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A. The Projects: Project Leaders and Project Titles.
Project 1. Robert
J. Parmer, M.D. (Department of Medicine).
Hereditary autonomic intermediate phenotypes in hypertension.
Project 2. Michael
G. Ziegler, M.D. (Department of Medicine).
Extra-adrenal epinephrine.
Project 3. Vivian
Y. Hook, Ph.D. (Department of Medicine).
Neuropeptide Y biosynthesis in chromaffin cells.
Project 4. Daniel
T. O'Connor, M.D. (Department of Medicine)
and Palmer Taylor, Ph.D. (Department of Pharmacology).
Formation and actions of a novel catecholamine release-inhibitory
peptide.
Projects 1 and 2 involve studies in intact
organisms, including humans (Projects 1 and 2) and rodents (Project
2). Projects 2 and 4 include cell and molecular biologic studies
of neurotransmitter biosynthesis, release, and actions in the
cleft.
These cellular or in vitro studies (Projects 2 and 4) exploit
biological materials (plasma, urine, sympathetic tissues) generated
in vivo by Projects 1 and 2 (human materials) and Project
2 (rodent materials).
1. Project 1 (also see here)
Project 1 (Hereditary autonomic intermediate phenotypes in hypertension; Robert J. Parmer, M.D.) has uncovered several novel autonomic phenotypes in pedigrees with multigenerational essential (hereditary) hypertension, with evidence that alterations in such phenotypes (such as blunted baroreceptor slope or augmented cardiac responses to alpha-2-adrenergic receptor blockade) are heritable; the clue of bimodality further suggests that such phenotypes may be subject to major gene (i.e., Mendelian) effects on heritability. Project 1 tests the hypothesis that such phenotypes are linked to (that is, co-segregate with) polymorphic alleles at candidate genetic loci, initially using non-parametric (sib-pair; allele-sharing) methods, as a first step in the search for hypertension predisposition loci. The linkage study focuses on particular candidate genes suggested by the adrenergic receptor phenotypes characterized by the four Projects (see the list of candidate loci, and their microsatellite PCR polymorphisms, in Core D (Molecular genetics and informatics). Biological materials (plasma, urine, and genomic DNA [from circulating leucocytes]) generated by Project 1 are exploited by Core B (Biochemistry), Core C (Adrenergic receptors and intracellular signaling), and Core D (Molecular genetics and informatics), as well as Projects 2, 3 and 4.
2. Project 2 (also see here)
Project 2 (Extra-adrenal epinephrine; Michael G. Ziegler, M.D.) pursues the observation that epinephrine has novel, alternative pathways and sites of formation (other than simply adrenal medullary chromaffin cells), and the hypothesis that such epinephrine may play a blood pressure-modulatory role, as well as a pathogenic role in some forms of hypertension.
3. Project 3 (also see here)
Project 3 (Neuropeptide Y biosynthesis in chromaffin cells; Vivian Y. Hook, Ph.D.) explores how pro-neuropeptide Y is proteolytically cleaved to form active NPY in catecholamine storage vesicles.
4. Project 4 (also see here)
Project 4 (A novel catecholamine release-inhibitory peptide; Daniel T. O'Connor, M.D., and Palmer Taylor, Ph.D.) explores the formation and actions of a novel, mid-molecule fragment of chromogranin A, a protein co-stored and co-released by exocytosis with catecholamines from their secretory granules. This fragment, bounded by pro-hormone convertase cleavage sites, is a potent (IC50 ~200 nM) inhibitor of catecholamine release, and seems to do so as a non-competitive nicotinic cholinergic antagonist. We have called it "catestatin."
B. The Core Units (also see here)
Core Leaders Themes/services.
A Daniel T. O'Connor, M.D. Administration.
B Michael G. Ziegler, M.D. Biochemistry.
C Paul A. Insel, M.D. Adrenergic receptors and intracellular
signaling.
D Bruce A. Hamilton, Ph.D./ Molecular genetics and informatics.
Douglas W. Smith, Ph.D.
