The present study was designed to investigate the role of a protein kinase C (PKC) isoform in the uncoupling of the mu-opioid receptor from G-proteins after repeated intrathecal injection of a selective mu-receptor agonist, [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO), in the spinal cord of mice. The activation of G-proteins by opioids was measured by monitoring the guanosine-5'-o-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Mice were injected intrathecally with saline or DAMGO once a day for 1-7 d. At 24 hr after every injection the spinal cord membranes were prepared for the assay. The enhanced [(35)S]GTPgammaS binding by mu-agonists DAMGO, endomorphin-1, or endomorphin-2 was attenuated clearly in spinal cord membranes obtained from mice that were treated intrathecally with DAMGO for 5 and 7 d, but not for 1 or 3 d. By contrast, no change in levels of [(35)S]GTPgammaS binding induced by the delta-receptor agonist SNC-80 or kappa-receptor agonist U-50,488H was noted in membranes obtained from mice that were treated with DAMGO. Concomitant intrathecal administration of a specific PKC inhibitor Ro-32-0432 with DAMGO blocked the attenuation of DAMGO-induced G-protein activation that was caused by chronic DAMGO treatment. Western blotting analysis showed that chronic DAMGO treatment increased the levels of PKCgamma, but not PKCalpha, PKCbetaI, and PKCbetaII isoforms, in spinal cord membranes. Furthermore, mice lacking PKCgamma failed to exhibit the desensitization of the DAMGO-stimulated [(35)S]GTPgammaS binding after repeated DAMGO injection. These findings indicate that repeated intrathecal administration of DAMGO may activate the PKCgamma isoform and in turn cause a desensitization of mu-receptor-mediated G-protein activation in the mouse spinal cord.