Review Article  |   January 2009
Modulation of Opioid Actions by Nitric Oxide Signaling
Author Affiliations & Notes
  • Noboru Toda, M.D., Ph.D.
  • Shiroh Kishioka, M.D., Ph.D.
  • Yoshio Hatano, M.D., Ph.D.
  • Hiroshi Toda, M.D., Ph.D.
  • * Professor Emeritus, Shiga University of Medical Science, Shiga, Japan, and Toyama Institute for Cardiovascular Pharmacology Research, Osaka, Japan. † Professor of Pharmacology, Department of Pharmacology, ‡ Professor of Anesthesiology, Department of Anesthesiology, Wakayama Medical University, Wakayama, Japan. § Head of Anesthesiology, Department of Anesthesiology, Kyoto Katsura Hospital, Kyoto, Japan.
Article Information
Review Article / Pain Medicine
Review Article   |   January 2009
Modulation of Opioid Actions by Nitric Oxide Signaling
Anesthesiology 1 2009, Vol.110, 166-181. doi:10.1097/ALN.0b013e31819146a9
Anesthesiology 1 2009, Vol.110, 166-181. doi:10.1097/ALN.0b013e31819146a9
THE labile molecule NO has been widely recognized to play pivotal roles in the regulation of physiologic functions; in contrast, it also participates in pathophysiological intervention. Nitric oxide (NO) synthesis was first found in vascular endothelial cells,1–4 central and peripheral nerve cells and fibers,5 and macrophages.6 Investigations on the functional role of this inorganic molecule have been extended to other organs and tissues in the whole body. The NO/cyclic guanosine monophosphate (cyclic GMP) signaling pathway contributes to mechanisms underlying the action of therapeutic agents, the most promising through mechanisms of enhancing the action of endogenous NO is phosphodiesterase-5 inhibitors such as sildenafil7 and its congeners.8 Morphine and other opioid agonists exert analgesia through μ-opioid receptors at spinal and multiple supraspinal sites. δ-Opioid receptor agonists also are potent analgesics in animals and humans. In animals, agonists for κ-receptors produce analgesia that is mediated at spinal sites. Some literature reports on opioid analgesics research have revealed that nitric oxide is involved in therapeutic actions but can also have untoward effects. Information concerning nitric oxide that undoubtedly plays important roles in modulating analgesic effects of opioids and their side effects would provide us with clues for establishing strategies for appropriate opioid therapy to enhance analgesic actions while minimizing tolerance, dependence, withdrawal syndrome, and other side effects.
Our previous review article9 summarized the involvement of nitric oxide in the actions of a variety of anesthetic agents without discussing the opioid analgesics, although these are useful for anesthesia and inevitably important for intolerable pain. The present article describes the involvement of endogenous nitric oxide in the antinociceptive effects of morphine and other related analgesics and the tolerance, dependence, and withdrawal syndrome associated with the use of these drugs.
Synthesis and Actions of NO
Nitric oxide is produced when l-arginine is transformed to l-citrulline through catalysis by nitric oxide synthase (NOS) in the presence of oxygen and cofactors. Ca2+is required for the activation of neuronal NOS (nNOS, NOS I) and endothelial NOS (NOS III) but not inducible NOS (iNOS, NOS II). nNOS, mostly a soluble enzyme, is constitutively expressed in the brain,5 peripheral nerves, and kidneys. Endothelial NOS is also constitutively expressed mostly in particulate fractions of the endothelial cell.10 iNOS is not constitutively expressed but is induced mainly in macrophages in response to bacterial lipopolysaccharide and cytokines. The synthesis of nitric oxide by these NOS isoforms is inhibited by l-arginine analogs, including N  G-monomethyl-l-arginine (l-NMMA),11 N  G-nitro-l-arginine (l-NA),12,13 l-NA methylester (l-NAME),12 and asymmetric dimethylarginine.14 7-Nitroindazol (7-NI) is one of the most promising nNOS inhibitors introduced.15 Aminoguanidine has a long history as a selective iNOS inhibitor.16 Nitro compounds, including nitroglycerin, sodium nitroprusside, and S-nitroso-N  -acetylpenicillamine, are capable of liberating nitric oxide and are called nitric oxide donors.
Endothelial nitric oxide causes vasodilatation, decreased vascular resistance, lowered blood pressure, inhibition of platelet aggregation and adhesion, inhibition of leukocyte adhesion and transmigration, and reduced vascular smooth muscle proliferation. Nitrovasodilators via  release of nitric oxide activate soluble guanylyl cyclase and produce cyclic GMP from guanosine triphosphate in smooth muscle cells. Methylene blue, oxyhemoglobin, and 1H[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one17 inhibit guanylyl cyclase activity. Accumulation of cyclic GMP causes activation of cyclic GMP-dependent protein kinase that is involved in the adenosine triphosphate (ATP)-sensitive K+-channel opening to produce spinal or peripheral antinociception18–20 and in Na+,K+-ATPase activation.21 Cyclic GMP-dependent protein kinase phosphorylates the serotonin transporter at Thr-276 and increases its activity by modifying the substrate permeation.22,23 Cyclic GMP is degraded by phosphodiesterase type 5 to 5′-GMP. Cyclic GMP is degraded by phosphodiesterase type-5 to 5′-GMP.
Nonadrenergic noncholinergic inhibitory responses to autonomic nerve stimulation are mainly mediated through nitric oxide synthesized by nNOS; Nitric oxide plays a crucial role as a neurotransmitter from the peripheral efferent nerves in cerebral blood vessels.24 Afferent nitrergic nerves control some aspects of sensory information processing. There is evidence that nitric oxide is the neurotransmitter released from primary sensory nerves that mediates mesenteric vasodilatation.25 Nitric oxide offers important roles in afferent signaling of pain through the dorsal horn of the spinal cord and in autonomic control through nitrergic innervation.26 Many of the homeostatic actions of spinal afferents are brought about by release of transmitters (nitric oxide and calcitonin gene-related peptide) from their peripheral endings.27 
Nitric oxide functions mainly as a neuromodulator in the brain. Nitric oxide signaling appears to be essential for neural plasticity; that is, long-term potentiation in the hippocampus and long-term depression in the cerebellum. Glutamate participates mainly in synaptic interactions; with the help of nitric oxide, the strength of excitatory input might be nonsynaptically signaled to the surrounding monoaminergic neurons in the brain. Nitric oxide formed by N  -methyl-d-aspartate (NMDA)-receptor activation diffuses to adjacent nerve terminals to modulate neurotransmitter release.28 Nitric oxide may act at several levels of the nervous system to develop hyperexcitability, resulting in hyperalgesia or allodynia.29,30 On the other hand, nitric oxide-releasing molecules, such as nitroparacetamol (NOX-701) resulting from the combination of paracetamol and a nitrooxybutyrol moiety, has been shown to be effective in acute nociception and in neuropathic pain.31,32 
Under pathologic conditions (e.g  ., during inflammation), high levels of nitric oxide are produced after iNOS expression is induced, mainly in macrophages. Nitric oxide possesses protective/destructive duality inherent in every other major component of the immune response. This labile molecule exerts beneficial effects by acting as an antibacterial, antiparasitic, antiviral agent or as a tumoricidal agent; on the other hand, high levels of nitric oxide, if uncontrolled, elicits detrimental effects that are produced because nitric oxide reacts with concomitantly produced superoxide anions, thereby generating highly toxic compounds such as peroxynitrite and hydroxyl radicals.
Effects of Morphine on NO Synthesis and Release
Morphine on NOS
Acute and chronic morphine treatment produced an increase in Ca2+-dependent NOS in mouse brain; the acute effect of morphine was blocked by coadministration of naloxone.33 In rat spinal cord, repeated administration of morphine increased the NOS mRNA level, the effect being accompanied by an increase in both the number of NOS-positive cells and the optical density of NOS-immunoreactivity, indicating that repeated morphine administration increases NOS biosynthesis.34 At 24 h after treatment with morphine (25 mg in pellet), there was decreased NOS activity in all brain regions and the spinal cord of the mouse, and NOS activity increased at 48 and 72 h after the treatment in both the cerebellum and cortex; implantation of a naltrexone pellet in conjunction with a morphine pellet blocked the changes in NOS activity.35 The authors suggested that the initial decrease in NOS activity is related to enhanced motor activity, whereas the increase in NOS activity is associated with tolerance-physical dependence development. Morphine increased the invertebrate immunocyte intracellular Ca2+level that was mediated by μ3-opioid receptors and was associated with stimulating nitric oxide production.36 The opioid stimulation of intracellular Ca2+levels appears to regulate constitutive NOS activity. Higher numbers of nNOS-positive cells were observed in the hippocampal dentate gyrus of wild-type mice repeatedly treated with either morphine or cocaine than in saline-treated wild-type mice; moreover, μ-opioid receptor knockout mice showed higher morphine- or cocaine-induced nNOS expression levels in the dentate gyrus than saline-treated wild-type mice.37 The knockout mice showed a higher morphine-induced nNOS expression level or a lower cocaine-induced nNOS expression level than morphine- or cocaine-treated wild-type mice. The authors suggested that morphine and cocaine sensitization is differentially regulated by the μ-opioid receptors in the knockout mice via  the nNOS systems in the dentate gyrus.
In contrast, Barjavel and Bhargava38 demonstrated that NOS activity, as determined by the rate of conversion of [3H]arginine into [3H]citrulline, was inhibited by the κ-opioid receptor agonist U-50,488H but only at a high concentration (0.1 mm) and was not affected by selective μ- and δ-opioid receptor agonists in rat cerebral cortex, indicating that drugs acting at μ-, δ-, and κ-receptors have no direct action on central NOS activity in vitro  . NOS activity was found to be unchanged in the brainstem and cerebellum of mice treated with morphine.39 Systemic administration of diacetylmorphine reduced the number of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive (nitric oxide-synthesizing) neurons in the rat brain raphe nuclei, and this effect was blocked by naloxone.40 Acute and chronic administration of morphine suppressed NADPH diaphorase-positive neurons in rat brain cervical nuclei, and naloxone reversed the morphine actions.41 
Morphine on NO Release
Acute exposure of human saphenous or internal thoracic artery endothelium or rat microvascular endothelial cells to morphine resulted in nitric oxide release via  the μ3-opiate receptor subtype.42,43 Intravenous morphine increased norepinephrine, acetylcholine, and nitrite in spinal dorsal horn microdialysate in anesthetized sheep, and these effects were antagonized by intrathecal injection of the α2adrenoceptor blocker idazoxan, atropine, or l-NMMA.44 Spinally released nitric oxide appears to play a role in the analgesic effects of systemic opioids. Fimiani et al  .45 noted that nitric oxide release was mediated through the μ3-opioid receptor, and morphine-stimulated nitric oxide release was higher in human surgical specimens of nonsmall-cell lung carcinoma than in those of the normal lung. Zhu et al  .46 provided evidence that morphine biosynthesis occurs in rat brain amygdala, and morphine releases nitric oxide in limbic tissues. Morphine and dopamine induced a transient surge of nitric oxide production in endometrial glandular epithelial cells, free of endothelial cells, isolated from human endometrial specimens.47 How increased nitric oxide release contributes to regulation of endometrial cell functions is indeed an intriguing mechanism to investigate. The μ-opioid receptor-specific antagonist β-funaltrexamine inhibited nitric oxide release from endomorphine 1–treated rodent and human immune cells.48 There was evidence suggesting that by inhibiting nNOS and reducing nitric oxide levels, asymmetric dimethylarginine decreases μ-opioid receptor constitutive activity in mice.49 
On the other hand, Pu et al  .50 obtained findings that suggest the existence of a dual-control mechanism composed of the excitatory NMDA and the inhibitory μ-opioid receptors in modulating cyclic GMP/nitric oxide release in the medial preoptic area of the rat brain.
Modulation of Morphine Actions by NO Analgesia
Supraspinal Site of Action
The central analgesic effect of morphine as tested by the rat paw pressure and tail flick tests was inhibited by intracerebroventricular injection of methylene blue and potentiated by an inhibitor of cyclic GMP phosphodiesterase, but it was not blocked by the NOS inhibitors l-NMMA and N  -iminoethyl-l-ornithine.51,52 Therefore, activation of the cyclic GMP system, not via  nitric oxide release, may be involved in the mechanism of the central analgesic effect of morphine. Antinociception induced by the muscarinic receptor agonist (+)-cis-dioxolane, but not β-endorphin, given supraspinally is likely mediated by the direct activation of an nitric oxide/cyclic GMP system, and the activation by the muscarinic agonist potentiates the antinociception induced by intracerebroventricular β-endorphin, but not that by μ-, δ-, or κ-opioid receptor agonists.53 Intracerebroventricular l-NA diminished the morphine-induced analgesia, and l-arginine administered by the same route increased the analgesic effect of morphine, indicating that increased nitric oxide synthesis may potentiate morphine analgesia.54 l-Arginine and the nitric oxide donor 3-morpholinosydnoimine intracerebroventricularly administered to mice produced antinociceptive effects that were blocked by naloxone and also by intracerebroventricular administration of a rabbit antiserum against rat dynorpohin 1-13; the antinociceptive effect of l-arginine was antagonized by an inhibitor of nNOS (table 1).55 The mechanisms for the antinociceptive action of l-arginine and 3-morpholinosydnoimine appear to be mediated by dynorphin and dependent on nitric oxide. Javanmardi et al  . 56 noted that mesencephalic morphine antinociception was reduced when MK-801 and l-NAME were microinjected sequentially into the rostral ventromedial medulla in rats, and this reduction was not significantly different from the effects of MK-801 or l-NAME alone, implying that NMDA receptors and nitric oxide production in the rostral ventromedial medulla modulate the transmission of opioid pain-inhibitory signals from the periaqueductal gray.
