Demonstration and Characterization of Opiate Inhibition of the Striatal Adenylate Cyclase

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Abstract
The conditions in which Leu5-enkephalin inhibition of striatal adenylate cyclase was observed were defined. It was determined that enkephalin inhibition was dependent on GTP. The apparent Km for GTP in opiate inhibition was determined to be 0.5 and 2 μM when 0.1 mM- and 0.5 mM-ATP were used as substrate. ITP, but not CTP or UTP, could substitute for GTP in the reaction. Though the addition of monovalent cations—Na+,K+, Li+, Cs+, and choline+—stimulated striatal adenylate cyclase activity, enkephalin inhibition of striatal adenylate cyclase did not require Na+ when theophylline was used as the phosphodiesterase inhibitor. Under optimal conditions, i.e., 20 μM-GTP and 100 mM-Na+, Leu5-enkephalin inhibited the striatal adenylate cyclase activity by 23–27%. When the enkephalin regulation of the cyclase activity was further characterized, it was observed that Leu5-enkephalin inhibited the rate of the enzymatic reaction. Kinetic analysis revealed that the opioid peptide decreases Vmax values but not the Km values for the substrates Mg2+ and Mg-ATP. Agents such as MnCl2, NaF, and guanyl-5′-ylimido-diphosphate, which directly activated the adenylate cyclase, antagonized the opiate inhibition. Levorphanol and (–)naloxone were more potent than dextrorphan and (+)naloxone in inhibiting adenylate cyclase and in reversing the enkephalin inhibition, respectively. There were differences in the potencies of various opiate peptides in their inhibition of striatal adenylate cyclase activity, with Met5- > Leu5-enkephalin > β-endorphin. The opiate receptor through which the enkephalin inhibition was observed is most likely δ in nature, since in the presence of either Na+ or K+, the magnitude of the alkaloid inhibition was reduced, whereas the peptide inhibition was either potentiated or not affected.