MOBILIZATION OF INTRACELLULAR CALCIUM BY INTRACELLULAR FLASH-PHOTOLYSIS OF CAGED DIHYDROSPHINGOSINE IN CULTURED NEONATAL RAT SENSORY NEURONS

The ability of dihydrosphingosine to release Ca2+ from intracellular s tores in neurones was investigated by combining the whole cell patch c lamp technique with intracellular flash photolysis of caged, N-(2-nitr obenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 mu M) wa s applied to the intracellular environment via the CsCl-based patch pi pette solution which also contained 0.3% dimethylformamide and 2 mM di thiothreitol. Cultured dorsal root ganglion neurones from neonatal rat s were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of di hydrosphingosine. Intracellular photorelease of dihydrosphingosine (ab out 5 mu M) was achieved using a Xenon flash lamp. Inward Ca2+-activat ed currents were evoked in 50 out of 57 neurones, the mean delay to cu rrent activation following photolysis was 82 +/- 13 s. The responses w ere variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 m s voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihyd rosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine curre nt had a reversal potential of -11 +/- 3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D- glucamine or application of the Cl- channel blocker niflumic acid (10 mu M) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 mu M rya nodine or extracellular application of 10 mu M dantrolene, but persist ed when Ca2+ free saline was applied to the extracellular environment. Intracellular application of uncaged dihydrosphingosine evoked respon ses which were attenuated by photolysis of the Gaged Ca2+ chelator Dia zo-2. Experiments also suggested that extracellular application of dih ydrosphingosine can activate membrane conductances. We conclude that d ihydrosphingosine directly or indirectly mobilises Ca2+ from ryanodine -sensitive intracellular stores in cultured sensory neurones.