E Vivian Y. Hook, Ph.D. Sympathochromaffin cell culture.
These core facilities provide the four Projects with access to catecholamine and vasoactive peptide measurement, receptor binding and post-receptor signal transduction assays, genotyping, DNA sequencing, informatic (sequence management) support, localization of specific mRNAs by in situ hybridization, and provision of uniform populations of catecholaminergic secretory cells.
C. Strategies for Achieving Objectives
We developed these 5 specific strategies, in order to maximize the interactive productivity of this Program group:
i. Focus. Each research Project is focused on one aspect of the sympathetic neuroeffector junction.
ii. Investigators. Participating Project or Core investigators were carefully chosen on the basis of past history of already cooperative efforts among the investigators in this Program application (Parmer, Ziegler, O'Connor/Taylor, Insel), or of reputation and curriculum vitae giving evidence of frequent and successful cooperative or collaborative efforts with others (Hook, Hamilton).
iii. Topics. Although each Project approaches the sympathetic neuroeffector junction in some way, each Project also has its own unique emphasis -- often a particular neurotransmitter or receptor. Thus, all interactions are likely to be complementary (or synergistic), rather than overlapping.
iv. Methods. Each Project or Core investigator brings a unique methodologic expertise to the Program. For example, Project 1 brings the unique resource of human pedigrees, while Project 4 brings expertise in secretory biology of the chromaffin cell. This availability of diverse methods, never found in any single laboratory, is a priceless resource to investigators desiring to broaden the significance of their findings.
v. Interest (or "affinity") groups. Already among these Project and Core directors, several interest or affinity groups have emerged over the past ~15 years, with collaborative studies frequently proceeding to co-authored publications:
vi. Core service ethic. Research core unit directorship is dispersed widely among the Program co-investigators -- Ziegler (Biochemistry), Hook (Sympathochromaffin cell culture), Insel (Adrenergic receptors and intracellular signaling), and Hamilton/Smith (Molecular genetics and informatics). Thus, reciprocal use of cores is likely to take place: investigator "A" will perform core services for investigator "B," and vice-versa. This should create and reinforce a spirit of cooperation, service and generosity among co-investigators; e.g., the recipient of core "B" service will soon be called upon to reciprocate with core "E" service.
D. Relation of each Project and Core to the Strategies
1. Four Projects (also see here)
Project 1 (Parmer), with its human hypertensive pedigrees, provides a focal point with in vivo sample generation for multiple Projects (Projects 2 [Ziegler], 3 [Hook], and 4 [O'Connor/Taylor]) and Cores (Core B/Biochemistry/Ziegler) with expertise in measurement of sympathetic junctional co-transmitters, as well as the adrenergic receptor core (Core C/Adrenergic receptors/Insel). Projects 1 and 2, assisted by and Cores B and C, will evaluate adrenergic receptor function (ligand and receptor) and gene expression. Core D will microsatellite-genotype pedigree members at candidate loci suggested by autonomic (biochemicaol and physiologic) phenotypes developed by each of the participating research Projects 1 and 4. Thus, Project 1 provides a crucial focal point for virtually the entire Program.
Project 2 (Ziegler), with its studies of epinephrine formation, provides expertise in catecholamine biosynthesis, release, measurement, and actions, crucial areas for Projects 1 (Parmer) and 4 (O'Connor/Taylor), and Core B (Biochemistry). Project 2 also provides the opportunity to test, in intact rodents, the cardiovascular (blood pressure and plasma catecholamine) effects of novel reagents generated by the other Projects, such as pro-NPY fragments (Project 3/Hook), and the nicotinic antagonist fragment of chromogranin A (Project 4, O'Connor/Taylor).
Project 3 (Hook) provides tools for examining proteolytic processing of catecholamine storage vesicle pro-hormones such as neuropeptide Y and chromogranin A. Such expertise will be invaluable in the NPY studies of Projects 1 (Parmer), and 2 (Ziegler), as well as the chromogranin A studies of Project 4 (O'Connor/Taylor). For example, Project 3 will provide expertise in NPY immunoassay for phenotyping of pedigree members in Project 1, pro-NPY fragments for effects on systemic cardiovascular function in Project 2, as well as local renal neuroeffector junctional activity in Project 2, and reagents (purified enzymes, cDNAs, inhibitors) to evaluate the route and mechanism of processing of the catestatin fragment out of its pro-hormone, chromogranin A (Project 4).
