11320 • The Journal of Neuroscience, November 2, 2016 • 36(44):11320–11330 Cellular/Molecular Nitric Oxide Modulates HCN Channels in Magnocellular Neurons of the Supraoptic Nucleus of Rats by an S-Nitrosylation-Dependent Mechanism X Melina Pires da Silva,1 Davi Jose´ de Almeida Moraes,1 Andre´ de Souza Mecawi,2 Jose´ Antunes Rodrigues,1 and X Wamberto Antonio Varanda1 1Department of Physiology, Ribeira˜o Preto Medical School, University of Sa˜o Paulo, 14049-900 Ribeira˜o Preto, Sa˜o Paulo, Brazil, and 2Department of Physiological Sciences, Biology Institute, Federal Rural University of Rio de Janeiro, 23890-000, Serope´dica, Rio de Janeiro, Brazil The control of the excitability in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus has been attributed mainly to synapticinputsfromcircunventricularorgans.However,nitricoxide(NO),agaseousmessengerproducedinthisnucleusduringisotonic and short-term hypertonic conditions, is an example of a modulator that can act directly on MNCs to modulate their firing rate. NO inhibits the electrical excitability of MNCs, leading to a decrease in the release of vasopressin and oxytocin. Although the effects of NO on MNCs are well established, the mechanism by which this gas produces its effect is, so far, unknown. Because NO acts independently of synaptic inputs, we hypothesized that ion channels present in MNCs are the targets of NO. To investigate this hypothesis, we used the patch-clamptechniqueinvitroandinsitutomeasurecurrentscarriedbyhyperpolarization-activatedandnucleotide-gatedcation(HCN) channelsandestablishtheirroleindeterminingtheelectricalexcitabilityofMNCsinrats.OurresultsshowthatblockadeofHCNchannels by ZD7288 decreases MNC firing rate with significant consequences on the release of OT and VP, measured by radioimmunoassay. NO induced a significant reduction in HCN currents by binding to cysteine residues and forming S-nitrosothiol complexes. These findings shed new light on the mechanisms that control the electrical excitability of MNCs via the nitrergic system and strengthen the importance of HCN channels in the control of hydroelectrolyte homeostasis. Key words: electrolyte homeostasis; electrophysiology; HCN channels; magnocellular neurons; nitric oxide; S-nitrosylation Significance Statement Cells in our organism live in a liquid environment whose composition and osmolality are maintained within tight limits. Magno- cellular neurons (MNCs) of the supra optic nucleus can sense osmolality and control the synthesis and secretion of vasopressin (VP) and oxytocin (OT) by the neurohypophysis. OT and VP act on the kidneys controlling the excretion of water and sodium to maintain homeostasis. Here we combined electrophysiology, molecular biology, and radioimmunoassay to show that the electri- calactivityofMNCscanbecontrolledbynitricoxide(NO),agaseousmessenger.NOreactswithcysteineresidues(S-nitrosylation) on hyperpolarization-activated and nucleotide-gated cation channels decreasing the firing rate of MNCs and the consequent secretion of VP and OT. Introduction mammalian species. The central control of hydroelectrolyte bal- Maintenance of hydroelectrolyte homeostasis requires complex ance involves highly organized hypothalamic structures that op- interactions between peripheral and CNS circuits in several erate to maintain the extracellular fluid osmolality within tight limits. Among these structures, the hypothalamic supraoptic nu- cleus (SON), composed of magnocellular neurosecretory cells Received May 15, 2016; revised Aug. 23, 2016; accepted Sept. 14, 2016. (MNCs), plays a critical role in the synthesis and secretion of Author contributions: M.P.d.S. and W.A.V. designed research; M.P.d.S., D.J.d.A.M., A.d.S.M., and J.A.R. per- vasopressin (VP) and oxytocin (OT). These neuropeptides are formed research; M.P.d.S. and W.A.V. analyzed data; M.P.d.S. and W.A.V. wrote the paper. M.P.d.S. was supported by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nível Superior fellowship. The labo- involved in several processes, including memory, vasoconstric- ratory of W.A.V. was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo Grant 2012/19750-7. tion, reproduction, and prevention of osmotic stress via renal D.J.d.A.M. was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo Grant 2013/10484-5. control of sodium and water reabsorption (Poulain et al., 1977; The authors declare no competing financial interests. Correspondence should be addressed to Dr. Melina Pires da Silva, Department of Physiology, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Av. Bandeirantes, 3900, 14049-900, Ribeira˜o Preto, Sa˜o Paulo, Brazil. DOI:10.1523/JNEUROSCI.1588-16.2016 E-mail: [email protected]. Copyright © 2016 the authors 0270-6474/16/3611320-11$15.00/0 Pires