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Determination of agmatine in brain and plasma using high-performance liquid chromatography with fluorescence detection
Abstract
Decarboxylated arginine, agmatine, is a neurotransmitter candidate for imidazoline receptors. A method is described to measure agmatine in rat brain and human plasma by isocratic high-performance liquid chromatography (HPLC) with flourescence detection and o-phthalaldehyde derivatization. Quantitation is based on the method of additions of internal agmatine spikes. This assay has sensitivity in the low picomole range and a detection limitof 100 fmol. The correlation coefficient for the agmatine standard curve was 0.999±0.001 S.D., and intra- and inter-assay C.V.s were less than 8%. The accuracy of our isocratic method compared favorably with a gradient HPLC protocol, originally developed for bacterial agmatine, which we modified for use with tissues. Agmatine concentrations in rat brain were proportioned similarly to the regional distribution of imidazoline-1 receptors. These methods can be used as reliable research tools in various biological samples.
... Disease grading was performed according to the GOLD 2017 report. Patients were classified as group A (low risk, few symptoms), group B (low risk, many symptoms), group C (high risk, few symptoms) and group D (high risk, many symptoms) according to the number of symptoms and exacerbations [18]. Thirty-five people who presented to Cumhuriyet University Medical Faculty Research and Training Hospital and did not have any systemic disease comprised the control group. ...
... The serums obtained were portioned for the determination of agmatine, telomerase, SIRT 1 and deubiquitinase, and stored at -40°C until the analysis was performed. In our study, agmatine levels were determined by high-performance liquid chromatography (HPLC) [18]. Sirtuin, deubiquitinase and telomerase levels were measured through the ELISA method. ...
... Plasma agmantine levels were determined at 450 nm emission and 350 nm excitation wave length using C18 column (250 Â 4 mm, Agilent Technologies, Palo Alto, CA) at HP Agilent 1260 HPLC series of High Pressure Liquid Chromatography (HPLC) system with the method which was first defined by Raasch, Regunathan, Li, and Reis (1995) in 1995 and modified by Feng, Halaris, and Piletz (1997) in 1997and Molderings et al. (2004 in 2004. ...
... Plasma agmantine levels were determined at 450 nm emission and 350 nm excitation wave length using C18 column (250 Â 4 mm, Agilent Technologies, Palo Alto, CA) at HP Agilent 1260 HPLC series of High Pressure Liquid Chromatography (HPLC) system with the method which was first defined by Raasch, Regunathan, Li, and Reis (1995) in 1995 and modified by Feng, Halaris, and Piletz (1997) in 1997and Molderings et al. (2004 in 2004. ...
Objectives: Agmatine is a cationic amine resulting from the decarboxylation of l-arginine. Agmatine has neuroprotective, anti-inflammatory, anti-stress, and anti-depressant properties. In this study, plasma agmatine, arginine decarboxylase, and agmatinase levels were measured during manic episode and remission period in patients with bipolar disorder.
Methods: Thirty healthy volunteers and 30 patients who meet Bipolar Disorder Manic Episode diagnostic criteria were included in the study. Additionally, the changes in the patient group between manic episode and remission period were examined. We evaluated the relationship between levels of l-arginine and arginine decarboxylase in the agmatine synthesis pathway, and level of agmatinase that degrades agmatine.
Results: Levels of agmatine and l-arginine were significantly increased than control group during manic episode (p < .01). All parameters were increased during manic episode compared to remission period (p < .05). Agmatinase was significantly decreased both during manic episode (p < .01) and remission period (p < .05) in comparison to the control group. Arginine decarboxylase levels did not show a significant difference between the groups (p > .05).
Conclusions: This study indicate that there may be a relationship between bipolar disorder and agmatine and its metabolic pathway. Nonetheless, we believe more comprehensive studies are needed in order to reveal the role of agmatine in etiology of bipolar disorder.
• Key points
• Agmantine, agmatinase, l-arginine and arginine decarboxylase levels in BD have not been explored before.
• Various neuro-chemical mechanisms act to increase agmatine in BD; however, agmatine could have elevated to compensate agmatine deficit prior to the manifestation of the disease as in schizophrenia.
• Elevated agmatine degradation resulting from excess expression of agmatinase which is suggested to be effective in pathogenesis of mood disorders was compensated by this way.
• Elevated agmatine may be one of the causes which play a role in mania development.
• Elevated agmatine levels are also suggested to trigger psychosis and be related with the etiology of manic episode and lead to BD.
... The striatum has a concentration of 430 ng/g and the hypothalamus show levels of 490 ng/g of this polyamine (Zhu et al., 2007). In a study by Feng et al. (1997) different concentrations of agmatine was also reported in rat brain, as follows: pons/medulla (1105 ng/g), frontal cortex (760 ng/g), midbrain (873 ng/g), hypothalamus (844 ng/g), hippocampus (611 ng/g), cerebellum (331 ng/ g). Conversely, a much lower agmatine concentration (2.40 ng/g) was reported in total brain by Raasch et al. (1995). ...