Table 1. Evidence for NO as an Analgesic Mediator at Supraspinal, Spinal, and Peripheral Sites in Experimental Animals 
Image not available
Table 1. Evidence for NO as an Analgesic Mediator at Supraspinal, Spinal, and Peripheral Sites in Experimental Animals 
On the other hand, l-NAME produced an opioid-independent antinociception in the mouse, probably by a direct effect within the brain.57 In awake dogs, l-NA or morphine perfused through the fourth ventricle increased the nociceptive threshold, and combined morphine/l-NA perfusions produced a greater antinociceptive effect than seen when morphine was given alone, suggesting that endogenous nitric oxide, produced at supraspinal sites, acts as a nociceptive mediator.58 Additional information in large mammals is required to extrapolate data to humans. In mice, chronic intraperitoneal administration of l-arginine decreased the antinociceptive response to subcutaneously administered morphine-6-β-d-glucuronide, a potent metabolite of morphine, whereas the response to intracerebroventricularly administered morphine-6-β-d-glucuronide was unaffected by l-arginine treatment; the decreased antinociceptive response to subcutaneous morphine-6-β-d-glucuronide was reversed by l-NA, suggesting that the decreased antinociceptive response of peripherally administered morphine-6-β-d-glucuronide by l-arginine may be related to a decrease in morphine-6-β-d-glucuronide entry into brain structures responsible for antinociceptive action.59 Similar results were also obtained with morphine.60,61 Bhargava et al  .62 obtained evidence suggesting that chronic intraperitoneal administration of l-arginine reduced the antinociceptive effect of morphine by increasing brain NOS activity and by decreasing the concentration of morphine in certain brain regions and the spinal cord in mice. Acute activation of the nitric oxide system by l-arginine administration attenuated morphine antinociception, possibly by inhibiting its uptake in central sites (midbrain and spinal cord) involved in antinociceptive actions.63 Streptozotocin-induced diabetes in mice markedly decreased the antinociceptive effect of intracerebroventricularly administered morphine and increased the urinary nitrite concentration; administration of aminoguanidine improved the effect of morphine and attenuated the increase in urinary nitrite concentration, indicating that an increase in nitric oxide formation by iNOS may be responsible for the observed decrease in antinociceptive effect of morphine in diabetic mice.64 
Spinal Site of Action
Nitric oxide acts as a modulator of dorsal horn spinal cord nociceptive pathways. NOS immunoreactivity was present in both humans and rats with similar distribution, being present in primary sensory neurons of dorsal root ganglia and their afferent terminals in the dorsal horn of spinal cord.65 nNOS-immunoreactive interneurons were found in the superficial layer of the dorsal horn and the intermediolateral cell column.
Kolesnikov et al  .66 noted that an antisense probe selectively targeting nNOS-2 blocked morphine analgesia and suggested that the facilitating nNOS-2 system predominates at the spinal level over the supraspinal level. Intravenous administration of morphine produced antinociception in rats, and an α2-adrenoceptor antagonist, NOS inhibitors, and a nitric oxide scavenger that were intrathecally injected produced attenuation of morphine-induced antinociception.67 It appears that a spinal α2-adrenergic mechanism is involved in antinociception from intravenously administered morphine and that spinal nitric oxide mediates antinociception produced by morphine. Spinally applied bovine milk-derived lactoferrin (BLF) produced μ-opioid receptor-mediated analgesia that was reversed by coadministration of l-NAME in the rat formalin test, and it potentiated the analgesia induced by morphine, suggesting that BLF acts as an enhancer of the spinal μ-opioidergic system via  a nitric oxide-mediated mechanism.68 Rats rendered diabetic with streptozotocin developed a mechanical hyperalgesia, and intrathecal [D-Pen2,D-Pen5]-enkephalin, a δ-opioid receptor agonist, increased the withdrawal threshold in response to noxious pressure in diabetic rats to a greater extent than in normal rats; intrathecal l-NMMA or the nitric oxide scavenger carboxy PTIO diminished the analgesic action of the δ-opioid receptor agonist in both normal and diabetic rats.69 Spinal endogenous nitric oxide seems to contribute to the analgesic action of intrathecal [D-Pen2,D-Pen5]-enkephalin in both normal and diabetic neuropathic pain conditions. Table 1summarizes the data indicating that morphine and nitric oxide show antinociceptive effects and that inhibitors of the nitric oxide/cyclic GMP pathway attenuate the analgesic effect of morphine or other opioids.
In contrast, there are findings indicating that NOS inhibition results in analgesic action and potentiates morphine-induced antinociception (table 2). Morphine or l-NAME given intrathecally, epidurally, or intravenously produced antinociceptive effects as assessed by tail flick latency in response to thermal stimulation, and coadministration of small doses of l-NAME and morphine produced reductions of the median effective doses for morphine.70 l-NAME given via  three different routes appears to have a synergistic antinociceptive interaction with morphine in response to thermal stimulation. l-NAME and morphine coadministered intrathecally elicited a profound and long-lasting antinociception, which was abolished by intrathecal administration of the NO donor 3-morpholinosydnoimine.71 Intrathecally injected 7-NI and l-NAME enhanced antinociception induced by morphine or by agonists of μ- and δ-opioid receptors in rat tail-flick, paw pressure, and formalin tests; however, coadministration of l-NAME and a κ-opioid receptor agonist produced antinociception in the paw pressure test only, showing that the inhibition of spinal NOS appears to potentiate the μ- and δ-mediated spinal antinociception and, to a lesser extent, κ-mediated spinal antinociception.72 The use of the nNOS-selective inhibitor 7-NI excludes the possible involvement of cerebral vasoconstriction and systemic blood pressure increase in the enhancing effect of NOS inhibition on morphine actions. l-NAME lost the ability to potentiate the analgesic actions of intrathecally administered morphine in nNOS null-mutant mice, and the heme oxygenase inhibitor no longer potentiated morphine-induced analgesia in mice lacking a functional heme oxygenase gene.73 In addition, the intrathecal injection of the cyclic GMP analog caused hyperalgesia in the hot plate assay; in spinal cord slices from either nNOS or heme oxygenase null-mutant mice, morphine did not stimulate cyclic GMP production. The authors suggested that spinal monoxide generation modifies the acute analgesic actions of morphine.
Table 2. Evidence for NO as an Algesic Mediator at Supraspinal, Spinal, or Systemic Sites in Experimental Animals 
Image not available
Table 2. Evidence for NO as an Algesic Mediator at Supraspinal, Spinal, or Systemic Sites in Experimental Animals 
Behavioral responses, such as vocalization and agitation, to intrathecal injection of high-dose morphine in rats were not reversed by naloxone but were inhibited by pretreatment with NMDA-receptor antagonists and l-NAME; the intrathecal injection of morphine evoked increases in nitric oxide metabolites and glutamine in the extracellular fluid of dorsal spinal cord that were reduced by antagonists against l-NAME and NMDA receptors, suggesting that the excitatory action of high-dose intrathecal morphine may be mediated by an NMDA–nitric oxide cascade in the spinal cord.74 These authors75 also noted that injections of formalin into the plantar surface of the paw evoked a biphasic spinal release of nitrite/nitrate and a transient release of glutamate; these effects, together with flinching and licking/biting, were reduced by intrathecal, combined administration of l-NAME and morphine, suggesting that l-NAME may enhance morphine-induced antinociception through an increased inhibition of nitrite/nitrate and glutamate releases evoked by formalin injection at the spinal cord level.
Peripheral Site of Action
Acetylcholine and sodium nitroprusside, which releases nitric oxide nonenzymatically, caused antinociception in the rat paw made hyperalgesic with prostaglandin E2, and these analgesic effects were enhanced by intraplantar injection of the inhibitor of cyclic GMP phosphodiesterase MY5445 and blocked by the guanylyl cyclase inhibitor methylene blue; the analgesia induced by acetylcholine, but not sodium nitroprusside, was blocked by l-NMMA, and l-arginine had no effect on prostaglandin-induced hyperalgesia but caused analgesia in paws inflamed with carrageenin.76 Peripheral analgesia induced by acetylcholine and morphine was potentiated by MY5445 and blocked by l-NMMA, whereas central morphine analgesia was potentiated by MY5445 but not affected by l-NMMA, suggesting that nitric oxide causes peripheral analgesia via  stimulation of the nitric oxide/guanylyl cyclase system and that the central analgesic effect of morphine is associated with activation of the cyclic GMP system that is not mediated by nitric oxide.51 Involvement of the l-arginine/nitric oxide/cyclic GMP pathway in peripheral morphine analgesia was also reported.77,78 FK409, a nitric oxide releaser, alone had no effect on the number of flinches induced by formalin injection in rats; however, when administered after intraplantar morphine, FK409 depressed the agitation behavior, and this inhibitory effect was reversed by naloxone and carboxy-PTIO.79 In the rat formalin test, morphine (10 μg, ipsilateral intraplantar injection) produced antinociception. However, contralateral injection of morphine did not produce any antinociceptive effect, an indication that the local administration of morphine did not result in a systemic drug distribution.80 Moreover, in the dipyrone-morphine combination study, the dose of morphine used (1.25 μg) was lower than the dose used for the study with morphine alone. Coadministration of sildenafil, an inhibitor of phosphodiesterase-5, enhanced the antinociceptive effect of morphine; and pretreatment of the paw with l-NAME, 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or naloxone blocked the effect of the combination of sildenafil and morphine, suggesting that sildenafil itself produces antinociception and increases that induced by morphine, probably through the inhibition of cyclic GMP degradation.81 Sildenafil also exhibited an antinociceptive effect against the paw pressure test in rats and the writhing test in mice, and pretreatment with l-NAME, methylene blue, or naloxone (intraplantar injection) blocked the effect of the sildenafil-morphine combination.82 Peripheral coadministration into the paw of morphine with a subeffective dose of BLF produced a potentiated antinociceptive effect compared to that of morphine alone in the rat formalin test, and this potentiating effect was reversed by l-NAME and a μ-opioid receptor antagonist.83 Local peripheral injection of codeine produced an antinociception in the rat formalin test, and local pretreatment of paws with l-NAME, methylene blue, ATP-sensitive K+channel inhibitors (glybenclamide and tolbutamide), nonselective voltage-dependent K+channel inhibitors (4-aminopyridine and tetraethylammonium), or naloxone prevented codeine-induced antinociception.84 Codeine appears to activate the opioid receptor- nitric oxide-cyclic GMP-K+channels pathway to produce its effect.
Stanojevic et al  .85 obtained data suggesting that the rat strain (Albino Oxford and Dark Agout)–dependent opposing effects of β-endorphin on paw inflammation are mediated through δ- and κ-opioid receptors and probably involve changes in the production of reactive oxygen species.
Actions of Systemically Administered Morphine
On the basis of the phenyl benzoquinone-induced abdominal constriction test86 in mice, morphine, mepyramine (H1-receptor antagonist), and l-arginine produced antinociception; l-arginine increased the antinociceptive effect of morphine and mepyramine, and l-NAME decreased the antinociception induced by these agents in combination with l-arginine.87 It appears that morphine and mepyramine produce peripheral antinociception through involvement of the l-arginine/nitric oxide cascade or other related pathways of nociceptive processes induced by nitric oxide. Brief and continuous footshock stress (3 min) induced a naloxone-insensitive antinociception that was not altered by either l-NAME, aminoguanidine, or l-arginine in mice; in contrast, prolonged and intermittent footshock (30 min) induced a naloxone-reversible antinociceptive effect that was blocked by l-NAME but not by aminoguanidine or l-arginine. In addition, morphine increased the antinociceptive effect of prolonged footshock, and this increase was inhibited by l-NAME but not by aminoguanidine.88 Based on these observations, the authors concluded that nitric oxide of constitutive origin may be selectively involved in an opioid-mediated type of footshock stress antinociception in mice.
However, there is evidence suggesting that nitric oxide counteracts the analgesic actions of morphine. l-Arginine administered orally or intraperitoneally, but not intracerebroventricularly, reduced the antinociceptive effect of morphine assessed in mice by using the hot plate, tail flick, and acetic acid-induced writhing tests; l-NMMA and l-NAME reversed the effects of l-arginine.89 Although l-NAME alone did not show any antinociceptive activity, it potentiated morphine-induced analgesia in mice; l-NAME dramatically augmented the analgesic effect of morphine in the late dark period at 19 h after the lights were turned on.90 Morphine, l-NAME, or both increased the nociceptive threshold for a criterion response to thermal stimuli for rats, and exposure to a magnetic field abolished the analgesic effects of morphine or l-NAME when applied separately but not when injected together relative to rats that had received these drugs and had been exposed to the sham field.91 Homayoun et al  . 92 demonstrated that acute administration of cyclocsporin A, known to decrease nitric oxide production in nervous tissues, or l-NAME enhanced the antinociception induced by administration of morphine, whereas chronic pretreatment with cyclosporine A or l-NAME did not affect morphine-induced antinociception. l-NAME, but not the iNOS inhibitor l-canavanine, enhanced the pain threshold and potentiated morphine-induced analgesia in mice.93 l-NAME changed the nonanalgesic effect of codeine to highly significant analgesia, and naloxone abolished the l-NAME-codeine–induced analgesia, indicating that the nitric oxide modulatory effect on the opioid analgesic codeine may be exerted, at least in part, through opioid receptors.94 Experimental diabetes in mice decreased the antinociceptive effect of morphine, and green tea extract reversed the morphine analgesia; coadministration of green tea extract and l-NAME further increased the antinociceptive effect of morphine, the stimulating effect being attenuated by administration of l-arginine. In addition, diabetes increased plasma nitrite levels that were decreased by green tea extract.95 Increased nitric oxide formation may be responsible for the decreased antinociceptive effect of morphine in diabetic mice, and green tea extract appears to restore the antinociceptive effect of morphine by inhibition of nitric oxide production.
The analgesic effect of morphine injected intraperitoneally exhibited biologic time-dependent differences in the thermally induced algesia in mice, whereas exogenously administered peroxynitrite exhibited either an algesic or analgesic effect, depending on the circadian time of its injection; concomitant administration of peroxynitrite and morphine reduced morphine-induced analgesia.96 
Human Studies
Randomized studies on patients with cancer pain who reported pain despite taking 80–90 mg of oral morphine daily indicated that the daily consumption of oral morphine on day 30 was greater in the group that received an additional 20 mg of oral morphine and the group that received 500 mg of oral dipyrone at 6-h intervals than in the group that received a 5-mg nitroglycerin (nitric oxide donor) patch daily and the group that received 0.5 mg/kg oral ketamine at 12-h intervals, suggesting that low-dose ketamine and transdermal nitroglycerin are effective coadjuvant analgesics.97 In conjunction with their opioid tolerance-sparing function, joint delivery of ketamine or nitric oxide donors with opiates may be of benefit in cancer pain management. In another randomized, double-blind study on cancer pain patients98 who complained of pain despite taking 80 to 90 mg of oral morphine daily and were allowed to freely manipulate their daily morphine consumption at the time the test drug (placebo patch or 5 mg/24-h nitroglycerin patch) was administered, these authors found that daily consumption of oral morphine was smaller in the nitroglycerin group compared with the placebo group, when evaluated on the fourteenth day after initiation of the study; patients from the placebo group in general complained of somnolence, compared with the nitroglycerin group. Delivery of nitric oxide donors together with opioids may be of significant benefit in cancer pain management. This idea was further evaluated by Iohom et al  .99 who performed studies on patients undergoing breast surgery with axillary clearance who were randomly allocated into one of two groups: group S received a standard intraoperative and postoperative analgesic regimen (morphine, diclofenac, dextropropoxyphene + acetaminophen), and group N received a continuous paravertebral block (for 48 h) and acetaminophen and parecoxib. They found that 80% of the patients in group S and no patient in group N developed chronic postsurgical pain, and the plasma concentrations of nitrite/nitrate were greater in group N compared with group S 48 h postoperatively. Whether increased nitric oxide production is involved in the analgesic efficacy could not be determined.
Figure 1summarizes the scheme of NMDA-receptor stimulation that mediates nNOS activation via  an increase in intracellular Ca2+concentrations and thereby the release of nitric oxide that modifies the opioidergic analgesic cascade. The nNOS activity and nitric oxide release are enhanced mainly by μ-opioid receptor stimulation. Up-to-date reports in the literature on studies employing experimental animals have led to the conclusion that nitric oxide appears to participate in potentiation of analgesia induced by morphine administered to peripheral sites (paw) via  μ-opioid receptors; the nitric oxide/cyclic GMP pathway may be involved (table 1). However, some studies with intrathecally administered morphine and NOS inhibitors have led to conclusions that endogenous nitric oxide, possibly formed through nNOS, plays significant roles as an antinociceptive agent and/or an enhancer of morphine antinociception (table 1), but other researchers report that NOS inhibitors enhance the antinociceptive effect of morphine or other opioids (table 2). Differences in animal species, NOS inhibitors, and nitric oxide scavengers employed in the various experiments do not appear to account for the opposite findings. A difference is only seen in diabetic mice, in which nitric oxide derived from iNOS acts as a nociceptive when morphine is administered intraventricularlly.54 Nitric oxide formed in large amounts by iNOS may contribute to lowering the pain threshold. Sousa and Prado100 noted that low doses of the nitric oxide donor 3-morpholinosydnoimine reduced and higher doses enhanced or had no effect on the mechanical allodynia evoked by chronic ligature of rat sciatic nerve, suggesting that nitric oxide produces a dual effect in a model of neuropathic pain. However, it might be too speculative to postulate that excessive concentrations of nitric oxide produced through iNOS or nNOS via  activation of NMDA receptors during nociceptive stimuli (tail-flick, hot plate tests mainly used in studies appearing in table 2) participate in promoting nociception. At present, there is only limited information available to support the beneficial use of nitric oxide as an analgesic in patients. However, data on patients are quite important because one frequently faces the problem of species variations (primate vs  . subprimate mammals) in the actions and mechanisms of action of endogenous molecules or therapeutic compounds. More data on healthy individuals and patients are required to introduce a qualified strategy of opioidergic analgesic treatment.
Fig. 1. Possible mechanism underlying interactions between μ-opioid receptor agonists and nitric oxide (NO) generated  via  NMDA-receptor stimulation in antinociceptive processes. Effects of NO on tolerance to and dependence on opioids and their withdrawal syndrome are also presented. NMDA-R,  N  -methyl-d-aspartate-receptor; opioid-R, opioid receptor; [Ca2+]i, intracellular concentrations of Ca2+; CaM, calmodulin; BH4, tetrahydrobiopterin; NADPH, reduced nicotinamide adenine dinucleotide phosphate; +, stimulation; –, inhibition; 7-NI, 7-nitroindazol; ADMA, asymmetrical dimethyl arginine; L-NA, NG-nitro-l-arginine; L-NAME, L-NA methylester; nNOS, neuronal nitric oxide synthase. 
Fig. 1. Possible mechanism underlying interactions between μ-opioid receptor agonists and nitric oxide (NO) generated  via  NMDA-receptor stimulation in antinociceptive processes. Effects of NO on tolerance to and dependence on opioids and their withdrawal syndrome are also presented. NMDA-R,  N  -methyl-d-aspartate-receptor; opioid-R, opioid receptor; [Ca2+]i, intracellular concentrations of Ca2+; CaM, calmodulin; BH4, tetrahydrobiopterin; NADPH, reduced nicotinamide adenine dinucleotide phosphate; +, stimulation; –, inhibition; 7-NI, 7-nitroindazol; ADMA, asymmetrical dimethyl arginine; L-NA, NG-nitro-l-arginine; L-NAME, L-NA methylester; nNOS, neuronal nitric oxide synthase. 
Fig. 1. Possible mechanism underlying interactions between μ-opioid receptor agonists and nitric oxide (NO) generated  via  NMDA-receptor stimulation in antinociceptive processes. Effects of NO on tolerance to and dependence on opioids and their withdrawal syndrome are also presented. NMDA-R,  N  -methyl-d-aspartate-receptor; opioid-R, opioid receptor; [Ca2+]i, intracellular concentrations of Ca2+; CaM, calmodulin; BH4, tetrahydrobiopterin; NADPH, reduced nicotinamide adenine dinucleotide phosphate; +, stimulation; –, inhibition; 7-NI, 7-nitroindazol; ADMA, asymmetrical dimethyl arginine; L-NA, NG-nitro-l-arginine; L-NAME, L-NA methylester; nNOS, neuronal nitric oxide synthase. 
The analgesic response to subcutaneous morphine given daily was abolished within 5 days in mice, and coadministration of l-NA with morphine prevented the development of tolerance for at least 11 days.101 Together with the finding that the NMDA-receptor antagonist MK-801 prevented the development of tolerance to morphine, the authors suggested that morphine tolerance involves the activation of NMDA receptors followed by the subsequent release of nitric oxide. Preventive effects of competitive and noncompetitive NMDA antagonists and l-NA against the development of morphine tolerance were also reported.102 Administration of l-NA or l-NAME along with morphine prevented the development of tolerance to morphine and also attenuated some signs of morphine dependence in mice.103 Downregulation by antisense treatment of nNOS-1 prevented the development of morphine tolerance in mice.66 Neurons in rat locus coeruleus expressed opioid receptors, and these neurons exhibited tolerance to chronic administration of opioids; the average median effective dose (obtained from curves for morphine dose vs  . single cell extracellular activity response) for morphine of the locus coeruleus cells from rats who received l-NA injections and morphine pellets was similar to that in cells from control rats, and the median effective dose of cells from morphine pelleted rats who received saline injections was substantially higher.104 Nitric oxide inhibition appears to attenuate the development of tolerance to morphine in locus coeruleus neurons. Cyclosporin A that decreases nitric oxide production in nervous tissues, l-NAME, and a combination of the two at per se  noneffective doses inhibited the induction and expression of tolerance to morphine-induced antinociception, and aminoguanidine did not alter morphine tolerance, suggesting the involvement of decreasing nitric oxide production through constitutive NOS, but not iNOS, in the modulation of morphine tolerance.105 
After implantation of morphine pellets in mice, the analgesic response was abolished on the third day, coadministration of l-NA with the pellets markedly retarded the development of tolerance, and l-NA slowly reversed preexisting tolerance; l-NA did not prevent tolerance to analgesia mediated by a κ1 or κ3 agonist.106 Multiple injections of [D-Pen2,D-Pen5]enkephalin (δ1-opioid receptor agonist), deltorphin II (δ2-opioid receptor agonist), or morphine resulted in the development of tolerance to their analgesic action in mice; concurrent administration of l-NA or l-NMMA had no effect on the development of [D-Pen2,D-Pen5]enkephalin or deltorphin tolerance but inhibited the development of morphine tolerance.107,108 Development of tolerance to the antinociceptive activity of morphine and the κ-opioid receptor agonist U-50,488H was inhibited by 7-NI, which did not modify the development of tolerance to the activity of [D-Pen2,D-Pen5]enkephalin in mice.109 It appears that inhibition of nNOS activity inhibits tolerance to the antinociceptive activity of μ- and κ- opioid receptor agonists but not δ-opioid receptor agonists. Development of acute antinociceptive tolerance to intracerebroventricular morphine in mice was blocked by l-NAME, l-NMMA, 7-NI, and 3-bromo-7-NI and also by a guanylyl cyclase inhibitor.110 Nitric oxide formed by nNOS acting through the cyclic GMP pathway appears to mediate the development of acute antinociceptive tolerance. Subchronic administration of 7-NI attenuated the development of morphine tolerance to the cellular and analgesic effects of μ-opioid receptor agonists in rats.111 Herraez-Baranda et al  .112 provided evidence indicating that κ-opioid receptors and nNOS in the same intracellular network of the rat periaqueductal gray appears to control the development of morphine tolerance and dependence. Blockade of nitric oxide overproduction, the consequence of NMDA-receptor activation by aminoguanidine via  inhibition of iNOS, attenuated the development of morphine tolerance and dependence.113 
In mice implanted subcutaneously with morphine pellets, NOS activity measured from the rate of conversion of [3H]arginine to [3H]citrulline in the cerebral cortex and cerebellum increased; morphine-implanted mice had higher Vmax values, but the Km values did not differ from those of control mice.114 Chronic treatment with morphine seems to increase NOS activity in the brain without modifying its substrate affinity. l-Arginine accelerated tolerance when it was coadministered with morphine and decreased morphine’s potency in mice when given alone.39 In superfused rat hippocampal slices, the amplitude of population spikes recorded by a glass microelectrode was increased by morphine; after continuous morphine superfusion, this effect was deteriorated (i.e  ., tolerance developed), which also increased the nitrite level in the superfusate; cosuperfusion of l-arginine with morphine further increased the nitrite level and facilitated the development of morphine tolerance.115 Morphine administration increased the nitric oxide production in hippocampal microdialysate in rats; the time course of altered nitric oxide production coincided with the development of antinociceptive tolerance.116 After sustained morphine administration, nNOS-deficient mice exhibited less tolerance development compared to the control group, although measurable tolerance still occurred; mice deficient in endothelial NOS showed a degree of tolerance similar to that of the control animals.117 In addition, tolerance development appears to be predominantly a nitric oxide-mediated process, but it is likely augmented by a secondary (non–nitric oxide) pathway. Heinzen et al  . 118 provided evidence for important nitric oxide-induced alterations in μ-opioid receptor functionality that directly lead to the development of opioid antinociceptive tolerance. Asymmetric dimethylarginine, a major circulating form of methylarginines in humans and animals, competes with l-arginine for the active site of NOS isoforms.119 Kielstein et al  .120 noted that increased nitric oxide production in mice resulted in an enhanced development of tolerance to morphine; nitric oxide increased constitutive μ-receptor activity; 7-NI attenuated morphine withdrawal in opioid-dependent rats. It was hypothesized that by inhibiting nNOS and reducing nitric oxide levels, asymmetric dimethylarginine may decrease μ-opioid receptor constitutive activity, resulting in alteration of the analgesic dose–response curve of morphine. According to Muscoli et al  .,121 morphine-induced antinociceptive tolerance in mice was associated with increased formation of proinflammatory cytokines and oxidative DNA, and inhibition of nitric oxide synthesis or removal of superoxide blocked these biochemical changes and inhibited the development of tolerance; coadministration of morphine with a peroxynitrite decomposition catalyst attenuated protein nitration and the observed biochemical changes and prevented the development of tolerance. Peroxynitrite decomposition catalysts may have therapeutic potential as adjuncts to opioids in relieving suffering from chronic pain.
Chronic administration of morphine resulted in the development of tolerance to the analgesic action of morphine, and l-NMMA attenuated the tolerance in rats at a higher dose than in mice.122 Induction phase l-arginine slowed the development of opioid tolerance and physical dependence, while l-NAME and l-NMMA led to a higher degree of tolerance but had no effect on the development of physical dependence; expression-phase NOS inhibition attenuated morphine tolerance and reduced the incidence of withdrawal signs.123 A 5-day treatment with increasing doses of morphine in rats induced antinociceptive tolerance, which was attenuated by l-NAME, whereas tolerance to the effect of morphine on thyrotropin (decrease) and prolactin (increase) levels was not modified by l-NAME.124 Both nitric oxide-dependent and nitric oxide-independent mechanisms may be involved in the development of tolerance to the various effects of morphine. Additional experimentation is needed to determine the mechanism underlying the nitric oxide action on morphine tolerance.
On the other hand, combined administration of BLF with morphine retarded the development of tolerance to morphine in mice; this effect of BLF was partially blocked by l-NAME or methylene blue and completely blocked by 7-NI, suggesting that BLF blocks the development of tolerance to morphine, possibly via  the selective activation of nNOS.125 
Spinal Tolerance
Intrathecal coadministration of l-NAME with spinal morphine produced only a small attenuation of tolerance in rats, showing little effect of nitric oxide on morphine tolerance at spinal sites.126 There was evidence indicating that spinal cyclooxygenase activity, and to a lesser extent NOS activity, may contribute to the development and expression of opioid tolerance.127 In a rat spinal model, coadministration of MK-801 inhibited the development of morphine tolerance; the binding affinity of [3H]MK-801 was higher in lumbar spinal cords of morphine-tolerant rats than control rats, and constitutive expression of nNOS protein was also higher in the morphine-tolerant group, this upregulation being prevented by MK-801.128 The authors suggested that morphine tolerance affects NMDA-receptor binding activity and increases nNOS expression in rat spinal cord. Pretreatment with midazolam inhibited the development of acute and chronic morphine tolerance in mice, and this was reversed by intrathecal injection of l-arginine; in chronic morphine-tolerant rats, midazolam decreased formalin-induced expression of Fos and Fos/NADPH diaphorase double-labeled neurons in the contralateral spinal cord and NADPH diaphorase-positive neurons in the bilateral spinal cord.129 It appears that decreases in both the activity and expression of NOS (nNOS and iNOS) contribute to the inhibitory effect of midazolam on the development of morphine tolerance. Intrathecal 7-NI attenuated not only the development of morphine antinociceptive tolerance, but also the activation of p38 mitogen-activated protein kinase in the spinal microglia induced by chronic intrathecal administration of morphine.130 Attenuation of morphine tolerance by nNOS inhibition appears to be associated with reducing p38 mitogen-activated protein kinase activation in the spinal microglia. On the basis of studies measuring the spinal gene expression of heme oxygenase, NOS, soluble guanylyl cyclase, and cyclic GMP-dependent protein kinase, Liang and Clark131 noted that the carbon monoxide/ nitric oxide-cyclic GMP signaling pathway was upregulated after chronic morphine exposure in mice. Xu et al  .132 provided evidence suggesting that activations of metabotropic glutamate receptor subtype-5 and NMDA receptors occur after the appearance of antinociceptive tolerance to morphine in rats and that the activations of these receptors appear to play a role in the development of tolerance and expression of spinal NOS through increased concentration of [Ca2+]i and activation of protein kinase C.
In summary, morphine tolerance involves the activation of NMDA receptors followed by subsequent release of nitric oxide formed by nNOS but not by iNOS (fig. 1); the nitric oxide/carbon monoxide-cyclic GMP pathway may participate in the induced tolerance. Morphine tolerance also appears to be partly mediated by nitric oxide-independent mechanisms.
Opioid tolerance and dependence are distinct phenomena, developing independently of each other.133,134 Administration of l-NA reduced dependence to morphine in mice with implanted morphine pellets and reversed previously established dependence.106 Chronic administration of morphine resulted in the development of physical dependence as evidenced by the appearance of a variety of symptoms including a stereotyped jumping response following naltrexone injection; concurrent treatment with l-NMMA inhibited the naltrexone-induced jumping response, but not other responses.122 l-NAME attenuated the expression phase of morphine dependence, but it did not modify the induction phase of morphine dependence and tolerance in mice.93 Acute and chronic administration of agmatine that inhibits NOS activity135 prevented morphine dependence/withdrawal in wild-type mice; in contrast, agmatine reduced only peripheral signs, not the central signs, of morphine physical dependence in nNOS knockout mice, indicating that the action of agmatine in reducing the central signs requires functional nNOS.136 In addition to inhibition of NOS activity, the effects of agmatine are mediated through imidazoline receptors, α2-adrenoceptors, and blockade of NMDA receptors that may involve peripheral signs of morphine dependence.
On the other hand, Pataki and Telegdy54 reported that neither l-NA nor l-arginine affected the signs of morphine dependence, as assessed by naloxone-precipitated withdrawal in mice. In morphine-dependent rats, 7-NI did not affect naloxone’s discriminative stimulus effects, but it decreased naloxone-induced weight loss and abolished expression of withdrawal signs, such as diarrhea, scream on touch, tremor, and “wet dog” like shaking, suggesting different mechanisms for subjective and somatic components of opioid withdrawal.137 In morphine-dependent rats, pretreatment with l-NA potentiated the cataleptic response to morphine and blocked morphine-induced hyperthermia.138 In the induction phase of morphine dependence, the α2-adrenoceptor agonist clonidine intensified and yohimbine attenuated the degree of dependence; l-NAME did not affect the development of dependence, but it potentiated the effect of clonidine.139 The α2-adrenergic pathway seems to functionally link with the nitric oxide pathway in the modulation of opioid dependence.
In heroin abusers, the levels of lipoperoxides in plasma and erythrocytes and the plasma level of nitric oxide increased with prolonged abuse and with increased daily quantity, whereas the plasma levels of vitamins C and E and β-carotene and the erythrocyte levels of superoxide dismutase, catalase, and glutathione peroxidase decreased.140 The balance between oxidation and antioxidation in the heroin abusers appears to be seriously impaired.
In summary, NOS inhibitors prevent morphine dependence; central signs of morphine dependence may be associated with nitric oxide derived from nNOS (fig. 1). Some investigators report that NOS inhibitors do not affect or even potentiate the signs of morphine dependence.
Withdrawal Syndrome
l-NA and l-NAME intraperitoneally administered 1 h before naloxone reduced “wet dog” shakes and weight loss that were evoked in morphine-dependent rats given naloxone141 or decreased naloxone-precipitated withdrawal jumping and diarrhea in morphine-dependent mice,142 suggesting the involvement of nitric oxide in morphine withdrawal syndrome. NOS inhibition may contribute to treatment of the opioid withdrawal syndrome. Similar results were also obtained in studies using l-NAME and isosorbide dinitrate, a nitric oxide donor, injected shortly before naloxone in morphine-dependent rats.143 Thorat et al  .144 obtained data suggesting that NOS inhibitors may be more beneficial than NMDA-receptor antagonists in managing the symptoms of morphine abstinence syndrome. l-NA and 7-NI attenuated naloxone-precipitated withdrawal signs, such as rearing, jumping, ptosis, rhinorrhoea, and irritability on touch, in morphine-dependent rats.145 Mainly central, but not endothelial, nitric oxide may be involved in the expression of some opioid withdrawal symptoms. l-NA, l-NAME, 7-NI, and N  5-(1-iminoethyl)-l-omithine, a potent inhibitor of endothelial NOS, administered shortly before morphine withdrawal produced decreases in weight loss, diarrhea, wet dog shakes, and grooming; 7-NI also reduced mastication, salivation, and genital effects, and clonidine produced similar effects to 7-NI, indicating that the nNOS inhibitor attenuates more signs of opioid withdrawal than inhibitors of other types of NOS without causing hypertension.146 Insofar as hypertension is a component of opioid withdrawal in humans, the ability of 7-NI to attenuate morphine withdrawal in rats without eliciting a vasopressor response suggests that 7-NI may have human therapeutic potential.147 These authors148 also obtained data suggesting that constitutive NOS isoforms, but not iNOS, have a primary role in nitric oxide-mediated processes that modulate the opioid withdrawal syndrome in the rat. There was evidence suggesting that nNOS and phospholipase A2, but not iNOS, play an important role in the expression of morphine-induced withdrawal syndrome in mice, possibly by increasing free radicals.149 When administered intracerebroventricularly, both l-NA and l-NMMA inhibited natrexone-induced stereotyped jumping behavior in morphine-dependent mice.150 Brain nitric oxide likely plays an important role in the expression of behavioral signs of morphine withdrawal syndrome. In morphine-dependent mice, subcutaneous l-NAME reduced the number of escape jumps and other motor symptoms of abstinence, together with a decrease in NOS activity in the cerebellum, indicating a hyperactivity of the l-arginine/nitric oxide pathway in opiate withdrawal.151 Pretreatment with subcutaneous lamotrigine, a new antiepileptic compound, reversed the withdrawal-induced increase in cerebellar Ca2+-dependent NOS activity and reduced the number of escape jumps and other motor symptoms of abstinence; MK-801 also showed similar effects on cerebellar NOS activity and motor symptoms.152 Treatment of morphine-dependent rats with l-NAME enhanced osmotically-stimulated oxytocin secretion during naloxone-precipitated withdrawal, and sodium nitroprusside inhibited oxytocin neurons during naloxone-precipitated morphine withdrawal.153 
Systemic and intracerebroventricular pretreatment of rats with l-NAME in a dose sufficient to inhibit brain NOS activity blocked the naloxone-precipitated locus coeruleus withdrawal response as measured with an in vivo  voltammetric approach, and it reduced the naloxone-induced increase in the catechol oxidation current signal.154 nitric oxide appears to play an intermediary role in the locus coeruleus neuronal hyperactivity associated with both acute and chronic morphine withdrawal. Jhamandas et al  .155 provided evidence by way of NADPH-diaphorase histochemistry for the activation of select populations of nitric oxide-synthesizing neurons in the paraventricular and supraoptic nuclei, and to a lesser extent in the brainstem nucleus tractus solitarius, during the opioid withdrawal syndrome. The soluble guanylyl cyclase may play an intermediary role in the genesis of locus coeruleus neuronal hyperactivity and behavioral signs of morphine withdrawal.156 In anesthetized rats chronically treated with morphine, intraperitoneal l-NAME attenuated some signs of opioid withdrawal and also reduced the withdrawal-induced hyperactivity of locus coeruleus neurons; intraperitoneal 7-NI caused a complete blockade of the withdrawal-induced hyperactivity, and application of 7-NI to the vicinity of the locus coeruleus also caused a partial blockade, leading to the conclusion that opioid withdrawal may to be mediated by nitric oxide acting as an intermediate messenger in the locus coeruleus.157 In morphine-treated freely moving rats, acute pretreatment with 7-NI or l-NA-p-nitroaniline (nNOS inhibitors) before naltrexone challenge attenuated the behavioral expression of morphine withdrawal and reduced the withdrawal-induced increase in 3,4-dihydroxyphenylacetic acid in the rat locus coeruleus, suggesting a role for nitric oxide in the expression of morphine withdrawal syndrome that may be mediated, at least in part, by locus coeruleus noradrenergic neurons.158 Other noradrenergic nuclei may also be involved in the action of NOS inhibition.
Mice first made dependent to morphine that were then withdrawn by removal of pellets followed by a sublethal dose of lipopolysaccharide exhibited 100% lethality, and these animals had elevated serum tumor necrosis factor-α and nitric oxide levels and depressed interleukin-12 levels compared to controls; anti–tumor necrosis factor-α antibody given at the same time as the lipopolysaccharide challenge afforded protection to morphine-withdrawn mice.159 Morphine withdrawal may sensitize the animals to lipopolysaccharide lethality via  increased production of tumor necrosis factor-α.
