Erythropoietin (EPO) reduced Ca2؉ -induced glutamate (Glu) release from cultured cerebellar granule neurons. Inhibition was also produced by EPO mimetic peptide 1 (EMP1), a small synthetic peptide agonist of EPO receptor (EPO-R), but not by iEMP1, an inactive analogue of EMP1. EPO and EMP1 induced autophosphorylation of Janus kinase 2 (JAK2), a tyrosine kinase that associates with EPO-R. Furthermore, genistein, but not genistin, antagonized both the phosphorylation of JAK2 and the suppression of Glu release induced by EPO and EMP1. During chemical ischemia, substantial amounts of Glu were released from cultured cerebellar and hippocampal neurons by at least two distinct mechanisms. In the early phase, Glu release occurred by exocytosis of synaptic vesicle contents, because it was abolished by botulinum type B neurotoxin (BoNT/B). In contrast, the later phase of Glu release mainly involved a BoNT/B-insensitive non-exocytotic pathway. EMP1 inhibited Glu release only during the early exocytotic phase. A 20-min exposure of hippocampal slices to chemical ischemia induced neuronal cell death, especially in the CA1 region and the dentate gyrus, which was suppressed by EMP1 but not iEMP1. However, EMP1 did not attenuate neuronal cell death induced by exogenously applied Glu. These results suggest that activation of EPO-R suppresses ischemic cell death by inhibiting the exocytosis of Glu.Brain ischemia induces delayed neuronal cell death, especially in the CA1 region of the hippocampus (1). Glutamate (Glu) 1 and related excitatory amino acids are well-known neurotoxins, and their extracellular levels increase dramatically in the course of brain ischemia. The excitotoxicity hypothesis suggests that, in neuronal hypoxia/ischemia, neurodegeneration can be triggered by cytoplasmic Ca 2ϩ overload, which occurs when N-methyl-D-aspartate receptors are overstimulated by excessive Glu (2). Numerous pharmacological approaches have been explored to prevent or attenuate neuronal cell death in ischemia, however, no satisfactory methods have been developed (3-5).The hematopoietic growth factor erythropoietin (EPO) is a primary regulator of mammalian erythropoiesis and is produced by kidney and liver in an oxygen-dependent manner (6, 7). The EPO receptor (EPO-R) is a member of the type 1 superfamily of single-transmembrane cytokine receptors. EPO binding to EPO-R induces receptor oligomerization and subsequent activation by autophosphorylation of Janus kinase 2 (JAK2), a protein-tyrosine kinase that associates with EPO-R. JAK2 phosphorylates a cytoplasmic transcription factor called signal transducer and activator of transcription (STAT), leading to translocation of STAT into the nucleus and regulation of transcription (8). EPO and EPO-R are also expressed in mammalian brain, where hypoxia strongly stimulates EPO production (9, 10). Although activation of EPO-R has been suggested to play a neuroprotective role (10, 11), little is known about the action of EPO in the nervous system. Recently, we showed that EPO inhibits Ca 2ϩ -induced d...

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