Ligand Interaction with Opiate Receptor, and Receptor-Effector Coupling in Brain Slices (Adenylate Cyclase Inhibition, Subtypes).

Molecular mechanisms for opiate action were investigated in morphologically intact cellular preparations, focusing on ligand binding and coupling of the opiate receptor to the putative effector adenylate cyclase. Evidence was provided that agonists promote the interaction of opiate receptors with adenylate cyclase, and that a high affinity GTPase is involved in the coupling process. In addition, heterogeneity in the receptor binding of putative u and k opiate agonists was demonstrated. In striatal slices from rat brain, opiates inhibited PGE(,2)-induced cAMP formation. The Ic50 values for morphine, levorphanol and ethylketocyclazocine (EKC) were 110 nM, 80 nM and 25 nM, respectively. The inhibition fulfilled criteria for mediation by opiate receptors. Neither antagonists nor dextrorotatory opiate isomers were inhibitory. On the other h and , antagonists blocked inhibition by agonists and displayed partial selectivity toward putative agonists of the u and k type. In isolated striatal membranes, opiates stimulated a low K(,m) GTPase. The EC50 values for levorphanol and morphine were 0.09 uM and 3.8 uM, respectively. The enhancement was stereospecific, limited to agonists and blocked by opiate antagonists. In membranes isolated from opiate-dependent rats, GTPase activity was reduced up to 40% relative to controls, while in membranes from opiate-abstinent rats, activity was intermediate. Based on kinetic analysis of the data, a model was proposed which describes the reciprocal relationship between adenylate cyclase and GTPase in mediating opiate receptor binding. A methodology for the assessment of lig and binding to opiate receptors in brain slices was developed. The binding of ('3)H-etorphine, ('3)H-dihydromorphine and ('3)H-ethylketocyclazocine was characterized by linear Scatchard plots, a receptor density of 200-400 fmole/mg protein and K(,D) values of 2-5 nM. The parameters for opiate receptor binding in slices isolated from dependent rats were not significantly different from those obtained with normal animals. Using the approach of competitive displacement, heterogeneity in the receptor binding of putative u and k opiate agonists was demonstrated. The data for both morphine (u) and EKC (k), but not for etorphine, were best fit by a model postulating the existence of two populations of binding sites with markedly different affinities.PhDBiochemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/160089/1/8422191.pd