Requirement for Nitric Oxide in Retinal Neuronal Cell Death Induced by Activated Mu ¨ller Glial Cells O. Goureau, F. Re ´gnier-Ricard, and Y. Courtois De ´veloppement, Vieillissement et Pathologie de la Re ´tine, U. 450, INSERM, affilie ´e CNRS, Association Claude Bernard, Paris, France Abstract: Retinal Mu ¨ ller glial cells express the inducible isoform (-2) of nitric oxide (NO) synthase (NOS) in vitro after stimulation by lipopolysaccharide (LPS) and inter- feron- (IFN-) or in vivo in some retinal pathologies. Because NO may have beneficial or detrimental effects in the retina, we have used cocultures of retinal neurons with retinal Mu ¨ ller glial (RMG) cells from mice disrupted for the gene of NOS-2 [NOS-2 (-/-)] to clarify the role of NO in retinal neurotoxicity. We first demonstrated that NO produced by activated RMG cells was not toxic for RMG cells themselves. Second, the NO released from LPS/IFN--stimulated RMG cells induced neuronal cell death, because no neuronal cell death has been ob- served in cocultures with RMG cells from NOS-2 (-/-) mice and because inhibition of NOS-2 induction by trans- forming growth factor- or blockade of NO release by different NOS inhibitors prevented neuronal cell death. Addition of urate, a peroxynitrite scavenger, or superox- ide dismutase partially prevented neuronal cell death in- duced by NO, whereas the presence of a poly(ADP- ribose) synthetase inhibitor, caspase inhibitors, or a guanylate cyclase inhibitor had no significant effect on cell death. These results demonstrated that a large re- lease of NO from RMG cells is responsible for retinal neuronal cell death in vitro, suggesting a neurotoxic role for NO and peroxynitrite during retinal inflammatory or degenerative diseases, where RMG cells were activated. Key Words: Nitric oxide—Retina—Lipopolysaccharide— Interferon-—Glial cells—Knock-out mice. J. Neurochem. 72, 2506 –2515 (1999). Nitric oxide (NO) is an important signaling molecule that mediates various essential physiological processes, including neurotransmission, vasodilatation, and host cell defense (Christopherson and Bredt, 1997; MacMick- ing et al., 1997; Nathan, 1997). NO is synthesized from L-arginine by NO synthase (NOS), a family of enzymes with distinct functional, biochemical, and regulatory properties (Fo ¨rstermann et al., 1994; Knowles and Moncada, 1994; Marletta, 1994). The constitutive NOS isoforms, originally described in endothelial cells and in neurons, produce small amounts of NO in response to an increase in intracellular calcium level (Christopherson and Bredt, 1997). The cytokine-inducible NOS (iNOS or NOS-2), whose expression requires protein synthesis, has been demonstrated and cloned in a wide variety of mammalian cells (Nathan, 1997). The role of a sustained NO production by NOS-2 is well known in murine macrophages, where NO is responsible for their cyto- static and cytolytic activities toward invading organisms (Fang, 1997; MacMicking et al., 1997). In addition, NO generated by NOS-2 is also involved in some pathophys- iological states, generally related to local and systemic inflammation (Knowles and Moncada, 1994; Nathan, 1997). In the retina NOS-2 is expressed in retinal pigmented epithelial, pericytes, and retinal Mu ¨ ller glial (RMG) cells in vitro after stimulation by cytokines (Goureau et al., 1993a, 1994a,b; Liversidge et al., 1994; Sparrow et al., 1994; Chakravarthy et al., 1995; Cotinet et al., 1997). Furthermore, we have reported that NOS-2 could be expressed in the retina from AIDS patients during cyto- megalovirus retinitis (Dighiero et al., 1994) and in the rat retina during endotoxin-induced uveitis (EIU) (Goureau et al., 1995; Jacquemin et al., 1996). More recently, the expression of NOS-2 has been related to rat retinal isch- emia–reperfusion injury (Hangai et al., 1996) and found in experimental autoimmune uveitis (EAU) induced by Received November 26, 1998; revised manuscript received January 29, 1999; accepted February 1, 1999. Address correspondence and reprint requests to Dr. O. Goureau at De ´veloppement, Vieillissement et Pathologie de la Re ´tine, INSERM U. 450, 29 rue Wilhem, 75016 Paris, France. The present address of Dr. F. Re ´gnier-Ricard is INSERM U. 363, Ho ˆpital Cochin, Paris, France. Abbreviations used: DAPI, 4,6-diamidino-2-phenylindole; DETA- NONOate, 2,2'-(hydroxynitrosohydrazino)bisethanamine; DMEM, Dulbecco’s modified Eagle’s medium; EAU, experimental autoim- mune uveitis; EIU, endotoxin-induced uveitis; IFN-, interferon-; LPS, lipopolysaccharide; MAP-2, microtubule-associated protein-2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; L-NIL, L-N 6 -(1-iminoethyl)lysine; L-NMMA, N G -monomethyl-L-argi- nine; NO, nitric oxide; NOS, nitric oxide synthase; PARS, poly(ADP- ribose) synthetase; PBS, phosphate-buffered saline; RMG, retinal Mu ¨l- ler glial; SIN-1, 3-morpholinosydnonimine; SOD, superoxide dis- mutase; TGF-, transforming growth factor-; WT, wild-type. 2506 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry