Soundar Regunathan

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.

Regulation of Inducible Nitric Oxide Synthase and Agmatine Synthesis in Macrophages and Astrocytes

Abstract: 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.

Effect of Agmatine against Cell death induced by NMDA and glutamate in neurons and PC12 cells

Abstract1. 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.2. Methods: Cell death in neurons, cultured from neonatal rat cortex, was induced by incubating with (a) NMDA (100 μM) 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 μ 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.3. 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.4. 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.

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.

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: 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, 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.

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.

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 β‐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.

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.

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 μg/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 β‐tubulin III. However, 21‐day treatment (50 μg/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.