KAPPA OPIOID RECEPTORS AND TURNOVER IN THE BRAIN

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The overall objective of this proposal is to investigate kappa opioid receptor-stimulated Phosphoinositide (PI) turnover in several areas in the rat brain. The proposal derives from the recent discovery in this laboratory that kappa (but neither mu nor delta) opioid receptors stimulate PI turnover in hippocampal slices. This proposal is aimed at extending the kappa receptor-mediated PI turnover response to other areas of the brain. The proposed work will constitute the first characterization of opioid receptor-mediated PI turnover and may provide the first biochemical evidence that PI turnover is the primary second messenger system associated with kappa receptor stimulation in the brain and perhaps in other tissues as well. The pharmacology of the response will be compared in different brain areas, measuring the ability of kappa-selective agonists and antagonists to activate or inhibit the activation of the response. Mu and delta- selective agonists and antagonists will be tested as well to establish selectivity. The level of the kappa receptor-mediated PI turnover response will be compared with the distribution of kappa receptor in the brain. It is proposed to use recently available selective kappa ligands for autoradiography and a state-of-the-art image analysis system to determine kappa receptor distribution, which has been problematic. The measurement of kappa opioid receptor-mediated PI turnover in hippocampal slices required extensive optimization of the assay. To increase further the sensitivity of the assay, the separation and quantitation of individual inositol phosphates by HPLC and radiochemical flow detection is proposed. An autosampler and radiochemical flow detector are requested to accomplish this aim. Finally, as a measure of G-protein coupling to kappa receptors, it is proposed to measure kappa receptor-stimulated, low Km GTPase in different areas of the brain, comparing the GTPase response with PI turnover and receptor distribution. The overall significance of the proposed work is that it will establish a link between kappa receptors and PI turnover, which previously has not been associated with any opioid receptor subtype. The work may provide evidence for a biochemical response underlying the behavioral effects of kappa receptor activation in the CNS.