Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of G(s). Measurements of intracellular free calcium concentration ([Ca²⁺](i)) reveal that mastoparan induces a dramatic elevation of [Ca²⁺](i) that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca²⁺](i) may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca²⁺](i) is maintained in the absence of extracellular Ca²⁺, suggesting that the rise of [Ca²⁺](i) is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca²⁺](i) stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca²⁺](i) and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca²⁺](i) and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca²⁺](i). Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca²⁺ release from intracellular stores, probably via activation of PLC and IP₃. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.