... It is synthesized, stored in synaptic vesicles (Otake et al., 1998), and released by depolarization (Reis and Regunathan, 1999), is enzymatically degraded by agmatinase (Sastre et al., 1996), and may undergo reuptake (Reis and Regunathan, 1998a;Sastre et al., 1997). Moreover, it binds with high affinity to some cell-surface receptors (Li et al., 1994; and attains brain concentrations similar to neurotransmitters, such as dopamine (Feng et al., 1997;Raasch et al., 1995). It has also been reported that agmatine possesses neuroprotective properties against oxidative stress, mitochondrial dysfunction, excitotoxicity, inflammatory and apoptotic conditions (Arndt et al., 2009;Condello et al., 2011;Fairbanks et al., 2000;Feng et al., 2002;Gilad et al., 1996;Hong et al., 2007;Horvath et al., 1999;Taksande et al., 2015;Yu et al., 2000). ...
Agmatine is a neuromodulator that regulates multiple neurotransmitters and signaling pathways. Several studies have focused on elucidating the mechanisms underlying the neuroprotective effects of this molecule, which seems to be mediated by a reduction in oxidative damage, neuroinflammation, and proapoptotic signaling. Since these events are implicated in acute and chronic excitotoxicity-related disorders (ischemia, epilepsy, traumatic brain injury, spinal cord injury, neurodegenerative, and psychiatric disorders) as well as in nociception, agmatine has been proposed as a therapeutic strategy for the treatment of central nervous system (CNS) disorders. Agmatine also stimulates the expression of trophic factors and adult neurogenesis, contributing to its ability to induce endogenous repair mechanisms. Therefore, considering its wide range of biological effects, this review summarizes the current knowledge about its protective and regenerative properties in the CNS.
... According to the neurodevelopmental hypothesis, schizophrenia is defined as a severe mental illness which leads to the complex table in the brain by creating a problem in almost all functions in the brain such as thinking, perception, cognitive functions, and mood. Because schizophrenia starts at an early age (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) for men, while in women 25-35 years of age) by showing a chronic course. For many years, it causes the quality of life of patients; it is one of the most important disability diseases in the world that limits a person's movements, senses, or activities [1]. ...
... Thus, agmatine has been receiving more attention polyamine than spermidine in schizophrenia. In fact, although agmatine is found in the brain as a very little amount, present endogenous agmatine is sufficient to interact with the receptors [19,20]. Agmatine is synthesised with arginine Agmatine mediated hypertonic stress development in Schizophrenia: a Novel study ...
The aim of this study was to investigate precursors such as agmatine, ornithine involved in the synthesis of polyamines, enzymes level of these pathways and their correlation with diseases. In this study, 37 patients with schizophrenia and 37 healthy individuals no systemic disease (diabetes, hypertension, schizophrenia and mental illness) were taken as the control group. Determination of Ornithine decarboxylase, Arginine Decarboxylase, and Agmatinase levels were determined according to the each standard enzyme curve graph by using ELISA kits, Arginase activity, and ornithine levels were measured spectrophotometrically. When arginase activity was compared to the control group, it was determined that arginase activity was dropped significantly in the patient group (p=0.001 p<0.05). On the other hand, statistically significant increase in ornithine and arginine decarboxylase levels was found (p=0.001 p<0.05) (p=0.044 p<0.05) respectively. Even though the increase in ornithine decarboxylase and agmatinase levels was found, it was not statistically significant (p>0.05). The increase in ornithine levels in schizophrenia is mediated by increasing levels of agmatine just as hypo glutamatergic hypothesis put forward in schizophrenia. As a result, it can be suggested that agmatine may play a fundamental role in the development of osmolarity and hypertonic stress in schizophrenia and may cause a negative impact on the prognosis of the disease
... Although for a long time, it was considered as a constituent of bacteria, plants and invertebrates, later evidences suggested that agmatine exists also in mammalian tissues including brain.[2][3][4] It is widely distributed in the brain and its concentration is similar to the other neurotransmitters concentrations.[3][4][5] Because agmatine is synthesized in the brain, stored in synaptic vesicles, accumulated by uptake, released by depolarization and inactivated by agmatinase, it is considered to be a putative neurotransmitter.[6] ...
... Agmatine-like immunoreactivity has been detected in axon terminals of CA1 area, synapsing with pyramidal cells.[12] This agmatine is suggested to be co-stored with glutamate in hippocampal synaptic vesicles.[5] In hippocampal slices, agmatine superfusion decreases CA1 neural discharges.[13] ...
Reports on agmatine are controversial showing that it may improve memory, it can deteriorate memory and some did not notice any interference with learning and memory. In the present study, the effect of directly intra-CA1 agmatine microinjection on water maze learning and memory has been assessed.
The cannuls were implanted in hippocampal CA1 regions of rats in a sterotaxic frame after general anesthesia. After one week recovery period, the animals were assessed in the reference memory version of water maze. Agmatine (1, 10, 100 or 200 μg/0.5 μl) or saline were infused 20 minutes before or immediately after training.
Agmatine-treated rats did not show any significant difference neither in water maze acquisition nor in consolidation task in comparison with control and sham groups.
Agmatine does not affect water maze learning and memory.
... Biological significance of agmatine W. Raasch et al animals designed to investigate this question have not been performed until now. In humans, substantially higher plasma concentrations (47 ng ml 71 ) were determined when compared to rats (Feng et al., 1997). The reasons underlying this large dierence remain to be clari®ed. ...
