This proposal seeks to define the relationship between guanyl nucleotides and opiate receptor function in brain. The proposal derives from our recent demonstration of specific opiate-stimulated GTPase activity in brain together with an emerging conceptual framework suggesting that neurotransmitters coupled either negatively or positively to cyclic AMP production have an absolute requirement for GTP and, further, that the hydrolysis of GTP to GDP by GTPase terminates the coupling between hormones and adenylate cyclase. The primary goal of the proposed work is to test the hypothesis that GTP regulates the coupling between a certain class of opioid receptors and its recently demonstrated inhibition of adenylate cyclase in rat brain. The work is focused on the GTP-binding protein complex, Ni, that is thought to mediate the coupling between the receptor and its effector. Two properties of the Ni protein -- GTP hydrolysis, which inactivates the coupling between hormone and the adenylate cyclase system and GTP for GDP exchange, which reactivates the coupling mechanism -- will be measured. Investigation of the pharmacological profile and kinetics of these two parameters in brain may answer the question of which opioid receptor subtype(s) is linked to adenylate cyclase and, further, may suggest regulation by N proteins of other receptor subtypes not linked to adenylate cyclase. These studies will define opioid receptor subtypes in a manner that is new, fundamentally different from and complementary to the current concepts of opioid receptor subtypes based on receptor binding studies. It is expected that the enkephalin-preferring """"""""delta"""""""" receptor will be one of the subtypes linked to adenylate cyclase and thus regulated by GTP. Measurement of receptor binding using radiolabeled ligands under GTP hydrolytic conditions is expected to resolve the apparent discrepancy between the lower potency of opioids for stimulating GTPase compared with their binding to receptors. The examination of opioid-stimulated GTPase and GTP-GDP exchange in tolerant-dependent animals may suggest new approaches to opiate addiction based on the Ni protein.
