Nitric oxide is produced in the brain by the neuronal nitric oxide synthase (nNOS) and carries out a wide range of functions by acting as a neurotransmitter-like molecule. Gonadal hormones are involved in the regulation of the brain nitrergic system. We have previously demonstrated that estradiol, via classical estrogen receptors (ERs), regulates NOS activity in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus, acting through both ERα and ERβ. Magnocellular and parvocellular neurons in the SON and PVN also express the G protein-coupled ER (GPER). In this study, we have assessed whether GPER is also involved in the regulation of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in the SON and PVN. Adult female ovariectomized rats were treated with G1, a selective GPER agonist, or with G1 in combination with G15, a selective GPER antagonist. G1 treatment decreased NADPH-diaphorase expression in the SON and in all PVN subnuclei. The treatment with G1 + G15 effectively rescued the G1-dependent decrease in NADPH-diaphorase expression in both brain regions. In addition, the activation of extracellular signal-regulated kinase (ERK) 1/2, one of the kinases involved in the GPER-dependent intracellular signaling pathway and in NOS phosphorylation, was assessed in the same brain nuclei. Treatment with G1 significantly decreased the number of p-ERK 1/2-positive cells in the SON and PVN, while the treatment with G1 + G15 significantly recovered its number to control values. These findings suggest that the activation of GPER in the SON and PVN inhibits the phosphorylation of ERK 1/2, which induces a decrease in NADPH-diaphorase expression.

Keywords:
NADPH-diaphorase, G protein-coupled estrogen receptors, Extracellular signal-regulated kinase, Nitric oxide synthase, Estrogen, Hypothalamus, Paraventricular nucleus, Supraoptic nucleus
1.
Garthwaite J, Boulton CL: Nitric oxide signaling in the central nervous system. Annu Rev Physiol 1995;57:683-706.
2.
Gotti S, Sica M, Viglietti-Panzica C, Panzica G: Distribution of nitric oxide synthase immunoreactivity in the mouse brain. Microsc Res Tech 2005;68:13-35.
3.
Rodrigo J, Springall DR, Uttenthal O, Bentura ML, Abadia-Molina F, Riveros-Moreno V, Martínez-Murillo R, Polak JM, Moncada S: Localization of nitric oxide synthase in the adult rat brain. Philos Trans R Soc Lond B Biol Sci 1994;345:175-221.
4.
Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH: Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci USA 1991;17:7797-7801.
5.
Vincent SR, Kimura H: Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 1992;46:755-784.
6.
Otukonyong EE, Okere CO, Johnstone LE, Murata T, Kaba H, Higuchi T: Effect of suckling on NADPH-diaphorase (nitric oxide synthase, NOS) reactivity and NOS gene expression in the paraventricular and supraoptic nuclei of lactating rats. J Neuroendocrinol 2000;12:1001-1008.
7.
Beijamini V, Guimarães FS: c-Fos expression increase in NADPH-diaphorase positive neurons after exposure to a live cat. Behav Brain Res 2006;170:52-61.
8.
Mueller PJ, Foley CM, Heesch CM, Cunningham JT, Zheng H, Patel KP, Hasser EM: Increased nitric oxide synthase activity and expression in the hypothalamus of hindlimb unloaded rats. Brain Res 2006;1115:65-74.
9.
Boccoli J, Loidl CF, Lopez-Costa JJ, Creydt VP, Ibarra C, Goldstein J: Intracerebroventricular administration of Shiga toxin type 2 altered the expression levels of neuronal nitric oxide synthase and glial fibrillary acidic protein in rat brains. Brain Res 2008;1230:320-333.
10.
Heesch CM, Zheng H, Foley CM, Mueller PJ, Hasser EM, Patel KP: Nitric oxide synthase activity and expression are decreased in the paraventricular nucleus of pregnant rats. Brain Res 2009;1251:140-150.
11.
Vega C, Moreno-Carranza B, Zamorano M, Quintanar-Stéphano A, Méndez I, Thebault S, Martínez de la Escalera G, Clapp C: Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei. Am J Physiol Regul Integr Comp Physiol 2010;299:R1701-R1708.
12.
Naseh M, Vatanparast J: Enhanced expression of hypothalamic nitric oxide synthase in rats developmentally exposed to organophosphates. Brain Res 2014;1579:10-19.
13.
Panzica GC, Viglietti-Panzica C, Sica M, Gotti S, Martini M, Pinos H, Carrillo B, Collado P: Effects of gonadal hormones on central nitric oxide producing systems. Neuroscience 2006;138:987-995.
14.
Tobet S, Knoll JG, Hartshorn C, Aurand E, Stratton M, Kumar P, Searcy B, McClellan K: Brain sex differences and hormone influences: a moving experience? J Neuroendocrinol 2009;4:387-392.
15.
Gotti S, Martini M, Viglietti-Panzica C, Miceli D, Panzica G: Effects of estrous cycle and xenoestrogens expositions on mice nitric oxide producing system. Ital J Anat Embryol 2010;115:103-108.
16.
Grassi D, Lagunas N, Amorim M, Pinos H, Panzica G, Garcia-Segura LM, Collado P: Role of oestrogen receptors on the modulation of NADPH-diaphorase-positive cell number in supraoptic and paraventricular nuclei of ovariectomized female rats. J Neuroendocrinol 2013;25:244-250.
17.
Merchenthaler I, Lane MV, Numan S, Dellovade TL: Distribution of estrogen receptor alpha and beta in the mouse central nervous system: in vivo autoradiographic and immunocytochemical analyses. J Comp Neurol 2004;473:270-291.
18.
Brailoiu E, Dun SL, Brailoiu GC, Mizuo K, Sklar LA, Oprea TI, Prossnitz ER, Dun NJ: Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system. J Endocrinol 2007;193:311-321.
19.
Hazell GG, Yao ST, Roper JA, Prossnitz ER, O'Carroll AM, Lolait SJ: Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues. J Endocrinol 2009;2:223-236.
20.
Srivastava DP, Evans PD: G-protein oestrogen receptor 1: trials and tribulations of a membrane oestrogen receptor. J Neuroendocrinol 2013;25:1219-1230.
21.
Carmeci C, Thompson DA, Ring HZ, Francke U, Weigel RJ: Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer. Genomics 1997;45:607-617.
22.
Filardo EJ, Quinn JA, Bland KI, Frackelton AR: Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 2000;14:1649-1660.
23.
Filardo EJ, Thomas P: Minireview: G protein-coupled estrogen receptor-1, GPER-1: its mechanism of action and role in female reproductive cancer, renal and vascular physiology. Endocrinology 2012;153:2953-2962.
24.
Filardo EJ, Quinn JA, Frackelton AR, Bland KI: Estrogen action via the G protein-coupled receptor, GPR30: stimulation of adenylyl cyclase and cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis. Mol Endocrinol Baltim 2002;16:70-84.
25.
Scaling AL, Prossnitz ER, Hathaway HJ: GPER mediates estrogen-induced signaling and proliferation in human breast epithelial cells and normal and malignant breast. Horm Cancer 2014;5:146-160.
26.
Petrie WK, Dennis MK, Hu C, Dai D, Arterburn JB, Smith HO, Hathaway HJ, Prossnitz ER: G protein-coupled estrogen receptor-selective ligands modulate endometrial tumor growth. Obstet Gynecol Int 2013;2013:472720.
27.
Tang H, Zhang Q, Yang L, Dong Y, Khan M, Yang F, Brann DW, Wang R: Reprint of ‘GPR30 mediates estrogen rapid signaling and neuroprotection'. Mol Cell Endocrinol 2014;389:92-98.
28.
Lamprecht MR, Morrison B: GPR30 activation is neither necessary nor sufficient for acute neuroprotection by 17β-estradiol after an ischemic injury in organotypic hippocampal slice cultures. Brain Res 2014;1563:131-137.
29.
Lindsey SH, Cohen JA, Brosnihan KB, Gallagher PE, Chappell MC: Chronic treatment with the G protein-coupled receptor 30 agonist G-1 decreases blood pressure in ovariectomized mRen2 Lewis rats. Endocrinology 2009;150:3753-3758.
30.
Feldman RD, Gros R, Ding Q, Hussain Y, Ban MR, McIntyre AD, Hegele RA: A common hypofunctional genetic variant of GPER is associated with increased blood pressure in women. Br J Clin Pharmacol 2014;78:1441-1452.
31.
Cheng WH, Lu PJ, Ho WY, Tung CS, Cheng PW, Hsiao M, Tseng CJ: Angiotensin II inhibits neuronal nitric oxide synthase activation through the ERK1/2-RSK signaling pathway to modulate central control of blood pressure. Circ Res 2010;106:788-795.
32.
Dennis MK, Burai R, Ramesh C, Petrie WK, Alcon SN, Nayak TK, Bologa CG, Leitao A, Brailoiu E, Deliu E, Dun NJ, Sklar LA, Hathaway HJ, Arterburn JB, Oprea TI, Prossnitz ER: In vivo effects of a GPR30 antagonist. Nat Chem Biol 2009;5:421-427.
33.
Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates, hard cover edition. New York, Academic Press, 2013.
34.
Watson RE, Wiegand SJ, Clough RW, Hoffman GE: Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides 1986;7:155-159.
35.
Ruiz-Palmero I, Hernando M, Garcia-Segura LM, Arevalo MA: G protein-coupled estrogen receptor is required for the neuritogenic mechanism of 17β-estradiol in developing hippocampal neurons. Mol Cell Endocrinol 2013;372:105-115.
36.
Bredt DS, Glatt CE, Hwang PM, Fotuhi M, Dawson TM, Snyder SH: Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 1991;7:615-624.
37.
Armstrong WE, Warach S, Hatton GI, McNeill TH: Subnuclei in the rat hypothalamic paraventricular nucleus: a cytoarchitectural, horseradish peroxidase and immunocytochemical analysis. Neuroscience 1980;5:1931-1958.
38.
Arévalo R, Sánchez F, Alonso JR, Carretero J, Vázquez R, Aijón J: NADPH-diaphorase activity in the hypothalamic magnocellular neurosecretory nuclei of the rat. Brain Res Bull 1992;28:599-603.
39.
Hatakeyama S, Kawai Y, Ueyama T, Senba E: Nitric oxide synthase-containing magnocellular neurons of the rat hypothalamus synthesize oxytocin and vasopressin and express Fos following stress stimuli. J Chem Neuroanat 1996;11:243-256.
40.
Grassi D, Lagunas N, Amorin M, Pinos H, Panzica GC, Garcia-Segura LM, Collado P: Estrogenic regulation of NADPH-diaphorase in the supraoptic and paraventricular nuclei under acute osmotic stress. Neuroscience 2013;248:127-135.
41.
Kitamura N, Araya R, Kudoh M, Kishida H, Kimura T, Murayama M, Takashima A, Sakamaki Y, Hashikawa T, Ito S, Ohtsuki S, Terasaki T, Wess J, Yamada M: Beneficial effects of estrogen in a mouse model of cerebrovascular insufficiency. PLoS One 2009;4:e5159.
42.
Liu J, Hu P, Qi XR, Meng FT, Kalsbeek A, Zhou JN: Acute restraint stress increases intrahypothalamic oestradiol concentrations in conjunction with increased hypothalamic oestrogen receptor β and aromatase mRNA expression in female rats. J Neuroendocrinol 2011;23:435-443.
43.
El-Emam Dief A, Caldwell JD, Jirikowski GF: Colocalization of p450 aromatase and oxytocin immunostaining in the rat hypothalamus. Horm Metab Res 2013;45:273-276.
44.
Shughrue P, Scrimo P, Lane M, Askew R, Merchenthaler I: The distribution of estrogen receptor-beta mRNA in forebrain regions of the estrogen receptor-alpha knockout mouse. Endocrinology 1997;138:5649-5652.
45.
Sawchenko PE, Swanson LW: Immunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat. J Comp Neurol 1982;205:260-272.
46.
Sawchenko PE, Swanson LW: The organization of forebrain afferents to the paraventricular and supraoptic nuclei of the rat. J Comp Neurol 1983;218:121-144.
47.
Voisin DL, Simonian SX, Herbison AE: Identification of estrogen receptor-containing neurons projecting to the rat supraoptic nucleus. Neuroscience 1997;78:215-228.
48.
Grassi D, Amorim MA, Garcia-Segura LM, Panzica G: Estrogen receptor alpha is involved in the estrogenic regulation of arginine vasopressin immunoreactivity in the supraoptic and paraventricular nuclei of ovariectomized rats. Neurosci Lett 2010;474:135-139.
© 2016 S. Karger AG, Basel
2016
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.