Spinal Sites of Action
Intrathecal administration of NMDA receptor antagonists, MK-801 and AP-7, reduced the expression of naloxone-precipitated cardiovascular and behavioral symptoms; l-NAME produced l-arginine-reversible inhibition of the cardiovascular component of withdrawal, but it had no effect on the expression of behavioral signs; in contrast, l-NA and l-NMMA inhibited only the expression of the behavioral signs.160 Both spinal NMDA receptors and a nitric oxide–generating system are suggested to play a role in the expression of both the cardiovascular and behavioral components of naloxone-precipitated withdrawal. However, the fact that different structural analogs of l-arginine have different profiles of activity is puzzling. Naloxone-precipitated morphine withdrawal increased the expression of Fos protein, NADPH-diaphorase-positive neurons, and Fos/NADPH diaphorase double-labeled neurons in all the laminae of the rat spinal cord; intrathecal injection of nNOS antisense oligonucleotides inhibited the increase in Fos expression and NMDA1A-receptor mRNA expression during morphine withdrawal and decreased the scores of morphine withdrawal symptoms.161 The authors concluded that nitric oxide seems to mediate the increase of Fos protein and NMDA1A-receptor mRNA expression in the spinal cord during morphine withdrawal. On the basis of immunohistochemical studies, morphine induced c-Fos expression in the striatum, cerebral cortex, and midline/intralaminar nuclei of the thalamus in rats; expression in the striatum, but not the thalamus or cortex, was blocked by 7-NI, and there was no colocalization of c-Fos and nNOS in any brain region, suggesting a role for nNOS in the neural circuits activated by morphine.162 Cao et al  .163 provided evidence supporting the idea that cross talk between nitric oxide and the extracellular signal regulated kinase 1 and 2 signaling pathway mediates morphine withdrawal and withdrawal-induced spinal neuronal sensitization in morphine-dependent rats. Intrathecal injection of muscarinic M2-receptor antisense oligonucleotides decreased the scores of morphine withdrawal symptoms; the expression of nNOS-positive neurons in the locus coeruleus increased in morphine-dependent rats and increased to a greater extent during morphine withdrawal, and intrathecal injection of M2antisense oligonucleotide inhibited the increase in nNOS expression.164 M2muscarinic receptors of the spinal cord appear to mediate the increase of nNOS expression in the locus coeruleus during morphine withdrawal.
As presented so far, naloxone-precipitated withdrawal responses are attenuated by NOS inhibitors, possibly via  the activation of guanylyl cyclase; nNOS-selective inhibitors are more effective than inhibitors of other types of NOS isoforms (fig. 1) and do not raise systemic blood pressure; these effects may be therapeutically beneficial for the prevention of withdrawal syndrome.
Summary and Conclusion
The current article includes a summary of the interactions between morphine/other opioids and nitric oxide in antinociception, dependence, tolerance, and withdrawal syndrome. Some investigators have reported that NOS inhibitors attenuate the antinociceptive effects of morphine; in contrast, others have shown that NOS inhibition augments morphine-induced analgesia in experimental animals. Limited studies on humans suggest the involvement of nitric oxide in morphine-induced antinociception. Dependence on morphine is either enhanced or inhibited by endogenous nitric oxide; however, tolerance to morphine and morphine withdrawal syndrome are potentiated by nitric oxide, as so far reported. Although the reason for the controversial results on morphine-nitric oxide interactions in experimental animals remains to be determined, information included in the present review should contribute to the construction of advanced strategies for therapy with morphine, with the goals of effectively eliminating nociception and minimizing side effects. However, further, consolidated studies on human materials, healthy individuals, and patients are eagerly awaited.
Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature (Lond) 1980; 288:373–6Furchgott, RF Zawadzki, JV
Furchgott RF: Studies on relaxation of rabbit aorta by sodium nitrite: The basis for the proposal that the acid-activatable inhibitory factor from bovine retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide, Vasodilatation: Vascular Smooth Muscle, Autonomic Nerves, and Endothelium. Edited by Vanhoutte PM, New York, Raven Press, 1988, pp. 401–14Furchgott, RF Vanhoutte PM New York Raven Press
Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G: Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A 1987; 84:9265–9Ignarro, LJ Buga, GM Wood, KS Byrns, RE Chaudhuri, G
Palmer RM, Ferrige AB, Moncada S: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature (Lond) 1987; 327:524–6Palmer, RM Ferrige, AB Moncada, S
Bredt DS, Snyder GH: Isolation of nitric oxide synthetase, a calmodulin- requiring enzyme. Proc Natl Acad Sci U S A 1990; 87:682–5Bredt, DS Snyder, GH
Hebel JM, White KA, Marletta MA: Purification of the inducible murine macrophage nitric oxide synthase: identification as a flavo-protein. J Biol Chem 1991; 266:22789–91Hebel, JM White, KA Marletta, MA
Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, MuirheadG, Naylor AM, Osterloch IH, Gengell C: Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res 1996; 8:47–52
Toda N, Ayajiki K, Okamura T: Nitric oxide and penile erectile function. Pharmacol Ther 2005; 106:233–66Toda, N Ayajiki, K Okamura, T
Toda N, Toda H, Hatano Y: Nitric oxide: Involvement in the effects of anesthetic agents. Anesthesiology 2007; 107:822–42Toda, N Toda, H Hatano, Y
Förstermann U, Pollock JS, Schmidt HH, Heller M, Murad F: Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc Nat Acad Sci U S A 1991; 88:1788–92Förstermann, U Pollock, JS Schmidt, HH Heller, M Murad, F
Palmer RMJ, Rees DD, Ashton DS, Moncada S: L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun 1988; 153:1251–6Palmer, RMJ Rees, DD Ashton, DS Moncada, S
Rees DD, Palmer RMJ, Schulz R, Hodson HF, Moncada S: Characterization of three inhibitors of endothelial nitric oxide synthase in vivo  and in vitro  . Br J Pharmacol 1990; 101:746–52Rees, DD Palmer, RMJ Schulz, R Hodson, HF Moncada, S
Toda N, Minami Y, Okamura T: Inhibitory effects of L-NG-nitro-arginine on the synthesis of EDRF and the cerebroarterial response to vasodilator nerve stimulation. Life Sci 1990; 47:345–51Toda, N Minami, Y Okamura, T
Vallance P, Leone A, Calver A, Collier J, Moncada S: Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis. J Cardiovasc Pharmacol 1992; 20(Suppl12):S60–2Vallance, P Leone, A Calver, A Collier, J Moncada, S
Moore PK, Babbedge RC, Wallace P, Gaffen ZA, Hart SL: 7-Nitroindazole, an inhibitor of nitric oxide synthase, exhibits antinociceptive activity in the mouse without increasing blood pressure. Br J Pharmacol 1993; 108:296–7Moore, PK Babbedge, RC Wallace, P Gaffen, ZA Hart, SL
Griffiths MJ, Messent M, MacAllister RJ, Evans TW: Aminoguanidine selectively inhibits inducible nitric oxide synthase. Br J Pharmacol 1993; 110:963–8Griffiths, MJ Messent, M MacAllister, RJ Evans, TW
Garthwaite J, Southam E, Boulton CL, Nielsen EB, Schmidt K, Mayer B: Potential and selective inhibition of nitric oxide sensitive guanylate cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-on. Mol Pharmacol 1995; 48:184–8Garthwaite, J Southam, E Boulton, CL Nielsen, EB Schmidt, K Mayer, B
Mixcoatl-Zecuatl T, Flores-Murrieta FJ, Granados-Soto V: The nitric oxide-cyclic GMP-protein kinase G-K+ channel pathway participates in the antiallodynic effect of spinal gabapentin. Eur J Pharmacol 2006; 531:87–95Mixcoatl-Zecuatl, T Flores-Murrieta, FJ Granados-Soto, V
Brito GA, Sachs D, Cunha FQ, Vale ML, Lotufo CM, Ferreira SH, Ribeiro RA: Peripheral antinociceptive effect of pertussis toxin: activation of the arginine/NO/cGMP/PKG/ATP-sensitive K channel pathway. Eur J Neurosci 2006; 24:1175–81Brito, GA Sachs, D Cunha, FQ Vale, ML Lotufo, CM Ferreira, SH Ribeiro, RA
Oritz MI, Medina-Tat DA, Sarmiento-Heredia D, Palma-Martinez J, Ganados-Soto V: Possible activation of the NO-cyclic GMP-protein kinase G-K+ channels pathway by gabapentin on the formalin test. Pharmacol Biochem Behav 2006; 83:420–7Oritz, MI Medina-Tat, DA Sarmiento-Heredia, D Palma-Martinez, J Ganados-Soto, V
Scavone C, Munhoz CD, Kawamoto EM, Glezer I, de Sa Lima L, Marcourakis T, Markus RP: Age-related changes in cyclic GMP and PKG-stimulated cerebellar Na,K-ATPase activity. Neurobiol Aging 2005; 26:907–16Scavone, C Munhoz, CD Kawamoto, EM Glezer, I de Sa Lima, L Marcourakis, T Markus, RP
Ramamoorthy S, Samuvel DJ, Buck ER, Rudnick G, Jayanthi LD: Phosphorylation of threonine residue 276 is required for acute regulation of serotonin transporter by cyclic GMP. J Biol Chem 2007; 282:11639–47Ramamoorthy, S Samuvel, DJ Buck, ER Rudnick, G Jayanthi, LD
Zhang YW, Gesmonde J, Ramamoorthy S, Rudnick G: Serotonin transporter phosphorylation by cGMP-dependent protein kinase is altered by a mutation associated with obsessive compulsive disorder. J Neurosci 2007; 27:10878–86Zhang, YW Gesmonde, J Ramamoorthy, S Rudnick, G
Toda N, Okamura T: Possible role of nitric oxide in transmitting information from vasodilator nerve to cerebral arterial muscle. Biochem Biophys Res Commun 1990; 170:308–13Toda, N Okamura, T
Zheng Z, Shimamura K, Anthony TL, Travagli RA, Kreulen DL: Nitric oxide is a sensory nerve neurotransmitter in the mesenteric artery of guinea pig. J Auton Nerv Syst 1997; 29:137–44Zheng, Z Shimamura, K Anthony, TL Travagli, RA Kreulen, DL
Zochodne DW, Levy D: Nitric oxide in damage, disease and repair of the peripheral nervous system. Cell Mol Biol 2005; 51:255–67Zochodne, DW Levy, D
Holzer P: Efferent-like roles of afferent neurons in the gut: blood flow regulation and tissue protection. Auton Neurosci 2006; 125:70–5Holzer, P
Kiss JP, Vizi ES: Nitric oxide: a novel link between synaptic and nonsynaptic transmission. Trends Neurosci 2001; 24:211–5Kiss, JP Vizi, ES
Riedel W, Neeck G: Nociception, pain, and antinociception: current concepts. Z Rheumatol 2001; 60:404–15Riedel, W Neeck, G
Levy D, Zochodne DW: NO pain: potential roles of nitric oxide in neuropathic pain. Pain Pract 2004; 4:11–8Levy, D Zochodne, DW
Bargaud JL, Riffaud JP, Del Soldato P: Nitric-oxide releasing molecules: a new class of drugs with several major indications. Curr Pharm Des 2002; 8:201–13Bargaud, JL Riffaud, JP Del, Soldato P
Romero-Sandoval EA, Curros-Criado MM, Gaitan G, Molina Herrero JF: Nitroparacetamol (NCX-701) and pain: first in a series of novel analgesics. CNS Drug Rev 2007; 13:279–95Romero-Sandoval, EA Curros-Criado, MM Gaitan, G Molina Herrero, JF
Leza JC, Lizasoain I, San-Martin-Clark O, Lorenzo P: Morphine-induced changes in cerebral and cerebellar nitric oxide synthase activity. Eur J Pharmacol 1995; 285:95–8Leza, JC Lizasoain, I San-Martin-Clark, O Lorenzo, P
