Agmatine reduces alcohol drinking and produces antinociceptive effects in rodent models of alcohol use disorder.

Alcohol use disorder (AUD) is a chronic, relapsing disorder characterized by an escalation of drinking and the emergence of negative affective states over time. Within this framework, alcohol may be used in excessive amounts to alleviate withdrawal-related symptoms, such as hyperalgesia. Future effective therapeutics for AUD may need to exhibit the ability to reduce drinking as well as to alleviate co-morbid conditions such as pain, and to take mechanistic sex differences into consideration. Agmatine is an endogenous neuromodulator that has been previously implicated in the regulation of reward and pain processing. In the current set of studies, we examined the ability of agmatine to reduce escalated ethanol drinking in complementary models of AUD where adult male and female mice and rats were made dependent via chronic, intermittent ethanol vapor exposure (CIE). We also examined the ability of agmatine to modify thermal and mechanical sensitivity in alcohol-dependent male and female rats. Agmatine reduced alcohol drinking in a dose-dependent fashion, with somewhat greater selectivity in alcohol-dependent female mice (versus non-dependent female mice), but equivalent efficacy across male mice and both groups of male and female rats. In mice and female rats, this efficacy did not extend to sucrose drinking, indicating some selectivity for ethanol reinforcement. Female rats made dependent on alcohol demonstrated significant hyperalgesia symptoms, and agmatine produced dose-dependent antinociceptive effects across both sexes. While additional mechanistic studies into agmatine are necessary, these findings support the broad-based efficacy of agmatine to treat co-morbid excessive drinking and pain symptoms in the context of AUD.

Sex and the Lab: An Alcohol-Focused Commentary on the NIH Initiative to Balance Sex in Cell and Animal Studies.

In May 2014, Dr. Francis Collins, the director of U.S. National Institutes of Health (NIH), and Dr. Janine Clayton, the director of the U.S. National Institutes of Health Office of Research on Women's Health, published a commentary in the journal Nature announcing new policies to ensure that preclinical research funded by the NIH considers both males and females. While these policies are still developing, they have already generated great interest by the scientific community and triggered both criticism and applause. This review provides a description and interpretation of the NIH guidelines, and it traces the history that led to their implementation. As expected, this NIH initiative generated some anxiety in the scientific community. The use of female animals in the investigation of basic mechanisms is perceived to increase variability in the results, and the use of both sexes has been claimed to slow the pace of scientific discoveries and to increase the cost at a time characterized by declining research support. This review discusses issues related to the study of sex as a biological variable (SABV) in alcohol studies and provides examples of how researchers have successfully addressed some of them. A practical strategy is provided to include both sexes in biomedical research while maintaining control of the research direction. The inclusion of sex as an important biological variable in experimental design, analysis, and reporting of preclinical alcohol research is likely to lead to a better understanding of alcohol pharmacology and the development of alcohol use disorder, may promote drug discovery for new pharmacotherapies by increasing scientific rigor, and may provide clinical benefit to women's health. This review aims to promote the understanding of the NIH's SABV guidelines and to provide alcohol researchers with a theoretical and practical framework for working with both sexes in preclinical research.

Neural mechanisms of pain and alcohol dependence.

An association between chronic pain conditions and alcohol dependence has been revealed in numerous studies with episodes of alcohol abuse antedating chronic pain in some people and alcohol dependence emerging after the onset of chronic pain in others. Alcohol dependence and chronic pain share common neural circuits giving rise to the possibility that chronic pain states could significantly affect alcohol use patterns and that alcohol dependence could influence pain sensitivity. The reward and emotional pathways that regulate drug/alcohol addiction also mediate chronic pain. For example, pain-evoked activation of brain learning and brain reward circuitry may modulate cortical processing of pain and central sensitization mediated by mesocorticolimbic circuitry. Imbalance and reorganization of amygdala-mPFC interactions may not only be important for persistent pain, but also for disorders characterized by the abnormal persistence of emotional-affective states such as drug and alcohol addiction. Further studies are necessary to understand how these neural circuits are regulated in comorbid conditions of alcoholism and chronic pain. In addition, long term alcohol use could induce pain symptoms and may exacerbate chronic pain arising from other sources. While prior studies have established a role of neuroendocrine stress axis mediators in alcohol abuse and neurotoxic effects, these studies have not explored the distinction between the individual impact of alcohol and stress hormones. Future studies should explore the mechanisms mediating the contribution of alcohol and stress axis hormones on pain, an important question in our understanding of the neurobiology of alcohol abuse and chronic pain.

