Vitamins and hormones, volume 96

Cover photo credit:
Girouard H., and Tabatabaei S.N.
Nitric Oxide and Cerebrovascular Regulation
Vitamins and Hormones (2014) 96, pp. 347–386
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CONTRIBUTORS
Ashok Aiyar

Stanley S. Scott Cancer Center, Louisiana State University, Health Sciences Center, New
Orleans, Louisiana, USA
Alexis Bavencoffe
Center for Neuroscience and Pain Research, Department of Anesthesiology and
Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston,
Texas, USA
Karin C. Calaza
Programa de Neurocieˆncias, and Departamento de Neurobiologia, Instituto de Biologia,
Universidade Federal Fluminense, Nitero´i, RJ, Brazil
Paula Campello-Costa
Programa de Neurocieˆncias, and Departamento de Neurobiologia, Instituto de Biologia,
Universidade Federal Fluminense, Nitero´i, RJ, Brazil
Shao-Rui Chen
Center for Neuroscience and Pain Research, Department of Anesthesiology and
Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston,
Texas, USA
Angela Cheung
Division of Developmental Neurobiology, MRC National Institute for Medical Research,
Mill Hill, London, United Kingdom
Marcelo Cossenza
Programa de Neurocieˆncias, Instituto de Biologia, Nitero´i, RJ, and Departamento de
Fisiologia e Farmacologia, Instituto Biome´dico, Universidade Federal Fluminense,
Rio de Janeiro, Brazil
Ivan C.L. Domith
Programa de Neurocieˆncias, Instituto de Biologia, Universidade Federal Fluminense,
Nitero´i, RJ, Brazil
Thaı´sa G. Encarnac¸a˜o
Programa de Neurocieˆncias, Instituto de Biologia, Universidade Federal Fluminense,
Nitero´i, RJ, Brazil
David A. Geller

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
He´le`ne Girouard
Department of Pharmacology, Faculty of Medicine, Universite´ de Montre´al, and Research
Center of the Institut Universitaire de Ge´riatrie de Montre´al, Montreal, Quebec, Canada

xiii


xiv

Contributors

Luis F.H. Gladulich
Programa de Neurocieˆncias, Instituto de Biologia, Universidade Federal Fluminense,
Nitero´i, RJ, Brazil
William P. Gray
Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff,
United Kingdom
Zhong Guo
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
Bradford G. Hill
Diabetes and Obesity Center, Institute of Molecular Cardiology; Department of Physiology
and Biophysics, and Department of Biochemistry and Molecular Biology, University of
Louisville School of Medicine, Louisville, Kentucky, USA
Michael A. Hough
School of Biological Sciences, University of Essex, Colchester, United Kingdom
Yao Hu
Institute for Stem Cells and Neural Regeneration, School of Pharmacy, Nanjing Medical
University, Nanjing, China
H.S. Jeffrey Man

Institute of Medical Science; Li Ka Shing Knowledge Institute, St. Michael’s Hospital, and
Divisions of Respirology and Critical Care Medicine, Department of Medicine, University
of Toronto, Toronto, Ontario, Canada
Jisha Joshua
Department of Medicine, University of California, San Diego, California, USA
Hema Kalyanaraman
Department of Medicine, University of California, San Diego, California, USA
Sangwon F. Kim
Department of Psychiatry and Pharmacology, Center for Neurobiology and Behavior, The
Perelman School of Medicine at University of Pennsylvania, Philadelphia, Pennsylvania,
USA
Peter Kruzliak
Department of Cardiovascular Diseases, International Clinical Research Center, St. Anne’s
University Hospital, Brno, Czech Republic
Nisha Marathe
Department of Medicine, University of California, San Diego, California, USA
Philip A. Marsden
Institute of Medical Science; Li Ka Shing Knowledge Institute, St. Michael’s Hospital, and
Division of Nephrology, Department of Medicine, University of Toronto, Toronto,
Ontario, Canada
Junko Maruyama
Department of Anesthesiology and Critical Care Medicine, Mie University School of
Medicine, and Department of Clinical Engineering, Suzuka University of Medical Science,
Mie, Japan


Contributors

xv

Kazuo Maruyama
Department of Anesthesiology and Critical Care Medicine, Mie University School of
Medicine, and Department of Clinical Engineering, Suzuka University of Medical Science,
Mie, Japan
Henrique R. Mendonc¸a
Programa de Neurocieˆncias, Instituto de Biologia, Universidade Federal Fluminense,
Nitero´i, RJ, Brazil
Roberto Paes-de-Carvalho
Programa de Neurocieˆncias, and Departamento de Neurobiologia, Instituto de Biologia,
Universidade Federal Fluminense, Nitero´i, RJ, Brazil
Hui-Lin Pan
Center for Neuroscience and Pain Research, Department of Anesthesiology and
Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston,
Texas, USA
Renate B. Pilz
Department of Medicine, University of California, San Diego, California, USA
Camila C. Portugal
Programa de Neurocieˆncias, Instituto de Biologia, Universidade Federal Fluminense,
Nitero´i, RJ, Brazil
Brian E. Sansbury
Diabetes and Obesity Center, Institute of Molecular Cardiology, and Department of
Physiology and Biophysics, Louisville, Kentucky, USA
Gary Silkstone
School of Biological Sciences, University of Essex, Colchester, United Kingdom
Renato Socodato
Programa de Neurocieˆncias, Instituto de Biologia, Universidade Federal Fluminense,
Nitero´i, RJ, Brazil
Seyed Nasrollah Tabatabaei
Department of Computer and Software Engineering and Institute of Biomedical
 cole Polytechnique de Montre´al