Project 4 (O'Connor/Taylor) evaluates a peptide ("catestatin") which inhibits nicotinic cholinergic-stimulated catecholamine release from chromaffin cells and sympathetic axons. Measurement of this peptide will provide a likely autonomic junctional phenotype for Project 1 (Parmer), a reagent for evaluation of effects on post-ganglionic sympathetic axon function in vivo in experimental animals (Project 2 [Ziegler]), and an antagonist for domain mapping on nicotinic subunits (Taylor). The involvement of Palmer Taylor provides the opportunity to document the mechanism of action of peptides and other endogenous modulators of nicotinic cholinergic control of catecholamine release, using expressed chimeric nicotinic receptor pentamers. The interaction of Drs. O'Connor and Taylor is immediately apparent, while nicotinic control of chromaffin cell release of NPY and its pro-hormone processing enzymes will be explored in Project 3 (Hook), and nicotinic control of sympathoadrenal catecholamine release in vivo is made possible by Project 2 (Ziegler).
The service of each of the five Core units to each of the four research Projects is explicitly detailed in outline form after the Core unit texts (here).
Core A (Administration) provides the necessary infrastructure for the smooth interaction of these research Projects and Cores. It facilitates communications among the Projects and Cores, and provides for periodic meetings, as well as internal and external reviews of our overall progress. It provides biological sample databasing and archiving for inter-Project and Project-Core phenotyping efforts.
Core B (Biochemistry) allows each of the Projects (1 through 4) access to standardized measurements for evaluation of the activity of the neuroeffector junction. This service will allow comparability of the studies in the 4 Projects, facilitating our ability to merge data and protocols.
Core C (Adrenergic receptors and intracellular signaling) allows each of the research Projects access to tools to characterize catecholamine actions (receptor and post-receptor) within the sympathetic neuroeffector junction. In addition, this Core assists Project 1 in evaluating functional and structural alterations at two adrenergic receptors (ADRB2 and ADRA1B) encoded on a human chromosomal region (5q31-q32) genetically linked to altered adrenergic prressor responses in the hypertensive pedigrees (Project 1).
Core D (Molecular genetics and informatics) allows us to verify linkage (co-segregation) between neuroeffector junctional phenotypes (such as responses to alpha-2-adrenergic blockade in Project 1) and microsatellite alleles at genetic loci suggested by each of the research Projects (see Table in Core D). After linkage, physical mapping is undertaken at specific candidate loci. As sequence information at candidate genetic loci emerges, an informatics component allows intelligent management and interpretation of the accumulated information. In addition, the Core provides each Project access to molecular biological tools to characterize their particular transmitter, enzyme, or receptor system. A good example is mRNA detection by in situ hybridization, to detect the cellular/microanatomic distribution of PNMT transcripts (Project 2), NPY transcripts (Project 3), chromogranin A transcripts (Project 4), and nicotinic cholinergic receptor subunits transcripts (Project 4).
Core E (Sympathochromaffin cell culture). Several Projects require cultured preparations of catecholamine-secreting cells, such as primary cultures of bovine adrenal chromaffin cells, PC12 pheochromocytoma cells, or sympathetic nerves (superior cervical ganglion cells). Such cells will be used by Project 2 (manipulation of PNMT expression), Project 3 (secretagogue alteration of NPY processing), Project 4 (catestatin effect on chromaffin cell and sympathetic neuronal catecholamine release; neuronal nicotinic cholinergic control of catecholamine secretion). Having well-defined secreting cell preparations, uniform across several Projects, will enhance our ability to collaborate, merge protocols and results, and co-publish.
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Latest modification: December, 1999
If you have comments or queries, send email to Doug Smith