MODULATION OF CA2-DEPENDENT CURRENTS IN METABOLICALLY STRESSED CULTURED SENSORY NEURONS BY INTRACELLULAR PHOTORELEASE OF ATP()

1 The whole cell recording technique was used to study high voltage-ac tivated Ca2+ currents and Ca2+-activated Cl- tail currents from cultur ed neonatal dorsal root ganglion neurones of the rat which were metabo lically stressed. The neurones were metabolically stressed with 2-deox y-D-glucose (5 mM) for 30 min to 3 h. The aim of the project was to ex amine the actions of intracellular photorelease of ATP on the properti es of Ca2+-dependent currents and determine if the effects of metaboli c stress could be reversed. 2 The mean duration of Ca2+-activated Cl- tail currents was significantly increased by metabolic stress and this effect was reversed by intracellular photorelease of approximately 30 0 mu M ATP. Intracellular photolysis of 'caged' photolabile compounds was achieved with a xenon flash lamp. 3 Intracellular photorelease of ATP and adenosine 3':5'-cyclic monophosphate (cyclic AMP) (about 40 mu M) also accelerated the inactivation of high voltage-activated Ca2+ c urrents evoked by 500 ms depolarizing step commands from - 90 mV to 0 mV. This effect was prevented by intracellular application of the calc ineurin (protein phosphatase-2B) inhibitor cyclosporin A (14 nM) and c yclophilin A (50 nM) either applied together or individually. In contr ast the protein phosphatase 1 and 2A inhibitor, calyculin A, increased voltage-activated Ca2+ currents, but failed to prevent enhanced inact ivation induced by intracellular photorelease of ATP. Intracellular ph otorelease of ATP had no effect on Ca2+ currents recorded from control neurones which were not metabolically stressed and supplied with gluc ose and ATP in the extracellular and patch pipette solutions respectiv ely. 4 In conclusion, intracellular photorelease of ATP increases the decay of Ca2+-activated Cl- tail currents in metabolically stressed ne urones suggesting that the efficiency of intracellular Ca2+ buffering was improved. Additionally, an ATP/cyclic AMP-dependent component of h igh voltage-activated Ca2+ current inactivation which is mediated by c alcineurin is revealed following photolysis of 'caged' ATP or cyclic A MP in metabolically stressed neurones.