... A similar structureactivity relationship was also reported concerning the MAO inhibitory activity of agmatine . Since the agmatine concentration (100 mM) required for an eective NMDA receptor blockade is relatively high, one has to question the physiological signi®cance of this eect, especially against the background of published agmatine tissue concentrations Feng et al., 1997;Stickle et al., 1996). However, since agmatine is not uniformly distributed in the CNS, but preferentially distributed in certain areas and subcellular structures (Otake et al., 1998;, a concentration adequate to block NMDA might be reached, providing that the agmatine becomes released. ...
... The flow rate was set to be 1 mL/min. Agmatine amounts were measured by comparing plasma sample chromatograms and standard chromatograms [13]. ...
ABSTRACT
Objectives: Since we assumed that endometriosis is a benign cell division disorder, our study
was conducted to investigate the effects of the relationships between polyamine synthesis and
angiogenesis in the formation of endometriosis.
Material and methods: Thirty-five patients with endometriosis and 35 healthy female
women were included in the study. The patient and the control groups were compared
regarding the blood levels of agmatine, argininecarboxylase (ADC), ornithinecarboxylase
(ODC), agmatinase, arginase, ornithine, and the vascular endothelial growth factor (VEGF).
Results: There is a statistically significant difference between the patient and the control
groups regarding the agmatinase, arginase and VEGF levels (higher in the patient group) (p <
0.05). There is no statistically significant difference between the patient and the control
groups regarding the ODC, ornithine and the ADC levels (p > 0.05). There is a statistically
significant difference between the patient and the control groups regarding the agmatine levels
(higher in the control group) (p < 0.05).
Conclusions: The increase in the serum levels of polyamine synthesis enzymes may
contribute to the formation of endometriosis. It is anticipated that the study of the relationship
between enzymes and molecules in the polyamine synthesis pathway and angiogenesis in
patients with endometriosis will contribute to the literature.
Key words: angiogenesis; apoptosis; endometriosis; polyamine
... Agmatine is a polyamine formed via decarboxylation of l-arginine by the enzyme arginine decarboxylase. It is widely distributed in the brain with a concentration similar to the other neurotransmitter concentrations (Feng et al., 1997;Piletz et al., 2013). Considering that agmatine can be synthesized in the brain, stored in synaptic vesicles, accumulated by uptake, released by depolarization and inactivated by agmatinase, it is considered to be a putative neurotransmitter (Reis and Regunathan, 2000;Piletz et al., 2013). ...
Agmatine, a polyamine derived from L-arginine, has been suggested to modulate memory. However, the available evidence regarding the effect of agmatine on the memory of intact animals is contradictory. This study aimed to assess the dose-response effect of subchronic agmatine on passive avoidance memory and anxiety-like parameters of elevated plus maze in adult intact mice. Furthermore, considering the roles of Akt/GSK-3β signaling pathway in memory and Alzheimer's disease, the hippocampal contents of phosphorylated and total forms of Akt and GSK-3β proteins were determined using the western blot technique. Agmatine was administered intraperitoneally at the doses of 10, 20, 30, 40 and 80 mg/kg/daily to adult male NMRI mice for 10 days after which the behavioral assessments were performed. Upon completion of the passive avoidance test, the hippocampi were removed for western blot analysis to detect the phosphorylated and total levels of Akt and GSK-3β proteins. Results showed the biphasic effect of agmatine on passive avoidance memory; in lower doses (10, 20 and 30 mg/kg), agmatine impaired memory whereas in higher ones (40 and 80 mg/kg) improved it. Though, agmatine in none of the doses affected animals' anxiety-like parameters in an elevated plus maze. Moreover, the memory-improving doses of agmatine augmented Akt/GSK-3β pathway. This study showed the biphasic effect of agmatine on passive avoidance memory and an augmentation of hippocampal Akt/GSK-3β signaling pathway following the memory-improving doses of this polyamine.
... In this context, agmatine has been considered a potential therapeutic tool for the management of diverse CNS disorders (for review, see Neis et al., 2017). Agmatine is an endogenous polyamine synthesized from L-arginine decarboxylation and is widely distributed in the CNS (Feng, et al., 1997;Otake et al., 1998;Raasch et al., 1995), including cortical and subcortical areas typically associated with ADHD. It binds to imidazoline receptors, inhibits glutamate N-methyl-d-aspartate (NMDA) receptors, and inhibits neuronal nitric oxide synthase (Halaris & Piletz, 2007;Reis & Regunathan, 2000). ...
Attention Deficit Hyperactivity Disorder (ADHD) is a highly prevalent and disabling disorder that frequently persists into adulthood. Many patients are considered nonresponders to typical pharmacological treatments due to insufficient symptoms' reduction or the inability to tolerate the side effects of these medications. Agmatine is an endogenous neuromodulator with emotional- and cognitive-enhancing properties that arises as a promising agent to manage several Central Nervous System disorders. Here, we investigated the effects of chronic treatment with agmatine on behavioral impairments exhibited by adult Spontaneously Hypertensive Rats (SHR), an animal model for the study of ADHD. Adult male Wistar and SHR (3-4 months old) received intraperitoneal (i.p.) treatment with saline (NaCl 0.9%) or agmatine (30 mg/kg/day) during 20 consecutive days and were evaluated in a battery of behavioral tasks. Agmatine treatment improved olfactory and recognition memory impairments of SHR evaluated in the olfactory discrimination, object recognition, and social recognition memory tasks. In addition, agmatine administration improved the cognitive flexibility in the water maze test. Agmatine did not alter SHR's locomotor activity and hedonic-like behaviors observed in the open-field and splash tests, respectively. No changes were observed in SHR's systolic blood pressure following agmatine treatment. This study provides the first evidence that agmatine improves olfactory and cognitive impairments observed in an animal model of ADHD. (PsycInfo Database Record (c) 2021 APA, all rights reserved).