Machelska H, Ziolkowska B, Mika J, Prezewlocka B, Prezewlocki R: Chronic morphine increases biosynthesis of nitric oxide synthase in the rat spinal cord. Neuroreport 1997; 8:2743–7Machelska, H Ziolkowska, B Mika, J Prezewlocka, B Prezewlocki, R
Kumar S, Bhargava HN: Time course of the changes in central nitric oxide synthase activity following chronic treatment with morphine in the mouse: reversal by naltrexone. Gen Pharmacol 1997; 223–7Kumar, S Bhargava, HN
Nieto-Fernandez FE, Mattocks D, Cavani F, Salzet M, Stefano GB: Morphine coupling to invertebrate immunocyte nitric oxide release is dependent on intracellular calcium transients. Comp Biochem Physiol B Biochem Mol Biol 1999; 123:295–9Nieto-Fernandez, FE Mattocks, D Cavani, F Salzet, M Stefano, GB
Yoo JH, Cho JH, Lee SY, Lee S, Loh HH, Ho IK, Jang CG: Differential effects of morphine- and cocaine-induced nNOS immunoreactivity in the dentate gyrus of hippocampus of mice lacking μ-opioid receptors. Neurosci Lett 2006; 395:98–102Yoo, JH Cho, JH Lee, SY Lee, S Loh, HH Ho, IK Jang, CG
Barjavel MJ, Bhargava HN: Effect of opioid receptor agonists on nitric oxide synthase activity in rat cerebral cortex homogenate. Neurosci Lett 1994; 181:27–30Barjavel, MJ Bhargava, HN
Babey AM, Kolesnikov Y, Cheng J, Inturrisi CE, Trifilletti RR, Pasternak GW: Nitric oxide and opioid tolerance. Neuropharmacology 1994; 33:1463–70Babey, AM Kolesnikov, Y Cheng, J Inturrisi, CE Trifilletti, RR Pasternak, GW
Dyuizen IV, Motavkin PA, Shorin VV: Dynamics of NADPH diaphorase activity in raphe neurons during chronic treatment with opiates. Bull Exp Biol Med 2001; 132:918–20Dyuizen, IV Motavkin, PA Shorin, VV
Dyuizen IV, Deridovich II, Kurbatskii RA, Shorin VV: NO-ergic neurons of the cervical nucleus of the rat brain in normal conditions and after administration of opiates. Neurosci Behav Physiol 2004; 34:621–6Dyuizen, IV Deridovich, II Kurbatskii, RA Shorin, VV
Stefano GB, Hartman A, Bilfinger TV, Magazine HI, Liu Y, Casares F, Goligorsky MS: Presence of the μ3 opiate receptor in endothelial cells. Coupling to nitric oxide production and vasodilation. J Biol Chem 1995; 270:30290–3Stefano, GB Hartman, A Bilfinger, TV Magazine, HI Liu, Y Casares, F Goligorsky, MS
Stefano GB, Salzet M, Bilfinger TV: Long-term exposure of human blood vessels to HIV gp120, morphine, and anandamide increases endothelial adhesion of monocytes: uncoupling of nitric oxide release. J Cardiovac Pharmacol 1998; 31:862–9Stefano, GB Salzet, M Bilfinger, TV
Xu Z, Tong C, Pan HL, Cerda SE, Eisenach JC: Intravenous morphine increases release of nitric oxide from spinal cord by an a-adrenergic and cholinergic mechanism. J Neurophysiol 1997; 78:2072–8Xu, Z Tong, C Pan, HL Cerda, SE Eisenach, JC
Fimiani C, Arcuri E, Santoni A, Rialas CM, Bilfinger TV, Peter D, Salzet B, Stefano GB: μ3 Opiate receptor expression in lung and lung carcinoma: ligand binding and coupling to nitric oxide release. Cancer Lett 1999; 146:45–51Fimiani, C Arcuri, E Santoni, A Rialas, CM Bilfinger, TV Peter, D Salzet, B Stefano, GB
Zhu W, Ma Y, Bell A, Esch T, Guarna M, Bilfinger TV, Bianchi E, Stefano GB: Presence of morphine in rat amygdala: evidence for the μ3 opiate receptor subtypes via  nitric oxide release in limbic structures. Med Sci Monit 2004; 10:BR433–9Zhu, W Ma, Y Bell, A Esch, T Guarna, M Bilfinger, TV Bianchi, E Stefano, GB
Tseng L, Mazella J, Goligorsky MS, Rialas CM, Stefano GB: Dopamine and morphine stimulate nitric oxide release in human endometrial glandular epithelial cells. J Soc Gynecol Investig 2000; 7:343–7Tseng, L Mazella, J Goligorsky, MS Rialas, CM Stefano, GB
Sarić A, Balog T, Sobocanec S, Marotti T: Endomorphine 1 activates nitric oxide synthase 2 activity and downregulates nitric oxide synthase 2 mRNA expression. Neuroscience 2007; 144:1454–61Sarić, A Balog, T Sobocanec, S Marotti, T
Kielstein A, Tsikas D, Galloway GP, Mendelson JE: Asymmetric dimethylarginine (ADMA)—a modulator of nociception in opiate tolerance and addiction? Nitric Oxide 2007; 17:55–9Kielstein, A Tsikas, D Galloway, GP Mendelson, JE
Pu S, Horvath TL, Diano S, Naftolin F, Kalra PS, Kalra SP: Evidence showing that β-endorphin regulates cyclic guanosine 3′,5′-monophosphate (cGMP) efflux: anatomical and functional support for an interaction between opiates and nitric oxide. Endocrinology 1997; 138:1537–43Pu, S Horvath, TL Diano, S Naftolin, F Kalra, PS Kalra, SP
Ferreira SH, Duarte ID, Lorenzetti BB: Molecular base of acetylcholine and morphine analgesia. Agents Actions Supple 1991; 32:101–6Ferreira, SH Duarte, ID Lorenzetti, BB
Duarte ID, Ferreira SH: The molecular mechanism of central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway. Eur J Pharmacol 1992; 221:171–4Duarte, ID Ferreira, SH
Xu JY, Tseng LF: Role of nitric oxide/cyclic GMP in i.c.v. administrated β-endorphin- and (+)-cis-dioxolane-induced antinociception in the mouse. Eur J Pharmacol 1994; 262:223–31Xu, JY Tseng, LF
Pataki I, Telegdy G: Further evidence that nitric oxide modifies acute and chronic morphine actions in mice. Eur J Pharmacol 1998; 357:157–62Pataki, I Telegdy, G
Chung E, Burke B, Bieber AJ, Doss JC, Ohgami Y, Quock RM: Dynorphin-mediated antinociceptive effects of L-arginine and SIN-1 (an NO donor) in mice. Brain Res Bull 2006; 70:245–50Chung, E Burke, B Bieber, AJ Doss, JC Ohgami, Y Quock, RM
Javanmardi K, Parviz M, Sadr SS, Keshavarz M, Minaii B, Dehpour AR: Involvement of N-methyl-D-aspartate receptors and nitric oxide in the rostral ventromedial medulla in modulating morphine pain-inhibitory signals from the periaqueductal grey matter in rats. Clin Exp Pharmacol Physiol 2005; 32:585–9Javanmardi, K Parviz, M Sadr, SS Keshavarz, M Minaii, B Dehpour, AR
Moore PK, Oluyomi AO, Babbedge RC, Wallace P, Hart SL: L-NG-nitro arginine methyl ester exhibits antinociceptive activity in the mouse. Br J Pharmacol 1991; 102:198–202Moore, PK Oluyomi, AO Babbedge, RC Wallace, P Hart, SL
Pelligrino DA, Laurito CE, VadeBoncouer TR: Nitric oxide synthase inhibition modulates the ventilatory depression and antinociceptive actions of fourth ventricular infusions of morphine in the awake dog. Anesthesiology 1996; 85:1367–77Pelligrino, DA Laurito, CE VadeBoncouer, TR
Bhargava HN, Bian JT: Effects of acute and chronic administration of L-arginine on the antinociceptive action of morphine-6-β-D-glucuronide. Pharmacology 1997; 55:165–72Bhargava, HN Bian, JT
Bhargava HN, Bian JT: NG-nitro-L-arginine reversed L-arginine induced changes in morphine antinociception and distribution of morphine in brain regions and spinal cord of the mouse. Brain Res 1997; 749:351–3Bhargava, HN Bian, JT
Bian JT, Bhargava HN: Effect of chronic administration of L-arginine, NG-nitro-L-arginine or their combination on morphine concentration in peripheral and urine of the mouse. Gen Pharmacol 1998; 30:753–7Bian, JT Bhargava, HN
Bhargava HN, Bian JT, Kumar S: Mechanism of attenuation of morphine antinociception by chronic treatment with L-arginine. J Pharmacol Exp Ther 1997; 281:707–12Bhargava, HN Bian, JT Kumar, S
Bhargava HN, Bian JT: Effects of acute administration of L-arginine on morphine antinociception and morphine distribution in central and peripheral tissues of mice. Pharmacol Biochem Behav 1998; 61:29–33Bhargava, HN Bian, JT
Grover VS, Sharma A, Singh M: Role of nitric oxide in diabetes-induced attenuation of antinociceptive effect of morphine in mice. Eur J Pharmacol 2000; 399:161–4Grover, VS Sharma, A Singh, M
Terenghi G, Riveros-Moreno V, Hudson LD, Ibrahim NB, Polak JM: Immunohistochemistry of nitric oxide synthase demonstrates immunoreactive neurons in spinal cord and dorsal ganglia of man and rat. J Neurol Sci 1993; 118:34–7Terenghi, G Riveros-Moreno, V Hudson, LD Ibrahim, NB Polak, JM
Kolesnikov YA, Pan YX, Babey AM, Jain S, Wilson R, Pasternak GW: Functionally differentiating two neuronal nitric oxide synthase isoforms through antisense mapping: evidence for opposing NO actions on morphine analgesia and tolerance. Proc Natl Acad Sci U S A 1997; 94:8220–5Kolesnikov, YA Pan, YX Babey, AM Jain, S Wilson, R Pasternak, GW
Song HK, Pan HL, Eisenach JC: Spinal nitric oxide mediates antinociception from intravenous morphine. Anesthesiology 1998; 89:215–21Song, HK Pan, HL Eisenach, JC
Hayashida K, Takeuchi T, Shimizu H, Ando K, Harada E: Lactoferrin enhances opioid-mediated analgesia via  nitric oxide in the rat spinal cord. Am J Physiol 2003; 285:R302–5Hayashida, K Takeuchi, T Shimizu, H Ando, K Harada, E
Chen SR, Pan HL: Spinal nitric oxide contributes to the analgesic effect of intrathecal [d-pen2,d-pen5]-enkephalin in normal and diabetic rats. Anesthesiology 2003; 98:217–22Chen, SR Pan, HL
Yamaguchi H, Naito H: Antinociceptive synergistic interaction between morphine and Nω-nitro-L-arginine methyl ester on thermal nociceptive tests in the rats. Can J Anaesth 1996; 43:975–81Yamaguchi, H Naito, H
Przewlocki R, Machelska H, Przewlocka B: Inhibition of nitric oxide synthase enhances morphine antinociception in the rat spinal cord. Life Sci 1993; 53:PL1–5Przewlocki, R Machelska, H Przewlocka, B
Machelska H, Labuz D, Prezewlocki R, Prezewlocka B: Inhibition of nitric oxide synthase enhances antinociception mediated by mu, delta, and kappa opioid receptors in acute and prolonged pain in the rat spinal cord. J Pharmacol Exp Ther 1997; 282:977–84Machelska, H Labuz, D Prezewlocki, R Prezewlocka, B
Li X, Clark JD: Spinal cord nitric oxide synthase and heme oxygenase limit morphine induced analgesia. Mol Brain Res 2001; 95:96–102Li, X Clark, JD
Watanabe C, Sakurada T, Okuda K, Sakurada C, Ando R, Sakurada S: The role of spinal nitric oxide and glutamate in nociceptive behaviour evoked by high-dose intrathecal morphine in rats. Pain 2003; 106:269–83Watanabe, C Sakurada, T Okuda, K Sakurada, C Ando, R Sakurada, S
Watanabe C, Okuda K, Sakurada C, Ando R, Sakurada T, Sakurada S: Evidence that nitric oxide-glutamate cascade modulates spinal antinociceptive effect of morphine: a behavioural and microdialysis study in rats. Brain Res 2003; 990:77–86Watanabe, C Okuda, K Sakurada, C Ando, R Sakurada, T Sakurada, S
Durate ID, Lorenzetti BB, Ferreira SH: Peripheral analgesia and activation of the nitric oxide-cyclic GMP pathway. Eur J Pharmacol 1990; 186:289–93Durate, ID Lorenzetti, BB Ferreira, SH
Ferreira SH, Duarte ID, Lorenzetti BB: The molecular mechanism of action of peripheral morphine analgesia: stimulation of the cGMP system via  nitric oxide release. Eur J Pharmacol 1991b; 201:121–2Ferreira, SH Duarte, ID Lorenzetti, BB
Granados-Soto V, Rufino MO, Gomes Lopes LD, Ferreira SH: Evidence for the involvement of the nitric oxide-cGMP pathway in the antinociception of morphine in the formalin test. Eur J Pharmacol 1997; 340:177–80Granados-Soto, V Rufino, MO Gomes Lopes, LD Ferreira, SH
Nozaki-Taguchi N, Yamamoto T: The interaction of FK409, a novel nitric oxide releaser, and peripherally administered morphine during experimental inflammation. Anesth Analg 1998; 86:367–73Nozaki-Taguchi, N Yamamoto, T
Aquirre-Banuelos P, Granados-Soto V: Evidence for a peripheral mechanism of action for the potentiation of the antinociceptive effect of morphine by dipyrone. J Pharmacol Toxicol Methods 1999; 42:79–85Aquirre-Banuelos, P Granados-Soto, V
