Arachidonic acid increases choline acetyltransferase activity in spinal cord neurons through a protein kinase C-mediated mechanism Malgorzata Chalimoniuk,* ,  Kelley King-Pospisil,* Ward A. Pedersen,à Andrzej Malecki,* , § Edward Wylegala,¶ Mark P. Mattson,** Bernhard Hennig  and Michal Toborek* *Department of Surgery, University of Kentucky Medical Center, Lexington, Kentucky, USA  Department of Cellular Signaling, Medical Research Center, Polish Academy of Science, Warsaw, Poland àCenter for Aging, Alzheimer’s Disease and Neurodegenerative Disorders, Creighton University Medical Center, Omaha, Nebraska, USA §Department of Pharmacology, Silesian Medical University, Katowice, Poland ¶Department of Ophthalmology, Railway Hospital, Katowice, Poland **Laboratory of Neurosciences, National Institute of Aging, Baltimore, Maryland, USA   College of Agriculture, University of Kentucky, Lexington, Kentucky, USA Abstract Arachidonic acid (AA) plays an important role as a signaling factor in the CNS. Therefore, exposure to AA may affect cho- linergic neurons in the spinal cord. To test this hypothesis, mRNA expression and activity of choline acetyltransferase (ChAT) was measured in cultured spinal cord neurons treated with increasing concentrations (0.1–10 lM) of AA. Exposure to AA increased mRNA levels and activity of ChAT in dose- and time-dependent manners. The most marked effect of AA on ChAT expression was observed in spinal cord neurons treated with 10 lM AA for 1 h. To study the mechanisms associated with these effects, ChAT mRNA levels and activity were measured in cultured spinal cord neurons exposed to AA and inhibitors of protein kinase C (PKC), such as 1-(5-iso- quinolinesulfonyl)-2-methylpiperazine dichloride (H-7) and chelerythrine. Inhibition of PKC completely prevented an AA-induced increase in ChAT expression. In addition, exposure of spinal cord neurons to phorbol-12-myristate-13-acetate (PMA), an activator of PKC, mimicked AA-induced stimulation of ChAT activity. The AA-mediated increase in ChAT mRNA levels and activity was also prevented by treatments with EGTA, indicating the role of calcium metabolism in induction of this enzyme. In contrast, treatments with 7-nitroindazole (7-NI, a specific inhibitor of neuronal nitric oxide synthase), sodium vanadate (NaV, a non-specific inhibitor of phosphatases), and N-acetyl-cysteine (NAC, an antioxidant) had no effect on AA-induced changes in ChAT activity. The protein synthesis inhibitor cycloheximide completely blocked AA-mediated increase in ChAT activity. These results indicate that the AA-evoked increase in ChAT activity in spinal cord neurons is mediated by PKC, presumably at the transcriptional level. Keywords: arachidonic acid, choline acetyltransferase, pro- tein kinase C, spinal cord neurons. J. Neurochem. (2004) 90, 629–636. Received February 26, 2004; revised manuscript received March 26, 2004; accepted March 26, 2004. Address correspondence and reprint requests to Michal Toborek, Molecular Neuroscience and Vascular Biology Laboratory, Department of Surgery, Division of Neurosurgery, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536, USA. E-mail: mjtobo00@uky.edu Abbreviations used: AA, arachidonic acid; ChAT, choline acetyl transferase; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dichlo- ride; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetyl-cysteine; NaV, sodium vanadate; 7-NI, 7-nitroindazole; NMDA, N-methyl-D- aspartate; nNOS, neuronal nitric oxide synthase; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate. Journal of Neurochemistry , 2004, 90, 629–636 doi:10.1111/j.1471-4159.2004.02535.x Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 629–636 629