Mechanism of Attenuation of Morphine Antinociception by Chronic Treatment with L-Arginine HEMENDRA N. BHARGAVA, JING-TAN BIAN and SHAILENDRA KUMAR

The effects of twice-daily injections of L-arginine or D-arginine (200 mg/kg i.p.) for 4 days on morphine-induced antinociception, brain nitric oxide synthase activity and brain and serum distribution of morphine and brain m-opioid receptors labeled with [H][D-Ala,MePhe,Gly-ol]enkephalin were determined in male Swiss-Webster mice. Chronic treatment with L-arginine, but not D-arginine, decreased the antinociceptive response to morphine in mice, increased the activity of nitric oxide synthase in the midbrain and decreased brain levels of morphine, compared with vehicle-injected controls. Significant decreases in morphine levels were observed in midbrain, pons and medulla, hippocampus, striatum and spinal cord of L-arginine-treated mice, in comparison with vehicle-injected mice. However, the levels of morphine in cortex, amygdala and hypothalamus of L-arginineor D-arginine-treated mice did not differ from those of vehicle-injected controls. Acute treatment with L-arginine (200 mg/kg i.p.) or D-arginine (200 mg/kg i.p.) did not modify either morphine antinociception or morphine distribution in brain regions or the spinal cord. Chronic administration of L-arginine or D-arginine did not alter the Bmax or Kd values of [H][D-Ala,MePhe,Gly-ol]enkephalin binding to the mouse brain membranes. These results suggest that chronic treatment with L-arginine reduces the antinociceptive effect of morphine by increasing brain nitric oxide synthase activity and by decreasing the concentration of morphine in certain brain regions and spinal cord. Considerable evidence suggests that NMDA/NO pathways may be involved in the acute and chronic actions of opioid drugs that are used primarily for the relief of moderateto severe-intensity pain. Opioid drugs produce their actions by interacting with three major types of opioid receptors, namely m, d and k, with morphine or DAMGO, [D-Pen,DPen]enkephalin and U-50,488H, respectively, being the prototypical agonists. NO, a second messenger involved in the regulation of cell function, is formed enzymatically from Larginine by NOS after the activation of the NMDA receptor (Moncada et al., 1991). NOS is inhibited by several L-arginine derivatives, such as N-nitro-L-arginine and L-NMMA (Rees et al., 1990; Thorat et al., 1994). Drugs that modify the concentration of NO in the central nervous system appear to modify opioid-induced antinociception. Furthermore, NO is involved in nociceptive processes, particularly in the spinal cord (Haley et al., 1992). Synaptic transmission in the central and peripheral nervous system seems to be modulated by NO (Meller and Gebhart, 1993). NO donors such as 3-morpholino-sydnonimine, sodium nitroprusside and hydroxylamine given i.t. induced hyperalgesia in rats, as measured by the tail-flick test, whereas NOS inhibitors such as L-NAME given i.t. or i.c.v. elicited a slight antinociception. However, the effects of other routes of administration of NO donors or NOS inhibitors were not determined (Przewlocka et al., 1994). In the abdominal constriction test, L-arginine had no effect but L-NAME and L-NMMA by themselves produced marked antinociception, as evidenced by inhibition of the abdominal constriction response in mice (Moore et al., 1991; Mustafa, 1992). In the tail-flick test, L-NAME, L-NMMA and L-arginine had no effect (Dambisya and Lee, 1995). In another study, i.c.v. administration of L-arginine (30 ng/mouse) elicited antinociception in mice, as assessed by the tail-flick test. The antinociceptive action of L-arginine was attributed to the formation of kyotorphin (L-Tyr-L-Arg) in the brain (Kawabata et al., 1994, 1996). In another study, L-arginine was also shown to produce antinociception by itself and to antagonize i.c.v. administered bradykinin-induced antinociception in mice (Germany et al., 1996). Thus, L-arginine appears to produce nociception and antinociception in tests that are sensitive to weak analgesic agents. On the other hand, acute administration of L-arginine, the precursor of NO, p.o. or i.p. but not i.c.v. reduced morphine antinociception in mice. These effects were observed at 300 and 1000 mg/kg doses of Received for publication September 20, 1996. 1 These studies were supported by Research Scientist Development Award K02-DA00130 to H.N.B. from the National Institute on Drug Abuse, National Institutes of Health. ABBREVIATIONS: AUC, area under the time-response curve; DAMGO, [d-Ala,MePhe,Gly-ol]enkephalin; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; i.t., intrathecal(ly); L-NAME, N-nitro-L-arginine methyl ester; NMDA, N-methyl-D-aspartate; L-NMMA, N-monomethylL-arginine; NO, nitric oxide; NOS, nitric oxide synthase; RIA, radioimmunoassay. 0022-3565/97/2812-0707$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 281, No. 2 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 281:707–712, 1997 707 at A PE T Jornals on N ovem er 7, 2016 jpet.asjournals.org D ow nladed from L-arginine and were reversed by the NOS inhibitors L-NAME and L-NMMA (Brignola et al., 1994). Intrathecal administration of L-NAME has been shown to enhance morphine antinociception in rats (Przewlocki et al., 1993), suggesting involvement of the spinal NO system. The effect of chronic administration of L-arginine on morphine antinociception has not been determined. Although, in the aforementioned studies, NO was implicated in the acute and chronic actions of opioid drugs, other factors, such as the activity of NOS and the distribution of opioid agents in the central nervous system, have received little attention. In the present studies, the effects of chronic administration of Larginine or D-arginine on the antinociceptive activity of morphine have been determined. The effects of such treatments on NOS activity and the distribution of morphine in certain brain regions, spinal cord and serum were evaluated. As controls, the effect of acute administration of L-arginine on morphine antinociception and its distribution in brain regions and spinal cord have also been determined. Finally, the effect of chronic administration of L-arginine or D-arginine on the binding of [H]DAMGO to brain membranes was also assessed.