Mixcoatl-Zecuatl T, Aguirre-Banuelos P, Granadoes-Soto V: Sildenafil produces antinociception and increases morphine antinociception in the formalin test. Eur J Pharmacol 2000; 400:81–7Mixcoatl-Zecuatl, T Aguirre-Banuelos, P Granadoes-Soto, V
Jain NK, Patil CS, Singh A, Kulkarni SK: Sildenafil, a phosphodiesterase5 inhibitor, enhances the antinociceptive effect of morphine. Pharmacology 2003; 67:150–6Jain, NK Patil, CS Singh, A Kulkarni, SK
Hayashida K, Takeuchi T, Harada E: Lactoferrin enhances peripheral opioid-mediated antinociception via  nitric oxide in rats. Eur J Pharmacol 2004; 484:175–81Hayashida, K Takeuchi, T Harada, E
Oritz MI, Castro-Olguin J, Pena-Samaniego N, Castaneda-Hernandez G: Probable activation of the opioid receptor-nitric oxide-cyclic GMP-K+ channels pathway by codeine. Pharmacol Biochem Behav 2005; 82:695–703Oritz, MI Castro-Olguin, J Pena-Samaniego, N Castaneda-Hernandez, G
Stanojevic S, Mitic K, Vujic V, Kovacevic-Jovanovid V, Dimitrijevic M: Beta-endorphine differentially affects inflammation in two inbred rat strains. Eur J Pharmacol 2006; 549:157–65Stanojevic, S Mitic, K Vujic, V Kovacevic-Jovanovid, V Dimitrijevic, M
Okun R, Liddon SC, Lasagnal L: The effects of aggregation, electric shock, and adrenergic blocking drug on inhibition of the “writhing syndrome.” J Pharmacol Exp Ther 1963; 139:107–9Okun, R Liddon, SC Lasagnal, L
Abacioglu N, Ozmen R, Cakici I, Tunctan B, Kanzik I: Role of L-arginine/nitric oxide pathway in the antinociceptive activities of morphine and mepyramine in mice. Arzneimittelforschung 2001; 51:977–83Abacioglu, N Ozmen, R Cakici, I Tunctan, B Kanzik, I
Homayoun H, Khavandgar S, Dehpour AR: The selective role of nitric oxide in opioid-mediated foodshock stress antinociception in mice. Physiol Behav 2003; 79:567–73Homayoun, H Khavandgar, S Dehpour, AR
Brignola G, Calignano A, Di Rosa M: Modulation of morphine antinociception in the mouse by endogenous nitric oxide. Br J Pharmacol 1994; 113:1372–6Brignola, G Calignano, A Di, Rosa M
Guney HZ, Gorgun CZ, Tunctan B, Uludag O, Hodoglugil U, Abacioglu N, Zengil H: Circadian-rhythm-dependent effects of L-NG-nitroarginine methyl ester (L-NAME) on morphine-induced analgesia. Chronobiol Int 1998; 15:283–9Guney, HZ Gorgun, CZ Tunctan, B Uludag, O Hodoglugil, U Abacioglu, N Zengil, H
Dixon SJ, Persinger MA: Suppression of analgesia in rats induced by morphine or L-NAME but not both drugs by micro Tesla, frequency-modulated magnetic fields. Int J Neurosci 2001; 108:87–97Dixon, SJ Persinger, MA
Homayoun H, Khavandgar S, Namiranian K, Dehpour AR: The effect of cyclosporine A on morphine tolerance and dependence: involvement of L-arginine/nitric oxide pathway. Eur J Pharmacol 2002; 452:67–75Homayoun, H Khavandgar, S Namiranian, K Dehpour, AR
Ozek M, Uresin Y, Gungor M: Comparison of the effects of specific and nonspecific inhibition of nitric oxide synthase on morphine analgesia, tolerance and dependence in mice. Life Sci 2003; 72:1943–51Ozek, M Uresin, Y Gungor, M
Khattab MM, El-Hadiyah TM, Al-Shabanah OA, Raza M: Modification by L-NAME of codeine induced analgesia: possible role of nitric oxide. Receptors Channels 2004; 10:139–45Khattab, MM El-Hadiyah, TM Al-Shabanah, OA Raza, M
Singal A, Anjaneyulu M, Chopra K: Modulatory role of green tea extract on antinociceptive effect of morphine in diabetic mice. J Med Food 2005; 8:386–91Singal, A Anjaneyulu, M Chopra, K
Altug S, Uludag O, Tunctan B, Cakici I, Zengil H, Abacioglu N: Biological time-dependent difference in effect of peroxynitrite demonstrated by the mouse hot plate pain model. Chronobiol Int 2006; 23:583–91Altug, S Uludag, O Tunctan, B Cakici, I Zengil, H Abacioglu, N
Lauretti GR, Lima IC, Reis MP, Prado WA, Pereira NL: Oral ketamine and transdermal nitroglycerin as analgesic adjuvants to oral morphine therapy for cancer pain management. Anesthesiology 1999; 90:1528–33Lauretti, GR Lima, IC Reis, MP Prado, WA Pereira, NL
Lauretti GR, Perez MV, Reis MP, Pereira NL: Double-blind evaluation of transdermal nitroglycerine as adjuvant to oral morphine for cancer pain management. J Clin Anesth 2002; 14:83–6Lauretti, GR Perez, MV Reis, MP Pereira, NL
Iohom G, Abdalla H, O’Brien J, Szarvas S, Larney V, Buckley E, Butler M, Shorten GD: The associations between severity of early postoperative pain, chronic postsurgical pain and plasma concentration of stable nitric oxide products after breast surgery. Anesth Analg 2006; 103:995–1000Iohom, G Abdalla, H O’Brien, J Szarvas, S Larney, V Buckley, E Butler, M Shorten, GD
Sousa AM, Prado WA: The dual effect of a nitric oxide donor in nociception. Brain Res 2001; 897:9–19Sousa, AM Prado, WA
Kolesnikov YA, Pick CG, Pasternak GW: NG-nitro-L-arginine prevents morphine tolerance. Eur J Pharmacol 1992; 221:399–400Kolesnikov, YA Pick, CG Pasternak, GW
Elliott K, Minami N, Kolesnikov YA, Pasternak GW, Inturrisi CE: The NMDA receptor antagonists, LY274614 and MK-804, and the nitric oxide synthase inhibitor, NG-nitro-L-arginine, attenuate analgesic tolerance to the μ-opioid morphine but not to κ opioids. Pain 1994; 56:69–75Elliott, K Minami, N Kolesnikov, YA Pasternak, GW Inturrisi, CE
Majeed NH, Przewlocka B, Machelska H, Przewlocki R: Inhibition of nitric oxide synthase attenuates the development of morphine tolerance and dependence in mice. Neuropharmacology 1994; 33:189–92Majeed, NH Przewlocka, B Machelska, H Przewlocki, R
Highfield DA, Grant S: NG-nitro-L-arginine, an NOS inhibitor, reduces tolerance to morphine in the rat locus coeruleus. Synapse 1998; 29:233–9Highfield, DA Grant, S
Homayoun H, Khavandgar S, Namiranian K, Dehpour AR: The effect of cyclosporine A on morphine tolerance and dependence: involvement of L-arginine/nitric oxide pathway. Eur J Pharmacol 2002; 452:67–75Homayoun, H Khavandgar, S Namiranian, K Dehpour, AR
Kolesnikov YA, Pick CG, Ciszewska G, Pasternak GW: Blockade of tolerance but not to κ opioids by a nitric oxide synthase inhibitor. Proc Natl Acad Sci USA 1993; 90:5162–6Kolesnikov, YA Pick, CG Ciszewska, G Pasternak, GW
Bhargava HN, Zhao GM: Effect of nitric oxide synthase inhibition on tolerance to the analgesic action of D-Pen2,D-Pen5 enkephalin and morphine in the mouse. Neuropeptides 1996; 30:219–23Bhargava, HN Zhao, GM
Zhao GM, Bhargava HN: Nitric oxide synthase inhibition attenuates tolerance to morphine but not to [D-Ala2,Glu4]deltorphin II, a δ2-opioid receptor agonist in mice. Peptides 1996; 17:619–23Zhao, GM Bhargava, HN
Bhargava HN, Cao YJ, Zhao GM: Effect of 7-nitroindazole on tolerance to morphine, U-50,488H and [D-Pen2,D-Pen5]enkephalin in mice. Peptides 1997; 18:797–800Bhargava, HN Cao, YJ Zhao, GM
Xu JY, Hill KP, Bidlack JM: The nitric oxide/cyclic GMP system at the supraspinal site is involved in the development of acute morphine antinociceptive tolerance. J Pharmacol Exp Ther 1998; 284:196–201Xu, JY Hill, KP Bidlack, JM
Santamarta MT, Ulibarri I, Pineda J: Inhibition of neuronal nitric oxide synthase attenuates the development of morphine tolerance in rats. Synapse 2005; 57:38–46Santamarta, MT Ulibarri, I Pineda, J
Herraez-Baranda LA, Carretero J, Gonzalez-Sarmiento R, Laorden ML, Milanes MV, Rodriguez RE: Evidence of involvement of the nNOS and the κ-opioid receptor in the same intracellular network of the rat periaqueductal gray that controls morphine tolerance and dependence. Brain Res Mol Brain Res 2005; 137:166–73Herraez-Baranda, LA Carretero, J Gonzalez-Sarmiento, R Laorden, ML Milanes, MV Rodriguez, RE
Abdel-Zaher AO, Hamdy MM, Aly SA, Abdel-Hady RH, Abdel-Rahman SRH: Attenuation of morphine tolerance and dependence by aminoguanidine in mice. Eur J Pharmacol 2006; 540:60–6Abdel-Zaher, AO Hamdy, MM Aly, SA Abdel-Hady, RH Abdel-Rahman, SRH
Bhargava HN, Kumar S, Barjavel MJ: Kinetic properties of nitric oxide synthase in cerebral cortex and cerebellum of morphine tolerant mice. Pharmacology 1998; 56:252–6Bhargava, HN Kumar, S Barjavel, MJ
Lue WM, Su MT, Lin WB, Tao PL: The role of nitric oxide in the development of morphine tolerance in rat hippocampal slices. Eur J Pharmacol 1999; 383:129–35Lue, WM Su, MT Lin, WB Tao, PL
Heinzen EL, Pollack GM: Pharmacodynamics of morphine-induced neuronal nitric oxide production and antinociceptive tolerance development. Brain Res 2004; 1023:175–84Heinzen, EL Pollack, GM
Heinzen EL, Pollack GM: The development of morphine antinociceptive tolerance in nitric oxide synthase-deficient mice. Biochem Pharmacol 2004; 67:735–41Heinzen, EL Pollack, GM
Heinzen EL, Booth RG, Pollack GM: Neuronal nitric oxide modulates morphine antinociceptive tolerance by enhancing constitutive activity of the μ-opioid receptor. Biochem Pharmacol 2005; 69:679–88Heinzen, EL Booth, RG Pollack, GM
Leiper J, Vallance P: Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res 1999; 43:542–8Leiper, J Vallance, P
Kielstein A, Tsikas D, Galloway GP, Mendelson JE: Asymmetric dimethylarginine (ADMA)—a modulator of nociception in opiate tolerance and addiction? Nitric Oxide 2007; 17:55–9Kielstein, A Tsikas, D Galloway, GP Mendelson, JE
Muscoli C, Cuzzocrea S, Ndengele MM, Mollace V, Porreca F, Fabrizi F, Esposito E, Masini E, Matsuschak GM, Salvemini D: Therapeutic manipulation of peroxynitrite attenuates the development of opoiate-induced antinociceptive tolerance in mice. J Clin Invest 2007; 117:3530–9Muscoli, C Cuzzocrea, S Ndengele, MM Mollace, V Porreca, F Fabrizi, F Esposito, E Masini, E Matsuschak, GM Salvemini, D
Bhargava HN: Attenuation of tolerance to, and physical dependence on, morphine in the rat by inhibition of nitric oxide synthase. Gen Pharmacol 1995; 26:1049–53Bhargava, HN
Dambisya YM, Lee TL: Role of nitric oxide in the induction and expression of morphine tolerance and dependence in mice. Br J Pharmacol 1996; 117:914–8Dambisya, YM Lee, TL
Rauhala P, Idanpaan-Heikkila JJ, Tuominen RK, Mannisto PT: N-nitro-L-arginine attenuates development of tolerance to antinociceptive but not to hormonal effects of morphine. Eur J Pharmacol 1994; 259:57–64Rauhala, P Idanpaan-Heikkila, JJ Tuominen, RK Mannisto, PT
Tsuchiya T, Takeuchi T, Hayashida K, Shimizu H, Ando K, Harada E: Milk-derived lactoferrin may block tolerance to morphine analgesia. Brain Res 2006; 1068:102–8Tsuchiya, T Takeuchi, T Hayashida, K Shimizu, H Ando, K Harada, E
Dunbar S, Yaksh TL: Effect of spinal infusion of L-NAME, a nitric oxide synthase inhibitor, on spinal tolerance and dependence induced by chronic intrathecal morphine in the rat. Neurosci Lett 1996; 207:33–6Dunbar, S Yaksh, TL
Powell KJ, Hosokawa A, Bell A, Sutak M, Milne B, Quirion R, Jhamandas K: Comparative effects of cyclo-oxygenase and nitric oxide synthase inhibition on the development and reversal of spinal opioid tolerance. Br J Pharmacol 1999; 127:631–44Powell, KJ Hosokawa, A Bell, A Sutak, M Milne, B Quirion, R Jhamandas, K
Wong CS, Hsu MM, Chou YY, Tao PL, Tung CS: Morphine tolerance increases [3H]MK-904 binding affinity and constitutive neuronal nitric oxide synthase expression in rat spinal cord. Br J Anaesth 2000; 85:587–91Wong, CS Hsu, MM Chou, YY Tao, PL Tung, CS
