1985 — 1991 |
Krause, James E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Substance P Biosynthesis and Axonal Transport in the Cns
This renewal application is a continuation of our studies designed to described the molecular mechanisms of tachykinin peptide biogenesis, axonal transport and turnover in the central nervous system (CNS) of the rat. The tachykinin family of peptides are neurotransmitter candidates in many neuronal systems and include Substance P (SP), Neurokinin A (NKA) and Neurokinin B (NKB). We have recently documented the existence of one rat preprotachykinin gen and three mRNAs derived from it that encode SP and NKA, and have further evidence of another distinct gene responsible for NKB production. We shall continue the molecular cloning of the genes, and mRNAs encoding these peptides to deduce their pathway of biogenesis. Analysis of the putative gene regulatory regions will allow for an analysis of tissue-specific expression of the genes and the mRNA primary structures will allow deduction of the precursor proteins encoding the tachykinins. The intracellular location and precise biochemical nature of the preprotachykinin processing events will be examined in cell-free translation systems, in large dense core secretory vesicles isolated from CNS regions, in selected cell lines infected with recombinant vaccinia vectors, and in discrete CNS projections in vivo. The regulation of tachykinin peptide biosynthesis and turnover during development and as a consequence of neurotransmitter input and gonadal steroid action will also be investigated in the basal ganglia, hypothalamic- anterior pituitary axis and ventral medulla-sympathetic preganglionic neuronal system. These studies will provide a basis for investigating whether aberrations in the biosynthesis or processing in peptidergic neurons are responsible for, or associated with, neurodegenerative and psychiatric disorders in which CNS peptides are implicated. As such, these investigations will provide a better understanding of basal ganglia SP/NKA neuron and their reported relationship to the Parkinson's disease and Huntington's chorea.
|
1 |
1992 — 1996 |
Krause, James E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Substance P Biosynthesis and Functions in the Cns
This renewal is a continuation of studies examining the molecular mechanisms of tachykinin peptide and receptor synthesis, function and regulation. Members of this peptide family are neurotransmitter candidates in many neuronal systems, and consist of substance P (SP), neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK) and neuropeptide gamma (NPgamma), which act at three characterized G-protein coupled receptors, called the SP receptor (SPR), NKAR and NKBR. We have characterized mechanisms of SP biosynthesis and regulation by examining the structure of the SP/NKA gene, the three mRNAs produced via differential RNA splicing, secretory pathway targeting of the translated precursors, and precursor processing mechanisms. We have also characterized the SPR receptor cDNA and gene by molecular cloning and functional expression. In this proposal, we shall pursue four specific aims to examine peptide receptor responses, to investigate cellular and molecular mechanisms regulating receptor sensitivity, and to evaluate regulation of peptide synthesis and receptor responsiveness in a physiological system. 1) Structure-activity relationships of the rat SPR will be determined by examining high affinity agonist binding of chimeric, deletion and site-directed mutant SPR forms. 2) The synthesis, assembly and turnover of the SPR will be examined in native SPR bearing cell lines in pulse-chase experiments using site-directed anti-SPR antisera, and the effects of cellular activation by second messages on these processes will be further assessed. 3) Agonist-dependent second messenger systems regulating SPR gene expression and mRNA turnover in native SPR bearing cell lines will be investigated to explore the hypothesis that acute and chronic SPR regulation involve transcriptional mechanisms. 4) SP synthesis, turnover and receptor functions will be studied in spinal sensory systems in vivo. Spinal sensory systems related to nociception will be activated using chemogenic or other pain models (formalin test, adjuvant induced polyarthritis, partial sciatic nerve ligation) and function, turnover and regulation of tachykinin peptides and receptors will be assessed. These studies will provide a basis for understanding the molecular actions of tachykinins at target sites and their regulation. They also provide a genetic basis for investigating whether aberrations in the biosynthesis of SP and its receptor are responsible for or associated with sensory and neurodegenerative disorders in which CNS peptides are implicated.
|
1 |