da Silva et al. • NO Modulates HCN Channels in MNCs J. Neurosci., November 2, 2016 • 36(44):11320–11330 • 11321 Antunes-Rodrigues et al., 2004). Their release is directly corre- Isolation of magnocellular neurons. MNCs were isolated from cylindri- lated with MNC firing rate. cal punches made laterally to the optic chiasm in hypothalamic coronal Although the control of MNC electrical excitability can be slices (230-␮m-thick), using a 16 gauge sharpened needle. The tissue influenced by synaptic inputs (Bourque, 2008), local modulators fragments containing the SON were washed and incubated in Hanks solution (in mM) as follows: 121 NaCl, 4 KCl, 5 NaHCO 10 HEPES, 10 have been shown to directly interfere in the final responses of 3, glucose (constantly gassed with 100% oxygen; pH 7.35, osmolality 295 these cells (Ruginsk et al., 2015). Indeed, previous reports from mosm/kg H2O) and trypsin (7 mg/ml, Sigma Chemical) for 30 min at our group (Ventura et al., 2008; da Silva et al., 2013) and others 37°C. Then, the tissue fragments were washed again with Hanks solution (Ventura et al., 2005; Coletti et al., 2015; Reis et al., 2015) indicate and incubated for 10 min with 0.05% trypsin inhibitor (Sigma Chemi- the participation of gaseous signaling molecules in the control of cal). Subsequently, the solution was substituted with Neurobasal Me- MNC excitability and neurohypophyseal secretion, both under dium supplemented with 2% B27 (Serum Free, Invitrogen), 0.5 mM physiological conditions and during short-term increases in glutamine, 1% penicillin and streptomycin, and the cells were gently plasma osmolality. One of these gaseous messengers is nitric ox- dispersed by aspiration and flushing with fire-polished Pasteur pipettes ide (NO). NO decreases MNC excitability and the release of neu- with decreasing diameters. The cells were then plated on plastic dishes ropeptides (Ventura et al., 2005; Reis et al., 2010), although the with glass bottoms, and electrophysiology was performed after 24 h. The corresponding underlying mechanism remains unknown. OT and/or VP phenotype of the isolated neurons was recognized using Members of the cyclic nucleotide-gated family of ion channels, single-cell qRT-PCR (for details, see below). In situ brainstem-hypothalamus preparation. In this set of experiments, such as the hyperpolarization-activated and nucleotide-gated cation we simultaneously measured electrophysiological parameters and hor- (HCN) channel, are strong candidates for controlling MNC excit- mone release using a precollicularly decerebrated preparation, as origi- ability (Ghamari-Langroudi and Bourque, 2000). First described in nally described by Antunes et al. (2006). Briefly, Wistar rats (100–120 g) the heart (DiFrancesco, 1993) and later in the CNS (Pape, 1996), were deeply anesthetized with halothane (Itapira), bisected below the HCN channels are involved in numerous intrinsic neuronal func- fifth rib and decorticated. At this point, anesthesia was terminated. The tions, such as regulation of resting membrane potential, spontane- preparation was transferred to a recording chamber, and a double-lumen ous firing in pacemaker cells, synaptic transmission, and regulation catheter was placed into the aortic arch through the left ventricle, for of membrane resistance (Kase and Imoto, 2012). Four subtypes of hypothalamus perfusion with ACSF solution containing the following HCN channels (HCN1-HCN4) are expressed in mammals with (in mM): 121 NaCl, 4 KCl, 10 glucose, 26 NaHCO3, 2 CaCl2,1 MgCl2 (pH 7.35, osmolality 295 mosm/kg H O) saturated with 95% O /5% CO . ϳ60% molecular identity between them. Once open, these channels 2 2 2 ϩ Ficoll (molecular weight 20,000; 1.25%) was added as an oncotic agent allow the flow of a cationic current, termed I , carried by both Na h and vecuronium bromide (4 ␮g/ml Crista´lia, Itapira) as a muscle relax- PNaϩ and Kϩ with permeability ratio ϭ 0.2 to 0.4 (Pape, 1996). In ant. The superior vena cava was cannulated to collect samples from the PKϩ hypophyseal circulation for measurement of peptide concentrations by addition to being voltage-dependent, HCN channels are also known radioimmunoassay (see below). to be controlled by cAMP and cGMP (Biel et al., 2009). The latter is MNCs intrinsic electrical activity was measured with the blind patch- also the major second messenger used by NO (Denninger and clamp technique in the whole-cell configuration (one neuron per prep- Marletta, 1999; Matulef and Zagotta, 2003; Zagotta et al., 2003). aration). Biocytin (0.2%, Invitrogen) was added to the patch pipette Based on this evidence, the present study aimed to analyze the mech- solution to label the recorded neurons by immunofluorescence (for de- anisms by which NO acts on HCN channels