Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxin

A channel open on the membrane can be formed by palytoxin (PTX). Ten nanomolar PTX caused an irreversible increase in the cytosolic calcium concentration ([Ca(2+)](c)), which was abolished in the absence of external calcium. The increase was eliminated by saxitoxin (STX) and nifedipine (NIF). Calcium rise is secondary to the membrane depolarization. PTX effect on calcium was dependent on extracellular Na(+). Li(+) decreased the PTX-evoked rise in [Ca(2+)](c); replacement of Na(+) by N-methyl-D-glucamine (NMDG) abolished PTX-induced calcium increase. [Ca(2+)](c) increase by PTX was strongly reduced after inhibition of the reverse operation of the Na(+)/Ca(2+) exchanger, in the presence of antagonists of excitatory amino acid (EAA) receptors, and by inhibition of neurotransmitter release. PTX did not modify calcium extrusion by the plasma membrane Ca(2+)-ATPase (PMCA), because blockade of the calcium pump increased rather than decreased the PTX-induced calcium influx. Extracellular levels of glutamate and aspartate were measured by HPLC and exocytotic neurotransmitter release by determination of synaptic vesicle exocytosis using total internal reflection fluorescence microscopy (TIRFM). PTX caused a concentration-dependent increase in EAA release to the culture medium. Ten nanomolar PTX decreased cell viability by 30% within 5 min. PTX-induced calcium influx involves three pathways: Na(+)-dependent activation of voltage-dependent sodium channels (VDSC) and voltage-dependent calcium channels (VDCC), reverse operation of the Na(+)/Ca(2+) exchanger, and indirect activation of EAA receptors through glutamate release. The neuronal injury produced by the toxin could be partially mediated by the PTX-induced overactivation of EAA receptors, VDSC, VDCC and the glutamate efflux into the extracellular space.