29th Annual Meeting of the American Society of Photobiology

Downtown Marriot

Chicago, Il.

Depletion of ATP and cytosolic Ca2+ accelerate PDT-induced firing inhibition and death of isolated nerve cell

Abstract-
Using isolated crayfish stretch receptor neuron (SRN) as a model we studied the role of bioenergetic processes and Ca²⁺ in photodynamic cell inactivation. After 30-min control firing recording, SRN was incubated 30 min with 100 nM sulphonated aluminum phthalocyanine Photosens and then irradiated by He-Ne laser (632.8 nm; 0.3 W/cm²) until firing abolition. Photosensitization gradually inhibited firing until its irreversible cessation. As shown cytochemically, impairment of bioelectric activity was accompanied with photoinactivation of succinate dehydrogenase. Energetic substrates (glucose, sodium succinate, sodium pyruvate and malate) slowed down photoinduced firing inhibition and increased SRN lifetime. Oppositely, inhibitors of different bioenergetic processes: glycolysis (sodium iodoacetate, 2-deoxy-D-glucose), Krebs cycle (sodium malonate), oxidative phosphorylation (2,4-dinitrophenol), and respiratory chain (antimycin A, sodium amital, rotenone, sodium azide) accelerated PDT-induced firing inhibition and shortened neuron lifetime. Therefore, regardless of the stage of the bioenergetic pathway, ATP depletion endowed SRN with higher sensitivity to PDT, whereas bioenergetic substrates protected neurons from photodynamic inactivation. Pharmacological agents increasing cytosolic Ca²⁺ concentration (calcium ionophore ionomycin, Ca²⁺ releasers thapsigargin and caffeine, extracellular ATP, 3-fold extracellular Ca²⁺ concentration, or cytosolic calcium buffering with 0.01 mM BAPTA-AM) precipitated PDT-induced firing inhibition and decreased neuron lifetime. Oppositely, agents decreasing intracellular Ca²⁺ concentration (blocker of calcium channels CdCl₂, inhibitor of intracellular calcium release dantrolene, prolonged action of caffeine or theophylline depleting Ca²⁺ stores) slowed down photoinduced firing inhibition and increased neuron lifetime. Therefore, intracellular Ca²⁺ as well as depletion of ATP accelerated PDT-induced irreversible abolition of SRN firing, which was considered as a functional sign of the cell death. Supported by Competion Center for Fundamental Science at SpbGU and RFBR.

Keywords: PDT, neuron, ATP, calcium