The propose research in project 5 is based on two working hypotheses: (a) spinal nicotinic cholinergic neurotransmission is a critical pathway in the maintenance of blood pressure and in mediating pressor responses elicited by external stimulae; (b) certain hypertensive phenotypes with a genetic basis result from hyper-excitability of the nicotinic system which amplifies responses to central stimulation through control by presynaptic receptors. Thus, hypertension and enhanced startle responses may be critical manifestations of hyper-excitability spinal nicotinic receptor regulated pathways. In the past award period we have mapped some of the critical pathways involved in the spinal pressor response, described differential responses to nicotinic agonists and examined amino acid release and nicotinic receptor densities in spontaneously hypertensive rat (SHR) and control (WKY) rats. We plan to extend these studies to recombinant inbred strains and to hypertension models that do not have a primary, central neurogenic component in order to determine whether nicotinic hyperexcitability cosegregates with hypertension and whether it is confined to neurogenic hypertension with a genetic origin. Recently, we developed an intrathecal microdialysis system that enables injection of nicotinic agonists and measurement of released neurotransmitters into the intrathecal space in apposition to the injection site. This cellular endpoint of neurotransmitter release will be correlated with radioligand detection of receptors by autoradiography in spinal sections and with the pharmacologic endpoints. These techniques will enable us to extend our analysis to the cellular and molecular levels. These studies will initially be directed to completing our analysis of nicotinic agonist mediated release of excitatory amino acids and then extending the studies to release of acetylcholine, substance P and catecholamines. We propose to identify cellular mediators such as intracellular Ca2+ and regulators of gene expression that may be responsible for the enhanced excitability of the spinal responses. These studies will interface with Projects 1 and 2. Specific procedures for ablation of primary afferents, ascending and descending pathways and interneurons have been developed and the influence of these interventions will be examined on the responses in the intact animal, the cellular response of transmitter release and the loss of nicotinic receptor subtypes and their localization. Second, we will examine the specificity and subtypes of nicotinic receptors involved through the use of receptor selective ligands, immunoprecipitation, and autoradiography and plan to correlate this information with the loci of genes affecting hypertension. Third, receptor subtype expression in the spinal cord will be correlated with the mRNA species through message protection and in situ hybridization.
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