Agmatine levels in the cerebrospinal fluid of normal human volunteers.

Agmatine is an amine formed by the decarboxylation of l-arginine by the enzyme arginine decarboxylase. The fact that exogenous agmatine modulates morphine analgesia and dependence raises the question of whether the biosynthesis of endogenous agmatine is regulated during chronic pain. As a first step to understand the biological role of agmatine in human neurological and psychiatric conditions, this study was aimed to determine the levels of cerebrospinal fluid (CSF) agmatine in normal individuals. The levels of agmatine in the CSF and blood were measured by high-performance liquid chromatography (HPLC) method. Samples of CSF and blood were collected from a total of 10 participants for this study. The CSF agmatine levels ranged from 24.3 to 54.0 ng/mL, whereas the plasma agmatine levels were from 8.4 to 65.1 ng/mL. The mean values with standard error for blood and CSF agmatine were 33.8 +/- 16.6 and 40.4 +/- 9.1, respectively. The statistical analysis of these 10 samples indicated no correlation between blood and CSF samples (r = .29); however, removing one outlier improved the correlation (r = .6). From this study, the authors conclude that human CSF agmatine levels can be measured by HPLC with precision and that a possible correlation exists between plasma and CSF agmatine levels. This study provides basis for future studies in human chronic pain conditions.

Repeated immobilization stress alters rat hippocampal and prefrontal cortical morphology in parallel with endogenous agmatine and arginine decarboxylase levels.

Agmatine, an endogenous amine derived from decarboxylation of L-arginine catalyzed by arginine decarboxylase, has been proposed as a neurotransmitter or neuromodulator in the brain. In the present study, we examined whether agmatine has neuroprotective effects against repeated immobilization-induced morphological changes in brain tissues and possible effects of immobilization stress on endogenous agmatine levels and arginine decarboxylase expression in rat brains. Sprague-Dawley rats were subjected to 2h immobilization stress daily for 7 days. This paradigm significantly increased plasma corticosterone levels, and the glutamate efflux in the hippocampus as measured by in vivo microdialysis. Immunohistochemical staining with beta-tubulin III showed that repeated immobilization caused marked morphological alterations in the hippocampus and medial prefrontal cortex that were prevented by simultaneous treatment with agmatine (50mg/kg/day), i.p.). Likewise, endogenous agmatine levels measured by high-performance liquid chromatography in the prefrontal cortex, hippocampus, striatum and hypothalamus were significantly increased by immobilization, as compared to controls. The increased endogenous agmatine levels, ranging from 92 to 265% of controls, were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. These results demonstrate that the administration of exogenous agmatine protects the hippocampus and medial prefrontal cortex against neuronal insults caused by repeated immobilization. The parallel increase in endogenous brain agmatine and arginine decarboxylase protein levels triggered by repeated immobilization indicates that the endogenous agmatine system may play an important role in adaptation to stress as a potential neuronal self-protection mechanism.

Exogenous agmatine has neuroprotective effects against restraint-induced structural changes in the rat brain.

Agmatine is an endogenous amine derived from decarboxylation of arginine catalysed by arginine decarboxylase. Agmatine is considered a novel neuromodulator and possesses neuroprotective properties in the central nervous system. The present study examined whether agmatine has neuroprotective effects against repeated restraint stress-induced morphological changes in rat medial prefrontal cortex and hippocampus. Sprague-Dawley rats were subjected to 6 h of restraint stress daily for 21 days. Immunohistochemical staining with beta-tubulin III showed that repeated restraint stress caused marked morphological alterations in the medial prefrontal cortex and hippocampus. Stress-induced alterations were prevented by simultaneous treatment with agmatine (50 mg/kg/day, i.p.). Interestingly, endogenous agmatine levels, as measured by high-performance liquid chromatography, in the prefrontal cortex and hippocampus as well as in the striatum and hypothalamus of repeated restraint rats were significantly reduced as compared with the controls. Reduced endogenous agmatine levels in repeated restraint animals were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. Moreover, administration of exogenous agmatine to restrained rats abolished increases of arginine decarboxylase protein levels. Taken together, these results demonstrate that exogenously administered agmatine has neuroprotective effects against repeated restraint-induced structural changes in the medial prefrontal cortex and hippocampus. These findings indicate that stress-induced reductions in endogenous agmatine levels in the rat brain may play a permissive role in neuronal pathology induced by repeated restraint stress.