... Therefore, the agmatinergic system is regarded as a candidate to pharmacologically exert neuromodulatory and neuroprotective effects, as summarized in some recent review articles [28,36]. Abundantly found in the hippocampus [13,40], agmatine has been postulated to affect learning and memory and there is a possibility that this polyamine could have advantage in AD amnesia [39]. In this regard, agmatine has shown protective effects against memory impairment in radial arm maze task [4] and depression induced by beta amyloid administration [20]. ...
β-Amyloid peptide (Aβ), the major element of senile plaques in Alzheimer’s disease (AD), has been found to accumulate in brain regions critical for memory and cognition. Deposits of Aβ trigger neurotoxic events which lead to neural apoptotic death. The present study examined whether agmatine, an endogenous polyamine formed by the decarboxylation of l-arginine, possesses a neuroprotective effect against Aβ-induced toxicity. Primary rat hippocampal cells extracted from the brains of 18–19-day-old embryos were exposed to 10 µM of Aβ (25–35) in the absence or presence of agmatine at 150 or 250 µM. Additionally, the involvement of Akt (Protein Kinae B), GSK-3β (glycogen synthase kinase 3-β), ERK (Extracellular Signal-Regulated Kinase) and TNF-α (Tumor necrosis factor-α) in the agmatine protection against Aβ-induced neurotoxicity was investigated. Agmatine significantly prevented the effect of Aβ exposure on cell viability and caspase-3 assays. Furthermore, agmatine considerably restored Aβ-induced decline of phospho-Akt and phospho-GSK and blocked Aβ-induced increase of phospho-ERK and TNF-alpha. Taken together, these findings might shed light on the protective effect of agmatine as a potential therapeutic agent for AD.
... Reactions were stopped with the addition of 1/5 volume of 40% trichloroacetic acid. The concentration of the agmatine product was determined by HPLC as described previously [38,39]. ...
Mercury compounds are well-known toxic environmental pollutants and potently induce severe neurotoxicological effects in human and experimental animals. Previous studies showed that one of the mechanisms of mercury compounds neurotoxicity arose from the over-activation of the N-methyl D-aspartate (NMDA)-type glutamate receptor induced by increased glutamate release. In this work, we aimed to investigate the molecular mechanisms of Hg compounds neurotoxicities by identifying their biological targets in cells. Firstly, the inhibitory effects of four Hg compounds, including three organic (methyl-, ethyl- and phenyl-mercury) and one inorganic (Hg²⁺) Hg compounds, on the activity of arginine decarboxylase (ADC), a key enzyme in the central agmatinergic system, were evaluated. They were found to inhibit the ADC activity significantly with methylmercury (MeHg) being the strongest (IC50=7.96 nM). Furthermore, they showed remarkable inhibitory effects on ADC activity in PC12 cells (MeHg > EtHg > PhHg > HgCl2), and led to a marked loss in the level of agmatine, an endogenous neuromodulatory and neuroprotective agent that selectively blocks the activation of NMDA receptors. MeHg was detected in the immunoprecipitated ADC from the cells, providing unequivocal evidence for the direct binding of MeHg with ADC in the cell. Molecular dynamics simulation revealed that Hg compounds could form the coordination bond not only with cofactor PLP of ADC, but also with substrate arginine. Our finding indicated that MeHg could attenuate the neuroprotective effects of agmatine by the inhibition of ADC, a new cellular target of MeHg, which might be implicated in molecular mechanism of MeHg neurotoxicity.
... Agmatine is a naturally-occurring compound and it is present in mammal tissues. For example, plasma agmatine in both rats (0.45 ng/ml) and humans (47 ng/ml) is well above detectable levels as a result of endogenous release (Raasch et al., 1995;Feng et al., 1997). ...
Methamphetamine abuse remains an alarming public heath challenge, with no approved pharmacotherapies available. Agmatine is a naturally occurring cationic polyamine that has previously been shown to attenuate the rewarding and psychomotor-sensitizing effects of methamphetamine. This study examined the effects of agmatine on the discriminative stimulus and hyperthermic effects of methamphetamine. Adult male rats were trained to discriminate 0.32 mg/kg methamphetamine from saline. Methamphetamine dose dependently increased drug-associated lever responding. The nonselective dopamine receptor antagonist haloperidol (0.1 mg/kg) significantly attenuated the discriminative stimulus effects of methamphetamine (5.9-fold rightward shift). Agmatine (10-100 mg/kg) did not substitute for methamphetamine, but significantly attenuated the stimulus effects of methamphetamine, leading to a maximum of a 3.5-fold rightward shift. Acute 10 mg/kg methamphetamine increased the rectal temperature by a maximum of 1.96±0.17°C. Agmatine (10-32 mg/kg) pretreatment significantly attenuated the hyperthermic effect of methamphetamine. Agmatine (10 mg/kg) also significantly reversed methamphetamine-induced temperature increase. Together, these results support further exploration of the value that agmatine may have for the treatment of methamphetamine abuse and overdose.
... Agmatine has been traditionally quantified employing liq-uid chromatography (LC) with fluorescence detection [12,13], capillary electrophoresis [14], and immunoassays [15], all of which lack the sensitivity and selectivity of a tandem mass spectrometry-based approach. Additionally, the short-lived nature of agmatine in biologically relevant matrices re-quires rapid, high-throughput analysis with the use of isotopi-cally labeled internal standards to accurately quantify this compound. ...
A method has been developed for the direct determination of agmatine in bacterial culture supernatants using isotope dilution ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (UPLC-MS/MS). Agmatine determination in bacterial supernatants is comprised of spiking culture or isolate supernatants with a fixed concentration of uniformly labeled (13)C5,(15)N4-agmatine (synthesized by decarboxylation of uniformly labeled (13)C6,(15)N4-arginine using arginine decarboxylase from Pseudomonas aeruginosa) as an internal standard, followed by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBDF) to improve the reversed-phase chromatographic retention characteristics of agmatine, as well as the selectivity and sensitivity of UPLC-MS/MS detection of this amine in complex biologically derived mixtures. Intrasample precisions for measurement of agmatine in culture supernatants average 4.1 % (relative standard deviation). Calibration curves are linear over the range 5 nM to 10 μM, and the detection limit is estimated at 1.5 nM. To demonstrate the utility of the method, agmatine levels in supernatants of overnight cultures of wild-type (UCBPP-PA14), as well as arginine decarboxylase and agmatine deiminase mutant strains of P. aeruginosa strain UCBPP-PA14 were measured. This method verified that the mutant strains are lacking the specific metabolic capabilities to produce and metabolize agmatine. In addition, measurement of agmatine in supernatants of a panel of clinical isolates from patients with cystic fibrosis revealed that three of the P. aeruginosa isolates hyper-secreted agmatine into the supernatant, hypothesized to be a result of a mutation in the aguA gene. Because agmatine has potential inflammatory activities in the lung, this phenotype may be a virulence factor for P. aeruginosa in the lung environment of cystic fibrosis patients.
... Bioactive Amines: Aspects of Quality and Safety in Food 141 the central nervous system [50][51][52]. ...
Presented in several types of food, bioactive amines are described as organic bases of low molecular weight. They have vasoactive, psychoactive and toxicological characteristics and constitute a potential health risk. The con-centration of amines formed in foods depends on the type of microorganisms present, the action of decarboxylase enzymes produced by microorganisms on specific amino acids and favorable conditions for enzymatic activity. The presence of these chemical metabolites has been suggested as a quality indicator in routine analyzes for food production and marketing monitoring. The detection of bioactive amines can be performed by chromatographic methods, fluorometric and enzymatic kits. Bioactive amine formation can be prevented mainly through the adoption of good manufacturing practices, but the industry can also use other methods such as temperature control in the production chain, modified atmosphere packaging and food irradiation. This review aims to ad-dress the formation of bioactive amines in foods, emphasizing the formation and classification of these metabo-lites, aspects related to health, acceptable limits, detection methods and control methods used in the industry to ensure food safety and quality. The success of this approach is linked to the importance of bioactive amines as quality indicators, as well as the discussion on the development of methodologies for determining these sub-stances and discussion of acceptable parameters in food.
... The plasma agmatine level was determined using an HPLC system, as previously described (Raasch et al., 1995;Feng et al., 1997). The HPLC system (HP 1100 series, HewlettePackard, Palo 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 PIAT2092_proof ■ 23 April 2013 ■ 2/7 Alto, CA) consisted of a quaternary pump (HP, G1311A), a fluorescence detector (HP, G1321 A), and an autosampler (HP, G1329A). ...
Agmatine is an endogenous substance, synthesized from l-arginine, and it is proposed to be a new neurotransmitter. Preclinical studies indicated that agmatine may have an important role in the pathophysiology of schizophrenia. This study was organized to investigate plasma agmatine in patients with schizophrenia and in healthy controls. Eighteen patients with schizophrenia and 19 healthy individuals constituted the subjects. Agmatine levels in the plasma were measured using the HPLC method. The S100B protein level, which is a peripheral biomarker for brain damage, was also measured using the ELISA method. While plasma levels of agmatine in patients with schizophrenia were significantly increased (p < 0.0001) compared to those of healthy individuals (control), there were no significant changes in the levels of S100B protein (p = 0.660). An ROC (receiver operating characteristic) curve analysis revealed that measuring plasma agmatine levels as a clinical diagnostic test would significantly differentiate between patients with schizophrenia and those in the control group (predictive value: 0.969; p < 0.0001). The predictive value of S100B measurements was not statistically significant (p > 0.05). A multiple regression analysis revealed that the age of the patient and the severity of the illness, as indicated by the PANSS score, significantly contributed the plasma agmatine levels in patients with schizophrenia. These results support the hypothesis that an excess agmatine release is important in the development of schizophrenia. The findings also imply that the plasma agmatine level may be a potential biomarker of schizophrenia.
... Agmatine is widely distributed in the brain, and its concentration is similar to that of the classic neurotransmitters (Raasch et al., 1995;Feng et al., 1997;Otake et al., 1998). Reis et al. (1998) demonstrated agmatine-like immunoreactivity in axon terminals that synapse with pyramidal cells in the CA1 sub-region of the hippocampus. ...
... The system dominating at high concentrations (0.2±2 mm) could represent passive diffusion or a carrier-mediated uptake with a K M greater than 2 mm. As the concentration of agmatine in biological fluids is two orders lower [4,29,30], a physiological role is unlikely for this component. Using N 5 -(hexamethylene)amiloride, a known inhibitor of putrescine uptake [24], we have shown that the high-affinity component is saturable, with a very low K M (0.03 mm). ...
Rat hepatocytes in culture take up [14C]-agmatine by both a high-affinity transport system [KM = 0.03 mm; Vmax = 30 pmol·min·(mg protein)−1] and a low-affinity system. The high-affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4-guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C-4-aminobutyrate is also accumulated in the presence of an inhibitor of 4-aminobutyrate transaminase.
... The source of circulating AGM remains undefined. In humans, higher plasma concentrations (47 ng/mL) were determined in comparison to rats [13] . The reasons underlying this large difference remain to be clarified. ...
To investigate the effect of administration of agmatine (AGM) on gastric protection against ischemia reperfusion (I/R) injury.
Three groups of rats (6/group); sham, gastric I/R injury, and gastric I/R + AGM (100 mg/kg, i.p. given 15 min prior to gastric ischemia) were recruited. Gastric injury was conducted by ligating celiac artery for 30 min and reperfusion for another 30 min. Gastric tissues were histologically studied and immunostained with angiopoietin 1 (Ang-1) and Ang-2. Vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) were measured in gastric tissue homogenate. To assess whether AKt/phosphatidyl inositol-3-kinase (PI3K) mediated the effect of AGM, an additional group was pretreated with Wortmannin (WM) (inhibitor of Akt/PI3K, 15 μg/kg, i.p.), prior to ischemic injury and AGM treatment, and examined histologically and immunostained. Another set of experiments was run to study vascular permeability of the stomach using Evan's blue dye.
AGM markedly reduced Evan's blue dye extravasation (3.58 ± 0.975 μg/stomach vs 1.175 ± 0.374 μg/stomach, P < 0.05), VEGF (36.87 ± 2.71 pg/100 mg protein vs 48.4 ± 6.53 pg/100 mg protein, P < 0.05) and MCP-1 tissue level (29.5 ± 7 pg/100 mg protein vs 41.17 ± 10.4 pg/100 mg protein, P < 0.01). It preserved gastric histology and reduced congestion. Ang-1 and Ang-2 immunostaining were reduced in stomach sections of AGM-treated animals. The administration of WM abolished the protective effects of AGM and extensive hemorrhage and ulcerations were seen.
AGM protects the stomach against I/R injury by reducing vascular permeability and inflammation. This protection is possibly mediated by Akt/PI3K.
... Agmatine is an amine formed by the decarboxylation of l-arginine by the enzyme arginine decarboxylase (ADC). While long recognized to be synthesized and stored in plants, bacteria, and invertebrates (Tabor and Tabor, 1984), agmatine and its biosynthetic enzyme were discovered in mammals, originally in rat brain (Li et al., 1994), later in other tissues and serum (Feng et al., 1997; Lortie et al., 1996; Raasch et al., 1995). Agmatine binds to imidazoline and α2-adrenoceptors and proposed as an endogenous ligand for imidazoline receptors (Li et al., 1994; Piletz et al., 1995). ...
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.
... The two L3 enzymes had similar pH optima, pH 9 for agmatinase in L3 T. circumcincta and pH 8.5 in H. contortus (Fig. 3B); there was marked loss of activity above the optimum pH. Although agmatinase is now recognised as a neu-ronal enzyme in mammals (Li et al., 1994(Li et al., , 1995Feng et al., 1997), it is more universally present in non-mammalian tissues (Tabor and Tabor, 1984). Arginase and agmatinase belong to the same enzyme family and are capable of using either arginine or agmatine as substrate (Ahn et al., 2004). ...
The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn(2+) and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.
... Accumulating evidence indicates that agmatine is a novel putative neurotransmitter, because it is synthesized in neurons, stored in synaptic vesicles, accumulated by uptake, released presynaptically by depolarization, and inactivated by agmatinase (for reviews see Halaris and Piletz, 2007;Reis and Regunathan, 2000). In the brain, its concentration is similar to that of the classic neurotransmitters (Feng et al., 1997;Raasch et al., 1995). Agmatine binds to imidazoline and a 2 -adrenonergic receptors, evokes a noncompetitive voltage-and concentration-dependent block of the N-methyl-D-aspartate (NMDA) receptor ionophore, regulates the production of nitric oxide (NO) and polyamines by influencing the catalytic activity of nitric oxide synthase (NOS) and ornithine decarboxylase, and regulating polyamine uptake (for reviews see Halaris and Piletz, 2007;Reis and Regunathan, 2000;Satriano, 2003). ...
Agmatine, a metabolite of L-arginine, is considered as a novel putative neurotransmitter. It has been detected in axon terminals that synapse with pyramidal cells in the hippocampus, a brain region that is critically involved in spatial learning and memory. However, the role of agmatine in learning and memory is poorly understood. Recently, we demonstrated water maze training-induced increases in tissue levels of agmatine in the CA1 subregion of the hippocampus. This finding has raised an issue whether an endogenous agmatine could directly participate in learning and memory processes as a neurotransmitter. In the present study, quantitative immunogold-labeling and electron-microscopical techniques were used to analyze the levels of agmatine in CA1 stratum radiatum (SR) terminals (n = 600) of male Sprague-Dawley rats that had been trained to find a hidden escape platform in the water maze (WM) task or forced to swim (SW) in the pool with no platform presented. Agmatine levels were significantly increased by ∼85% in the synaptic terminals of SR of trained WM group compared with the SW control group (all P < 0.001). These results, for the first time, demonstrate spatial learning-induced elevation in agmatine levels at synapses in the hippocampus and provide evidence of its participation in learning and memory processing as a novel neurotransmitter.
