On the molecular level, the actions of opiates and endogenous opioid peptides are mediated by a receptor recognition site connected to a membrane signal transduction system in which the signal for receptor binding is transmitted into a biological response. In many cases, this transduction system consists of a second messenger system such as cyclic AMP. These systems may not only be responsible for some of the acute actions of opiates, but regulation of the efficacy of these systems may play an important role in the development of opiate tolerance and physical dependence. This project will explore the properties of one of these second messenger systems, opioid-inhibited adenylyl cyclase, in rat brain membranes and in neuronal cell culture models. Previous studies have shown that opioid-inhibited adenylyl cyclase decreases phosphorylation of synapsin, a major phosphoprotein in brain which helps to mediate neurotransmitter release. These studies will explore the role of opioid- inhibited synapsin phosphorylation in both the acute actions of opioids and in the development of tolerance. The role of receptor-mediated changes in pro-enkephalin mRNA levels will be explored in primary neuronal cultures. The regulation of efficacy in this system will be explored by examining mechanism of opioid efficacy increase in NG108-15 cells by low pH pretreatment. These latter experiments will also use antisense probes to G-S proteins to determine the role that G-S plays in mediating the efficacy of G-i-linked receptors. Finally, the role of low affinity opioid receptors, which exist under physiological conditions in brain, will be examined by their distribution and their reactivity to specific opioid alkylating agents. These studies will provide molecular clues to mechanisms of opioid signal transduction in neurons.
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