336 SB Yoo, et al DISCUSSION We have previously demonstrated that nNOS gene ex- pression in the hypothalamic PVN changes depending on feeding conditions; i.e., it is decreased during food depriva- tion and restored following refeeding, and that pCREB may be involved in the PVN-nNOS expression [3,5]. The up- stream promoter of nNOS gene contains cAMP response el- ement (CRE) [9]. In vitro studies have reported that nNOS expression is regulated by calcium influx through a CREB family transcription factor-dependent mechanism [11], and that cAMP markedly induces nNOS expression in human A673 neuroepithelial cells expressing CREB, and the in- duction is enhanced in A673 cells transfected with CREB [23]. In vivo study showed that the hypothalamic cAMP lev- els are decreased during food deprivation and restored by food consumption [16]. In this study, icv administration of cAMP-dependent PKA activator Sp-cAMP increased not on- ly pCREB levels but also nNOS expression in the PVN of food deprived rats, and cAMP antagonist/PKA inhibitor Rp-cAMP [16] did not affect the fasting-induced down-regu- lation of the PVN-nNOS. Together, it is suggested that the regulation of the PVN-nNOS expression depending on feed- ing conditions may be mediated by cAMP/PKA pathway. Previous studies have suggested that the plasma gluco- corticoids may suppress nNOS expression in the PVN dur- ing food deprivation, via down-regulation of CREB phos- phorylation [3,4,8], and that glucocorticoids dis-inhibition is required, although it is not sufficient, to restore the fast- ing-induced down-regulation of the PVN-nNOS and -pCREB [5,14]. However, in this study, Sp-cAMP administration in- creased both nNOS and pCREB in the PVN of food deprived rats, despite the fasting-induced elevation of the plasma corticosterone was still persisted. In vitro studies have re- ported that glucocorticoids regulate phosphorylation of Ser- 133 residue in CREB, a critical step in activation of CREB [12,13], and interfere with the cAMP-induced DNA binding of CREB [24]. Underlying mechanism by which Sp-cAMP increased pCREB and nNOS in the PVN of food deprived rats despite of the glucocorticoids inhibition is currently not clear. It should be noticed that cAMP agonist used in this study Sp-cAMP is a more potent PKA activator with higher affinity to the regulatory subunit of PKA and more resis- tant to phosphodiesterase than endogenous cAMP [16,25], which might have allowed Sp-cAMP to overcome the gluco- corticoids inhibition. Our previous study suggested that refeeding-induced nNOS expression in the PVN is a nutrient directed event; i.e., refeeding with non-caloric palatable food mash failed to restore the fasting-induced decreases not only of the PVN-nNOS but also of -pCREB [5]. Many studies have ex- amined the effects of various nutrients on NOS expression, however, those studies have been mostly focused on endo- thelial or inducible NOS, but not nNOS [26]. In vitro stud- ies have demonstrated that glucose, the major substrate for cerebral energy metabolism, increases the intracellular cAMP content [27], and activates cAMP/PKA pathway [28]. Also, an intragastric infusion of glucose induced c-Fos ex- pression, conventional marker for neuronal activation, in the PVN of food deprived rats [29]. Together with the cur- rent results, it is suggested that postprandial glucose may induce nNOS expression in the PVN at refeeding following food deprivation, by activating the cAMP/PKA pathway. In this study, food deprivation down-regulated nNOS ex- pression both in the parvocelluar and the magnocellular di- visions of PVN; however, Sp-cAMP restored nNOS expres- sion only in the parvocelluar PVN, but not in the magnocel- lular PVN. Furtheremore, Sp-cAMP following food depriva- tion increased pCREB levels only in the parvocellular PVN, but not in the magnocellular PVN. Our previous study showed that CREB phosphorylation is associated with nNOS down- regulation in the parvocellular PVN, but not in the magno- cellular PVN, during food deprivation. That is, the fasting- induced nNOS down-regulation was observed not only in the parvocellular PVN but also in the magnocellular PVN; however, food deprivation reduced pCREB only in the par- vocellular PVN [3]. Thus, it is concluded that nNOS ex- pression in the magnocellular PVN, not likely in the parvo- cellular PVN, may not be mediated by cAMP/PKA pathway. Nitric oxide (NO) is an important biological messenger that acts in a vast array of physiological processes [30]. In the nervous system, neuronal nitric oxide synthase (nNOS) accounts for the majority of the physiologic actions of NO [31,32]. Many investigations have shown that nNOS ex- pression is dynamically regulated by both physiological and pathophysiological stimuli [31,33-35]. Feeding-related chan- ges of nNOS expression in the PVN may be a part of the regulatory pathway for autonomic changes associated with feeding status, such as heart rate and blood pressure [36]. The PVN nitric oxide has been suggested to be involved in the regulation of autonomic functions [37,38], and re- ported to modulate vasopressin release [39]. Vasopressin is known to be involved in the control of blood pressure and heart rate as well as in the regulation of fluid and elec- trolyte balance [40]. Food deprivation decreases the hypo- thalamic contents of vasopressin [41] and refeeding acti- vates vasopressin neurons in the PVN [15]. Overall, it is postulated that feeding-related changes in nNOS expres- sion in the PVN may play a role in the regulatory control for blood pressure and heart rate, at least partly by modu- lating the activity of vasopressin neurons. ACKNOW LEDGEMENTS This study was supported by grants from the Brain Research Center of the 21st Century Frontier Research Program (2012K001115) and the National Research Foun- dation (2010-0003642) funded by the Korea Government (Ministry of Education, Science and Technology). REFERENCES 1. Ueta Y, Levy A, Chowdrey HS, Lightman SL. Inhibition of hypothalamic nitric oxide synthase gene expression in the rat paraventricular nucleus by food deprivation is independent of serotonin depletion. J Neuroendocrinol. 1995;7:861-865. 2. O’Shea RD, Gundlach AL. Food or water deprivation modulate nitric oxide synthase (NOS) activity and gene expression in rat hypothalamic neurones: correlation with neurosecretory acti- vity? J Neuroendocrinol. 1996;8:417-425. 3. Kim YM, Lee JY, Choi SH, Kim DG, Jahng JW. RU486 blocks fasting-induced decrease of neuronal nitric oxide synthase in the rat paraventricular nucleus. Brain Res. 2004;1018:221-226. 4. Jahng JW, Lee JH, Kim GT, Kim YM, Houpt TA, Kim DG. Adrenalectomy abolishes fasting-induced down-regulation of NADPH-diaphorase in the rat paraventricular nucleus. Yonsei Med J. 2004;45:123-128. 5. Jahng JW, Lee JY, Yoo SB, Kim YM, Ryu V, Kang DW, Lee JH. Refeeding-induced expression of neuronal nitric oxide syn- thase in the rat paraventricular nucleus. Brain Res. 2005;1048: