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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2018 Aug 12;175(19):3758–3772. doi: 10.1111/bph.14445

Stimulation of brain nicotinic acetylcholine receptors activates adrenomedullary outflow via brain inducible NO synthase‐mediated S‐nitrosylation

Youichirou Higashi 1, Takahiro Shimizu 1,✉, Masaki Yamamoto 1, Kenjiro Tanaka 2, Toshio Yawata 3, Shogo Shimizu 1, Suo Zou 1, Tetsuya Ueba 3, Kazunari Yuri 2, Motoaki Saito 1
PMCID: PMC6135790  PMID: 30007012

Abstract

Background and Purpose

We have demonstrated that i.c.v.‐administered (±)‐epibatidine, a nicotinic ACh receptor (nAChR) agonist, induced secretion of noradrenaline and adrenaline (catecholamines) from the rat adrenal medulla with dihydro‐β‐erythroidin (an α4β2 nAChR antagonist)‐sensitive brain mechanisms. Here, we examined central mechanisms for the (±)‐epibatidine‐induced responses, focusing on brain NOS and NO‐mediated mechanisms, soluble GC (sGC) and protein S‐nitrosylation (a posttranslational modification of protein cysteine thiol groups), in urethane‐anaesthetized (1.0 g·kg−1, i.p.) male Wistar rats.

Experimental Approach

(±)‐Epibatidine was i.c.v. treated after i.c.v. pretreatment with each inhibitor described below. Then, plasma catecholamines were measured electrochemically after HPLC. Immunoreactivity of S‐nitrosylated cysteine (SNO‐Cys) in α4 nAChR subunit (α4)‐positive spinally projecting neurones in the rat hypothalamic paraventricular nucleus (PVN, a regulatory centre of adrenomedullary outflow) after i.c.v. (±)‐epibatidine administration was also investigated.

Key Results

(±)‐Epibatidine‐induced elevation of plasma catecholamines was significantly attenuated by L‐NAME (non‐selective NOS inhibitor), carboxy‐PTIO (NO scavenger), BYK191023 [selective inducible NOS (iNOS) inhibitor] and dithiothreitol (thiol‐reducing reagent), but not by 3‐bromo‐7‐nitroindazole (selective neuronal NOS inhibitor) or ODQ (sGC inhibitor). (±)‐Epibatidine increased the number of spinally projecting PVN neurones with α4‐ and SNO‐Cys‐immunoreactivities, and this increment was reduced by BYK191023.

Conclusions and Implications

Stimulation of brain nAChRs can induce elevation of plasma catecholamines through brain iNOS‐derived NO‐mediated protein S‐nitrosylation in rats. Therefore, brain nAChRs (at least α4β2 subtype) and NO might be useful targets for alleviation of catecholamines overflow induced by smoking.

Abbreviations

CRF

corticotropin‐releasing factor

DMF

N,N‐dimethylformamide

eNOS

endothelial NOS

IML

intermediolateral cell column

iNOS

inducible NOS

nAChR

nicotinic ACh receptor

nNOS

neuronal NOS

PVN

paraventricular nucleus

SNO‐Cys

S‐nitrosylated cysteine

TBS

Tris‐buffered saline

Introduction

Cigarette smoking has been shown to be a major risk factor for cardiovascular diseases including hypertension (Hering et al., 2010; Virdis et al., 2010). (−)‐Nicotine is one of the components of cigarette smoke, and it has been known to centrally exert hypertension. For example, exposure to (−)‐nicotine vapour increased mean arterial blood pressure due to the disruption of the homeostasis of the renin‐angiotensin system in the brain (Yue et al., 2018), and centrally administered (−)‐nicotine induced hypertension through release of noradrenaline in the hypothalamic paraventricular nucleus (PVN) (Harland et al., 1989; Sharp and Matta, 1993), a control centre of the sympatho‐adrenomedullary system (Pyner, 2009), and caused a pressor response and increase in plasma catecholamines (adrenaline and noradrenaline) through brain http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=76 (nAChRs) (Kiritsy‐Roy et al., 1990; Buccafusco and Yang, 1993; Tseng et al., 1993). We have demonstrated that (i) central administration of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5348, a non‐selective nAChR agonist (Lembeck, 1999), resulted in the secretion of both catecholamines from the adrenal medulla in rats, as demonstrated by the disappearance of the secretion in bilateral adrenalectomized rats (Shimizu and Yokotani, 2009) and that (ii) the (±)‐epibatidine‐induced secretion was attenuated by central pretreatment with a selective α4β2 nAChR antagonist in rats (Shimizu et al., 2011). These lines of evidence indicate that stimulation of brain α4β2 nAChRs can mainly induce activation of central adrenomedullary outflow.

Previously, we reported that activation of central adrenomedullary outflow induced by the centrally administered neuropeptide bombesin was suppressed by central pretreatment with http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5213, a non‐selective inhibitor of http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=253, and carboxy‐PTIO, an http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2509 scavenger, in rats (Yokotani et al., 2005; Lu et al., 2008; Tanaka et al., 2012). In addition, centrally administered SIN‐1, an NO donor, elevated plasma catecholamines, and the response was abolished by centrally administered carboxy‐PTIO in rats (Murakami et al., 1998). These findings suggest that brain NOS‐mediated NO production might be involved in the activation of central adrenomedullary outflow induced by centrally administered (±)‐epibatidine.

NO is produced from L‐arginine by NOS, which has three isoforms: neuronal, endothelial and inducible NOS (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1251&familyId=253&familyType=ENZYME, eNOS and http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1250&familyId=253&familyType=ENZYME) (Costa et al., 1996). Under normal physiological conditions, the constitutively expressed nNOS is the most abundant isoform in the mammalian brain (Bredt and Snyder, 1989; Vincent and Kimura, 1992). By contrast, iNOS is usually undetectable in the healthy brain, although it can be up‐regulated in response to pathological stimuli such as pro‐inflammatory cytokines (Nathan and Xie, 1994). NO exerts its functions including acting as a neurotransmitter/neuromodulator (Pacher et al., 2007; Tegeder et al., 2011; Garthwaite, 2016) through two major mechanisms. One is the NO‐induced activation of http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=939 (sGC), thereby producing cGMP and activating http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=287 (Kots et al., 2009). Another mechanism is mediated through S‐nitrosylation, which is a posttranslational modification of protein cysteine residues that alters the functions of many target proteins (Jaffrey et al., 2001; Hess et al., 2005; Wolhuter and Eaton, 2017; Koriyama and Furukawa, 2018).

