolic Ca(2+) and protein kinase C activity in human cerebrocortical synaptosomes

Protein kinase C (PKC) is an important family of kinases regulated by lipid second messengers and cofactors that interact with cellular membranes. Both Ca(2+)-dependent and -independent isoforms of PKC have been described in rat cerebrocortical presynaptic nerve terminals (synaptosomes). In the present study, synaptosomes were prepared from human cerebral cortex obtained from standard temporal lobe specimens removed due to epilepsy. In order to measure free cytosolic Ca(2+) ([Ca(2+)](i)) and PKC activity continuously, the synaptosomes were loaded with the fluorescent probes fura-2 and fim-1. Membrane depolarisation by 4-aminopyridine (4-AP) 1 mM increased the [Ca(2+)](i) fluorescence by 14.4+/-2.2% and the PKC activity fluorescence by 16.7+/-1.6%. Partial depolarisation with 4-AP 0.3 mM increased the [Ca(2+)](i) fluorescence by 9.0+/-1.5% and the PKC activity fluorescence by 4.5+/-0.7%. When CaCl(2) was omitted from the media, PKC activity fluorescence increased by 7.9+/-1.2% subsequent to stimulation with 4-AP 1 mM. This method is thus well suited for studying presynaptic [Ca(2+)](i) and PKC activity involved in neurotransmission, both under physiological conditions and under the influence of neuropharmacological agents.