Research report
Spinal cord nitric oxide synthase and heme oxygenase limit morphine induced analgesia

https://doi.org/10.1016/S0169-328X(01)00251-0Get rights and content

Abstract

Spinal cord tissue contains two enzyme systems capable of producing monoxide gasses which in turn are linked to the stimulation of soluble guanylate cyclase, nitric oxide synthase (NOS) which produces NO and heme oxygenase (HO) which produces CO. Reports from several laboratories link these two enzyme systems to pain of inflammatory and neuropathic etiologies. Additional studies have demonstrated that the activation of the NOS system by morphine limits the spinal analgesic action of this drug. In this study we first employed the hot plate model of pain to demonstrate that the NOS inhibitor l-NAME and the HO inhibitor Sn-P potentiate the analgesic actions of intrathecally administered morphine while having no intrinsic analgesic action at the doses used. We then determined that l-NAME loses its ability to potentiate morphine in nNOS null-mutant mice, while Sn-P no longer potentiates morphine in mice lacking a functional HO-2 gene. The intrathecal injection of the cGMP analog 8-Br cGMP caused hyperalgesia in the hot plate assay. Focusing on the possible involvement of cGMP metabolism, we documented that morphine stimulates cGMP production in a spinal cord slice model in a concentration dependent and naloxone reversible manner. Both l-NAME and Sn-P were potent inhibitors of morphine-stimulated cGMP production. Buffer containing either CO or the NO donor compound SNAP stimulated cGMP production as well. In spinal cord slices from either nNOS or HO-2 null-mutant animals morphine did not stimulate cGMP production. Taken together our data suggest that spinal monoxide generation modifies the acute analgesic actions of morphine.

Introduction

A great deal of data exist demonstrating roles for the spinal cord nitric oxide synthase (NOS) enzyme system in nociceptive and analgesic pathways. For example, the inhibition of NOS expressed in spinal cord tissue, predominantly neuronal NOS (nNOS), by the intrathecal injection of selective NOS inhibitors has been demonstrated to reduce pain behaviors in models of neuropathic and inflammatory pain [25], [29], [35], [41]. Likewise, the activation of NOS after acute spinal morphine administration seems to limit the analgesic efficacy of this clinically important opioid in what might be considered an opponent process to the analgesic effects [23], [39]. Still other studies have demonstrated a critical role for spinal cord NOS in the acquisition of analgesic tolerance to morphine [8], [10], [27]. Finally, the NOS enzyme system appears to participate in the hyperalgesia which results from the abrupt cessation of opioid administration [18]. Although some important exceptions exist, the spinal cord NOS enzyme system has often be implicated in mechanisms facilitating nociception or limiting opioid analgesia.

Over the last few years a similar picture has emerged for the other major monoxide producing enzyme system, heme oxygenase (HO). Again, the systemic or intrathecal administration of HO inhibitors seems to reduce pain behaviors measured in models of neuropathic and inflammatory pain [19], [20], [40], probably by inhibiting the constitutively expressed HO-2 isozyme [19]. Though no reports exist to date demonstrating a causal link between HO enzymatic activity and morphine tolerance, it has been demonstrated that spinal cord HO enzymatic activity and expression of HO-2 increase after chronic exposure to morphine [21]. As is the case for NOS, the HO enzyme system participates in the hyperalgesia caused by the abrupt cessation of opioid administration [18]. Thus, the HO enzyme system also seems to be linked to the facilitation of nociception if not the limitation of analgesia. Interestingly, both NOS and HO produce monoxide products, NO and CO, respectively, which stimulate soluble guanylate cyclase and increase intracellular cGMP levels [3], [15], [17], [30]. The intrathecal administration of cGMP causes hyperalgesia [12], possibly by stimulation of certain populations of spinal cord neurons [26]. Thus, the stimulation of cGMP production by the NOS and HO enzyme systems may partially or fully explain their pronociceptive actions.

The studies presented below were designed to address the hypothesis that the NOS and HO enzyme systems limit the analgesic effects of morphine by a mechanism involving the production of cGMP in spinal cord tissue. Using wild type and null-mutant mice, we first describe behavioral observations supporting this hypothesis and then turn to a spinal cord slice model to examine pertinent biochemical issues.

Section snippets

Animals

All animal protocols were approved by our institution’s Subcommittee on Animal Studies and conformed to standards suggested by the International Association for the Study of Pain (IASP). Male mice of the C57Bl/6 strain were purchased from Charles River (Freemont, CA, USA) and were kept 6/cage using a 12 h–12 h light–dark cycle. Food and water were provided ad libitum. Mice were used at 12–16 weeks of age. Null-mutant HO-2 knockout mice in the C57Bl/6 background were the kind gift of Dr. Phyllis

Results

Both the HO inhibitor Sn-P and the NOS inhibitor l-NAME reduced formalin-induced hind paw licking. In mice who received only intrathecal saline, formalin injection caused 67±5 s of licking behavior during phase I and 165±13 s during phase II. As shown in Fig. 1, i.t. injection of 5 μg l-NAME left phase I licking time unchanged, but reduced phase II time to 18% of control. The i.t. injection of 700 ng Sn-P reduced phase II licking time to a similar extent, but also reduced phase I licking as we

Discussion

The data presented here support the hypothesis that morphine stimulates spinal cord cGMP production by activation of nNOS and HO-2 thus reducing the overall level of analgesia obtained. The specific behavioral evidence in support of this hypothesis is that: (1) agents which block enzymes linked to the stimulation of cGMP production like nNOS and HO-2 also increase the analgesic effects of morphine, (2) cGMP analogs reduce analgesia from morphine and can cause hyperalgesia and (3) mice lacking

References (41)

Cited by (39)

  • Remote intrathecal morphine preconditioning confers cardioprotection via spinal cord nitric oxide/cyclic guanosine monophosphate/protein kinase G pathway

    2015, Journal of Surgical Research
    Citation Excerpt :

    NO acts as a modulator of dorsal horn spinal cord nociceptive pathways. Activation of NOS by intrathecal administration with morphine limits the spinal analgesic action of this drug [11]. It is well known that morphine shows antinociceptive effects and that inhibitors of the NO/cGMP pathway attenuate the analgesic effect of morphine or other opioids [17,18].

  • Effects of selective and non-selective inhibitors of nitric oxide synthase on morphine- and endomorphin-1-induced analgesia in acute and neuropathic pain in rats

    2013, Neuropharmacology
    Citation Excerpt :

    Many studies supporting our results indicate that morphine-induced analgesia is enhanced after co-administration of NOS inhibitors (Xu and Tseng, 1995; Luo et al., 1995; Machelska et al., 1997b; Ozek et al., 2003). Our study is in line with that of Li and Clark (2001), who demonstrated that i.t. administration of the nonselective NOS inhibitor (l-NAME) potentiates the analgesic actions of morphine, but this effect is absent in NOS1 null-mutant mice. Our study supports the results of Xu and Tseng (1995), who reported that i.t. injections of Nω-nitro-l-arginine, haemoglobin, methylene blue (an inhibitor of guanylate cyclase) and/or NOS (Luo et al., 1995) potentiate the antinociception induced by i.c.v. morphine administration.

View all citing articles on Scopus
View full text