Role of Spinal Nitric Oxide in the Inhibitory Effect of [D-Pen, D-Pen]-Enkephalin on Ascending Dorsal Horn Neurons in Normal and Diabetic Rats

Intrathecal [D-Pen,D-Pen]-enkephalin (DPDPE; a -opioid agonist) has a profound antinociceptive effect in neuropathic pain. Spinal nitric oxide (NO) has been implicated in the analgesic effect of several G protein-coupled receptor agonists. Little, however, is known about the role of spinal NO in the inhibitory effect of DPDPE on spinal dorsal horn neurons. In the present study, we determined the role of NO in the inhibitory effect of DPDPE on ascending dorsal horn neurons in normal rats and in a rat model of diabetic neuropathic pain. Single-unit activity of ascending dorsal horn neurons was recorded in anesthetized rats. The responses of dorsal horn neurons to graded mechanical stimuli and von Frey filaments were determined before and after local spinal application of 0.1 to 5 M DPDPE. The influence of an NO synthase inhibitor, 1-(2-trifluoromethylphenyl) imidazole (TRIM; 30 M), on the effect of DPDPE was then studied in separate groups of dorsal horn neurons in normal and diabetic rats. DPDPE inhibited the response of dorsal horn neurons in both normal and diabetic rats in a concentrationdependent fashion. The inhibitory effect of 1 M DPDPE was abolished by 1 M naltrindole, a -opioid antagonist. Furthermore, the inhibitory effect of DPDPE on the evoked response of dorsal horn neurons was largely eliminated by TRIM in normal and diabetic rats. These data suggest that DPDPE has a profound inhibitory effect on dorsal horn neurons in normal and diabetic rats. Spinal endogenous NO is essential for the inhibitory effect of DPDPE on ascending dorsal horn neurons in both normal and diabetic rats. The spinal cord dorsal horn is an important site for transmission and modulation of nociception. Spinally administered and -opioid receptor agonists produce potent analgesia (Heyman et al., 1987; Malmberg and Yaksh, 1992; Hurley et al., 1999). This is consistent with high levels of and -opioid receptors in the spinal cord (Gouarderes et al., 1985; Dickenson et al., 1987; Besse et al., 1991). Diabetic neuropathic pain, however, is often poorly relieved by -opioid receptor agonists in patients (Arner and Meyerson, 1988; Wright, 1994). Also, experiments performed in the rat model of diabetic neuropathic pain have consistently shown a reduced analgesic effect of -opioid agonists (Courteix et al., 1994; Malcangio and Tomlinson, 1998; Zurek et al., 2001). The underlying mechanisms of neuropathic pain are complex and probably include both the peripheral and central components. The presence of hypersensitivity of spinothalamic tract dorsal neurons has been demonstrated in a rat model of diabetic neuropathic pain (Chen and Pan, 2002). Thus, pharmacological suppression of hypersensitivity of dorsal horn neurons represents an important strategy for treatment of this neuropathic pain condition. On the other hand, the -opioid receptor agonist [D-Pen,DPen]-enkephalin (DPDPE) probably is an important alternative for the treatment of neuropathic pain because of its increased analgesic potency, lowered abuse potential, and fewer adverse effects compared with the -opioids (Quock et al., 1999). In this regard, DPDPE produces an antinociceptive effect both at the spinal and supraspinal levels (Heyman et al., 1987; Kamei et al., 1992; Malmberg and Yaksh, 1992; Stewart and Hammond, 1993; Takemori and Portoghese, 1993; Hurley et al., 1999). The antinociceptive effect of intrathecal DPDPE has been demonstrated in neuropathic pain caused by diabetic neuropathy and sciatic nerve injury in rodents (Kamei et al., 1992; Mika et al., 2001). The increased analgesic potency of DPDPE in diabetic animals indicates that it has a potential for treatment of neuropathic pain in diabetic patients. It is well established that the effect of -opioid agonists is dependent on the coupling to inhibitory G This study was supported by Grants GM64830 and NS41178 cofunded by the National Institutes of Health and the Juvenile Diabetes Foundation International. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.040865. ABBREVIATIONS: DPDPE, [D-Pen,D-Pen]-enkephalin; NO, nitric oxide; NOS, nitric-oxide synthase; aCSF, artificial cerebrospinal fluid; nNOS, neuronal nitric-oxide synthase; TRIM, 1-(2-trifluoromethylphenyl) imidazole; imp, impulses. 0022-3565/02/3033-1021–1028$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 303, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 40865/1025754 JPET 303:1021–1028, 2002 Printed in U.S.A. 1021 at A PE T Jornals on Sptem er 7, 2016 jpet.asjournals.org D ow nladed from proteins, and stimulation of -opioid receptors reduces intracellular cAMP levels and modulates the voltage-gated calcium and potassium channels (Quock et al., 1999). The analgesic mechanisms of DPDPE in the spinal cord, however, are not yet fully known, and the effect of DPDPE on dorsal horn projection neurons in neuropathic pain has not been examined previously. Nitric oxide (NO) is involved in the antinociceptive effect of peripherally applied -opioid agonists in a rat model of inflammatory pain (Nozaki-Taguchi and Yamamoto, 1998b). Administration of NO donors also can enhance the analgesic effects of peripherally administered morphine (Nozaki-Taguchi and Yamamoto, 1998a). Nitric-oxide synthase (NOS)-containing neurons are present in the superficial layers of the spinal dorsal horn in rats (Valtschanoff et al., 1992). Some inhibitory interneurons in the dorsal horn are also known to contain neuronal NOS (Valtschanoff et al., 1992). Spinal endogenous NO is an important mediator for the analgesic actions of intrathecal muscarinic and 2 receptor agonists in rats (Iwamoto and Marion, 1994; Pan et al., 1998). Furthermore, spinal NO contributes to the antinociceptive action of systemic morphine in normal rats (Song et al., 1998). Little, however, is known about the role of endogenous NO in the effect of DPDPE on dorsal horn neurons in diabetic neuropathic pain. In the present study, we first examined the effect of DPDPE on spinal dorsal horn projection neurons in normal rats and in a rat model of diabetic neuropathic pain. Subsequently, the role of endogenous NO in the inhibitory effect of DPDPE on dorsal horn neurons was investigated in normal and diabetic rats. Materials and Methods