Korean J Physiol Pharmacol Vol 16: 333－337, October, 2012 http://dx.doi.org/10.4196/kjpp.2012.16.5.333 cAMP/PKA Agonist Restores the Fasting-Induced Down-Regulation of nN OS Expression in the Paraventricular N ucleus Sang Bae Yoo1,*, Seoul Lee2,*, Joo Young Lee1, Bom-Taeck Kim3, Jong-Ho Lee1, and Jeong W on Jahng1 1Dental Research Institute, Department of Oral & Maxillofacial Surgery, Seoul National University School of Dentistry, Seoul 110-768, 2Department of Pharmacology, Wonkwang University School of Medicine, Iksan 570-749, 3Department of Family Practice and Community Health, Ajou University School of Medicine, Suwon 443-721, Korea Gene expression of neuronal nitric oxide synthase (nNOS) changes in the hypothalamic paraven- tricular nucleus (PVN) depending on feeding conditions, which is decreased during food deprivation and restored by refeeding, and phosphorylated cAMP response element binding protein (pCREB) was suggested to play a role in its regulation. This study was conducted to examine if the fasting-induced down-regulation of the PVN-nNOS expression is restored by activation of cAMP-dependent protein kinase A (cAMP/PKA) pathway. Freely moving rats received intracerebroventricular (icv) injection of cAMP/PKA activator Sp-cAMP (40 nmol) or vehicle (sterilized saline) following 48 h of food deprivation. One hour after drug injections, rats were transcardially perfused with 4% paraformaldehyde, and the PVN tissues were processed for nNOS or pCREB immunohistochemistry. Sp-cAMP significantly increased not only nNOS but also pCREB immunoreactivities in the PVN of food deprived rats. Fasting- induced down-regulation of the PVN-nNOS was restored by 1 h after the icv Sp-cAMP. Results suggest that cAMP/PKA pathway may mediate the regulation of the PVN-nNOS expression depending on different feeding conditions. Key Words: Food deprivation, Gene expression, Neuronal nitric oxide synthase, Paraventricular nucleus, Rat INTRODUCTION Food deprivation decreases mRNA level of neuronal nitric oxide synthase (nNOS), the intracellular intensity of NADPH- diaphorase (NADPH-d) staining, a marker for nNOS activity in the brain, and the apparent number of nNOS immunor- eactive cells in the hypothalamic paraventricular nucleus (PVN) [1-5]. Plasma glucocorticoids, which are elevated during food deprivation [3,5-7], appear to mediate the fast- ing-induced down-regulation of nNOS in the hypothalamic PVN. We have reported that depletion of endogenous gluco- corticoids by adrenalectomy abolishes fasting-induced down- regulation of nNOS in the rat PVN, and dexamethasone decreases the PVN-nNOS in both adrenalectomized and in- tact rats [4,8]. Furthermore, treatment with a glucocorti- coids receptor antagonist RU486 during food deprivation Received June 4, 2012, Revised August 3, 2012, Accepted August 21, 2012 Corresponding to: Jeong Won Jahng, Dental Research Institute, Seoul National University School of Dentistry, 275-1, Yeongeon-dong, Jongno-gu, Seoul 110-768, Korea. (Tel) 82-2-2072-0739, (Fax) 82-2- 766-4948, (E-mail) jwjahng@snu.ac.kr *These authors contributed equally to this work. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 333 blunted the fasting-induced decrease of nNOS in the PVN [3]. These reports together suggested that plasma gluco- corticoids may suppress nNOS expression in the PVN dur- ing food deprivation, via its receptor-mediated pathway. However, the upstream promoter of nNOS does not carry glucocorticoid response element, but carries cAMP response element [9-11]. In vitro studies have reported that nNOS expression is regulated by a cAMP response element-bind- ing protein (CREB) family transcription factor-dependent mechanism [11], and that phosphorylation of Ser-133 resi- due in CREB, a critical step in the activation of CREB, is regulated by glucocorticoids [12,13]. We have reported that CREB phosphorylation in the PVN is markedly decreased by 48 h of food deprivation, and this reduction is inhibited by treatment with a glucocorticoid receptor antagonist RU486 [3]. Thus, it is suggested that the plasma glucocorti- coids may suppress nNOS expression in the PVN during food deprivation, via down-regulation of CREB phosphory- lation. Not only the fasting-induced decrease of the PVN-nNOS but also of the PVN-pCREB is restored shortly after refeed- ing [5], and the refeeding-induced increases of the hypo- thalamic CREB phosphorylation and nNOS expression are blocked by dexamethasone treatments [5,14]. Also, the fast- ABBREVIATIONS: nNOS, neuronal nitric oxide synthase; PVN, paraventricular nucleus; pCREB, phosphorylated cAMP response element binding protein; cAMP/PKA, cAMP-dependent protein kinase A; icv, intracerebroventricular; NADPH-d, NADPH-diaphorase; NO, nitric oxide.