Immunoneutralization of Agmatine Sensitizes Mice to -Opioid Receptor Tolerance

Systemically or centrally administered agmatine (decarboxylated arginine) prevents, moderates, or reverses opioid-induced tolerance and self-administration, inflammatory and neuropathic pain, and sequelae associated with ischemia and spinal cord injury in rodents. These behavioral models invoke the N-methyl-D-aspartate (NMDA) receptor/nitric-oxide synthase cascade. Agmatine (AG) antagonizes the NMDA receptor and inhibits nitric-oxide synthase in vitro and in vivo, which may explain its effect in models of neural plasticity. Agmatine has been detected biochemically and immunohistochemically in the central nervous system. Consequently, it is conceivable that agmatine operates in an anti-glutamatergic manner in vivo; the role of endogenous agmatine in the central nervous system remains minimally defined. The current study used an immunoneutralization strategy to evaluate the effect of sequestration of endogenous agmatine in acute opioid analgesic tolerance in mice. First, intrathecal pretreatment with an anti-AG IgG (but not normal IgG) reversed an established pharmacological effect of intrathecal agmatine: antagonism of NMDA-evoked behavior. This result justified the use of anti-AG IgG to sequester endogenous agmatine in vivo. Second, intrathecal pretreatment with the anti-AG IgG sensitized mice to induction of acute spinal tolerance of two -opioid receptor-selective agonists, [D-Ala,N-Me-Phe,Gly-ol]-enkephalin and endomorphin-2. A lower dose of either agonist that, under normal conditions, produces moderate or no tolerance was tolerance-inducing after intrathecal pretreatment of anti-AG IgG (but not normal IgG). The effect of the anti-AG IgG lasted for at least 24 h in both NMDA-evoked behavior and the acute opioid tolerance. These results suggest that endogenous spinal agmatine may moderate glutamate-dependent neuroplasticity. Decarboxylated arginine (agmatine) has been identified in the mammalian central nervous system (CNS) both biochemically (Li et al., 1994; Fairbanks et al., 2000) and neuroanatomically (Fairbanks et al., 2000; Goracke-Postle et al., 2006). Agmatine was discovered as a clonidine-displacing substance (Li et al., 1994) that bound but did not activate or inhibit -2 adrenergic receptors (Pinthong et al., 1995). However, agmatine also acts as an N-methyl-D-aspartate (NMDA) receptor antagonist at polyamine site (Ki 15 M) (Gibson et al., 2002) and the MK-801 binding site (Reynolds, 1990). Agmatine also inhibits (Galea et al., 1996) or inactivates (Demady et al., 2001) nitric-oxide synthase. Electrophysiological (Yang and Reis, 1999) and pharmacological (Fairbanks et al., 2000; Roberts et al., 2005) evidence supports agmatine antagonism/inhibition at both NMDA receptor and nitric-oxide synthase, proteins known as essential components of glutamatergic neurotransmission. This dual activity raises the question as to whether agmatine functions endogenously as an anti-glutamatergic neuromodulator. This proposed role for agmatine is also suggested from reports describing agmatine-mediated inhibition of opioid tolerance (Kolesnikov et al., 1996; Fairbanks and Wilcox, 1997), opioid self-administration (Morgan et al., 2002; Wade et al., 2008), inflammationand neuropathy-induced hyperalgesia (FairThis work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants K01-DA00509, R21-DA15387, R21DA023545] (to C.A.F); and National Institutes of Health National Research Service Award [Grant F31-DA021054] (predoctoral fellowship to C.L.W.). L.L.E. was supported by a Melendy research training grant from the College of Pharmacy, University of Minnesota. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.155424. ABBREVIATIONS: CNS, central nervous system; NMDA, N-methyl-D-aspartate; MK-801, 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); AG, agmatine; Endo-2, endomorphin-2; %MPE, percentage maximal possible effect; CI, confidence intervals; GP, guinea pig; ELISA, enzyme-linked immunosorbent assay; IR, immunoreactivity; NeuN, neuronal nuclei; SR 141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride. 0022-3565/09/3312-539–546$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 155424/3525107 JPET 331:539–546, 2009 Printed in U.S.A. 539 at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from banks et al., 2000), behavioral sequelae after spinal cord injury (Fairbanks et al., 2000; Gilad and Gilad, 2000; Yu et al., 2000) and evoked seizure (Feng et al., 2005). It has been suggested (Reis and Regunathan, 1998) that agmatine meets several criteria characteristic of an endogenous neuromodulator, including synthesis of agmatine in the brain (Reis and Regunathan, 1998), localization to neurons and synaptic vesicles (Otake et al., 1998; Goracke-Postle et al., 2006), transport into nerve terminals (Sastre et al., 1997; Goracke-Postle et al., 2006, 2007a,b), release by depolarization (Reis and Regunathan, 1998; Goracke-Postle et al., 2006, 2007b), transport into astrocytes (Regunathan et al., 1995), and enzymatic degradation by CNS agmatinase (Sastre et al., 1996). An important criterion yet to be tested includes a demonstration that endogenous agmatine performs the same physiological function as does exogenously administered agmatine. This criterion has been tested for a variety of neurotransmitters and neuropeptides, including -endorphin (Guerrero-Munoz et al., 1979), endomorphin (Zadina et al., 1997), anandamide (Devane et al., 1992), substance P (Share and Rackham, 1981), calcitonin gene-related peptide (CGRP) (Tan et al., 1994), Metand Leu-enkephalin (Mulder et al., 1984; Hardy and Haigler, 1985), and small molecules such as norepinephrine (Hardy and Haigler, 1985). In view of the ability of exogenously administered agmatine to prevent opioid analgesic tolerance (Nguyen et al., 2003), we hypothesized that endogenous agmatine participates in control of opioidergic processes. Previous studies of potential antinociceptive endogenous compounds have inferred the activity of these neuromodulators through the use of pharmacological antagonists in vivo. However, agmatine is itself a receptor antagonist and an enzyme inhibitor, rendering such a strategy inappropriate. We applied an immunoneutralization strategy using scavenger antisera to evaluate the physiological role of agmatine, a method previously used for other pharmacological antagonists (Vanderah et al., 1994; Tseng et al., 2000; Ohsawa et al., 2001). We hypothesized that sequestration of endogenous agmatine using a structure-specific anti-agmatine (AG) IgG antibody would invoke the induction of acute -opioid receptor tolerance at doses that normally are not tolerance-inducing. Our objective was to assess the impact of spinal delivery of anti-AG IgG on acute homologous tolerance induced by both [D-Ala,N-Me-Phe,Gly-ol]-enkephalin (DAMGO) and endomorphin-2 (Endo-2) to determine whether diminished availability of endogenous agmatine affected that process. We show that exogenously applied agmatine prevents the induction of DAMGOand Endo-2-induced acute spinal tolerance, as has been shown previously for morphine. The present study demonstrates that pretreatment with anti-AG IgG (but not normal IgG) increases DAMGOand Endo-2induced acute spinal tolerance, supporting the proposal that endogenous agmatine exerts a modifying affect on -opioid receptor acute tolerance and providing a mirror image parallel to the studies using exogenous agmatine in the same paradigm. Materials and Methods