Regunathan S. Agmatine: Biological Role and Therapeutic Potentials in Morphine Analgesia and Dependence. AAPS Journal. 2006; 8(3): E479-E484. DOI:  10.1208/aapsj080356

Correspondence to:
Soundar Regunathan
Tel: (601) 984-5471
Fax: (601) 984-5899
Email: sregunathan@psychiatry.umsmed.edu

Received: March 10, 2006;  Accepted: March 31, 2006;  Published: July 21, 2006

Abstract

Agmatine is an amine that is formed by decarboxylation of L-arginine by the enzyme arginine decarboxylase (ADC) and hydrolyzed by the enzyme agmatinase to putrescine. Agmatine binds to several target receptors in the brain and has been proposed as a novel neuromodulator. In animal studies, agmatine potentiated morphine analgesia and reduced dependence/withdrawal. While the exact mechanism is not clear, the interactions with N-methyl-D-aspartate (NMDA) receptors, α2-adrenergic receptors, and intracellular cyclic adenosine monophosphate (cAMP) signaling have been proposed as possible targets. Like other monoamine transmitter molecules, agmatine is rapidly metabolized in the periphery and has poor penetration into the brain, which limits the use of agmatine itself as a therapeutic agent. However, the development of agmatinase inhibitors will offer a useful method to increase endogenous agmatine in the brain as a possible therapeutic approach to potentiate morphine analgesia and reduce dependence/withdrawal. This review provides a succinct discussion of the biological role/therapeutic potential of agmatine during morphine exposure/pain modulation, with an extensive amount of literature cited for further details.

Keywords: agmatine, morphine, opioids, analgesia, withdrawal

Introduction

Agmatine is an amine that is formed by decarboxylation of L-arginine by the enzyme arginine decarboxylase (ADC) and hydrolyzed by the enzyme agmatinase (agmatine uryl hydrolase) to putrescine. After an initial report in 1994,¹ several laboratories confirmed the presence of agmatine in the brain and its interaction with several target receptors such as α2-adrenergic, imidazoline, and NMDA receptors.^1-4 These observations suggested that agmatine may have functions of a novel neurotransmitter/neuromodulator.⁵ Using specific antibodies to agmatine,⁶ we have shown by immunocytochemical studies that in the brain agmatine (1) is stored in the perikarya of a specific population of central neurons,⁷ and (2) is found in small vesicles of axon terminals^8,9 that form synaptic contacts and is presumably costored and released with traditional transmitters/modulators, including l-glutamate and arginine vasopressin.⁹ Agmatine is synthesized by a mammalian form of ADC^10,11 whose complementary DNA (cDNA) sequence has been recently identified.¹² Agmatine can be degraded by agmatinase in the brain¹³ and by diamine oxidase in peripheral tissues.¹⁴ The physiological role of agmatine in normal brain function is still unknown, in part because of the absence of adequate pharmacological tools to manipulate its synthesis and degradation. Moreover, since agmatine has several molecular targets and acts as an antagonist in most targets, it has been difficult to evaluate the function of endogenous agmatine in the whole organism. However, as discussed below, studies of the actions of exogenous agmatine have identified several intriguing neurally relevant functions of the amine that are of potential therapeutic importance. Agmatine administered intrathecally, locally or systemically, reduces the neuronal injury produced by excitotoxins,^15,16 global/focal ischemia,^17-19 spinal cord injury,^17,20 and hypoxic ischemic injury.²¹ Another notable effect of agmatine is its ability to reduce chemically and electrically induced convulsive seizures.^22-24 Agmatine has also been proposed as an adjunct in the treatment of several chronic pain syndromes, and as effective in facilitating the action of morphine while profoundly reducing the development of tolerance.²⁵ Agmatine administered intrathecally or intraperitoneally (IP) blocks the development of morphine tolerance^20,25 and inhibits naloxone-precipitated signs of morphine withdrawal in rats.^26-29 Agmatine also has notable effects on learning behavior in fear-conditioning models^30,31 and antidepressant-like effects in depression models.^32-34

Exogenous Agmatine, Morphine, and Analgesia

Several studies have reported the in vivo effects of agmatine on morphine analgesia/dependence, nociceptive responses, neuronal injury, and other behavioral effects in several animal models, as recently reviewed by Nguyen et al.²⁹ The first study, from the Pasternak laboratory, showed that agmatine at the dose of 10 mg/kg (subcutaneous) potentiated morphine analgesia and prevented the development of tolerance to chronic morphine.²⁵ Subsequently, several studies have confirmed the effects of agmatine on morphine pain tolerance and withdrawal symptoms with doses ranging from 1 to 25 mg/kg.^26,35,36 Most intriguing is agmatine’s ability to potentiate the analgesic effect of morphine while also reducing the withdrawal symptoms after chronic exposure.^35,37 Thus, acute injection of agmatine by the peripheral route increases the analgesic effects of morphine and reduces tolerance to repeated morphine injection. Agmatine has been shown to reduce symptoms of withdrawal from morphine as measured by physical dependence in animal models. The reduction of central symptoms required the expression of functional neuronal nitric oxide synthase activity.³⁸ In related studies, intravenous agmatine was shown to evoke the escalation of fentanyl, but not cocaine, self-administration.³⁹ Besides potentiating morphine analgesia, agmatine, by itself, also has noted effects on nociceptive responses.^20,27,40-43 For example, agmatine can act like an antihyperalgesic agent in reducing the mechanical and inflammation-induced hypersensitivity to pain stimulation.²⁰ It is also important to point out that in all animal studies reported, agmatine had no effect on normal behavior, motor activity, or cardiovascular parameters and had no other toxic effects in normal animals in doses up to 100 mg/kg.

Molecular Targets of Agmatine Action

As agmatine does not bind to opiate receptors, the analgesic effects and the reduction of withdrawal to morphine are not likely to be mediated by a direct effect on opiate receptors.⁴⁴ However, agmatine interacts with several other target proteins that could mediate its effects. Agmatine binds to NMDA receptors and acts as an antagonist at NMDA receptor channels,^8,16,45-47 and NMDA antagonists are known to block opioid withdrawal symptoms.⁴⁸ Agmatine also binds to α2-adrenergic receptors, and agonists of α2-adrenergic receptors have been known to inhibit opioid withdrawal. The activation of α2-adrenergic receptors by agonists like clonidine inhibits dependence and withdrawal. While agmatine was discovered because of its ability to bind to α2-adrenergic receptors,¹ several subsequent functional studies reported that agmatine is not an agonist at this site.^4,49 Moreover, administration of α2-adrenergic agonists like clonidine, while blocking opiate withdrawal, causes sympathetic inhibition and reduction in arterial pressure.^50-52 In several animal models, agmatine, administered intracerebro-ventricular (i.c.v.) or IP, has not been shown to lower arterial pressure,^53-55 thus ruling out the possibility of α2-adrenergic receptor activation in this action of agmatine. Thus, the actions of agmatine on morphine pain response and withdrawal are most likely mediated by its ability to block NMDA receptor channels.⁵⁶ Meanwhile, other studies have suggested that the blockade of NMDA receptors may not be the only mechanism by which agmatine regulates glutamatergic neurotransmission during acute or chronic morphine exposure. Agmatine has been shown to modulate presynaptic calcium channels,^57,58 and such an effect could lead to lower glutamate release after acute agmatine injection. Our recent results indicate that acute agmatine administration reduces extracellular glutamate during pentylenetetrazole (PTZ)-induced convulsive seizures as measured by in vivo microdialysis.⁵⁹ Thus, agmatine may also regulate the release of glutamate during morphine withdrawal.