Engineering, E
David Tate
Stanley S. Scott Cancer Center, Louisiana State University, Health Sciences Center, New
Orleans, Louisiana, USA
Douglas D. Thomas
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,
Chicago, Illinois, USA
Albert K.Y. Tsui
Li Ka Shing Knowledge Institute, and Department of Anesthesia, St. Michael’s Hospital,
Toronto, Ontario, Canada
Laura B. Valdez
Institute of Biochemistry and Molecular Medicine (IBIMOL), Physical Chemistry Division,
School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina


xvi

Contributors

Divya Vasudevan
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,
Chicago, Illinois, USA
Cecilia Vecoli
Institute of Clinical Physiology-CNR, Pisa, Italy
Michael T. Wilson
School of Biological Sciences, University of Essex, Colchester, United Kingdom
Jonathan Worrall
School of Biological Sciences, University of Essex, Colchester, United Kingdom
Tamara Zaobornyj
Institute of Biochemistry and Molecular Medicine (IBIMOL), Physical Chemistry Division,

School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
Arnold H. Zea
Stanley S. Scott Cancer Center, Louisiana State University, Health Sciences Center, New
Orleans, Louisiana, USA
Dong-Ya Zhu
Institute for Stem Cells and Neural Regeneration, and Department of Pharmacology, School
of Pharmacy, Nanjing Medical University, Nanjing, China


PREFACE
Nitric oxide (nitrogen monoxide, NO), a gaseous molecule, is derived from
the amino acid, L-arginine, in the human body. Its discovery was a great
surprise because other hormones and regulators in the body are made up
of proteins, lipid-derived compounds, and other molecules, none of which
are gaseous. NO affects a number of enzyme systems and binds to heme, a
cofactor in some important enzymes. In many ways, NO acts as a hormone
and regulates many processes. Its activities are generally beneficial. For
example, it is a powerful generator of vasodilation by suppressing vascular
smooth muscle contraction. Its action is rapid as it remains in the blood
for only seconds. In other activities, NO inhibits platelet aggregation and
the adhesion of leukocytes to endothelia. Poorly functioning pathways
involving NO are hallmarks in patients with various diseases, such as diabetes, atherosclerosis, and hypertension. Consequently, nitric oxide becomes a
substance of interest in therapeutic applications. Many of the properties and
actions of this new regulator, nitric oxide, are reviewed in this volume.
Reviews in this book have been ordered by first introducing the basic
aspects of nitric oxide followed by chapters that involve clinical concepts.
Accordingly, the first chapter is on the “Regulation of nociceptive transduction and transmission by nitric oxide” by A. Bavencoffe, S.-R. Chen, and
H.-L. Pan. The next offering is by Z. Guo and D.A. Geller entitled
“microRNA and human inducible nitric oxide synthase.” “Heart
mitochondrial nitric oxide synthase: a strategic enzyme in the regulation

of cellular bioenergetics” is authored by T. Zaobornyj and L.B. Valdez.
W.P. Gray and A. Cheung review “Nitric oxide regulation of adult
neurogenesis.” “Nitric oxide in the nervous system: biochemical, developmental, and neurobiological aspects” is the work of M. Cossenza, R.
Socodato, C.C. Portugal, I.C.L. Domith, L.F.H. Gladulich, T.G.
Encarnac¸a˜o, K.C. Kalaza, H.R. Mendonc¸a, P. Campello-Costa, and
R. Paes-de-Carvalho. Y. Hu and D.-Y. Zhu report on “Hippocampus
and nitric oxide.” “Nitric oxide and hypoxia signaling” is a review by
H.S.J. Man, A.K.Y. Tsui, and P.A. Marsden. M.A. Hough, G. Silkstone,
J. Worrall, and M.T. Wilson discuss “NO binding to the proapoptotic
cytochrome c–cardiolipin complex.” S.F. Kim writes on “The nitric
oxide-mediated regulation of prostaglandin signaling in medicine.”

xvii


xviii

Preface

“Nitric oxide as a mediator of estrogen effects in osteocytes” is the topic of
J. Joshua, H. Kalyanaraman, N. Marathe, and R.B. Pilz.
In aspects related to disease conditions, contributions begin with
“Insights into the diverse effects of nitric oxide on tumor biology” by D.
Vasudevan and D.D. Thomas. A.H. Zea, A. Aiyar, and D. Tate review
“Dual effect of interferon (IFNγ)-induced nitric oxide on tumorigenesis
and intracellular bacteria.” Next, B.E. Sansbury and B.G. Hill cover the
“Anti-obesogenic role of endothelial nitric oxide synthase.” H. Girouard
and S.N. Tabatabaei report on “Nitric oxide and cerebrovascular
regulation.” Then, C. Vecoli reports on “Endothelial nitric oxide synthase
gene polymorphisms in cardiovascular disease.” The “Role of nitric oxide in

pathophysiology and treatment of pulmonary hypertension” by P. Kruzliak,
J. Maruyama, and K. Maruyama is the final chapter.
The cover illustration is Fig. 14.2 of Chapter 14.
Helene Kabes and Mary Ann Zimmerman of Elsevier, Oxford, UK were
instrumental in the processing of these chapters.
GERALD LITWACK
North Hollywood, California
May 8, 2014


