Tolerance to morphine involves numerous adaptations at multiple levels of the signal transduction pathway. Studies suggest that changes likely involve desensitization of multiple opioid receptors, decreased G-protein coupling, decreased GTPase activity, increased adenylyl cyclase activity, increased cAMP-dependent kinase activity (PKA) increased protein kinase C activities (PKC), increased Ca2+ conductance, decreased potassium conductance and induction of immediate early genes. Adaptational changes lead to elevated intercellular free calcium levels, which could in turn contribute to the decreased efficacy of morphine in inhibiting neuronal firing and neurotransmitter release. Recent studies in our laboratory have implicated protein kinases, especially PKA and PKC, in the regulation of tolerance. Morphine tolerance can be reversed by PKC and PKA inhibitors within 30 min, when central administered together with morphine. The underlying mechanism that triggers the change in morphine induced antinociception as determined in the animal's response to pain, remains elusive. The rapid onset in the effect indicates that constant Phosphorylation of proteins by kinases is essential to maintain tolerance. PKA and PKC have numerous potential target proteins, which are involved in mu opioid receptor mediated antinociception, and thus, the inhibition of PKA or PKC could interfere at numerous sites in the signal transduction cascade. The fact that two kinase controlled by different effector enzymes independently regulate tolerance suggests that a common step within the signal transduction cascade is altered by both kinases. We propose to induce morphine- tolerance and its reversal in vivo, and use membrane preparations from tolerant and reversed-tolerant animals to characterize the effects of treatment at the receptor, G-protein and effector level, by a combination of pharmacological, functional, biochemical and immunochemical methods. These studies will provide a better understanding, which steps in the signal transduction cascade are crucial for the development and maintenance of tolerance.
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