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Title:The roles of nitric oxide,cGMP, and cGMP-dependent protein kinase in generation and modulation of circadian rhythms by the suprachiasmatic nucleus
Author(s):Weber, Edward Todd
Doctoral Committee Chair(s):Gillette, Martha U.
Department / Program:Molecular and Integrative Physiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Animal Physiology
Abstract:The hypothalamic suprachiasmatic nuclei (SCN) serve as the primary mammalian circadian pacemaker. Measurements from SCN in brain slice preparations from rats reveal circadian rhythms of intracellular levels of cGMP, and in $\rm\lbrack\gamma$-$\rm\sp{32}PO\sb4\rbrack$-orthophosphate incorporation into constant levels of a 75 kD protein in in vitro reactions stimulated by cGMP. This protein was identified as cGMP-dependent protein kinase (PKG) by immunoprecipitation and immunoblots using polyclonal antisera raised against this phosphoprotein extracted and purified from bovine brain. In vitro assays revealed a circadian rhythm of endogenous PKG activity in SCN from samples procured at different points in the circadian cycle which coincided with the rhythm of endogenous cGMP levels and PKG autophosphorylation.
In vivo studies were performed in which the nitric oxide synthase inhibitor, L-NAME, was injected into the third ventrical at the level of the SCN, 10 minutes prior to photic stimulation, and was shown to inhibit light-induced phase shifts of locomotor rhythms. The effect was reversible and dose-related, and could be outcompeted by addition of excess arginine. D-NAME did not block photic phase-shifts. Neither L-NAME nor arginine affected phase of rhythms in the absence of light. Arginine did not potentiate light-induced phase shifts. L-NAME did not block light induced expression of the intermediate-early gene product, Fos.
Finally, the putative regulation of cAMP-response element binding protein (CREB) by PKG was evaluated using several approaches. CREBtide, a 14-amino acid sequence of CREB surrounding the activation-specific phosphoacceptor ser-133, was phosphorylated by PKG at a rate comparable to that of the PKG-specific heptapeptide substrate sequence of histone used to characterize PKG kinetics. $\rm\lbrack\sp{32}PO\sb4\rbrack$-orthophosphate incorporation into a 43 kD protein corresponding to CREB on immunoblots was demonstrated in SCN following subjective nighttime stimulation with 8-Br-cGMP. Finally, specific phospho-CREB immunostaining in the SCN was significantly blocked by preincubation of the antiserum with CREBtide phosphorylated by PKG.
Issue Date:1995
Rights Information:Copyright 1995 Weber, Edward Todd
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9522186
OCLC Identifier:(UMI)AAI9522186

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