Based on these findings, we hypothesized that stimulation of brain nAChRs can activate the central adrenomedullary outflow via brain NO‐mediated mechanisms. In the present study, therefore, we examined (i) the involvement of brain NO/NOS isoforms and (ii) which NO‐mediated signalling, sGC/PKG and/or protein S‐nitrosylation, is involved, in the (±)‐epibatidine‐induced elevation of plasma catecholamines in rats.

Methods

Animals

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). All animal care and experimental procedures complied with the guiding principles for care and use of laboratory animals approved by Kochi University (#G‐5, H‐39), in accordance with the ‘Guidelines for proper conduct of animal experiments’ proposed by the Science Council of Japan; these guidelines conform to the standards of the ARRIVE and NIH. All efforts were made to minimize the suffering of the animals and the number of animals used to obtain reliable results. A total of 114 animals were used in the experiments described below. Eight‐week‐old male Wistar rats (Japan SLC Inc., Hamamatsu, Japan) weighing 200–250 g were housed, two per cage, and were maintained in an air‐conditioned room at 22–24°C under a 14/10 h light–dark cycle with lights on at 05:00 h and given food (laboratory chow, CE‐2; Clea Japan) and water ad libitum for more than 2 weeks. Upon reaching a body weight of 310–360 g, the rats were subjected to the following experiments. These rats were divided into 21 groups (16 for blood collection and five for immunohistochemical study) randomly and were used for experiments as described below. Because some groups previously reported that oestrogen can modulate responses to stress (Huang et al., 2008; Handa and Weiser, 2014), we could not exclude the possibility that oestrous cycle might affect brain nAChRs‐mediated activation of the sympatho‐adrenomedullary outflow, one of the responses to stress, in female rats. That is why we have investigated brain mechanisms for the nAChRs‐mediated activation only in male rats (Shimizu and Yokotani, 2009; Shimizu et al., 2011) and also used male rats in this study.

Experimental procedures for i.c.v. administration of drugs

In the morning (09:30–11:30 h), under urethane anaesthesia (1.0 g·kg−1, i.p.), the femoral vein was cannulated for saline infusion (1.2 mL·h−1) and the femoral artery was cannulated in order to collect blood samples. Subsequently, each rat was placed in the prone position in a stereotaxic apparatus for the brain (SR‐6R; Narishige, Tokyo, Japan) until the end of each experiment, as described previously (Shimizu et al., 2004). The skull was drilled for i.c.v. administration of drugs using a stainless‐steel cannula (outer diameter of 0.3 mm). The stereotaxic coordinates of the tip of the cannula were as follows (in mm): AP −0.8, L 1.5, V 4.0 (AP, anterior from the bregma; L, lateral from the midline; V, below the surface of the brain), according to the rat brain atlas (Paxinos and Watson, 2005). Three hours after the surgery, the steel cannula was inserted into the right lateral ventricle and each drug was administered as described below. The surgery and blood collections described below were performed under anaesthesia without recovery; therefore, we monitored sufficient levels of anaesthesia during experiments by confirming negative reflex responses to toe pinch every 30 min. If the level was insufficient, additional doses of urethane (0.05 g·kg−1 per injection, i.p.) were administered. In this study, urethane was administered to 111 rats of 114 rats, and the averaged total dose was 1.005 g·kg−1.

Drug administration

L‐NAME (a non‐selective inhibitor of NOS), carboxy‐PTIO (an NO scavenger), BYK191023 (a selective inhibitor of iNOS) and dithiothreitol (a thiol‐reducing reagent) were dissolved in 5 μL of sterile saline per animal, and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5113 (a selective inhibitor of nNOS) and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5234 (an inhibitor of sGC) were dissolved in 3 μL N,N‐dimethylformamide (DMF) per animal. Each reagent was i.c.v. administered using a cannula connected to a 10 μL Hamilton syringe, which was retained for 15 min to avoid the leakage of these reagents and then removed from the ventricle. Subsequently (±)‐epibatidine (an agonist of nAChRs) dissolved in DMF in a volume of 2.5 μL per animal was then i.c.v. administered into the ventricle using the cannula connected to a 10 μL Hamilton syringe, 30 min after application of L‐NAME, carboxy‐PTIO, BYK191023, 3‐bromo‐7‐nitroindazole or ODQ and 120 min after the application of dithiothreitol as this reagent induced a slight elevation in basal plasma levels of catecholamines. After the administration of (±)‐epibatidine, the cannula was retained until the end of the experiment. One hour after the (±)‐epibatidine administration, Cresyl Violet solution was injected through the cannula. Thereafter, the rats were decapitated under anaesthesia, and the brains were removed in order to confirm the exact location of the cannula inserted in the brain and to verify whether the solution had spread throughout the entire ventricular space. In the histochemical studies described below, confirmation of cannula location was performed by Nissl staining of brain frozen sections. Due to cannula misplacement, eight rats were excluded from the 114 rats used; therefore, the data were obtained from 106 rats.

Experimental groups for i.c.v. administration

The 75 rats placed in a stereotaxic apparatus were divided into 13 groups for 30 min pretreatment: (Figure 1), saline (5 μL per animal)‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 7), L‐NAME [0.37 μmol (100 μg) per animal]‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 5), L‐NAME [1.11 μmol (300 μg) per animal]‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 5); (Figure 2), saline (5 μL per animal)‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 5), carboxy‐PTIO [2.5 μmol (750 μg) per animal]‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 6); (Figure 3), saline (5 μL per animal)‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 6), BYK191023 [300 nmol (98.2 μg) per animal]‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 6), BYK191023 [1000 nmol (327 μg) per animal]‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 5); (Figure 4), DMF (3 μL per animal)‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 6), 3‐bromo‐7‐nitroindazole [300 nmol (72.6 μg) per animal]‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 6), 3‐bromo‐7‐nitroindazole [1000 nmol (242 μg) per animal]‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 5); (Figure 6), DMF (3 μL per animal)‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 8), ODQ [300 nmol (56.1 μg) per animal]‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 5).

Figure 1.

Figure 1

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Effect of L‐NAME on the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines. L‐NAME (a non‐selective inhibitor of NOS) (0.37 or 1.11 μmol per animal) or vehicle (V) (5 μL saline per animal) was i.c.v. administered 30 min before the administration of (±)‐epibatidine (Epi) (5 nmol per animal, i.c.v.). (A) Increments of plasma catecholamines (noradrenaline and adrenaline) above the basal level at each time point are expressed as pg·mL−1. ΔNoradrenaline and ΔAdrenaline: increments of noradrenaline and adrenaline above the basal level. Arrows indicate the administration of L‐NAME/V and Epi. (B) The AUC of the elevation of plasma catecholamines above the basal level for each group is expressed as pg·1 h−1. Each point represents the mean ± SEM. *P < 0.05, when compared, by use of the Bonferroni method, to the V‐ and Epi‐treated group. The number of animals per group is indicated in parentheses.

Figure 2.

Figure 2

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Effect of carboxy‐PTIO on the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines. Carboxy‐PTIO (PTIO) (an NO scavenger) (2.5 μmol per animal) or vehicle (V) (5 μL saline per animal) was i.c.v. administered 30 min before the administration of (±)‐epibatidine (Epi) (5 nmol per animal, i.c.v.). (A) Increments of plasma catecholamines above the basal level. Arrows indicate the administration of PTIO/V and Epi. (B) AUC of the elevation of plasma catecholamines above the basal level for each group. *P < 0.05, when compared, by use of Student's t‐test, to the V‐ and Epi‐treated group. The other conditions are the same as those of Figure 1.

Figure 3.

Figure 3

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Effect of BYK191023 on the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines. BYK191023 (BYK) (a selective inhibitor of iNOS) (300 or 1000 nmol per animal) or vehicle (V) (5 μL saline per animal) was i.c.v. administered 30 min before the administration of (±)‐epibatidine (Epi) (5 nmol per animal, i.c.v.). (A) Increment of plasma catecholamines above the basal level. Arrows indicate the administration of BYK/V and Epi. (B) AUC of the elevation of plasma catecholamines above the basal level for each group. *P < 0.05, when compared with the Bonferroni method to the V‐ and Epi‐treated group. The other conditions are the same as those of Figures 1 and 2.

Figure 4.

Figure 4

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Effect of 3‐bromo‐7‐nitroindazole on the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines. 3‐Bromo‐7‐nitroindazole (B‐NI) (a selective inhibitor of nNOS) (300 or 1000 nmol per animal) or vehicle (V) (3 μL DMF per animal) was i.c.v. administered 30 min before the administration of (±)‐epibatidine (Epi) (5 nmol per animal, i.c.v.). (A) Increment of plasma catecholamines above the basal level. Arrows indicate the administration of B‐NI/V and Epi. (B) AUC of the elevation of plasma catecholamines above the basal level for each group. *P < 0.05, when compared with the Bonferroni method to the V‐ and Epi‐treated group. The other conditions are the same as those of Figures 1, 2, 3.

Figure 6.

Figure 6

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Effect of ODQ on the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines. ODQ (an inhibitor of sGC) (300 nmol per animal) or vehicle (V) (3 μL DMF per animal) was i.c.v. administered 30 min before the administration of (±)‐epibatidine (Epi) (5 nmol per animal, i.c.v.). (A) Increment of plasma catecholamines above the basal level. Arrows indicate the administration of ODQ/V and Epi. (B) AUC of the elevation of plasma catecholamines above the basal level for each group. The other conditions are the same as those of Figures 1, 2, 3, 4.

The 16 rats placed in a stereotaxic apparatus were divided into three groups for 120 min pretreatment: (Figure 7), saline (5 μL per animal)‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 5); dithiothreitol [1.9 μmol (300 μg)·kg−1 body wt of animal and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 6); dithiothreitol [9.5 μmol (1500 μg) kg−1 body wt of animal]‐ and (±)‐epibatidine (5 nmol per animal)‐administered group (n = 5).

Figure 7.

Figure 7

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Effect of dithiothreitol (DTT) on the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines. DTT (a thiol‐reducing reagent) (1.9 and 9.5 μmol·kg−1 body wt of animal) or vehicle (V) (5 μL saline per animal) was i.c.v. administered 120 min before the administration of (±)‐epibatidine (Epi) (5 nmol per animal, i.c.v.). (A) Increment of plasma catecholamines above the basal level. Arrows indicate the administration of DTT/V and Epi. (B) AUC of the elevation of plasma catecholamines above the basal level for each group. *P < 0.05, when compared, by use of the Bonferroni method, to the V‐ and Epi‐treated group. The other conditions are the same as those of Figures 1, 2, 3, 4 and 6.

Measurement of plasma catecholamines

Blood samples (250 μL) were collected through the cannulated femoral artery at 0, 5, 10, 30 and 60 min after the administration of (±)‐epibatidine. The samples were preserved on ice during the experiments. Plasma was prepared immediately after the final sampling. http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=484 and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=509 were extracted from the plasma by the method of Anton and Sayre (1962) with a slight modification and were assayed electrochemically by HPLC (Shimizu et al., 2004).

Immunohistochemical study on the spinally projecting neurones of the hypothalamic PVN

For labelling presympathetic spinally projecting neurones in the hypothalamic PVN, a mono‐synaptic retrograde tracer Fast‐Blue (Polysciences, Warrington, PA, USA) was microinjected into the intermediolateral cell column (IML) of the thoracic spinal cord with a slight modification of previously reported methods (Shimizu et al., 2011, 2015; Tanaka et al., 2012, 2014). Briefly, under pentobarbital anaesthesia (50 mg·kg−1, i.p.), 15 rats were placed in a stereotaxic apparatus for the spinal cord (STS‐B; Narishige) until the end of surgery. The spinal cord was exposed by dorsal laminectomy through a back midline incision with an aseptic surgical procedure. Fast‐Blue (4% in sterile saline) was bilaterally microinjected into the IML (0.5 mm lateral from the midline and 1.0 mm below the surface of the spinal cord) at the T8 level in a volume of 200 nL on each side using a 33‐gauge stainless‐steel cannula (outer diameter of 0.2 mm) at a rate of 40 nL·min−1. Then, the muscle and skin were sutured. Afterwards penicillin G potassium (10 000 u·kg−1; Meiji Seika Pharma, Tokyo, Japan) was administered i.m., and the rats were returned to their homecages. The exact location of the spinal cord injection was verified by Nissl staining at the end of each experiment, as described below.

One week after the injection of Fast‐Blue, 15 rats were divided into five groups and 12 rats of four groups were placed in a stereotaxic apparatus for the brain, and the skull was drilled under urethane anaesthesia (1.0 g·kg−1, i.p.) as described above. Reagents were administered to these 12 rats of four groups as follows: DMF (2.5 μL per animal, i.c.v.)‐administered group (n = 3); (±)‐epibatidine (5 nmol per animal, i.c.v.)‐administered group (n = 3); saline (5 μL per animal, i.c.v.)‐ and (±)‐epibatidine (5 nmol per animal, i.c.v.)‐administered group (n = 3); BYK191023 (300 nmol per animal, i.c.v.)‐ and (±)‐epibatidine (5 nmol per animal, i.c.v.)‐administered group (n = 3). Saline or BYK191023 was injected as a pretreatment 30 min before the administration of (±)‐epibatidine as described above. One hour after the administration of (±)‐epibatidine or DMF, the rats were perfused through the left cardiac ventricle with 100 mL of 0.1 M PBS (pH 7.4) followed by 400 mL of ice‐cold 4% paraformaldehyde in 0.1 M phosphate buffer. The remaining three rats of one group were perfused similarly under pentobarbital anaesthesia (50 mg·kg−1, i.p.) without i.c.v. administration. Brains and spinal cords were immediately removed, post fixed in the same fixative overnight, equilibrated in 0.1 M phosphate buffer containing 20% sucrose at 4°C, coronally cut on a freezing cryostat (Cryostat HM505E, Thermo Scientific, Yokohama, Japan) at a thickness of 20 μm, and washed in 0.05 M Tris‐buffered saline (TBS; pH 7.4).

Immunohistochemical analysis was performed with a slight modification of previously reported methods (Shimizu et al., 2011, 2015; Tanaka et al., 2012, 2014). Free‐floating sections were incubated in a mixed diluent of a rabbit polyclonal antibody against http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=465 (1:1000; sc‐390 884, Santa Cruz Biotechnology, Santa Cruz, CA) (Qian et al., 2018), and a mouse monoclonal antibody against iNOS (1:50, sc‐7271, Santa Cruz Biotechnology) (Teitelbaum et al., 1999) or a mouse monoclonal antibody against S‐nitroso‐cysteine (1:1000; ab94930, Abcam, Cambridge, UK) (Bahnson et al., 2016) for 48 h at 4°C. After being washed in 0.05 M TBS, the sections were incubated in a mixed diluent of FITC‐conjugated donkey anti‐rabbit IgG (cat# 711‐095‐152) and Cy3‐conjugated donkey anti‐mouse IgG (cat# 715‐165‐150) antibodies (1:1000, respectively; Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h at room temperature in the dark, and washed in 0.05 M TBS again. All antisera were diluted in 0.05 M TBS containing 0.25% Triton X‐100 and 0.3% BSA. The sections were then mounted on silane‐coated slides and coverslipped with VECTASHIELD® mounting medium (H‐1000; Vector Laboratories, Burlingame, CA). Control experiments, which were performed by omitting primary antibodies as a test of cross‐reactivity of the secondary antibody, resulted in the absence of staining. Photographs from brain sections were captured using a digital camera (DP70; Olympus, Tokyo, Japan) attached to a fluorescent microscope (AX70; Olympus) with appropriate filter sets that allow the separate visualization of FITC (for nAChR α4 subunit), Cy3 (for iNOS or S‐nitroso‐cysteine) and UV excitation (for Fast‐Blue). Fast‐Blue was visible without the need of immunostaining under UV illumination. Representative photographs are shown as qualitative data in the present study.

The number of cells labelled with Fast‐Blue and the number of triple‐labelled cells (Fast‐Blue, nAChR α4 subunit and iNOS or S‐nitroso‐cysteine) were counted manually in the PVN bilaterally across two or three anatomically‐matched sections per animal. These quantitative data analyses were performed by investigators (M.Y. and S.S.) blinded to experimental conditions. Nuclei classification was identified according to the rat brain atlas (Paxinos and Watson, 2005).

Data analysis and statistics

Increments in plasma catecholamines above the basal level at each time point are expressed as pg·mL−1. The AUC is also expressed as pg·1 h−1. These calculations were performed by an investigator (S.S.) blinded to experimental conditions. The number of animals in each group is shown in all figures. All values are expressed as means ± SEM. The sample size in each experimental group for blood sampling was determined based on the expected difference in a desired endpoint measurement among the test and control groups, and the mean of the SDs for the groups observed in our previous studies (Shimizu and Yokotani, 2009; Shimizu et al., 2011). In the experiments consisting of 16 groups of rats, six rats per group were used at first for collection of blood samples. However, one or two more rats were added to the group pretreated with vehicle of L‐NAME (Figure 1) or ODQ (Figure 6), respectively, because of a high dispersion of values in each group. In addition, in eight out of the 16 groups, one rat per group was excluded due to cannula misplacement as described above. In the histochemical studies, the sample size in each experimental group was reduced to a minimum. Statistical differences were determined using repeated‐measure (treatment × time) or one‐way ANOVA, followed by post hoc analysis with the Bonferroni method. The post hoc analysis was performed when ANOVA achieved significance. When only two means were compared, Student's unpaired t‐test was used. P values less than 0.05 were taken to indicate statistical significance.

Materials

The following materials were used: (±)‐epibatidine dihydrochloride hydrate [(±)‐epibatidine] and L‐NAME (N ω‐nitro‐L‐arginine methyl ester) (Sigma Aldrich Fine Chemicals, St. Louis, MO, USA); carboxy‐PTIO [2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazole‐1‐oxyl‐3‐oxide, sodium salt] (Dojindo Molecular Technologies Inc., Kumamoto, Japan); BYK191023 [2‐[2‐(4‐methoxy‐2‐pyridinyl)ethyl]‐1H‐imidazo[4,5‐b]pyridine dihydrochloride] (Tocris Bioscience, Bristol, UK); 3‐bromo‐7‐nitroindazole and ODQ [1H‐(1,2,4)oxadiazolo(4,3‐a)quinoxalin‐1‐one] (Cayman Chemical, Ann Arbor, MI, USA); dithiothreitol (Wako, Osaka, Japan). All other reagents were of the highest grade available (Sigma Aldrich Fine Chemicals). The dosage of drugs was determined according to previous studies (Murakami et al., 1998; Lu et al., 2008; Pei et al., 2008; Shimizu and Yokotani, 2009; Tanaka et al., 2012) and our preliminary experiments.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a, 2017b).

Results

Central pretreatment with L‐NAME, a non‐selective NOS inhibitor, reduced the centrally administered (±)‐epibatidine‐induced elevation in plasma catecholamines

Since we previously reported that (±)‐epibatidine (1, 5 or 10 nmol per animal, i.c.v.) dose‐dependently elevated plasma levels of catecholamines (noradrenaline and adrenaline) in rats (Shimizu and Yokotani, 2009), we used a sub‐maximal dose of 5 nmol per animal in this study. Administration of (±)‐epibatidine (5 nmol per animal, i.c.v.) significantly elevated plasma levels of both catecholamines (adrenaline > noradrenaline) (Figure 1). Pretreatment with L‐NAME (0.37 and 1.11 μmol per animal, i.c.v.) dose‐dependently reduced the (±)‐epibatidine‐induced elevations (Figure 1). We had previously observed that centrally administered L‐NAME alone, at least at 1.11 μmol per animal, showed no effect on plasma levels of catecholamines (Lu et al., 2008). The actual values for noradrenaline and adrenaline at 0 min were 268 ± 43 and 142 ± 48 pg·mL−1 in the vehicle (5 μL saline per animal)‐pretreated group (n = 7), 393 ± 30 and 252 ± 43 pg·mL−1 in the L‐NAME (0.37 μmol per animal)‐pretreated group (n = 5), and 208 ± 31 and 161 ± 32 pg·mL−1 in the L‐NAME (1.11 μmol per animal)‐pretreated group (n = 5).

