The current amended application is for continuation of a project that has provided biochemical characterization as well as anatomical localization of posttranslational processing intermediates of the multidimensional tachykinin peptide substance P (SP). As a necessary complement to the peptide biochemistry and to gain additional information relating to tachykinin expression at an earlier stage of biosynthesis, we have recently developed an in situ hybridization histochemistry procedure to selectively monitor cellular distributions of the two major alternatively spliced species of mRNA encoding SP and related tachykinin peptides. Additionally, tachykinin-expressing sensory neurons of the dorsal root ganglia (DRG) are major sites of opioid action via high densities of opioid receptors. Furthermore, these neurons are responsive to a wide variety of exogenous opioids, and other pharmacological agents, involved in antinociceptive processes and in the etiology of tolerance and physical dependence. With these strong functional considerations in mind, the proposal will address a major testable hypothesis: TACHYKININ- AND OPIOID- EXPRESSING NEURAL SYSTEMS IN THE PRIMARY AFFERENT DRG-SPINAL CORD SYSTEM ARE FUNCTIONALLY INTERACTIVE IN THE MODULATION OF IMPORTANT PHYSIOLOGICAL PROCESSES. This underlying hypothesis will be examined through a series of integrated neurochemical, anatomical, and pharmacological studies of tachykinin systems as well as complementary neurochemical and anatomical studies of spinal opioid systems. Two major Specific Aims are defined: 1. To explore the functional aspects of neuropeptide processing and diversity from biochemical and cellular perspectives. In part A., we will perform initial in vitro binding analyses in an attempt to characterize a spinal cord receptor that recognizes the unamidated immediate precursor to SP, i.e., SP-Glycine (SP-G), as well as other intermediate processing forms of SP. In part B., we will define a valuable cell culture system by which we may investigate tachykinin expression, i.e., biosynthesis and turnover, coupled to evoked release of peptides. 2. To perform pharmacological and physiological studies to elucidate functional interactions between tachykinin and opioid systems in spinal and supraspinal pathways related to pain and analgesia. In part A., we will continue pharmacological studies designed to elucidate the biochemical mechanisms underlying the potentiation of opioid analgesia in two different types of pain paradigms by SP and SP-related peptides. We will also continue studies evaluating the role of SP and SP-related precursor peptides in opioid tolerance development at the spinal level. In part B., we will investigate the effects of afferent nerve stimulation on PPT gene expression in the DRG, and postsynaptically on PPT and opioid gene expression at spinal and supraspinal sites. Overall, the proposed studies will strengthen the biochemical and molecular foundations for research on functionally interactive tachykinin- and opioid-expressing neural systems, and elucidate some of the mechanisms by which narcotic agonists can influence sensory and affective processes.
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