Chronic treatment with glucocorticoids alters rat hippocampal and prefrontal cortical morphology in parallel with endogenous agmatine and arginine decarboxylase levels.

In the present study, we examined the possible effect of chronic treatment with glucocorticoids on the morphology of the rat brain and levels of endogenous agmatine and arginine decarboxylase (ADC) protein, the enzyme essential for agmatine synthesis. Seven-day treatment with dexamethasone, at a dose (10 and 50 mug/kg/day) associated to stress effects contributed by glucocorticoids, did not result in obvious morphologic changes in the medial prefrontal cortex and hippocampus, as measured by immunocytochemical staining with beta-tubulin III. However, 21-day treatment (50 mug/kg/day) produced noticeable structural changes such as the diminution and disarrangement of dendrites and neurons in these areas. Simultaneous treatment with agmatine (50 mg/kg/day) prevented these morphological changes. Further measurement with HPLC showed that endogenous agmatine levels in the prefrontal cortex and hippocampus were significantly increased after 7-day treatments with dexamethasone in a dose-dependent manner. On the contrary, 21-day treatment with glucocorticoids robustly reduced agmatine levels in these regions. The treatment-caused biphasic alterations of endogenous agmatine levels were also seen in the striatum and hypothalamus. Interestingly, treatment with glucocorticoids resulted in a similar change of ADC protein levels in most brain areas to endogenous agmatine levels: an increase after 7-day treatment versus a reduction after 21-day treatment. These results demonstrated that agmatine has neuroprotective effects against structural alterations caused by glucocorticoids in vivo. The parallel alterations in the endogenous agmatine levels and ADC expression in the brain after treatment with glucocorticoids indicate the possible regulatory effect of these stress hormones on the synthesis and metabolism of agmatine in vivo.

Agmatine: biological role and therapeutic potentials in morphine analgesia and dependence.

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, alpha2-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.

Agmatine protects against cell damage induced by NMDA and glutamate in cultured hippocampal neurons.

Agmatine is a polyamine and has been considered as a novel neurotransmitter or neuromodulator in the central nervous system. In the present study, the neuroprotective effect of agmatine against cell damage caused by N-methyl-D-aspartate (NMDA) and glutamate was investigated in cultured rat hippocampal neurons. Lactate dehydrogenase (LDH) activity assay, beta-tubulin III immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay were conducted to detect cell damage. Exposure of 12-day neuronal cultures of rat hippocampus to NMDA or glutamate for 1 h caused a concentration-dependent neurotoxicity, as indicated by the significant increase in released LDH activities. Addition of 100 microM agmatine into media ablated the neurotoxicity induced by NMDA or glutamate, an effect also produced by the specific NMDA receptor antagonist dizocilpine hydrogen maleate (MK801). Arcaine, an analog of agmatine with similar structure as agmatine, fully prevented the NMDA- or glutamate-induced neuronal damage. Spermine and putrescine, the endogenous polyamine and metabolic products of agmatine without the guanidine moiety of agmatine, failed to show this effect, indicating a structural relevance for this neuroprotection. Immunocytochemical staining and TUNEL assay confirmed the findings in the LDH measurement. That is, agmatine and MK801 markedly attenuated NMDA-induced neuronal death and significantly reduced TUNEL-positive cell numbers induced by exposure of cultured hippocampal neurons to NMDA. Taken together, these results demonstrate that agmatine can protect cultured hippocampal neurons from NMDA- or glutamate-induced excitotoxicity, through a possible blockade of the NMDA receptor channels or a potential anti-apoptotic property.

Expression of arginine decarboxylase in brain regions and neuronal cells.

After our initial report of a mammalian gene for arginine decarboxylase, an enzyme for the synthesis of agmatine from arginine, we have determined the regional expression of ADC in rat. We have analyzed the expression of ADC in rat brain regions by activity, protein and mRNA levels, and the regulation of expression in neuronal cells by RNA interference. In rat brain, ADC was widely expressed in major brain regions, with a substantial amount in hypothalamus, followed by cortex, and with least amounts in locus coeruleus and medulla. ADC mRNA was detected in primary astrocytes and C6 glioma cells. While no ADC message was detected in fresh neurons (3 days old), significant message appeared in differentiated neurons (3 weeks old). PC12 cells, treated with nerve growth factor, had higher ADC mRNA compared with naive cells. The siRNA mixture directed towards the N-terminal regions of ADC cDNA down-regulated the levels of mRNA and protein in cultured neurons/C6 glioma cells and these cells produced lower agmatine. Thus, this study demonstrates that ADC message is expressed in rat brain regions, that it is regulated in neuronal cells and that the down-regulation of ADC activity by specific siRNA leads to lower agmatine production.