... It was originally believed that agmatine was merely a metabolic intermediate in a pathway of polyamine biosynthesis that parallels another pathway by which putrescine is synthesized from ornithine by ornithine decarboxylase (ODC; EC 4.1.1.17). The recent finding that agmatine is synthesized, stored, and released in the brain and other cells and is present in serum [4] suggests that the amine may have other physiological functions, including as a neurotransmitter [5], a regulator of cellular proliferation [6,7], and in inflammation [8]. Thus, ADC plays a crucial role in determining the availability of agmatine in tissues and cells and in the circulation. ...
The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase's structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His(10)-ADC (77.3 kDa) was first purified by Ni-NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus (TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His(10) tag removed recombinant ADC (74.1 kDa)'s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6-9) and temperature (20-80 degrees C). Our research may facilitate further studies of ADC structure and function, including the determination of its crystal structure by X-ray diffraction.
Agmatine, a naturally occurring polyamine derived from arginine via arginine decarboxylase, has been shown to play multifaceted roles in the mammalian body, impacting a wide range of physiological and pathological processes. This comprehensive review delineates the significant insights into agmatine's pharmacological profile, emphasizing its structure and metabolism, neurotransmission and regulation, and pharmacokinetics and function. Agmatine's biosynthesis is highly conserved across species, highlighting its fundamental role in cellular functions. In the brain, comparable to established neurotransmitters, agmatine acts as a neuromodulator, influencing the regulation, metabolism, and reabsorption of neurotransmitters that are key to mood disorders, learning, cognition, and the management of anxiety and depression. Beyond its neuromodulatory functions, agmatine exhibits protective effects across various cellular and systemic contexts, including neuroprotection, nephroprotection, cardioprotection, and cytoprotection, suggesting a broad therapeutic potential.
The review explores agmatine's interaction with multiple receptor systems, including NMDA, α2-adrenoceptors, and imidazoline receptors, elucidating its role in enhancing cell viability, neuronal protection, and synaptic plasticity. Such interactions underpin agmatine's potential in treating neurological diseases and mood disorders, among other conditions. Furthermore, agmatine's pharmacokinetics, including its absorption, distribution, metabolism, and excretion, are discussed, underlining the complexity of its action and the potential for therapeutic application. The safety and efficacy of agmatine supplementation, demonstrated through various animal and human studies, affirm its potential as a beneficial therapeutic agent.
Conclusively, the diverse physiological and therapeutic effects of agmatine, spanning neurotransmission, protection against cellular damage, and modulation of various receptor pathways, position it as a promising candidate for further research and clinical application. This review underscores the imperative for continued exploration into agmatine's mechanisms of action and its potential in pharmacology and medicine, promising advances in the treatment of numerous conditions.
Agmatine, Polyamine, Cytoprotection, Neurotransmitter, Metabolism
Alzheimer's disease (AD) is one of the most prevalent neurodegenerative disease characterized by brain cholinergic dysfunction. Evidence suggests the impairment of memory retrieval phase in AD. It has been shown that CaMKII-α expressing neurons are selectively reduced in the hippocampus in AD brains. The present study aimed to investigate the effect of scopolamine on the memory retrieval phase and the hippocampal CaMKII-α signaling. In addition, the effect of sub-chronic administration of agmatine against scopolamine induced memory and possible hippocampal CaMKII-α deregulation was investigated in mice. Adult male NMRI mice were administered with agmatine at the doses of 5, 10, 20, 30 and 40 mg/kg/i.p. or saline for 11 days. Acquisition and retrieval tests of passive avoidance task were performed on days 10 and 11, respectively (30 Min following agmatine treatment). Scopolamine (1 mg/kg/i.p.) was administered once, 30 Min before retrieval test. Upon completion of the behavioral tasks, the hippocampi were isolated for western blot analysis to detect the phosphorylated and total levels of CaMKII-α and beta actin proteins. The results showed that scopolamine induced memory retrieval deficit and decreased the phosphorylated level of hippocampal CaMKII-α. Sub-chronic agmatine treatment at the dose of 40 mg/kg prevented scopolamine induced memory retrieval deficit and restored the level of hippocampal phosphorylated CaMKII-α. This study suggests that hippocampal CaMKII-α might play a role in scopolamine induced amnesia and sub-chronic agmatine prevents the impairing effect of scopolamine on the retrieval phase of memory and the phosphorylation of hippocampal CaMKII-α protein.
