The long-term objectives of this research are to determine the physiologic, cellular and molecular mechanisms regulating the processing of proTRH, a 255 amino acid polyprotein which contains 5 copies of a thyrotrophin-relating hormone (TRH) progenitor sequence flanked by paired basic residues, and the secretion of TRH in the various regions where TRH and its prohormone occur.
The specific aims of the project are to elucidate: i) the nature of the intermediates and final products of proTRH processing using pulse-chase studies in tumor cell lines transfected with an expression vector containing preproTRH cDNA and in rat perinatal pancreas in monolayer culture; ii) the mechanisms underlying the normal pattern of proTRH processing in specific regions of the CNS and its regulation; iii) the role of the thyroid axis and monoaminergic input in regulating proTRH processing and the subsequent secretion of TRH form the hypothalamus in vivo by hypophysial portal vessel cannulation. Stereotactic implantation of push-pull cannulae in both the pituitary, at the junction of the stalk, and in the olfactory bulb, will enable such regulation and its cellular specificity to be characterized in nonstressed awake animals by direct comparison of the hypothalamus with another CNS location. iv) The role of autocrine and paracrine factors in regulating the secretion of TRH and other proTRH- derived peptides from the Islets of Langerhans will be determined using fetal/neonatal pancreas in monolayer culture. v) Determination of whether histidyl proline diketopiperazine (HisProDKP) is derived directly from proTRH without prior formation of TRH, this biologically active cyclized dipeptize will be characterized in transfected cell lines and in rat pancreatic islets following streptozotocin treatment by RIA, immunohistochemistry and in situ hybridization. This research will provide information on the synthesis and metabolism of biologically active peptides in the neuroendocrine system of mammals and has relevance to the pathophysiology of nervous system, hypothalamic-pituitary disorders and diabetes mellitus in man.
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