Cao JL, Ding HL, He JH, Zheng LC, Duan SM, Zeng YM: The spinal nitric oxide involved in the inhibitory effect of midazolam on morphine-induced analgesia tolerance. Pharmacol Biochem Behav 2005; 80:493–503Cao, JL Ding, HL He, JH Zheng, LC Duan, SM Zeng, YM
Liu W, Wang CH, Cui Y, Mo LQ, Zhi JL, Sun SN, Wang YL, Yu HM, Zhao CM, Feng JQ, Chen PX: Inhibition of neuronal nitric oxide synthase antagonizes morphine antinociceptive tolerance by decreasing activation of p38 MAPK in the spinal microglia. Neurosci Lett 2006; 410:174–7Liu, W Wang, CH Cui, Y Mo, LQ Zhi, JL Sun, SN Wang, YL Yu, HM Zhao, CM Feng, JQ Chen, PX
Liang DY, Clark JD: Modulation of the NO/CO-cGMP signaling cascade during chronic morphine exposure in mice. Neurosci Lett 2004; 365:73–7Liang, DY Clark, JD
Xu T, Jiang W, Du D, Xu Y, Zhou Q, Pan X, Lou Y, Xu L, Ma K: Inhibition of MPEP on the development of morphine antinociceptive tolerance and the biosynthesis of neuronal nitric oxide synthase in rat spinal cord. Neurosci Lett 2008; 436:214–8Xu, T Jiang, W Du, D Xu, Y Zhou, Q Pan, X Lou, Y Xu, L Ma, K
Wüster M, Schulz R, Herz A: Opioid tolerance and dependence. Trend Pharmacol Sci 1985; 6:64–7Wüster, M Schulz, R Herz, A
Johnson SM, Fleming WW: Mechanisms of cellular adaptive sensitivity changes: applications to opioid tolerance and dependence. Pharmacol. Rev 1989; 41:435–88Johnson, SM Fleming, WW
Galea E, Regunathan S, Eliopoulos V, Feinstein DL, Reis DJ: Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation of arginine. Biochem J 1996; 316:247–9Galea, E Regunathan, S Eliopoulos, V Feinstein, DL Reis, DJ
Aricioglu F, Paul IA, Regunathan S: Agmatine reduces only peripheral-related behavior signs, not the central signs, of morphine withdrawal in nNOS deficient transgenic mice. Neurosci Lett 2004; 354:153–7Aricioglu, F Paul, IA Regunathan, S
Medvedev IO, Dravolina OA, Bespalov AY: Differential effects of nitric oxide synthase inhibitor, 7-nitroindazole, on discriminative stimulus and somatic effects of naloxone in morphine-dependent rats. Eur J Pharmacol 1999; 377:183–6Medvedev, IO Dravolina, OA Bespalov, AY
Afify EA, Daabees TT, Gabra BH, Abou Zeit-Har MS: Role of nitric oxide in catalepsy and hyperthermia in morphine-dependent rats. Pharmacol Res 2001; 44:533–9Afify, EA Daabees, TT Gabra, BH Abou Zeit-Har, MS
Zarrindast MR, Homayoun H, Khavandgar S, Fayaz-Dastgerdi M, Fayaz-Dastgerdi M: The effects of simultaneous administration of α2-adrenergic agents with L-NAME or L-arginine on the development and expression of morphine dependence in mice. Behav Pharmacol 2002; 13:117–25Zarrindast, MR Homayoun, H Khavandgar, S Fayaz-Dastgerdi, M Fayaz-Dastgerdi, M
Zhou JF, Yan ZF, Ruan ZR, Peng FY, Cai D, Yuan H, Sun L, Ding DY, Xu SS: Heroin abuse and nitric oxide, oxidation, peroxidation, lipoperoxidation. Biomed Environ Sci 2000; 13:131–9Zhou, JF Yan, ZF Ruan, ZR Peng, FY Cai, D Yuan, H Sun, L Ding, DY Xu, SS
Kimes AS, Vaupel DB, London ED: Attenuation of some signs of opioid withdrawal by inhibitors of nitric oxide synthase. Psychopharmacology 1993; 112:521–4Kimes, AS Vaupel, DB London, ED
Cappendijk SL, de Vries R, Dzoljic MR: Inhibitory effect of nitric oxide (NO) synthase inhibitors on naloxone-precipitated withdrawal syndrome in morphine-dependent mice. Neurosci Lett 1993; 162:97–100Cappendijk, SL de Vries, R Dzoljic, MR
Adams ML, Kalicki JM, Meyer ER, Cicero TJ: Inhibition of the morphine withdrawal syndrome by a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester. Life Sci 1993; 52:PL245–9Adams, ML Kalicki, JM Meyer, ER Cicero, TJ
Thorat SN, Barjavel MJ, Matwyshyn GA, Bhargava HN: Comparative effects of NG-monomethyl-L-arginine and MK-801 on the abstinence syndrome in morphine-dependent mice. Brain Res 1994; 642:153–9Thorat, SN Barjavel, MJ Matwyshyn, GA Bhargava, HN
Cappendijk SL, Duval SY, de Vries R, Dzoljic MR: Comparative study of normotensive and hypertensive nitric oxide synthase inhibitors on morphine withdrawal syndrome in rats. Neurosci Lett 1995; 183:67–70Cappendijk, SL Duval, SY de Vries, R Dzoljic, MR
Vaupel DB, Kimes AS, London ED: Comparison of 7-nitroindazole with other nitric oxide synthase inhibitors as attenuators of opioid withdrawal. Psychopharmacology 1995; 118:361–8Vaupel, DB Kimes, AS London, ED
Vaupel DB, Kimes AS, London ED: Nitric oxide synthase inhibitors. Preclinical studies of potential use for treatment of opioid withdrawal. Neuropsychopharmacology 1995; 13:315–22Vaupel, DB Kimes, AS London, ED
Vaupel DB, Kimes AS, London ED: Further in vivo  studies on attenuating morphine withdrawal: isoform-selective nitric oxide synthase inhibitors differ in efficacy. Eur J Pharmacol 1997; 324:11–20Vaupel, DB Kimes, AS London, ED
Mori T, Ito S, Matsubayashi K, Sawaguchi T: Comparison of nitric oxide synthase inhibitors, phospholipase A2 inhibitor and free radical scavengers as attenuators of opioid withdrawal syndrome. Behav Pharmacol 2007; 18:725–9Mori, T Ito, S Matsubayashi, K Sawaguchi, T
Bhargava HN, Thorat SN: Evidence for a role of nitric oxide of the central nervous system in morphine abstinence syndrome. Pharmacology 1996; 52:86–91Bhargava, HN Thorat, SN
Leza JC, Lizasoain I, Cuellar B, Moro MA, Lorenzo P: Correlation between brain nitric oxide synthase activity and opiate withdrawal. Naunyn Schmiedebergs Arch Pharmacol 1996; 353:349–54Leza, JC Lizasoain, I Cuellar, B Moro, MA Lorenzo, P
Lizasoain I, Leza JC, Cuellar B, Moro MA, Lorenzo P: Inhibition of morphine withdrawal by lamotrigine: involvement of nitric oxide. Eur J Pharmacol 1996; 299:41–5Lizasoain, I Leza, JC Cuellar, B Moro, MA Lorenzo, P
Bull PM, Ludwig M, Blackburn-Munro GJ, Delgado-Cohen H, Brown CH, Russell JA: The role of nitric oxide in morphine dependence and withdrawal excitation of rat oxytocin neurons. Eur J Neurosci 2003; 18:2545–51Bull, PM Ludwig, M Blackburn-Munro, GJ Delgado-Cohen, H Brown, CH Russell, JA
Hall S, Milne B, Jhamandas K: Nitric oxide synthase inhibitors attenuate acute and chronic morphine withdrawal response in the rat locus coeruleus: an in vivo  voltammetric study. Brain Res 1996; 739:182–91Hall, S Milne, B Jhamandas, K
Jhamandas JH, Harris KH, Petrov T, Jhamandas KH: Activation of nitric oxide-synthesizing neurons during precipitated morphine withdrawal. Neuroreport 1996; 7:2843–6Jhamandas, JH Harris, KH Petrov, T Jhamandas, KH
Sullivan ME, Hall SR, Milne B, Jhamandas K: Suppression of acute and chronic opioid withdrawal by a selective soluble guanylyl cyclase inhibitor. Brain Res 2000; 859:45–56Sullivan, ME Hall, SR Milne, B Jhamandas, K
Pineda J, Torrecilla M, Martin-Ruiz R, Ugedo L: Attenuation of withdrawal-induced hyperactivity of locus coeruleus neurons by inhibitors of nitric oxide synthase in morphine-dependent rats. Neuropharmacology 1998; 37:759–67Pineda, J Torrecilla, M Martin-Ruiz, R Ugedo, L
Javelle N, Berod A, Renaud B, Lambas-Senas L: NO synthase inhibitors attenuate locus coeruleus catecholamine metabolism and behavior induced by morphine withdrawal. Neuroreport 2002; 13:725–8Javelle, N Berod, A Renaud, B Lambas-Senas, L
Feng P, Meissler JJ, Adler MW, Eisenstein TK: Morphine withdrawal sensitizes mice to lipopolysaccharide: elevated TNF-α and nitric oxide with decreased IL12. J Neuroimmunol 2005; 164:57–65Feng, P Meissler, JJ Adler, MW Eisenstein, TK
Buccafusco JJ, Terry AV, Shuster L: Spinal NMDA receptor-nitric oxide mediation of the expression of morphine withdrawal symptoms in the rat. Brain Res 1995; 679:189–99Buccafusco, JJ Terry, AV Shuster, L
Gao JL, Zeng YM, Zhang LC, Gu J, Liu HF, Zhou WH, Yang GD: NO mediated increase of Fos protein and NMDA1A R mRNA expression in rat spinal cord during morphine withdrawal. Acta Pharmacol Sin 2001; 22:505–11Gao, JL Zeng, YM Zhang, LC Gu, J Liu, HF Zhou, WH Yang, GD
Harlan RE, Webber DS, Garcia MM: Involvement of nitric oxide in morphine-induced c-Fos expression in the rat striatum. Brain Res Bull 2001; 54:207–12Harlan, RE Webber, DS Garcia, MM
Cao JL, Liu HL, Wang JK, Zeng YM: Cross talk between nitric oxide and ERK1/2 signaling pathway in the spinal cord mediates naloxone-precipitated withdrawal in morphine-dependent rats. Neuropharmacology 2006; 51:315–26Cao, JL Liu, HL Wang, JK Zeng, YM
Chou WB, Zeng YM, Duan SM, Zhou WH, Gu J, Yang GD: M2 muscarinic receptor of spinal cord mediated increase of nNOS expression in locus coeruleus during morphine withdrawal. Acta Pharmacol Sin 2002; 23: 691–7Chou, WB Zeng, YM Duan, SM Zhou, WH Gu, J Yang, GD
Fig. 1. Possible mechanism underlying interactions between μ-opioid receptor agonists and nitric oxide (NO) generated  via  NMDA-receptor stimulation in antinociceptive processes. Effects of NO on tolerance to and dependence on opioids and their withdrawal syndrome are also presented. NMDA-R,  N  -methyl-d-aspartate-receptor; opioid-R, opioid receptor; [Ca2+]i, intracellular concentrations of Ca2+; CaM, calmodulin; BH4, tetrahydrobiopterin; NADPH, reduced nicotinamide adenine dinucleotide phosphate; +, stimulation; –, inhibition; 7-NI, 7-nitroindazol; ADMA, asymmetrical dimethyl arginine; L-NA, NG-nitro-l-arginine; L-NAME, L-NA methylester; nNOS, neuronal nitric oxide synthase. 
Fig. 1. Possible mechanism underlying interactions between μ-opioid receptor agonists and nitric oxide (NO) generated  via  NMDA-receptor stimulation in antinociceptive processes. Effects of NO on tolerance to and dependence on opioids and their withdrawal syndrome are also presented. NMDA-R,  N  -methyl-d-aspartate-receptor; opioid-R, opioid receptor; [Ca2+]i, intracellular concentrations of Ca2+; CaM, calmodulin; BH4, tetrahydrobiopterin; NADPH, reduced nicotinamide adenine dinucleotide phosphate; +, stimulation; –, inhibition; 7-NI, 7-nitroindazol; ADMA, asymmetrical dimethyl arginine; L-NA, NG-nitro-l-arginine; L-NAME, L-NA methylester; nNOS, neuronal nitric oxide synthase. 
Fig. 1. Possible mechanism underlying interactions between μ-opioid receptor agonists and nitric oxide (NO) generated  via  NMDA-receptor stimulation in antinociceptive processes. Effects of NO on tolerance to and dependence on opioids and their withdrawal syndrome are also presented. NMDA-R,  N  -methyl-d-aspartate-receptor; opioid-R, opioid receptor; [Ca2+]i, intracellular concentrations of Ca2+; CaM, calmodulin; BH4, tetrahydrobiopterin; NADPH, reduced nicotinamide adenine dinucleotide phosphate; +, stimulation; –, inhibition; 7-NI, 7-nitroindazol; ADMA, asymmetrical dimethyl arginine; L-NA, NG-nitro-l-arginine; L-NAME, L-NA methylester; nNOS, neuronal nitric oxide synthase. 
Table 1. Evidence for NO as an Analgesic Mediator at Supraspinal, Spinal, and Peripheral Sites in Experimental Animals 
Image not available
Table 1. Evidence for NO as an Analgesic Mediator at Supraspinal, Spinal, and Peripheral Sites in Experimental Animals 
Table 2. Evidence for NO as an Algesic Mediator at Supraspinal, Spinal, or Systemic Sites in Experimental Animals 
Image not available
Table 2. Evidence for NO as an Algesic Mediator at Supraspinal, Spinal, or Systemic Sites in Experimental Animals