Signal transduction within the central nervous system involves the recognition of a neurotransmitter or neuromodulator by a specific receptor and the subsequent activation (or inhibition) of a cellular event. These cellular events (ion translocation, neurotransmitter release, etc.) are often mediated by second messengers evoked as a result of receptor occupancy. Dr. Rasenick will determine how a number of components within the neuron are integrated with respect to the adenylate cyclase second messenger system. Cultured glial, neuronal or hybrid cells will be permeabilized with saponin, and the components of the adenylate cyclase system will be probed, in situ. Dr. Rasenick's laboratory has developed an assay for adenylate cyclase (as opposed to cAMP accumulation) in permeabilized cells; C6 NG.108.15 and NCB.20 cells will be used for the proposed experiments. Initially, Dr. Rasenick will study the kinetics for the receptor mediated activation of the adenylate cyclase system through the stimulatory GTP binding protein (GNs). The agonist catalyzed activation of GNs will be studied with a GTP.photoaffinity probe. This study also will examine the recently discovered interaction between GNs and the inhibitory GTP binding protein (GNi) both through kinetic experiments and by photoaffinity experiments. Dr. Rasenick has demonstrated that GTP analogues may be exchanged between GNs and GNi (as well as tubulin and GNi) in synaptic membranes; he further intends to examine the possible role of receptors in the nucleotide exchange process. The permeabilized cells provide the ideal milieu for such experiments; many of the elements which may act in concert to modify receptor responsiveness are retained in this system. As the saponin treated cells have holes sufficiently large to allow the passage of proteins and Fab fragments, a variety of experiments to probe the role of the cytoskeleton, as well as that of a newly discovered 32KDa GTP binding protein, are planned. Lastly, effects of morphological differentiation will be examined. This research provides the exciting possibility to investigate the cellular response to neurotransmitters in a environment resembling that of the intact neuron.