Central pretreatment with carboxy‐PTIO, an NO scavenger, reduced the centrally administered (±)‐epibatidine‐induced elevation in plasma catecholamines

In order to confirm whether NOS‐induced NO production contributes to the (±)‐epibatidine‐induced elevation of plasma catecholamines, we examined the effect of carboxy‐PTIO on the (±)‐epibatidine‐induced response. Pretreatment with carboxy‐PTIO (2.5 μmol per animal, i.c.v.) significantly reduced the (±)‐epibatidine (5 nmol per animal, i.c.v.)‐induced elevation (Figure 2). We had previously found that centrally administered carboxy‐PTIO alone, at least 2.5 μmol per animal, showed no effect on plasma levels of catecholamines (Tanaka et al., 2012). The actual values for noradrenaline and adrenaline at 0 min were 309 ± 46 and 181 ± 59 pg·mL−1 in the vehicle (5 μL saline per animal)‐pretreated group (n = 5), and 267 ± 13 and 84 ± 22 pg·mL−1 in the carboxy‐PTIO (2.5 μmol per animal)‐pretreated group (n = 6).

Centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines was reduced by BYK191023, a selective inhibitor of iNOS, but not by 3‐bromo‐7‐nitroindazole, a selective inhibitor of nNOS

We examined the involvement of two major brain NOS isozymes, iNOS and nNOS, in the (±)‐epibatidine‐induced elevation of plasma catecholamines by using BYK191023, a selective inhibitor of iNOS, and 3‐bromo‐7‐nitroindazole, a selective inhibitor of nNOS. In preliminary experiments, we confirmed that central administration of BYK191023 (1000 nmol per animal) or 3‐bromo‐7‐nitroindazole (1000 nmol per animal) alone did not affect plasma levels of catecholamines (data not shown). Pretreatment with BYK191023 (300 or 1000 nmol per animal, i.c.v.) significantly reduced the (±)‐epibatidine (5 nmol per animal, i.c.v.)‐induced elevation (Figure 3). On the other hand, 3‐bromo‐7‐nitroindazole (300 or 1000 nmol per animal, i.c.v.) showed no significant effect on the (±)‐epibatidine‐induced response (Figure 4). The actual values for noradrenaline and adrenaline at 0 min were 280 ± 48 and 157 ± 54 pg·mL−1 in the vehicle (5 μL saline per animal, i.c.v.)‐pretreated group (n = 6, Figure 3), 376 ± 60 and 162 ± 28 pg·mL−1 in the BYK191023 (300 nmol per animal)‐pretreated group (n = 6), 428 ± 8 and 281 ± 64 pg·mL−1 in the BYK191023 (1000 nmol per animal)‐pretreated group (n = 5), 181 ± 49 and 173 ± 27 pg·mL−1 in the vehicle (3 μL DMF per animal, i.c.v.)‐pretreated group (n = 6, Figure 4), 258 ± 47 and 110 ± 19 pg·mL−1 in the 3‐bromo‐7‐nitroindazole (300 nmol per animal)‐pretreated group (n = 6) and 350 ± 88 and 179 ± 69 pg·mL−1 in the 3‐bromo‐7‐nitroindazole (1000 nmol per animal)‐pretreated group (n = 5).

Immunohistochemical identification of nAChR α4 subunit and iNOS on the spinally projecting PVN neurones in the hypothalamus

In the rats microinjected with a retrograde tracer Fast‐Blue into the spinal cord, Fast‐Blue‐labelled neurones were abundantly observed in the dorsal cap and ventral part of the PVN (Figure 5A), in line with previous studies from this laboratory (Tanaka et al., 2012; Shimizu et al., 2015). However, these labelled neurones were not detected in other subnuclei such as the medial and lateral parts of the PVN (Figure 5A). Immunoreactivity of nAChR α4 subunit or iNOS was detected in the dorsal cap and ventral part of the PVN in rats untreated with drugs (Figure 5B). Triple‐labelled cells (nAChR α4 subunit, iNOS and Fast‐Blue) were observed in about 60–70% of Fast‐Blue‐labelled neurones in the dorsal cap and ventral part of the PVN (n = 3) (Figure 5C). The numbers of triple‐labelled cells were 7.3 ± 0.6 in the dorsal cap and 8.1 ± 1.5 in the ventral part, and those of nAChR α4 subunit‐ and iNOS‐positive and Fast‐Blue‐negative cells were 15.4 ± 3.5 in the dorsal cap and 17.0 ± 2.2 in the ventral part (n = 3).

Figure 5.

Figure 5

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Immunohistochemical identification of iNOS and nAChR α4 subunit on the spinally projecting hypothalamic PVN neurones. Rat brain samples were collected under pentobarbital anaesthesia (50 mg·kg−1, i.p.). (A) Left panel shows a schematic illustration of the PVN (−1.80 mm anterior from the bregma) based on the rat brain atlas of Paxinos and Watson (2005). Enlargement of the box in the scheme is shown in the right panel as a representative photograph of Fast‐Blue (FB)‐labelled neurones in the PVN. The FB‐labelled neurones were abundantly distributed in the PVN subdivisions dorsal cap (PaDC) and ventral part (PaV). (B) Photographs present the FB‐labelling (FB) and the immunoreactivity of nAChR α4 subunit (α4) and iNOS in the PaDC (upper) and the PaV (lower). Arrows indicate triple‐labelled neurones. Scale bar = 50 μm. (C) The number of FB‐labelled or triple‐labelled neurones (left side of the Y‐axis) and the ratio of triple‐labelled neurones to FB‐labelled neurones (right side of the Y‐axis). Values are expressed as mean ± SEM.

Central pretreatment with ODQ, a selective inhibitor of sGC, did not reduce the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines

We examined the contribution of the NO signalling pathway to the (±)‐epibatidine‐induced elevation of plasma catecholamines by using ODQ. Pretreatment with ODQ (300 nmol per animal, i.c.v.) showed no significant effect on the (±)‐epibatidine‐induced response (Figure 6). The actual values for noradrenaline and adrenaline at 0 min were 237 ± 46 and 134 ± 32 pg·mL−1 in the vehicle (3 μL DMF per animal, i.c.v.)‐pretreated group (n = 8), and 377 ± 47 and 113 ± 76 pg·mL−1 in the ODQ (300 nmol per animal)‐pretreated group (n = 5).

