The research to be performed centers on the concept that the rapid intracellular metabolism of arachidonic acid mediates responses to receptor activation in neural tissue and will investigate whether this is a feature of cholinergic neurotransmission in the brain. A long-term goal is to characterize physiological responses to cerebral cortical muscarinic stimulation, especially how these responses are perturbed in Alzheimer's Dementia, a disorder of cortical/hippocampal cholinergic innervation. Receptor-mediated guanosine cyclic monophosphate (cGMP) formation in murine neuroblastoma (clone N1E-115) is a useful indicator of receptor activation and is mediated by release of arachidonate from phospholipids and its metabolism by the lipoxygenase pathway. Receptor-mediated arachidonate metabolism thus may be a general mechanism of neurotransmission. Murine neuroblastoma cells in culture exhibit many properties of differentiated neurons and are a model system for biochemical studies of receptor function. Clone NIE-115 possesses several receptors that mediate cGMP or cAMP formation. The N1E-115 muscarine receptor has been particularly well-studied and provides an excellent system for the initial examination of the molecular nature of receptor-linked arachidonate metabolism, which can be extended to other receptors of N1E-115 cells and to studies in brain tissue. Excitotixic lesion of the rat nucleus basalis complex produces a neurochemical profile of presynaptic cholinergic deficits in rat cortex and hippocampus that is similar to that observed post-mortem in Alzheimer's Disease and provides a suitable animal model for studying the modulation of cortical/hippocampal muscarinic-linked arachidonate metabolism in the disease state. Studies of N1E-115 receptor will then provide the molecular pharmacologic background for the animal studies, which will lay emphasis on a key cholinergic pathway and its pathophysiology.
Specific aims i nclude detailed characterization of the pharmacology of receptor-linked arachidonate release in N1E-115 cells and brain synaptosome, assays of lipoxygenase activity and identification of products in both and guanylate cyclase, testing oxidized arachidonate derivatives for inhibition of the cascade in vivo and in vitro, and relating receptor desensitization to alterations in arachidonate metabolism. Methods include cell culture; radiochemical assays; thin-layer, high-pressure liquid, and gas chromatography; excitotoxin lesions to manipulate cholinergic pathways; and mass spectrometric identification of arachidonate metabolites. The project will be detailed investigation of receptor-linked arachidonate metabolism in neural tissue and is relevant to the pathology and treatment of Alzheimer's Dementia.
