Effects of calcium channel blockers on the kinetics of voltage-dependent changes in synaptosomal calcium concentrations.
Synaptosomal preparations from rat cerebral cortex have been used in stopped-flow fluorescence studies to measure rapid changes in intrasynaptosomal calcium concentrations upon depolarization. Synaptosomes were loaded with the fluorescent calcium chelating dye, Fura-2, by incubation with the membrane permeant acetoxymethyl ester derivative. Depolarization by elevated external K+ concentration resulted in a rapid increase in cytoplasmic Ca2+ as measured by a quench in Fura-2 fluorescence when excited at 390 nm. The fluorescence change could be reasonably fit by a single exponential process with an apparent rate of 10-15 s-1 and the magnitude of the response was voltage-dependent, increasing with increasing external K+ over the range of 5-30 mM. The observed quench was blocked by micromolar concentrations of the inorganic calcium channel blockers, Cd2+, Co2+ and La3+. Nimodipine, a dihydropyridine which blocks L-type calcium channels, inhibited only 10-15% of the flux response while nitrendipine had no consistent effect. omega-Conotoxin GVIA, a blocker of N-type channels in many species, had only a small inhibitory effect at high (1-10 microM) concentrations. The response was, however, inhibited by pre-incubation of the synaptosomes with venom of the funnel web spider. Agelenopsis aperta (0.1-300 micrograms/ml). Inhibition was observed with both a purified polyamine fraction (FTX) from the venom (IC50 = 4 nl/ml) and a purified peptide toxin, omega-AgaIVA (IC50 = 30 nM). These results indicate that voltage-dependent Ca2+ uptake by mammalian nerve terminals is mediated primarily by channels that are insensitive to dihydropyridines and omega-conotoxin GVIA but are sensitive to components of funnel web spider venom.
Thomas
M M
MM
Department of Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Canada.
Puligandla
P S
PS
Dunn
S M
SM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002132:Calcium Radioisotopes; D016257:Fura-2; D007700:Kinetics; D008024:Ligands; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D011073:Polyamines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013111:Spider Venoms / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism; D020867:omega-Agatoxin IVA
D002121:Calcium Channel Blockers; D002132:Calcium Radioisotopes; C071920:FTX, spider toxin; D008024:Ligands; D011073:Polyamines; D013111:Spider Venoms; D020867:omega-Agatoxin IVA; D002118:Calcium; D016257:Fura-2