We have investigated the signaling of OX 1 receptors to cell death using Chinese hamster ovary cells as a model system. OX 1 receptor stimulation with orexin-A caused a delayed cell death independently of cytosolic Ca 2؉ elevation. The classical mitogen-activated protein kinase (MAPK) pathways, ERK and p38, were strongly activated by orexin-A. p38 was essential for induction of cell death, whereas the ERK pathway appeared protective. A pathway often implicated in the p38-mediated cell death, activation of p53, did not mediate the cell death, as there was no stabilization of p53 or increase in p53-dependent transcriptional activity, and dominantnegative p53 constructs did not inhibit cell demise. Under basal conditions, orexin-A-induced cell death was associated with compact chromatin condensation and it required de novo gene transcription and protein synthesis, the classical hallmarks of programmed (apoptotic) cell death. However, though the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (Z-VAD-fmk) fully inhibited the caspase activity, it did not rescue the cells from orexin-A-induced death. In the presence of Z-VAD-fmk, orexin-A-induced cell death was still dependent on p38 and de novo protein synthesis, but it no longer required gene transcription. Thus, caspase inhibition causes activation of alternative, gene transcription-independent death pathway. In summary, the present study points out mechanisms for orexin receptor-mediated cell death and adds to our general understanding of the role of G-protein-coupled receptor signaling in cell death by suggesting a pathway from G-protein-coupled receptors to cell death via p38 mitogen-/stress-activated protein kinase independent of p53 and caspase activation.There is a growing interest in the ability of G-protein-coupled receptors (GPCRs) 2 to affect synaptic plasticity and cell differentiation as well as cell growth, death, and survival (reviewed in Refs. 1-3). However, the mechanisms of GPCR signaling in these processes are poorly characterized. The pathways engaged seem to include classical GPCR signals like second messenger-dependent activation of protein kinase A and C but also (for GPCRs) more novel signal transducers like small G-proteins (of Ras and Rho families), PI3K (phosphatidylinositol 3-kinase) and nonreceptor tyrosine kinases (e.g. Src).In this study we have focused on the signaling of a novel GPCR, OX 1 orexin receptor. Little is known about the intracellular signaling of OX 1 receptor and its cognate receptor, OX 2 receptor. Orexin receptor-expressing neuronal, endocrine, and muscle cells often seem to be excited by orexins, an effect that may be related to activation of Ca 2ϩ or nonselective cation channels (reviewed in Refs. 4 and 5). Recently, orexin receptors have been suggested to regulate cell plasticity. ERK (extracellular signal-regulated kinase) is strongly activated in CHO cells heterologously expressing OX 1 receptors (4, 6, 7). In the hippocampus, orexin receptors regulate synaptic plasticity (8). Lo...