CHAPTER ONE

Regulation of Nociceptive
Transduction and Transmission
by Nitric Oxide
Alexis Bavencoffe, Shao-Rui Chen, Hui-Lin Pan1
Center for Neuroscience and Pain Research, Department of Anesthesiology and Perioperative Medicine,
The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
1
Corresponding author: e-mail address:

Contents
1. Introduction
2. Role of NO in Nociceptive Transduction at the Periphery
3. Diverse Effects of NO on Ion Channels Expressed on Primary Sensory Neurons
3.1 Acid-sensing ion channels
3.2 Transient receptor potential channels
3.3 KATP channels
4. Role of NO in Regulating Nociceptive Transmission at the Spinal Cord Level
5. NO Reduces Excitatory, But Potentiates Inhibitory, Synaptic Transmission

in Spinal Cords
5.1 Glutamatergic input from primary afferent nerves
5.2 Voltage-activated calcium channels in sensory neurons
5.3 Synaptic NMDA receptors
5.4 Synaptic release of glycine
6. Conclusions and Future Directions
Acknowledgments
References

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Abstract
The potential involvement of nitric oxide (NO), a diffusible gaseous signaling messenger,
in nociceptive transduction and transmission has been extensively investigated. However, there is no consistent and convincing evidence supporting the pronociceptive
action of NO at the physiological concentration, and the discrepancies are possibly
due to the nonspecificity of nitric oxide synthase inhibitors and different concentrations

of NO donors used in various studies. At the spinal cord level, NO predominantly
reduces synaptic transmission by inhibiting the activity of NMDA receptors and glutamate release from primary afferent terminals through S-nitrosylation of voltageactivated calcium channels. NO also promotes synaptic glycine release from inhibitory

Vitamins and Hormones, Volume 96
ISSN 0083-6729
/>
#

2014 Elsevier Inc.
All rights reserved.

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Alexis Bavencoffe et al.

interneurons through the cyclic guanosine monophosphate/protein kinase G signaling
pathway. Thus, NO probably functions as a negative feedback regulator to reduce nociceptive transmission in the spinal dorsal horn during painful conditions.

1. INTRODUCTION
Pain receptors, also called nociceptors, are a group of sensory neurons
with specialized nerve endings widely distributed in the skin, deep tissues
(including the muscles and joints), and most of visceral organs. They respond
to tissue injury or potentially damaging stimuli by sending nerve signals to
the spinal cord and brain to begin the process of pain sensation. Nociceptors
are equipped with specific molecular sensors, which detect extreme heat or
cold and certain harmful chemicals. Mechanical nociceptors can also
respond to tissue-damaging stimuli, such as pinching the skin or overstretching the muscles. Activation of nociceptors generates action potentials,

which are propagated along the afferent nerve axons, especially unmyelinated C-fibers and thinly myelinated Aδ-fibers. At the spinal cord level, the
nociceptive nerve terminals release excitatory neurotransmitters to activate
their respective postsynaptic receptors on second-order neurons. In the spinal dorsal horn, both excitatory and inhibitory interneurons can augment or
attenuate nociceptive transmission (Cervero & Iggo, 1980; Zhou, Zhang,
Chen, & Pan, 2007, 2008). The nociceptive signal, encoding the quality,
location, and intensity of the noxious stimuli, is then conveyed via the
ascending pathway to reach various brain regions to elicit pain sensation.
Physiological pain responses normally protect us from tissue damage by
quickly alerting us to impending injury. Unlike acute physiological pain,
chronic pathological pain, including neuropathic and inflammatory pain,
is often associated with increased activity and responses of spinal dorsal horn
neurons, termed central sensitization (Woolf & Thompson, 1991; Xu,
Dalsgaard, & Wiesenfeld-Hallin, 1992). This phenomenon is the cellular
basis for hyperalgesia (increased pain response to a noxious stimulus) and
allodynia (painful sensation in response to a nonnoxious stimulus).
Nitric oxide (NO) is a membrane-permeable gaseous second messenger
involved in signal transduction. The physiological function of NO has been
shown in a large variety of cell types and tissues, including the immune system, blood vessels, endothelial cells, and neurons. NO is produced from
L-arginine by three major isoforms of nitric oxide synthase (NOS): neuronal


Nitric Oxide and Pain Regulation

3

NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial
NOS (eNOS or NOS3) (Alderton, Cooper, & Knowles, 2001; Knowles &
Moncada, 1994). Both nNOS and eNOS are constitutively expressed and
activated by Ca2+/calmoduline-dependent signaling, whereas iNOS is typically induced by immunostimulation, such as inflammatory cytokines and
bacterial endotoxins, independent of intracellular Ca2+ levels. Classically,

the intracellular NO effect is mediated by the NO-sensitive soluble guanylyl
cyclase (sGC). When activated, sGC converts guanosine triphosphates
(GTP) into cyclic guanosine monophosphates (cGMP). cGMP has different
targets such as serine/threonine protein kinases G (PKG-I and PKG-II),
cGMP-regulated phosphodiesterase, and cGMP-activated ion channels
(Ahern, Klyachko, & Jackson, 2002; Calabrese et al., 2007). In addition,
NO can promote a covalent and reversible posttranslational protein modification by interacting with the thiol side chain of cysteine residues. This
chemical reaction, named S-nitrosylation, occurs without the action of
any enzymes (Ahern et al., 2002; Choi et al., 2000).
The role of NO in pain signaling has been investigated in many studies
using rodent models and in humans. In this chapter, we critically review the
reported complex actions of NO in pain transduction and transmission. We
also present recent electrophysiological evidence showing that NO inhibits
nociceptive transmission at the spinal cord level and the signaling mechanisms involved.

2. ROLE OF NO IN NOCICEPTIVE TRANSDUCTION
AT THE PERIPHERY
The evidence about the role of NO in pain transduction is inconsistent
and conflicting. In human subjects, injection of the NO solution
(>0.7 mM) into the paravascular tissues and veins (Holthusen & Arndt,
1995) or the cutaneous tissue in the forearm (Holthusen & Arndt, 1994)
evokes pain in a dose-dependent fashion. However, the physiological relevance of high NO concentrations used in these studies is not clear, because
the tissue level of NO is <10 μM (Wink et al., 1993). On the other hand,
local infusion of the NO donor glyceryl trinitrate at the site of surgery produces a small analgesic effect in humans undergoing oral surgery (Hamza
et al., 2010). Also, transdermal nitroglycerine alone does not affect pain itself
but prolongs the analgesic effect of opioids in patients after knee surgery
(Lauretti, de Oliveira, Reis, Mattos, & Pereira, 1999). Others have reported
that inhaled NO can reduce pain intensity in patients with sickle cell crisis

4

Alexis Bavencoffe et al.

(Head et al., 2010) and that local spray of isosorbide dinitrate, an NO donor,
reduces pain and burning sensation, in diabetic patients (Yuen, Baker, &
Rayman, 2002). Nevertheless, the potential therapeutic effect of NO donors
for chronic pain treatment still needs to be confirmed in large scale clinical
trials.
It has been reported that local injection of NG-methyl-L-arginine
(L-NMA), an NOS inhibitor, attenuates mechanical hypersensitivity caused
by injection of prostaglandin E2 into the rat skin (Chen & Levine, 1999).
Also, systemic treatment with an NOS inhibitor, NG-nitro-K-arginine
methyl ester (L-NAME), attenuates tactile and cold allodynia in a rat model
of neuropathic pain induced by spinal nerve ligation (Yoon, Sung, &
Chung, 1998). However, others have shown that the antiallodynic effect
of L-NAME cannot be reversed by L-arginine (Lee, Singh, & Lodge,
2005). Furthermore, systemic treatment with other NOS inhibitors,
including 7-nitroindazole, N(omega)-nitro-L-arginine (L-NNA), aminoguanidine, and LY457963, does not affect allodynia in rats subjected to
spinal nerve ligation (Lee et al., 2005; Luo et al., 1999). Thus, it seems that
the commonly used NOS inhibitors, such as L-NAME, may affect targets
other than NOS. In support of this possibility, it has been demonstrated that
the arginine analogues with alkyl or aryl esterification, including L-NAME,
are in fact muscarinic receptor antagonists (Buxton et al., 1993; Chang,
Chen, & Hsiue, 1997). In addition, tactile allodynia caused by nerve injury
or tissue damage is attenuated in nNOS-knockout mice (Chu et al., 2005;
Guan, Yaster, Raja, & Tao, 2007). sGC-knockout mice also show reduced
nociceptive responses to tissue inflammation or nerve injury, but their
responses to acute pain are not affected (Schmidtko et al., 2008). It should
be acknowledged that in eNOS-, nNOS-, or iNOS-knockout mice, there

is a developmental compensatory increase in the expression of other NOS
isoforms in the spinal cord (Boettger et al., 2007; Tao et al., 2003). This compensatory upregulation of other NOS subtypes in specific NOS isoformknockout mice complicates the interpretation of the behavioral test results.
In contrast, it has been shown that local injection of an NO donor,
sodium nitroprusside, reduces mechanical hyperalgesia in the rat paw
induced by prostaglandin E2 injection (Durate, Lorenzetti, & Ferreira,
1990). Interestingly, topical application of a cream containing different concentrations of NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or isosorbide dinitrate, produces an opposite effect on allodynia in a rat model of
postoperative pain (Prado, Schiavon, & Cunha, 2002). At a low concentration, SNAP (1–10%) or isosorbide (2.5–5%) reduces allodynia through the


Nitric Oxide and Pain Regulation

5

guanylyl cyclase. However, at a high concentration, SNAP (30%) increases
allodynia induced by surgical incision, which is not blocked by a guanylyl
cyclase inhibitor (Prado et al., 2002). Notably, high concentrations of
NO may produce tissue-damaging effects via highly reactive oxidant species
such as peroxynitrite (Koppenol, Moreno, Pryor, Ischiropoulos, &
Beckman, 1992).

3. DIVERSE EFFECTS OF NO ON ION CHANNELS
EXPRESSED ON PRIMARY SENSORY NEURONS
3.1. Acid-sensing ion channels
Acid-sensing ion channels (ASIC) are expressed in small-diameter sensory
neurons, and their expression is increased during inflammation or by
proinflammatory mediators such as bradykinin and interleukin-1
(Krishtal, 2003; Mamet, Baron, Lazdunski, & Voilley, 2002). These channels are responsible for the ion conductance activated by extracellular protons in sensory neurons (Krishtal, 2003; Krishtal & Pidoplichko, 1980). NO
potentiates the activity of proton-gated ASIC channels when exposed to a
modest decrease of pH to 6.3, mimicking the conditions of inflammation
and anoxia, in neonatal rat dorsal root ganglion (DRG) neurons (Cadiou

et al., 2007). This effect seems to be independent of cGMP/PKG pathway
and requires an extracellular site of action, possibly S-nitrosylation. Topical
application of an NO donor, glyceryl trinitrate, significantly increases acidevoked pain but has no effects on heat or mechanical pain thresholds in the
skin of human volunteers (Cadiou et al., 2007).

3.2. Transient receptor potential channels
3.2.1 TRPV1
Transient receptor potential (TRP) channels are cationic nonselective channels, which respond to a broad range of stimuli such as temperature, protons,
osmolarity, mechanical stress, and painful stimuli. For example, transient
receptor potential vanilloid type 1 (TRPV1) channels are activated by noxious heat (>43  C), protons, and capsaicin, the active component of red chili
pepper. Opening of TRPV1 channels produces a sensation of burning pain
(Caterina et al., 1997, 2000). TRPV1 are expressed in small nociceptive
neurons and are upregulated under inflammatory conditions. TRPV1 can
be sensitized by proinflammatory mediators, including bradykinin and prostaglandins, resulting in enhanced pain sensation during inflammation.


6

Alexis Bavencoffe et al.

It has been reported that TRPV1 channels can be S-nitrosylated on two
cysteine residues (Cys616 and Cys621) in the N-terminal of the poreforming region in the S5–S6 linker (Yoshida et al., 2006). Applications of
SNAP or an S-nitrosylating agent (5-nitro-2-PDS) trigger Ca2+ influx via
TRPV1 channels in HEK293 cells. Proton and heat sensitivities of TRPV1
channels are also enhanced in the presence of SNAP and 5-nitro-2-PDS.
Deletion of the two cysteine residues reduces this enhancement without
affecting proton and heat sensitivity and the surface expression of TRPV1
channels (Yoshida et al., 2006). However, others have shown that SNAP
stimulates Ca2+ influx in mouse DRG neurons and in HEK293 cells
expressing TRPV1 channels only at a very high concentration (>1 mM)

(Miyamoto, Dubin, Petrus, & Patapoutian, 2009). In contrast, a recent study
has shown that low concentrations of NO donors, such as 100 μM sodium
nitroprusside (SNP) or 100 μM SNAP, inhibits TRPV1 channel activity
through activation of the cGMP/PKG pathway in rat DRG neurons
( Jin, Kim, & Kwak, 2012).
3.2.2 TRPA1
Originally named ANKTM1, TRPA1 channels are activated by cold
temperatures (<17  C), proinflammatory mediators, and chemical irritants such as mustard oil, wasabi, allicin, and formalin ( Jordt et al.,
2004). They are mainly coexpressed with TRPV1 channels in primary
sensory neurons. Topical applications of TRPA1 agonists induce spontaneous pain, cold allodynia, and heat/thermal hyperalgesia (Fernandes,
Fernandes, & Keeble, 2012; Nilius, Appendino, & Owsianik, 2012). It
has been reported that high concentrations of SNAP (2–3 mM) can activate TRPA1 channels in DRG neurons and in HEK293 cells (Miyamoto
et al., 2009).
3.2.3 TRPV3 and TRPV4
TRPV3 channels are heat-sensitive channels (>33  C) expressed in
keratinocytes and DRG neurons (Peier et al., 2002; Smith et al., 2002).
TRPV3 is also activated by eugenol, camphor, and inflammatory mediators
such as arachidonic acids (Hu et al., 2006). TRPV4 channels are activated by
warm temperature (>27  C), low pH, hypotonicity, and diacyl glycerol.
TRPV3-knockout mice exhibit a deficit in innocuous and noxious heat sensitivity (Moqrich et al., 2005). Both SNAP and 5-nitro-2-PDS seem to stimulate TRPV3 and TRPV4 channels expressed in HEK293 cells (Yoshida
et al., 2006). However, TRPV3 and TRPV4 do not have a major


Nitric Oxide and Pain Regulation

7

contribution to heat sensation (Huang, Li, Yu, Wang, & Caterina, 2011).
Their physiological relevance to nociceptive transduction in sensory neurons and the skin is not clear.

3.3. KATP channels
ATP-sensitive potassium channels are regulated by the ATP/ADP ratio in a
way that a drop of this ratio will activate these channels. Following their
opening, the efflux of potassium will induce a hyperpolarization, decreasing
the neuronal excitability. KATP channels also control neurotransmitter
release by regulating neuronal excitability (Yamada & Inagaki, 2005). KATP
channels are expressed in large DRG neurons and are activated by
S-nitrosylation of cysteine residues in their SUR1 subunit (Kawano et al.,
2009) or by the NO/cGMP/PKG pathway (Sachs, Cunha, & Ferreira,
2004). The cGMP/PKG/KATP signaling seems to be involved in the analgesic effects of noradrenaline (Romero, Guzzo, Perez, Klein, & Duarte,
2012), cannabinoid receptor 2 agonists (Negrete, Hervera, Leanez,
Martin-Campos, & Pol, 2011), and morphine (Cunha et al., 2010).

4. ROLE OF NO IN REGULATING NOCICEPTIVE
TRANSMISSION AT THE SPINAL CORD LEVEL
The spinal dorsal horn is a critical site for nociceptive transmission and
modulation. Both nNOS and sGC are present in the superficial spinal dorsal
horn (Ding & Weinberg, 2006; Schmidtko et al., 2008; Terenghi, RiverosMoreno, Hudson, Ibrahim, & Polak, 1993). It has been reported that
intrathecal injection of L-NAME decreases thermal hyperalgesia caused
by carrageenan in rats, but an nNOS inhibitor, 7-nitroindazole, has no
such effect (Osborne & Coderre, 1999). Also, mechanical hypersensitivity
induced by spinal nerve ligation or tissue inflammation is reduced by intrathecal administration of nNOS inhibitors (L-NAME or 7-nitroindazole)
(Chu et al., 2005; Guan et al., 2007). Moreover, it has been reported that
intrathecal injection of an nNOS inhibitor, N(omega)-propyl-l-arginine,
or an iNOS inhibitor, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine
hydrochloride, dose-dependently reduces thermal and mechanical hypersensitivity induced by partial sciatic nerve ligation in mice (Tanabe,
Nagatani, Saitoh, Takasu, & Ono, 2009). These studies suggest that spinal
NO may be involved in the potentiation of central sensitization after tissue
damage or nerve injury.

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Alexis Bavencoffe et al.

In contrast, other studies have shown that NO at the spinal cord
level plays a role in inhibition of nociceptive transmission. For instance,
intrathecal administration of the NO precursor L-arginine increases the
mechanical nociceptive withdrawal threshold in rats (Zhuo, Meller, &
Gebhart, 1993). Also, intrathecal administration of an NO donor,
3-morpholinosydnonimine (SIN-1; 0.1–100 μg), consistently produces an
antinociceptive effects in the rat tail-flick test in a dose-dependent manner
(Sousa & Prado, 2001). The analgesic effect of morphine also involves NO
since the antinociceptive action of morphine is attenuated by NOS inhibitors and NO scavengers (Song, Pan, & Eisenach, 1998). Furthermore,
intrathecal injection of an nNOS inhibitor (1,2-trifluoromethylphenyl
imidazole, TRIM) or an NO scavenger (carboxy-PTIO), reduces the antiallodinic effect of clonidine in rat model neuropathic pain caused by L5–L6
spinal nerve ligation (Pan, Chen, & Eisenach, 1998). We have shown that
intrathecal injection of 300 μg L-arginine or 100 μg SNAP significantly
increases the nociceptive threshold in response to a noxious pressure
stimulus n rats ( Jin, Chen, Cao, Li, & Pan, 2011). In addition, the antinociceptive effect of L-arginine and SNAP is abolished by cotreatment with
TRIM and carboxy-PTIO, respectively. These findings suggest that NO
inhibits nociceptive transmission at the spinal cord level.
Some studies have reported a dual effect of NO in regulating nociception
at the spinal cord level. Intrathecal injection of small doses of L-arginine
(10 μg) attenuates the nociceptive behaviors evoked by formalin injection
in rats, whereas L-arginine at high doses (250 μg) increases nociceptive
responses (Li & Qi, 2010). Similarly, intrathecal delivery of small doses
(0.1–0.25 μM) of a cGMP analogue, 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP), decreases nociceptive behaviors caused by formalin injection. But intrathecal injection of a high dose of 8-bromo-cGMP
(2.5 μM) increases nociceptive responses (Tegeder, Schmidtko, Niederberger,
Ruth, & Geisslinger, 2002). Furthermore, in a rat model of neuropathic

pain induced by sciatic nerve ligation, intrathecal administration of a low dose
of SIN-1 (0.1–2 μg) reduces mechanical allodynia (Sousa & Prado, 2001).
However, intrathecal injection of a high dose of SIN-1 (10–20 μg) increases
allodynia (Sousa & Prado, 2001). The discrepancy regarding the complex
function of NO in nociceptive processing may result from the use of different
animal models of pain, the nonspecificity of NOS inhibitors, and/or different
NO levels produced at the spinal cord level in various studies.
There are only a few published electrophysiological studies about the
effect of NO on spinal nociceptive neurons. Spinally administered NO


Nitric Oxide and Pain Regulation

9

donors predominantly reduce the firing activity of spinal dorsal horn neurons, and treatment with L-NAME or a selective sGC inhibitor,
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), increases the activity of nociceptive dorsal horn neurons (Hoheisel, Unger, & Mense, 2000,
2005; Pehl & Schmid, 1997). In contrast, administration of L-NAME or
7-nitroindazole via a microdialysis fiber inserted to the spinal cord tissues
blocks sensitization of spinothalamic tract neurons induced by intradermal
capsaicin injection (Lin et al., 1999). Notably, NOS inhibitors have no
effects on the responses of spinothalamic tract neurons to peripheral stimulation in the absence of capsaicin injection (Lin et al., 1999).
nNOS is the major isoform of NOS in the spinal dorsal horn, and its
expression level is increased during peripheral inflammation and nerve
injury (Dolan, Kelly, Huan, & Nolan, 2003; Ma & Eisenach, 2007). In
the central nervous system, the production of NO by nNOS requires Ca2+
influx, which is dependent on activation of postsynaptic NMDA receptors. When NO is produced postsynaptically after N-methyl-D-aspartate
(NMDA) receptor activation by glutamate release from nociceptive primary
afferent nerve terminals, NO could diffuse back to the presynaptic terminals
of primary afferent terminals in the dorsal horn to inhibit glutamate release

( Jin et al., 2011). Because increased NO production is a secondary response
to tissue/nerve injury-induced NMDA receptor activation, increased spinal
NO release and NOS expression levels under painful conditions should
not be considered supporting evidence for a pronociceptive role of NO.

5. NO REDUCES EXCITATORY, BUT POTENTIATES
INHIBITORY, SYNAPTIC TRANSMISSION IN
SPINAL CORDS
To further determine how NO controls excitatory and inhibitory synaptic transmission in the spinal dorsal horn, we conducted a series of patchclamp recording experiments using a spinal cord slice preparation, which
preserves the intrinsic connection between primary afferent terminals and
dorsal horn neurons. High-resolution whole-cell recording of postsynaptic
currents under voltage-clamp conditions provides a sensitive and accurate
measure of synaptic release of glutamate, GABA, and glycine in real-time.
Combined with pharmacologic and ionic manipulations, information is
obtained about the dynamics of transmitter–receptor interactions, the types
of postsynaptic receptors activated, the effects of drugs on transmission, and
the mechanisms of synaptic plasticity. Another advantage of this technique is


10

Alexis Bavencoffe et al.

that experiments are performed in spinal tissue slices in the absence of any anesthetics. In the sections below, we present electrophysiological evidence about
the role of NO in regulating synaptic transmission in the spinal dorsal horn.

5.1. Glutamatergic input from primary afferent nerves
Glutamate is the predominant excitatory neurotransmitter involved in nociceptive transmission. When nociceptors are activated, the primary afferent
nerves release glutamate at their central terminals to superficial dorsal horn
neurons of the spinal cord. Glutamate will then bind to its postsynaptic

ligand-gated channels, 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)
propanoic acid (AMPA) and NMDA receptors, to excite second-order neurons in the spinal dorsal horn. Calcium influx after activation of NMDA
receptors stimulates calcium/calmoduline-dependent signaling and nNOS
activity to generate NO (Rameau, Chiu, & Ziff, 2004). Released NO
can act paracrinally on neighboring neurons, glial cells or have a retrograde
presynaptic effect in the spinal cord.
L-arginine significantly decreases the frequency of spontaneous excitatory postsynaptic currents (EPSCs) but has no significant effect on the frequency or amplitude of miniature EPSCs of spinal dorsal horn neurons ( Jin
et al., 2011). To determine the role of NO in the control of glutamate release
from primary afferent terminals, the effect of L-arginine or SNAP on glutamatergic monosynaptic EPSCs evoked from the dorsal root is also examined. Both L-arginine and SNAP inhibit the amplitude of evoked
monosynaptic EPSCs of dorsal horn neurons, and their effects are abolished
by treatment with TRIM and carboxy-PTIO, respectively ( Jin et al., 2011).
These findings strongly suggest that NO inhibits glutamatergic synaptic
transmission between primary afferent nerves and second-order neurons
in the spinal dorsal horn.
Interestingly, sGC/cGMP is not involved in the NO effect on synaptic
glutamate release to dorsal horn neurons ( Jin et al., 2011).
N-ethylmaleimide (NEM) is a specific alkylating agent of cysteine sulfhydryls, covalently modifies protein sulfhydryl groups thereby preventing subsequent S-nitrosylation of proteins (Bolotina, Najibi, Palacino, Pagano, &
Cohen, 1994; Broillet & Firestein, 1996). In the presence of NEM,
L-arginine or SNAP has no effect on the amplitude of evoked EPSCs of
dorsal horn neurons ( Jin et al., 2011). These results suggest that NO probably inhibits glutamate release from primary afferent terminals through
S-nitrosylation of presynaptic proteins.


Nitric Oxide and Pain Regulation

11

5.2. Voltage-activated calcium channels in sensory neurons
High voltage-activated calcium channels (HVACCs) are essential for neurotransmitter release and for nociceptive transmission (Smith & Cunnane,
1997). Because NO inhibits evoked glutamate release from primary afferent

terminals through S-nitrosylation, NO may inhibit HVACCs in DRG neurons through S-nitrosylation. This notion is supported by the findings from
using acutely dissociated DRG neurons and HEK293 cells expressing
HVACCs ( Jin et al., 2011). For example, L-arginine or SNAP causes a large
decrease in the HVACC, but not T-type, currents in rat DRG neurons.
However, this effect was not affected by ODQ, which is consistent with
the report showing that NO-sensitive guanylyl cyclase is not expressed in
DRG neurons (Schmidtko et al., 2008). Importantly, NEM completely
blocks the inhibitory effect of L-arginine and SNAP on HVACCs in
DRG neurons and in HEK293 cells ( Jin et al., 2011). Therefore, NO likely
inhibits the activity of HVACCs in primary sensory neurons through
S-nitrosylation.

5.3. Synaptic NMDA receptors
NMDA receptors are involved in synaptic transmission and plasticity in
many chronic pain conditions (Zhou, Chen, & Pan, 2011). NO can inhibit
NMDA receptor currents in recombinant systems (Aizenman & Potthoff,
1999; Lei et al., 1992). In spinal dorsal horn neurons, SNAP reversibly
reduces the amplitude of NMDA receptor-mediated EPSCs evoked from
stimulation of spinal dorsal root and puff NMDA currents (Nicholson,
Dibb, & Renton, 2004). Furthermore, it has been shown that exogenous
and endogenous NO can inhibit NMDA receptor activity through
S-nitrosylation of the cysteine residue 399 on the NR2A subunit (Choi
et al., 2000). Because both HVACCs and NMDA receptors are critically
involved in nociceptive transmission, it seems reasonable to propose that
NO could act as a physiological brake to prevent over-excitation of spinal
dorsal horn neurons, caused by synaptic glutamate release and NMDA
receptor activation, during painful states.

