Neurochemical Research, VoL 18, No. 2, 1993, pp. 133-138 Differential Involvement of Protein Kinase C in Transmitter Release and Response to Excitatory Amino Acids in Cultured Cerebellar Neurons Maria Luisa Eboli,, 1,3 Maria Teresa Ciotti, z Delio Mercanti, z and Pietro Calissano z (Accepted May 18, 1992) Cerebellar granule cells cultured in the presence of a differentiating factor isolated from rabbit serum exhibit, at variance with those cultured in fetal calf serum, an almost complete resistance to excitatory aminoacid (EAA)-induced citotoxicity. We investigated the behaviour of protein kinase C (PKC), strongly implicated in EAA cytotoxicity, in the two types of culture. Phorbol esters, used to monitor the enzyme, enhanced the depolarization-evokedrelease of D-[3H]aspartate, but less effectively in factor-conditioned cells. EAAs increased phorbol esters binding in both cultures, but the effect was briefly lasting in factor-conditioned cells. The different behaviour of PKC is postulated to be causally related to different response to EAA of the cultures. KEY WORDS: Cerebellar granules; excitotoxicity; resistance; protein kinase C; translocation; transmitter release. INTRODUCTION Protein kinase C (PKC*; ATP: protein phospho- transferase, EC 2.7.1.37) consists of a family of closely related enzymes which ]have been largely implicated in a variety of physiological and pathological cellular re- sponses (1). In the central nervous system the partici- pation of PKC in several important physiological processes, such as neuronal excitability, ionic fluxes, transmitter release as well as dendritic and axonal out- growth, has been proposed (2-4). More recently, an ad- i Permanent address: Institute of General Pathology, Catholic Univer- sity, 00168 Rome, Italy. 2 Institute of Neurobiology, CNR, 00137 Rome, Italy. 3 To whom to address reprint requests. * Abbreviations used: PKC: protein kinase C; EAA: excitatory ami- noacids; BME: basal Eagle's medium; FCS: fetal caff serum; NOAC: neurite outgrowth adhesion complex; DIV: days in vitro; KR: Krebs- Ringer medium; TPA: phorbol 12-tetradecanoate 13-acetate; PdBu: 4-13-phorbol 12,13-dibutyrate. 133 ditional major role has been suggested in the pathogenesis of neuronal injury (5-7). Primary cultures of cerebellar granule cells have become a widely utilized tool for studying different as- pects of nerve cell biology. In these cells the down reg- ulation of PKC, obtained by prolonged treatment with phorbol esters, has been shown to protect against glu- tamate mediated neurotoxicity (8). This indirectly dem- onstrates the major role played by the enzyme in the excitatory amino acid (EAA) mediated cell death of these types of neurons (reviewed in ref.5). It has recently been shown that cerebellar granule cells may be cultured in a basal Eagle's medium (BME) in the presence of a protein complex isolated from rabbit serum. This complex has been defined as neurite out- growth adhesion complex, or NOAC as acronym, on the basis of its peculiar biological property. NOAC cultures express phenotypic markers to an extent comparable to those detectable in the presence of 10% fetal calf serum (FCS), but survive for much longer periods (up to one month). Moreover, NOAC culture exhibit an almost 0364-3190/93/0200-0133507.00/0 9 I993Plenum Publishing Corporation