Central pretreatment with dithiothreitol, a thiol‐reducing reagent, reduced the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines

Dithiothreitol can reduce oxidized cysteine, thereby reducing S‐nitrosylated proteins. Therefore, we examined the effect of the reagent on the (±)‐epibatidine‐induced elevation of plasma catecholamines in order to clarify the involvement of the post‐translational modification in the (±)‐epibatidine‐induced response. Pretreatment with dithiothreitol (1.9 and 9.5 μmol·kg−1 body wt of animal, i.c.v.) dose‐dependently reduced the (±)‐epibatidine (5 nmol per animal, i.c.v.)‐induced elevation (Figure 7). The actual values for noradrenaline and adrenaline at 0 min were 201 ± 50 and 182 ± 73 pg·mL−1 in the vehicle (5 μL saline per animal)‐pretreated group (n = 5), 198 ± 46 and 154 ± 33 pg·mL−1 in the dithiothreitol (1.9 μmol·kg−1 body wt of animal)‐pretreated group (n = 6) and 304 ± 24 and 99 ± 9 pg·mL−1 in the dithiothreitol (9.5 μmol·kg−1)‐pretreated group (n = 5).

Immunohistochemical identification of iNOS‐mediated S‐nitroso‐cysteine in nAChR α4 subunit‐positive spinally projecting PVN neurones in the hypothalamus after (±)‐epibatidine administration

In the ventral part of the PVN, the Fast‐Blue‐labelled neurones with nAChR α4 subunit‐immunoreactivity were abundantly observed in both vehicle‐1 (2.5 μL DMF per animal, i.c.v.)‐ and (±)‐epibatidine (5 nmol per animal, i.c.v.)‐administered groups (Figure 8A). These observations were also detected in the dorsal cap of the PVN (data not shown). Fast‐Blue‐labelled neurones with nAChR α4 subunit‐ and S‐nitroso‐cysteine‐immunoreactivities were detected in the (±)‐epibatidine‐administered group compared with the vehicle‐1‐administered group in the dorsal cap and ventral part of the PVN (Figure 8A and C). The percentage of triple‐labelled cells (nAChR α4 subunit, S‐nitroso‐cysteine and Fast‐Blue) in Fast‐Blue‐labelled neurones was 14.3 ± 3.6% in the dorsal cap of the PVN and 22.3 ± 4.2% in the ventral part of the PVN in the vehicle‐1‐administered group (n = 3), and 83.0 ± 5.3% and 81.4 ± 3.8% in the (±)‐epibatidine‐administered group (n = 3). In the (±)‐epibatidine‐administered group (n = 3), the numbers of triple‐labelled cells were 7.9 ± 0.9 in the dorsal cap and 12.5 ± 1.2 in the ventral part, and those of nAChR α4 subunit‐ and S‐nitroso‐cysteine‐positive and Fast‐Blue‐negative cells were 5.9 ± 1.5 in the dorsal cap and 3.0 ± 2.1 in the ventral part. In addition, triple‐labelled neurones with Fast‐Blue, nAChR α4 subunit and S‐nitroso‐cysteine were detected in animals treated with vehicle‐2 (5 μL saline per animal, i.c.v.) and (±)‐epibatidine (5 nmol per animal, i.c.v.), while these neurons were almost undetectable in animals pretreated with BYK191023 (300 nmol per animal, i.c.v.), a selective inhibitor of iNOS, in the dorsal cap and ventral part of the PVN (Figure 8B and D). The percentage of triple‐labelled cells (nAChR α4 subunit, S‐nitroso‐cysteine and Fast‐Blue) in Fast‐Blue‐labelled neurones was 71.2 ± 6.0% in the dorsal cap of the PVN and 74.0 ± 2.8% in the ventral part of the PVN in the vehicle‐2‐ and (±)‐epibatidine‐administered group (n = 3), and 20.6 ± 4.8% and 30.6 ± 5.4% in the BYK191023‐ and (±)‐epibatidine‐administered group (n = 3).

Figure 8.

Figure 8

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Immunohistochemical identification of iNOS‐dependent cysteine S‐nitrosylation in the spinally projecting and nAChR α4 subunit expressing neurones in the hypothalamic PVN after (±)‐epibatidine administration. Rat brain samples were collected under urethane anaesthesia (1.0 g·kg−1, i.p.). (A) Photographs present the Fast‐Blue (FB)‐labelling (FB) and the immunoreactivity of nAChR α4 subunit (α4) and S‐nitrosylated cysteine (SNO‐Cys) in the PVN ventral part of the vehicle‐1 (upper, V‐1; 2.5 μL DMF per animal, i.c.v.)‐ or (±)‐epibatidine (lower, Epi; 5 nmol per animal, i.c.v.)‐administered animals. (B) Photographs present the FB‐labelling (FB) and the immunoreactivity of α4 and SNO‐Cys in the PVN ventral part of animals pretreated with vehicle‐2 (upper, V‐2; 5 μL saline per animal, i.c.v.) or BYK191023 (a selective inhibitor of iNOS) (lower, BYK; 300 nmol per animal, i.c.v.) 30 min before the administration of Epi (5 nmol per animal, i.c.v.). Arrows indicate triple‐labelled neurones. Scale bar = 50 μm. (C and D) The number of FB‐labelled or triple‐labelled neurones in the dorsal cap and ventral part of the PVN (PaDC and PaV). Values are expressed as mean ± SEM.

Discussion

The present study demonstrated the following: (i) i.c.v.‐administered (±)‐epibatidine‐induced elevation of plasma catecholamines was mediated by iNOS‐derived NO production as demonstrated by carboxy‐PTIO, L‐NAME or BYK191023‐induced inhibition of the elevation; (ii) iNOS was co‐localized with nAChR α4 subunit in hypothalamic PVN neurones projecting to the spinal cord; (iii) NO‐mediated protein S‐nitrosylation, but not activation of sGC, in the brain can be involved in the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines as evidenced by dithiothreitol‐, but not ODQ, induced inhibition of the elevation; (iv) centrally administered (±)‐epibatidine induced immunoreactivity of S‐nitroso‐cysteine in nAChR α4 subunit‐positive spinally projecting neurones in the PVN with a BYK191023‐sensitive manner. Considering that plasma catecholamines elevated by (±)‐epibatidine are mainly derived from the rat adrenal medulla (Shimizu and Yokotani, 2009), the present data suggest that brain NO produced by iNOS may be involved in centrally administered (±)‐epibatidine‐induced activation of central adrenomedullary outflow through protein S‐nitrosylation in spinally projecting PVN neurones in rats.