5.4. Synaptic release of glycine
Both γ-aminobutryic acid (GABA) and glycine are the most predominant

inhibitory neurotransmitters involved in the regulation of nociceptive transmission at the spinal cord level (Zhou et al., 2007, 2008, 2012). Activation of
GABAA and glycine receptors normally causes chloride influx and


12

Alexis Bavencoffe et al.

hyperpolarization of mature neurons. Bath application of 100–400 μM
L-arginine concentration-dependently increases the frequency of glycinergic
inhibitory postsynaptic currents (IPSPs) of spinal dorsal horn neurons; and
such an effect is abolished by pretreatment with TRIM ( Jin et al., 2011).
SNAP at 100 μM also potentiates the frequency of glycinergic spontaneous
IPSCs of dorsal horn neurons, and this effect is blocked by carboxy-PTIO.
L-arginine still increases the frequency of glyinergic miniature IPSCs,
suggesting that NO acts at presynaptic terminals to potentiate synaptic glycine release in the spinal dorsal horn ( Jin et al., 2011). Interestingly, neither
L-arginine nor SNAP has any effect on the frequency and amplitude of
GABAergic spontaneous IPSCs of dorsal horn neurons ( Jin et al., 2011).
It has also been shown that NO promotes synaptic glycine, but not GABA,
release to sympathetic preganglionic neurons in the lateral spinal cord (Wu &
Dun, 1996).
Further studies reveal that the stimulating effect of L-arginine on glycinergic
sIPSCs is abolished by inhibition of sGC with ODQ. A membranepermeable cGMP analogue, 8-bromo-cGMP, significantly increases the
frequency, not the amplitude, of glycinergic spontaneous IPSCs. In addition,
Rp-8-Br-PET-cGMPS, a specific and membrane-permeable PKG inhibitor
blocks the potentiating effect of 8-bromo-cGMP or SNAP on glycinergic
spontaneous IPSCs ( Jin et al., 2011). Thus, the sGC/cGMP/PKG signaling
cascade mediates the potentiating effect of NO on synaptic glycine release
to spinal cord horn neurons.
Collectively, NO inhibits nociception transmission at the spinal cord

level by inhibiting excitatory glutamatergic input from primary afferent
nerves and by potentiating inhibitory glycine release. These electrophysiological data provide new insights into the underlying of cellular and signaling
mechanisms of NO in the inhibition of nociceptive transmission at the
spinal level.

6. CONCLUSIONS AND FUTURE DIRECTIONS
The controversial role of NO in nociceptive transduction and transmission has not been fully resolved. Despite various reports showing the
involvement of NO in the nociception and nociceptor activation, there is
no convincing evidence supporting the pronociceptive actions of NO at
the physiological concentration. The discrepancies are possibly due to the
nonspecificity of NOS inhibitors and different concentrations of NO donors
used in various studies. At the spinal cord level, NO predominantly reduces


Nitric Oxide and Pain Regulation

13

pain transmission by inhibiting the activity of NMDA receptors and voltageactivated calcium channels through S-nitrosylation and by facilitating glycine release from inhibitory interneurons via the cGMP/PKG pathway
(Fig. 1.1). This electrophysiological evidence indicates that NO likely

Figure 1.1 Diagram shows that NO inhibits synaptic transmission in the dorsal horn of
the spinal cord. Stimulation of primary sensory nerves by painful stimuli triggers action
potentials and opening of voltage-activated calcium channels, leading to calcium influx
and glutamate release from presynaptic nerve terminals. Released glutamate binds to
AMPA (not shown) and NMDA receptors present on postsynaptic dorsal horn neurons
to increase intracellular calcium levels and to recruit nNOS to the close proximity
of NMDA receptors by the scaffolding protein PSD95 and nNOS activation via
calcium/calmodulin-dependent signaling. Subsequently, nNOS converts L-arginine into
NO and citruline. NO then exerts a negative feedback action by inhibiting the NMDA

receptor activity via S-nitrosylation. NO also diffuses passively into the presynaptic terminal and inhibits the activity of high voltage-activated calcium channels to decrease
synaptic glutamate release. Moreover, NO diffuses into the neighboring glycinergic
interneurons and potentiates synaptic glycine release via stimulation of an sGC/PKGdependent pathway. Glycine activates postsynaptic glycine receptors, leading to chloride influx. Collectively, decreased glutamate release, inhibition of NMDA receptors, and
increased glycine release by NO result in hyperpolarization of the postsynaptic neuron,
thereby reducing nociceptive transmission at the spinal cord level. CaM, calmodulin;
cGMP, cyclic guanosine monophosphate; GABAR, γ-aminobutyric acid receptor; GlyR,
glycine receptor; GTP, guanosine triphosphate; HVACC, high voltage-activated calcium
channel; NMDAR, N-methyl-D-aspartate receptor; nNOS, neuronal nitric oxide synthase;
NO, nitric oxide; PKG, protein kinase G; PSD95, postsynaptic density protein 95; sGC,
soluble guanylyl cyclase; SN, S-nitrosylation.


14

Alexis Bavencoffe et al.

functions as a negative feedback regulator to reduce nociceptive transmission
in the spinal dorsal horn during various painful conditions. Because of the
potential confounding issues associated with pharmacological agents and
conventional NOS-knockout mice, further studies are warranted to use
siRNA and conditional knockout approaches to unambiguously validate
the physiological role of NO in the regulation and signaling of nociception
during acute and chronic pain conditions.

ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health Grant NS073935 and by the
N.G. and Helen T. Hawkins endowment (to H. -L. P.).

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