Xth International Symposium on Cholinergic Mechanisms 431 C.C. Felder”, J.H. Joonb, E.M. Brileyb, M.W. Wood” UNeuroscience Discovery, Eli Lilly Research Laboratories, Indiunrcpolis, IN 46285. USA ‘Lab oj Cellular and Moleculur Regulation, National Institute of Mental Health. Bethesdu. MD 20892, USA TNFa is a pleiotropic cytokine primarily secreted by activated and resident mdcrophages such as Kupffer cells and microglia (reviewed in 1). T’NFa mediates a wide spectrum of biological activity including cell death, cell proliferation, and lymphocyte activation, Two receptors for TNFa have been identified, the p55 and ~75, both of which have been cloned and sequenced. Most mam- malian cells express at least one of these receptors. Previous studies have shown that TNFa can stimulate the release of arachidonic acid presumably through activation of phospholipase A2 (2). Neural de- generation of cholinergic neurons such as occurs during Alzheimer’s disease. may involve cytokine-mediated inflammatory processes. A 85.kDa phospholipase A2, cPLA2, has been implicated in ischaemic neural loss (3) and is elevated in Alzheimer’s brains (4). We inves- tigated possible relationships between cytokine and muscarinic re- ceptor signal transduction mediated by phospholipase A2. We have previously demonstrated that the human melanoma cell line A2058 expresses only the m5 subtype of muscarinic re- ceptor (5). In A2058 cells, treatment with the cholinergic agonist, carbachol (CC) induces a 2-I-fold increase in arachidonic acid release over basal levels. CC-induced arachidonic acid release is blocked by the muscarinic antagonist, atropine. The CC-induced arachidonic acid release is potentiated 2-3-fold by pretreatment of A2058 cells with either of the inflammatory cytokines, tumor necrosis factor-a (TNFu) or interleukin 1-p (IL-I@). Enhance- ment of CC-induced arachidonic acid release by TNFa pretreat- ment peaks near 1 h, consistent with a transcriptionally derived event. Western analysis of IkBu, the inhibitory subunit of the NFKB complex, suggests that both cytokines are capable of ac- tivating tcB-driven transcription. Pretreatment of A2058 cells with either of the proteasome inhibitors, MG-I 15 or MG-132, blocks the cytokine-dependent degradation of B&a but does not affect the enhancement of CC-induced arachidonic acid release. Western analysis demonstrates that both cytokines can trigger the phos- phorylation (and activation) of ~38 MAP kinase. Furthermore, pretreatment of A2058 cells with the ~38 kinase inhibitor, SB202190, ablates cytokine-dependent augmentation without in- terfering with CC-induced AA release. Finally, no change in the phosphorylation state of the p44/42 MAP kinases follows cytokine treatment. Thus, inflammatory cytokines may regulate muscarinic signaling through the ~38 MAP kinase pathway. Additionally, this study provides evidence that potentiation of muscarinic receptor- mediated signal transduction may be a direct result of inflammation. References Vandenabeele P, Declercq W.. Beyaert R., and Fiers W. Two tumor necrosis factor receptors: structure and function. Trends m Cell Biol 5:392-399 (1995). Suffys P., Beyaert R., De V&k D., Vanhaesebroeck B., Van Roy E. Fiers W. Tumour-necrosis-factor-mediated cytotoxicity is correla- ted with phospholipaseA2 activity, but not wrth arachidonic acid release per se. Europ J Eiochem. 195:465-75 (1991). Bonventre J.V., Huang Z., Taberi M.R., O’Leary E., Li E., moskowitr M.A., Sapirstein A. Reduced fertihty and posrischaemic brain in- jury in mice deficient in cytosolic phospholipase A2. Nature 390:622-625 (1997). Stephenson D.T., Lemere CA., Selkoe D.J.. Clemens J.A. Cytosalic phospholipase A2 (cPLA2) immunoreactivity is elevated in Allhei- mer’s disease bram. Neurobiol of Disease 3:51-63 (1996). Kohn EC, Alessandro R.. Probst J., Jacobs W., Briley E.M., Felder CC. ldenttfication and molecular characterization of a m5 mus- carinic receptor in A2058 human melanoma cells: Coupling to in- hibition of adenylate cyclase and stimulation of phospholipase A2. J Biol Chem 271:17476-17484 (1996). of naive and chronic nicotine treated rats L. Friberg, I. Bednar, A. Nordberg Department of Clinical Neuroscience and Family Medicine, Division of Molecuiar NeurophornlacoloRy, Kurolinska Institute, Huddinge University Hospital, B84, S-141 86 Huddinge, Sweden Accumulating data indicate that the neuronal nicotinic receptors (nAChR) are involved in memory, learning and dependence pro- cesses in brain (Newhouse, 1997; Lindstrom, 1997). The reduced number of nAChRs in neurodegenerative disorders, such as Al- zheimer’s disease (Nordberg, 1992), motivates further research to understand the properties of the various nAChRs, their distribu- tion, physiological function and pharmacological properties. Three nAChRs binding sites have been found in brain using ni- cotinic receptor ligands such as nicotine, epibatidine and a-bun- garotoxin with different affinities to ct4, a3, a7 nAChRs subunits, The nAChRs belong to the family of ligand-gated ion channel receptors. The nAChRs consist of two type of homologous o. and B subunits. To date, eight a (a2-a9) and three B (B2-p4) subunits have been cloned which can appear in different combinations (Gotti et al., 1997) with probably different functional properites. Interactive mechanisms between the cholinergic and dopami- nergic systems seems to be important in learning, memory and dependence processes in which DA is suggested to play a role in attention and reward mechanisms (Wise, 1978; Beninger, 1983). Nicotine has been shown to stimulate dopamine release in brain (Wonnacott, 1997). The involvement of nAChRs is not fully understood although both a4, a3 nAChRs have been suggested to be involved.