Susan E. Leeman, PhD - US grants

Several series of experiments are planned in continuing work on two neural peptides, substance P and neurotensin, that have been isolated, sequenced, and synthesized in this laboratory. The studies outlined include a) mapping the distribution of these peptides in brain and peripheral nerve. b) delineation of substance P or neurotensin-containing tracts within the central nervous system. c) the release of substance P and neurotensin from neural tissue in vitro and in vivo. d) the chemical characterization of substance P and neurotensin in plasma. e) manipulation of the plasma levels of immunoreactive substance P and neurotensin.

This proposal requests support for a conference on "Substance P related peptides: Cellular and molecular physiology." The meeting will be held in Worcester, Massachusetts on July 19-21, 1990, and will bring together a large group of nationally and internationally recognized scientists working in the area, including physiologists, pharmacologists, biochemists and molecular biologists. The scope of the meeting will be on recent advances in the field. The structures, expression and regulation of the two preprotachykinin genes will be addressed, as will the different patterns of posttranslational processing of the multiple precursors. A major focus will be on the molecular biological, biochemical and pharmacological characterization of the multiple receptors for substance P and related peptides. Progress has continued on the development of antagonists permitting new studies on the physiological roles of substance P. Several highly selective peptide-based receptor antagonists have been defined over the past two years, and an exciting recent achievement has been the identification of non-peptide receptor antagonists. Several studies related to these antagonists will be reported at this meeting. Signal transduction mechanisms will be presented from biochemical and electrophysiological points of view. The anatomical localization of the peptides and their receptors, and their cognate mRNAs, as well as their colocalization with other neurotransmitter systems, will be addressed. Finally, the many functions of Substance P and related peptides in specific central nervous regions, in the sensory and the autonomic nervous systems, in the reproductive tract, and in regulation of immune function, will be covered. Substance P has been implicated in the transmission of painful stimuli, in inflammatory responses, in arthritis and in inflammatory bowel disease. Substance P fibers innervate the vasculature including the coronary circulation. Thus the possible clinical implications of substance P will be considered. It is hoped that this meeting will attract many younger persons including graduate students, postdoctoral fellows and junior faculty members to attend and participate in the scheduled poster sessions. The requested funds will used to help defray expenses for these younger scientists.

Neurons that synthesize the peptide neurotensin (NT) are particularly abundant in the preoptic area and may play an important role in regulating neuroendocrine function, many aspects of which are sexually differentiated. This proposal seeks to clarify the relationship between female-specific, estrogen-dependent expression of the neurotensin/neuromedin N (NT/N) gene in the rat preoptic area and female-specific regulation of luteinizing hormone (LH) secretion. A major objective of this proposal is to determine the extent to which estrogen can act directly on NT neurons in the preoptic area. To that end, in situ hybridization histochemistry will be used to determine the detailed regional distribution of neurons that express both NT/N mRNA and estrogen receptor mRNA in the male preoptic area and female preoptic area. A second major objective of this proposal is to determine the extent to which NT can act directly on neurons that synthesize gonadotropin-releasing hormone (GnRH). To that end, in situ hybridization histochemistry will be used to determine the detailed regional distribution of neurons that express both GnRH mRNA and NT receptor mRNA in the basal forebrain of the male and female. In addition, the effect of estrogen on the abundance of NT receptor mRNA in GnRH neurons will be determined. A third major objective of this proposal is to investigate the development of female-specific NT/N gene expression in the preoptic area. Developmental parameters to be investigated include: a) the age at which the distribution of NT/N mRNA in the preoptic area becomes sexually differentiated, b) the extent to which sexually differentiated expression of NT/N mRNA in the adult preoptic area is determined by neonatal androgen levels, and c) the age at which estrogen is able to alter the abundance of NT/N mRNA in the female preoptic area. The results of these developmental studies will help to clarify the role of NT neurons in the development of female-specific regulation of LH secretion. Identifying neuro- transmitters that regulate GnRH neurons will further our understanding of neural regulation of the menstrual cycle, may provide insight into its pathology, and may point the way toward novel therapeutic approaches.

DESCRIPTION (provided by applicant): Substance P (SP) is an 11 amino acid peptide that plays multiple physiological and pathological roles as a neurotransmitter, an autocrine agent, and a neurohormone. SP participates in diverse functions such as the transmission of pain, regulation of inflammation, and in mediating behavioral responses. The activities of SP depend upon interaction with its 7-transmembrane G-protein coupled receptor, the NK1 receptor (NK1R). The NH2 terminus has two consensus sequences for N glycosylation and there is good evidence that the NH2 terminus is glycosylated; however little is known of the chemical structures of the attached carbohydrates nor their function. This proposal addresses this gap in our knowledge and focuses on the chemistry and biological consequences of NK1R glycosylation. HT29 cells (human colonic cells) will be stably transfected with cDNA encoding wild type human NK1 receptors or mutant receptors with one, the other or both N-glycosylation sites altered. Methods for purification of receptors based on his-tagged techniques will be developed to extract these receptors in sufficient quantity and purity to permit mass spectrometric analysis of their carbohydrate moities. Several functional studies will be done to assess possible differences in wild type and mutant receptors including comparing a) association and disassociation rates of ligand binding to receptors; b) receptor internalization; c) and signal transduction mechanisms including activation of cAMP, phosphoinositide hydrolysis and transactivation of the MARK pathway. These pilot experiments may lead to new directions for understanding the role of glycosylation in SP-NK1R mediated responses that could be of therapeutic importance.

Binding; Binding (Molecular Function); CRISP; Carbohydrates; Cells; Characteristics; Computer Retrieval of Information on Scientific Projects Database; Consensus; Consensus Sequence; ConsensusSequence; Development; ERK 2; Euler-Gaddum Substance P; Extracellular Signal-Regulated Kinase 2; Funding; Grant; Institution; Investigators; Kassinin, 1-de-L-aspartic acid-2-de-L-valine-3-L-histidine-5-L-threonine-7-L-serine-; Laboratories; Ligands; Link; MAP Kinase 1; MAP Kinase 2; MAPK1; MAPK1 Mitogen-Activated Protein Kinase; MAPK2 Mitogen-Activated Protein Kinase; Maps; Metabolic Glycosylation; Method LOINC Axis 6; Methodology; Mitogen Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 2; Modeling; Molecular Interaction; NIH; NK-1 Receptors; NK1R; NKIR; National Institutes of Health; National Institutes of Health (U.S.); Neurokinin A; Neurokinin alpha; Neuromedin L; Neuropeptides; P42MAPK; PRKM1; Pattern; Play; Preparation; Receptor Protein; Receptors, Neurokinin-1; Research; Research Personnel; Research Resources; Researchers; Resources; Role; SP(1-11); SP-P Receptors; Signal Pathway; Site; Source; Structure; Substance K; Substance P; Substance P Receptor; TAC1R; TACR1; Tachykinin Receptor 1; United States National Institutes of Health; Work; base; glycosylation; mitogen-activated protein kinase p38; mutant; neurokinin 1; p38 MAP Kinase; p38 MAPK; p38 Protein Kinase; p38 SAPK; p42 MAP Kinase; p42 MAPK; p42(Mapk) Kinase; p42(Mitogen-Activated Protein Kinase); radioligand; receptor; receptor internalization; social role