Agmatine reduces extracellular glutamate during pentylenetetrazole-induced seizures in rat brain: a potential mechanism for the anticonvulsive effects.

Glutamate has been implicated in the initiation and spread of seizure activity. Agmatine, an endogenous neuromodulator, is an antagonist of NMDA receptors and has anticonvulsive effects. Whether agmatine regulate glutamate release, as measured by in vivo microdialysis, is not known. In this study, we used pentylenetetrazole (PTZ)-induced seizure model to determine the effect of agmatine on extracellular glutamate in rat brain. We also determined the time course and the amount of agmatine that reached brain after peripheral injection. After i.p. injection of agmatine (50 mg/kg), increase of agmatine in rat cortex and hippocampus was observed in 15 min with levels returning to baseline in one hour. Rats, naïve and implanted with microdialysis cannula into the cortex, were administered PTZ (60 mg/kg, i.p.) with prior injection of agmatine (100 mg/kg, i.p.) or saline. Seizure grades were recorded and microdialysis samples were collected every 15 min for 75 min. Agmatine pre-treatment significantly reduced the seizure grade and increased the onset time. The levels of extracellular glutamate in frontal cortex rose two- to three-fold after PTZ injection and agmatine significantly inhibited this increase. In conclusion, the present data suggest that the anticonvulsant activity of agmatine, in part, could be related to the inhibition glutamate release.

Agmatine attenuates stress- and lipopolysaccharide-induced fever in rats.

Physiological stress evokes a number of responses, including a rise in body temperature, which has been suggested to be the result of an elevation in the thermoregulatory set point. This response seems to share similar mechanisms with infectious fever. The aim of the present study was to investigate the effect of agmatine on different models of stressors [(restraint and lipopolysaccharide (LPS)] on body temperature. Rats were either restrained for 4 h or injected with LPS, both of these stressors caused an increase in body temperature. While agmatine itself had no effect on body temperature, treatment with agmatine (20, 40, 80 mg/kg intraperitoneally) dose dependently inhibited stress- and LPS-induced hyperthermia. When agmatine (80 mg/kg) was administered 30 min later than LPS (500 microg/kg) it also inhibited LPS-induced hyperthermia although the effect became significant only at later time points and lower maximal response compared to simultaneous administration. To determine if the decrease in body temperature is associated with an anti-inflammatory effect of agmatine, the nitrite/nitrate levels in plasma was measured. Agmatine treatment inhibited LPS-induced production of nitrates dose dependently. As an endogenous molecule, agmatine has the capacity to inhibit stress- and LPS-induced increases in body temperature.

Effect of agmatine on the development of morphine dependence in rats: potential role of cAMP system.

Agmatine is an endogenous amine derived from arginine that potentiates morphine analgesia and blocks symptoms of naloxone-precipitated morphine withdrawal in rats. In this study, we sought to determine whether treatment with agmatine during the development of morphine dependence inhibits the withdrawal symptoms and that the effect is mediated by cAMP system. Exposure of rats to morphine for 7 days resulted in marked naloxone-induced withdrawal symptoms and agmatine treatment along with morphine significantly decreasing the withdrawal symptoms. The levels of cAMP were markedly increased in morphine-treated rat brain slices when incubated with naloxone and this increase was significantly reduced in rats treated with morphine and agmatine. The induction of tyrosine hydroxylase after morphine exposure was also reduced in locus coeruleus when agmatine was administered along with morphine. We conclude that agmatine reduces the development of dependence to morphine and that this effect is probably mediated by the inhibition of cAMP signaling pathway during chronic morphine exposure.

Expression of human arginine decarboxylase, the biosynthetic enzyme for agmatine.

Agmatine, an amine formed by decarboxylation of L-arginine by arginine decarboxylase (ADC), has been recently discovered in mammalian brain and other tissues. While the cloning and sequencing of ADC from plant and bacteria have been reported extensively, the structure of mammalian enzyme is not known. Using homology screening approach, we have identified a human cDNA clone that exhibits ADC activity when expressed in COS-7 cells. The cDNA and deduced amino acid sequence of this human ADC clone is distinct from ADC of other forms. Human ADC is a 460-amino acid protein that shows about 48% identity to mammalian ornithine decarboxylase (ODC) but has no ODC activity. While naive COS-7 cells do not make agmatine, these cells are able to produce agmatine, as measured by HPLC, when transfected with ADC cDNA. Northern blot analysis using the cDNA probe indicated the expression of ADC message in selective human brain regions and other human tissues.

Agmatine reduces only peripheral-related behavioral signs, not the central signs, of morphine withdrawal in nNOS deficient transgenic mice.

Agmatine inhibits morphine tolerance/dependence and potentiates morphine analgesia. This study was designed to investigate whether neuronal nitric oxide mediates the actions of agmatine in morphine dependence by using mice lacking a functional form of this enzyme. Mice received agmatine just after the morphine pellet implantation for 3 days twice daily or single injection 30 min before naloxone. In both genotypes treated for 3 days with morphine pellets, naloxone administration precipitated clear signs of withdrawal. Both acute and chronic administration of agmatine reduced withdrawal signs in wild type mice and reduced only peripheral signs of morphine dependence in neuronal nitric oxide synthase knockout mice. Withdrawal signs, that are related to central nervous system activity were not affected. These findings indicate that neuronal nitric oxide synthase partly mediates the effects of agmatine in morphine physical dependence.

Effect of agmatine against cell death induced by NMDA and glutamate in neurons and PC12 cells.

AIMS: Agmatine is an endogenous guanido amine and has been shown to be neuroprotective in vitro and in vivo. The aims of this study are to investigate whether agmatine is protective against cell death induced by different agents in cultured neurons and PC12 cells. METHODS: Cell death in neurons, cultured from neonatal rat cortex, was induced by incubating with (a) NMDA (100 microM) for 10 min, (b) staurosporine (protein kinase inhibitor, 100 nM) for 24 h, and (c) calcimycin (calcium ionophore, 100 nM) for 24 h in the presence and absence of agmatine (1 micro M to 1 mM). Cell death in PC12 cells was induced by exposure to glutamate (10 mM), staurosporine (100 nM), and calcimycin (100 nM). The activity of lactate dehydrogenase (LDH) in the medium was measured as the marker of cell death and normalized to cellular LDH activity. RESULTS: Agmatine significantly reduced the medium LDH in NMDA-treated neurons but failed to reduce the release of LDH induced by staurosporin or calcimycin. In PC12 cells, agmatine significantly reduced LDH release induced by glutamate exposure, but not by staurosporine or calcimycin. Agmatine itself neither increased LDH release nor directly inhibited the enzyme activity. CONCLUSION: We conclude that agmatine protects against NMDA excitotoxicity in neurons and PC12 cells but not the cell death induced by protein kinase blockade or increase in cellular calcium.

Regulation of inducible nitric oxide synthase and agmatine synthesis in macrophages and astrocytes.

Agmatine is a novel endogenous guanido amine synthesized from arginine by arginine decarboxylase. Among several biologic effects, the ability of agmatine to protect against ischemic injury and chronic neuropathic pain is particularly interesting. Because inflammation is a common contributor to these conditions, we sought to determine if agmatine acts by decreasing the production of proinflammatory molecules such as nitric oxide and if agmatine synthesis is regulated by inflammatory stimuli. We tested whether agmatine affects astroglial and macrophage (RAW 264.7 cell line) nitric oxide synthase-2 (NOS-2) expression. NOS-2 was induced in these cells by incubation with lipopolysaccharide (LPS) plus three cytokines for astrocytes and LPS alone for RAW 264.7 cells in the presence and absence of varying concentrations of agmatine. NOS-2 activity was assessed after 24 hours by nitrite accumulation in the culture media. Agmatine dose-dependently inhibited nitrite accumulation, and shorter incubation with agmatine (1 and 4 hours) also caused significant reduction. Agmatine decreased the expression of NOS-2 activity and NOS-2 protein as determined by immunoblot analysis. Incubation of astrocytes and RAW 264.7 cells with LPS/cytokines for 2 hours resulted in an increase in arginine decarboxylase (ADC) activity, whereas longer-term incubation (12-17 hours) lowered ADC activity. Agmatine levels in these cells are increased after 6-hour incubation with LPS/cytokines. These results show that agmatine inhibits the production of nitric oxide by decreasing the activity of NOS-2 in macrophages and astroglial cells by decreasing the levels of NOS-2 protein. These findings provide a molecular basis for the neuroprotective and anti-inflammatory actions of agmatine.