In initial behavior studies in rats and mice, symptoms of withdrawal from morphine, induced by naloxone, were reduced by agmatine when it was injected with naloxone,^25,35 probably because the agmatine directly inhibited NMDA receptors. More recently, we showed that chronic injection of agmatine during morphine exposure (lasting 7 days), but not at the time of inducing withdrawal by naloxone, substantially reduced the withdrawal symptoms.³⁷ Since agmatine was not present during the induction of withdrawal, direct inhibition of NMDA receptors could not be involved in this mode of agmatine action. Therefore, it appears that agmatine, administered during the development of morphine dependence, blocks the events leading to a hyperexcitable state of the neurons during chronic morphine exposure, thereby reducing withdrawal. This idea was conceived based on several reports indicating intracellular effects of agmatine. These effects include inhibition of cellular proliferation in kidney and vascular smooth muscle cells,^60,61 inflammatory signaling in macrophages and glial cells,^62-64 and morphine-induced cAMP superactivation in NG108-15 cells⁶⁵ and the rat brain.³⁷

The cellular and molecular mechanisms for opiate tolerance/dependence and withdrawal have been fairly well documented. Electrophysiological and neurochemical studies have indicated that a hyperexcitable state of neurons in the locus coeruleus (LC), the ventral tegmental area (VTA), and the nucleus accumbens after chronic morphine exposure contributes to dependence and withdrawal. These changes occur because of a cycle of molecular events of higher phosphorylation and gene expression, resulting in adaptive upregulation of the cAMP system after chronic morphine abuse.⁶⁶ This upregulation has been shown in in vitro model systems including NG108-15 cells and cells transfected with μ-opioid receptors, as well as in in vivo animal models.^67-70 The resulting higher cAMP causes increased protein kinase A (PKA) activity, which phosphorylates several target proteins, including tyrosine hydroxylase (TH) and cAMP response element binding protein (CREB). The phosphorylated CREB subsequently acts as a transcription factor increasing the expression of several proteins, including adenylate cyclase and TH.⁷¹ There is also evidence that initial suppression of cAMP production by morphine could increase the expression of specific subunits of PKA.⁷¹ All these molecular changes initiate the cycle of events resulting in higher TH, adenylate cyclase, PKA, and several phosphorylated proteins, including membrane sodium channels, causing the hyperexcitable state of the neurons. We hypothesized that chronic administration of agmatine along with morphine interferes with some step in these intracellular signal transduction pathways, thereby reducing dependence/withdrawal. In fact, one previous study reported that agmatine inhibited the increase in cAMP in morphine-exposed NG108-15 cells when the cells were challenged with naloxone.⁶⁵ Our recent report confirmed this finding in brain cortical slices of rats chronically exposed to morphine and agmatine.³⁷ We have also observed that agmatine inhibits the higher expression of TH during chronic morphine exposure in rat LC and striatum (S. Regunathan and F. Aricoglu, unpublished data, December 2004). These initial findings support our hypothesis, but further studies are required to investigate how agmatine regulate the downstream events of cAMP signaling.

Morphine Exposure and Endogenous Agmatine

While exogenous agmatine is clearly effective in modulating opiate analgesia/dependence, whether endogenous agmatine has a similar function is not known. It is important to note that all the brain structures that are involved in drugs of abuse—the VTA, the nucleus accumbens, the amygdala, and the LC—contain substantial agmatine immunoreactive neurons.⁷ Agmatine and ADC activity are known to be regulated in certain other conditions, such as inflammatory neuropathic pain, ischemic stroke, and depression.^18,20,72,73 Our initial studies have indicated that agmatine levels and ADC activity are lower in the rat brain and other tissues after 3 days of exposure to morphine.⁷⁴ Studies on the in vivo release of agmatine during morphine exposure and withdrawal should provide some evidence for the role of endogenous agmatine in opiate drug abuse. However, the ultimate proof will be to show that increasing endogenous agmatine levels reduces symptoms of morphine withdrawal and decreasing these levels exacerbates symptoms of morphine withdrawal. Blocking the biosynthetic enzyme, ADC, could be the direct approach to reducing endogenous agmatine levels. Although no selective inhibitor of mammalian ADC is presently available, other means of decreasing ADC activity are feasible. For example, the cDNA sequence of mammalian ADC can be used to design RNA interference (small double-stranded RNA) to degrade ADC messenger RNA (mRNA), thereby lowering the expression of ADC activity and levels of agmatine. We have recently produced small interfering ribonucleic aid (siRNA) capable of reducing ADC mRNA levels and agmatine production in cultured neurons and glial cells.⁷⁵

Another approach that is currently being used successfully is blocking the action of endogenous agmatine by selective agmatine antibodies. In a recent study, Fairbanks et al showed that antiagmatine immunoglobulin G (IgG), but not normal IgG, reversed exogenous agmatine-mediated, but not MK801-mediated, inhibition of NMDA-evoked behavior in mice and induced tolerance to opioid agonists at lower doses.⁷⁶ These findings were interpreted to mean that sequestration of endogenous agmatine by agmatine-selective IgG increases the susceptibility to tolerance induced by opioid agonists. This strategy has been used previously⁷⁷ to show that an antiserum raised against [Leu5]enkephalin prevents the increase in morphine potency induced by exogenous i.c.v. administration of [Leu5]enkephalin. These results validate the approach of determining the physiological effects of inactivating endogenous agmatine by administering exogenous antisera.

Obviously, further studies using the approach of increasing or decreasing endogenous agmatine are required to establish the proof-of-concept that agmatine is an endogenous protective molecule against morphine/opiate dependence/withdrawal and that increasing its levels in the brain benefits the system. Such observations will also provide a basis for the use of drugs to increase endogenous agmatine levels as a way to potentiate morphine analgesia and reduce morphine dependence/tolerance. Although agmatine is effective in animal models, it is unlikely to be a useful drug candidate because of its rapid metabolism, high turnover, and poor penetration into the brain. Frequent administration of high doses of exogenous agmatine is required to observe the effect in animal models. Thus, increasing endogenous agmatine by other means will be a valuable way to sustain higher levels of agmatine in the brain. One approach would be the use of inhibitors of agmatinase, the major degradative enzyme for agmatine in the brain.⁷⁸ The inhibitors of mammalian agmatinase are currently being evaluated,³⁴ and these initial efforts have identified a certain class of compounds as potential targets. It is important to identify selective agmatinase inhibitors since many structural analogs of agmatine are also potent inhibitors of NMDA receptors. For example, arcaine (diguanido butane), a potent inhibitor of agmatinase,³⁴ is a well-known NMDA antagonist.⁴⁵ From our initial screening of selective compounds, we have identified 3-aminoprpopyl guanidine as a potent inhibitor of agmatinase in vitro with no binding to NMDA receptors.³⁴ Further studies are under way to determine whether agmatinase inhibitors can actually increase endogenous agmatine levels in the brain in vivo. The use of agmatinase inhibitors along with exogenous agmatine will be a very useful approach to sustaining higher levels of agmatine in the brain.

Conclusions

One of the most fascinating aspects of agmatine, an endogenous molecule, is its ability to potentiate the analgesic effect of morphine while also reducing morphine dependence and withdrawal symptoms. At the same time, agmatine has absolutely no effect in naive animals on behavior, locomotion, or cardiovascular functions. Here, therefore, we have the opportunity to manipulate a system that is activated only when the normal homeostasis of the brain/cells/neurons is altered, for example, in the hyperexcitable state after chronic morphine exposure. Moreover, as agmatine has multiple molecular targets with low affinity and, thus, is easily reversible in functional actions with no toxic effects, it has tremendous therapeutic potential. The use of agmatine by itself or along with selective agmatinase inhibitors will be a valuable therapeutic approach for several targets, including ischemic injury, convulsive seizures, and opiate analgesia with reduced risk of dependence.

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