In this study, we used a non‐selective nAChR agonist (±)‐epibatidine (Lembeck, 1999). We previously reported that centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines was supressed by central pretreatment with dihydro‐β‐erythroidine (a selective antagonist of α4β2 nAChRs) (Whiteaker et al., 1998), but not with methyllycaconitine (a selective antagonists of α7 nAChRs) (Davies et al., 1999), in rats (Shimizu et al., 2011), indicating that at lease brain α4β2, but not α7, nAChRs are involved in the (±)‐epibatidine‐induced response. In addition, we also reported that centrally administered RJR‐2403, a selective agonist of α4β2 nAChRs (Bencherif et al., 1996), at 5 μmol per animal elevated plasma catecholamines, while centrally administered (±)‐epibatidine induced similar elevation at much lower doses, 5 or 10 nmol per animal (Shimizu et al., 2011). In rat brain membranes, (±)‐epibatidine inhibits [3H]‐nicotine binding with a Ki value of 0.02 nM (Sharples et al., 2000), while RJR‐2403 inhibits [3H]‐nicotine binding with a Ki value of 26 nM (Bencherif et al., 1996). These findings indicate that (±)‐epibatidine is more effective than RJR‐2403 in the brain α4β2 nAChRs‐mediated elevation of plasma catecholamines. That is why we chose (±)‐epibatidine instead of the selective agonist RJR‐2403 in this study.

There are some reports showing a functional relationship between nAChRs and NO signalling in the brain. In the mouse cerebellum, nAChRs have a role in the cross‐tolerance between nicotine and ethanol‐induced ataxia through the NO/cGMP signalling pathway (Taslim et al., 2011). In the rat brainstem nucleus tractus solitarius, stimulation of nAChRs induces noradrenaline release in the hypothalamic PVN through NO production (Zhao et al., 2007) and decreases blood pressure through eNOS (Cheng et al., 2011). To our knowledge, however, there have been no reports demonstrating the involvement of NO in central activation of sympatho‐adrenomedullary outflow in response to stimulation of brain nAChRs by (±)‐epibatidine. In the present study, centrally pretreated L‐NAME, a representative and non‐selective inhibitor of NOS (Griffith and Kilbourn, 1996), and carboxy‐PTIO, scavenging NO without affecting NOS activity (Maeda et al., 1994), suppressed the (±)‐epibatidine‐induced elevation of plasma catecholamines in rats. These results indicate that endogenously produced brain NO is involved in the (±)‐epibatidine‐induced elevation of plasma catecholamines, in line with our previous reports showing that centrally administered L‐NAME and carboxy‐PTIO inhibited the elevation of plasma catecholamines induced by centrally administered neuropeptide bombesin in rats (Lu et al., 2008; Tanaka et al., 2012). These previous reports support the involvement of brain NO in elevation of plasma catecholamines.

Next, we attempted to characterize which NOS isoform is involved in the (±)‐epibatidine‐induced elevation of plasma catecholamines. In the present study, due to the lack of highly selective inhibitors of eNOS, we used a highly selective iNOS inhibitor BYK191023 and a selective nNOS inhibitor 3‐bromo‐7‐nitroindazole. BYK191023 exhibits IC50 values of 86, 17 000 and 162 000 nM for iNOS, nNOS and eNOS respectively (Strub et al., 2006). On the other hand, 3‐bromo‐7‐nitroindazole exhibits IC50 values of 0.29, 0.17 and 0.86 μM for iNOS, nNOS and eNOS respectively (Bland‐Ward and Moore, 1995). Central pretreatment with BYK191023, but not 3‐bromo‐7‐nitroindazole, effectively reduced the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines. These results suggest that at least iNOS‐mediated NO production in the brain can be involved in the (±)‐epibatidine‐induced elevation of plasma catecholamines.

The hypothalamic PVN has been recognized as a control centre of the sympatho‐adrenomedullary system (Pyner, 2009). PVN is one of the primary brain regions containing NOS‐positive neurones (Patel et al., 2001), which has been shown to play an important role in the regulation of sympatho‐adrenomedullary outflow in rodent models of diseases such as hypertension and heart failure (Zheng et al., 2011; Zhou et al., 2014). NOS‐positive neurones in the PVN have been also reported to regulate the outflow in response to centrally administered stress‐related neuropeptides such as corticotropin‐releasing factor (CRF) and bombesin (Yamaguchi et al., 2009; Tanaka et al., 2014) and to resistant stress exposure (Yamaguchi et al., 2010). However, it has been recognized that these NO‐mediated regulations of the sympatho‐adrenomedullary outflow are dependent on NOS isoforms. In a rodent model of insulin resistance, microinjection of selective inhibitors of nNOS and eNOS, but not iNOS, into the PVN significantly increased basal sympathetic nerve activity (Lu et al., 2015). Furthermore, it has been also reported that overexpression of nNOS in the PVN alleviated the enhancement in renal sympathetic nerve activity in hypertensive rats (Zheng et al., 2011). In contrast, an iNOS inhibitor attenuated centrally administered CRF‐induced elevation of plasma catecholamines (Okada et al., 2003). In addition, iNOS expressing in spinally projecting PVN neurones induced sympathetic activation in response to resistant stress exposure (Yamaguchi et al., 2010). In the present study, we microinjected Fast‐Blue, a mono‐synaptic retrograde tracer, into the rat spinal cord at the T8 level, in which sympathetic preganglionic neurones innervating the adrenal medulla are mainly located (Pyner and Coote, 1994, 2000; Shafton et al., 1998). In Fast‐Blue‐labelled spinally projecting PVN neurones expressing nAChR α4 subunit and iNOS were apparently detected. About one third of nAChR α4 subunit‐ and iNOS‐expressing cells were labelled with Fast‐Blue in the PVN, indicating that some parts of these double‐expressing PVN neurons are spinally projecting. Taken together with our pharmacological data obtained from BYK191023‐ or 3‐bromo‐7‐nitroindazole‐pretreated rats, iNOS in the spinally projecting PVN neurones may participate in elevation of plasma catecholamines in response to (±)‐epibatidine.

NO binds to the haem group of sGC, thereby producing cGMP, which in turn activates PKG (Kots et al., 2009). ODQ has been shown to be highly selective haem‐site inhibitor of sGC (Schrammel et al., 1996). Centrally administered ODQ at 1.3 nmol per animal induced larger decrease in arginine‐vasopressin and oxytocin gene expression in the rat hypothalamic PVN during sepsis, which induces massive production of NO (Oliveira‐Pelegrin et al., 2010). In the present study, however, centrally administered ODQ even at a higher dose (300 nmol per rat) had no effect on the (±)‐epibatidine‐induced elevation of plasma catecholamines, indicating the involvement of mechanisms other than a sGC‐dependent one in the (±)‐epibatidine‐induced elevation of plasma catecholamines.