Is agmatine an endogenous factor against stress?

Agmatine is an endogenous amine synthesized from the decarboxylation of arginine. A proposed intracellular role of agmatine is to balance the production of polyamines (a promitotic process) and nitric oxide (an inflammatory process). Agmatine is also released from neurons upon depolarization. We previously reported that agmatine concentrations are increased in rat pups' brains shortly after hypoxic-ischemia and in the plasma of depressed patients. Herein, male rats (270-290 g) were divided into four groups receiving different degrees of known stress: 2-hour restraint at 21 degrees C, 4-hour restraint at 21 degrees C, 4-hour restraint at 4 degrees C, and control rats only handled at 21 degrees C. Cortex, cerebellum, medulla, hippocampus, hypothalamus, and blood plasma samples were collected for determination of endogenous agmatine levels. No changes in agmatine levels were detected after 2-hour and 4-hour restraint at room temperature, but concentrations of agmatine were increased in all brain regions except cerebellum after 4-hour restraint in the cold. Plasma agmatine levels (ng/mL) were 6.8 +/- 0.6 in controls versus 58.1 +/- 12.8 in the 4-hour restraint-plus-cold group. Cortical agmatine levels (ng/g wet tissue) were 15.3 +/- 2.4 in controls versus 57.4 +/- 19.6 in the 4-hour restraint-plus-cold group. Therefore, endogenous agmatine was increased in response to cold-restraint stress, possibly as a neuroprotective agent.

Effect of chronic morphine treatment on the biosynthesis of agmatine in rat brain and other tissues.

Agmatine is an endogenous amine derived from the decarboxylation of arginine by arginine decarboxylase (ADC), and metabolized to putrescine by agmatinase. Exogenously administered agmatine has several biological actions including its ability to potentiate morphine analgesia and block symptoms of morphine tolerance/withdrawal in rats. To investigate the role of endogenous agmatine in this action, we sought to determine whether chronic exposure to morphine and induction of withdrawal modulate the synthesis of agmatine in rat brain and other tissues. Exposure of rats to morphine for three days significantly decreases the activity of ADC and the levels of agmatine in rat liver, kidney, brain, aorta and intestine with no changes in agmatinase activity. The precipitation of withdrawal syndrome by injecting naloxone further decreases ADC activity and agmatine levels in these tissues. We conclude that endogenous agmatine may play an important role in regulating morphine tolerance/dependence and withdrawal symptoms.

Imidazoline receptors in the heart: characterization, distribution, and regulation.

Imidazoline receptors were identified in cardiac tissues of various species. Imidazoline receptors were immunolocalized in the rat heart. Membrane binding and autoradiography on frozen heart sections using 0.5 nM para-iodoclonidine (125I-PIC) revealed that binding was equally and concentration-dependently inhibited by epinephrine and imidazole-4-acetic acid (IAA), implying 125I-PIC binding to cardiac alpha2-adrenergic and I1-receptors, respectively. After irreversible blockade of alpha2-adrenergic receptors, binding was inhibited by the selective I1-agonist, moxonidine, and the I1-antagonist, efaroxan, in a concentration-dependent (10-12 to 10-5 M) manner. Calculation of kinetic parameters revealed that in canine left and right atria, I1-receptor Bmax was 13.4 +/- 1.7 and 20.1 +/- 3.0 fmol/mg protein, respectively. Compared to age-matched normotensive Wistar Kyoto rats, I1-receptors were increased in 12-week-old hypertensive rat (SHR) right (22.6 +/- 0.3 to 43.7 +/- 4.4 fmol/unit area, p < 0.01) and left atria (13.3 +/- 0.6 to 30.2 +/- 4.1 fmol/unit area, p < 0.01). Also, compared to corresponding normal controls, Bmax was increased in hearts of hamsters with advanced cardiomyopathy (13.9 +/- 0.4 to. 26.0 +/- 2.3 fmol/unit area, p < 0.01) and in human ventricles with heart failure (12.6 +/- 1.3 to 35.5 +/- 2.9 fmol/mg protein, p < 0.003). These studies demonstrate that the heart possesses imidazoline I1-receptors that are up-regulated in the presence of hypertension or heart failure, which would suggest their involvement in cardiovascular regulation.