S‐nitrosylation is the covalent modification of protein cysteine thiol groups by NO to generate an S‐nitrosothiol (Jaffrey et al., 2001; Hess et al., 2005; Wolhuter and Eaton, 2017; Koriyama and Furukawa, 2018). This NO‐mediated posttranslational modification modifies the function of many target proteins including receptors (Choi et al., 2000), transcription factors (Yasinska and Sumbayev, 2003), enzymes (Numajiri et al., 2011) and ion channels (Kawano et al., 2009). Dithiothreitol is a reducer of oxidized sulfhydryl groups and is well known to reduce oxidized cysteine, thereby reducing S‐nitrosylated proteins. In fact, some groups have been using this reagent as an ‘inhibitor of protein S‐nitrosylation’ in vitro (Zhang et al., 2008) and in vivo (Pei et al., 2008) studies. And we previously confirmed that central pretreatment with dithiothreitol supressed centrally administered SIN‐1 (an NO donor)‐induced elevation of plasma catecholamines (Tanaka et al., 2012). In the present study, central pretreatment with dithiothreitol effectively reduced the centrally administered (±)‐epibatidine‐induced elevation of plasma catecholamines. In addition, in rats labelled of pre‐sympathetic neurones in the hypothalamic PVN with Fast‐Blue, centrally administered (±)‐epibatidine obviously induced immunoreactivity of S‐nitroso‐cysteine in these labelled neurones expressing nAChR α4 subunit. In response to (±)‐epibatidine, about a half of nAChR α4 subunit‐ and S‐nitroso‐cysteine‐positive cells in the dorsolateral part of the PVN and about 80% of these double‐positive cells in the ventral part of the PVN were labelled with Fast‐Blue, indicating that (±)‐epibatidine can induce protein S‐nitrosylation in most of nAChR α4 subunit‐expressing and spinally projecting PVN neurons. Furthermore, the induction of these triple‐labelled neurones (S‐nitroso‐cysteine, nAChR α4 subunit and Fast‐Blue) in response to (±)‐epibatidine was suppressed by central pretreatment with BYK191023, an inhibitor of iNOS. These findings suggest that protein S‐nitrosylation induced by brain iNOS‐derived NO might participate in elevation of plasma catecholamines in response to (±)‐epibatidine. In line with our previous report, centrally pretreated dithiothreitol inhibited the elevation of plasma catecholamines induced by centrally administered bombesin, which induced immunoreactivity of S‐nitroso‐cysteine in spinally projecting PVN neurones in rats (Tanaka et al., 2012), supporting the involvement of protein S‐nitrosylation in the brain, at least in the PVN, in elevation of plasma catecholamines. On the other hand, a limitation of this study is that it is unclear which protein is S‐nitrosylated in the brain during the (±)‐epibatidine‐induced elevation of plasma catecholamines and how the modified protein by NO regulates the outflow. Further studies are therefore needed to identify these target proteins in pre‐sympathetic neurones of the PVN during elevation of plasma catecholamines induced by stimulation of nAChRs in the PVN.

The advantage of the protein S‐nitrosylation has been reported that NO can be stored and transported long distance in its nitrosylated form (Hess et al., 2005; Wolhuter and Eaton, 2017) and that the modification is reversal (S‐nitrosylation and de‐nitrosylation) although binding of NO to cysteine occurs in a covalent way (Hess et al., 2005; Benhar et al., 2008). Therefore, it is possible that the protein S‐nitrosylation in local affects functional proteins in widespread brain areas and the reversibility allows NO to modulate these proteins depending on the redox state. Some groups reported that oxidative stress in the brain can induce activation of the sympatho‐adrenomedullary outflow (Hirooka, 2008; Su et al., 2014), and we showed a possibility that the activation can be induced by protein S‐nitrosylation in the pre‐sympathetic neurons in the hypothalamic PVN. Thus, the reversal protein S‐nitrosylation depending on the redox state in the brain might be a key regulator of the central sympatho‐adrenomedullary outflow.

In summary, we demonstrated that brain iNOS‐derived NO‐mediated protein S‐nitrosylation is probably involved in (±)‐epibatidine‐induced activation of central adrenomedullary outflow in rats. Because cigarette dependence is reported as a risk factor for cardiovascular disease including hypertension (McBride, 1992; Piano et al., 2010), the findings of our present study indicate that central nAChRs can be a novel therapeutic target for nicotine dependence‐mediated hypertension. However, in clinical studies with varenicline, a partial agonist of α4β2 nAChRs and an effective drug for smoking cessation, hypertension was reported as a side effect in some smokers (Rigotti et al., 2010; Ware et al., 2013). Therefore, further studies are needed to investigate the role of chronic stimulation of central nAChRs in the regulation of blood pressure and to compare which strategy is better for treating smoking‐mediated hypertension, central nAChRs themselves or signalling molecules downstream of central nAChRs including NO and S‐nitrosylated proteins in the brain.

Author contributions

Y.H. and T.S. created this research design. Y.H., T.S., M.Y., T.Y. and S.Z. performed animal experiments and the assays of plasma catecholamines. K.T. performed histological experiments. Y.H, T.S., M.Y., T.Y. and S.S. analysed data. Y.H. and T.S. interpreted results of experiments. Y.H., T.S., S.S., T.U., K.Y. and M.S. drafted the manuscript. Y.H., T.S., M.Y., K.T., T.Y., S.S., S.Z., T.U., K.Y. and M.S. approved final version of manuscript.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This http://onlinelibrary.wiley.com/doi/10.1111/bph.13405/abstract acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgements

The authors would like to thank K. Nakamura for technical assistance with the experiments. This work was supported in part by a grant from The Smoking Research Foundation in Japan, a Grant‐in‐Aid for Scientific Research (C) (no. 26460909 to T.S. and 17K09303 to T.S.) from the Japan Society for the Promotion of Science, a grant from The Japan Health Foundation and a grant from Narishige Neuroscience Research Foundation in Japan.

Higashi, Y. , Shimizu, T. , Yamamoto, M. , Tanaka, K. , Yawata, T. , Shimizu, S. , Zou, S. , Ueba, T. , Yuri, K. , and Saito, M. (2018) Stimulation of brain nicotinic acetylcholine receptors activates adrenomedullary outflow via brain inducible NO synthase‐mediated S‐nitrosylation. British Journal of Pharmacology, 175: 3758–3772. 10.1111/bph.14445.

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