Major depressive disorder (MDD) is a disabling and highly prevalent mood disorder as well as a common cause of suicide. Chronic stress, inflammation, and intestinal dysbiosis have all been shown to play crucial roles in the pathophysiology of MDD. Although conventional antidepressants are widely used in the clinic, they can take weeks to months to produce therapeutic effects. The discovery that ketamine promotes fast and sustaining antidepressant responses is one of the most important breakthroughs in the pharmacotherapy of MDD. However, the adverse psychomimetic/dissociative and neurotoxic effects of ketamine discourage its chronic use. Therefore, agmatine, an endogenous glutamatergic modulator, has been postulated to elicit fast behavioral and synaptogenic effects by stimulating the mechanistic target of rapamycin complex 1 signaling pathway, similar to ketamine. However, recent evidence has demonstrated that the modulation of the NLR family pyrin domain containing 3 inflammasome and gut microbiota, which have been shown to play a crucial role in the pathophysiology of MDD, may also participate in the antidepressant-like effects of both ketamine and agmatine. This review seeks to provide evidence about the mechanisms that may underlie the fast antidepressant-like responses of agmatine in preclinical studies. Considering the anti-inflammatory properties of agmatine, it may also be further investigated as a useful compound for the management of MDD associated with a pro-inflammatory state. Moreover, the fast antidepressant-like response of agmatine noted in animal models should be investigated in clinical studies.
Objectives:
The knowledge that agmatine is found in the human body has existed for several years; however, its role in sepsis has not yet been studied. In the present study, we investigate the role of agmatine in the progression and treatment of sepsis.
Design:
Clinical/laboratory investigations.
Setting:
Medical centers/University-based research laboratory.
Subjects:
Elective ICU patients with severe sepsis and healthy volunteers; C57BL/6 mice weighing 18-22 g.
Interventions:
Serum agmatine level and its associations with inflammatory markers were assessed in patients with sepsis. Agmatine was administered intraperitoneally to mice before a lipopolysaccharide challenge. Human peripheral blood mononuclear cells and murine macrophages were pretreated with agmatine followed by lipopolysaccharide stimulation.
Measurements and main results:
Serum agmatine levels were significantly decreased in patients with sepsis and lipopolysaccharide-induced mice, and correlated with Acute Physiology and Chronic Health Evaluation II score, procalcitonin, tumor necrosis factor-α, and interleukin-6 levels. In a therapeutic experiment, exogenous agmatine attenuated the cytokine production of peripheral blood mononuclear cells from patients with sepsis and healthy controls. Agmatine also exerted a significant beneficial effect in the inflammatory response and organ damage and reduced the death rate in lipopolysaccharide-induced mice. Imidazoline I2 receptor agonist 2-benzofuran-2-yl blocked the pharmacological action of agmatine; whereas, other imidazoline receptor ligands did not. Furthermore, agmatine significantly impaired the inflammatory response by inactivating nuclear factor-κB, but not protein 38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and inducible nitric oxide synthase signaling in macrophages. Activation of imidazoline I2 receptor or knockdown of ribosomal S6 kinase 2 counteracted the effects of agmatine on phosphorylation and degradation of inhibitor of nuclear factor-κBα.
Conclusions:
Endogenous agmatine metabolism correlated with the progression of sepsis. Supplemental exogenous agmatine could ameliorate the lipopolysaccharide-induced systemic inflammatory responses and multiple organ injuries through the imidazoline I2 receptor-ribosomal S6 kinase 2-nuclear factor-κB pathway. Agmatine could be used as both a clinical biomarker and a promising pharmaconutrient in patients with severe sepsis.
Agmatine (AgM, 100 mg/kg i.p.) effect was tested in parallel at two animal models of cerebral ischemia – rat MCAO model (60′/24 h, 60′/48 h, 90′/24 h, 90′/48 h) and gerbil global ischemia (10′) model, administrated 5 min after reperfusion. Aim was to evaluate AgM effect on functional outcome 24 and 48 h after MCAO on neurological and sensor-motor function, and coordination in rats. AgM administration significantly reduced infarct volume, improved neurological score and improved post-ischemic oxidative status. Results of behavioral tests (cylinder test, beam walking test, and adhesive removal test) have shown very effective functional recovery after AgM administration.
Efficiency of AgM administration in gerbils was observed in forebrain cortex, striatum, hippocampus, and cerebellum at the level of each examined oxidative stress parameter (nitric oxide level, superoxide production, superoxide dismutase activity, and index of lipid peroxidation) measured in four different time points starting at 3 h up to 48 h after reperfusion. The highest levels were obtained 6 h after the insult. The most sensitive oxidative stress parameter to AgM was nitric oxide.
Additionally, we performed pharmacological analysis of AgM on rat isolated common carotid arteries. The findings imply that mixed population of potassium channels located on the smooth muscle cells was involved in common carotid artery response to AgM, with predominance of inward rectifying K⁺ channels. In our comparative experimental approach, judged by behavioral, biochemical, as well as pharmacological data, the AgM administration showed an effective reduction of ischemic neurological damage and oxidative stress, hence indicating a direction towards improving post-stroke recovery.
Agmatine (decarboxylated L-arginine), an endogenous ligand at irnidazoline and α2-receptors, is widely distributed in organs and serum. Agmatine and its biosynthetic enzyme argininedecarboxylase are present in brain where it is synthesized and/or stored in neuronal and non-neuronal cells, can be released and taken back up into the cells, and is enzyrnatically degraded by agrnatinase. Hence, agmatine possesses the properties of a neurotransrnitter or neurornodulator. Agmatine is also a precursor of polyamine synthesis in o pathway heretofore not known in mammals. In addition, it is an endogenous inhibitor of nitric oxide synthesis. Clinical relevant properties are among others its neuroprotective effect and that it counteracts opioide tolerance.
In this study, a novel process for enzymatic production of agmatine from L-arginine using recombinant arginine decarboxylase (ADC) was established. The speA gene encoding ADC was expressed in Escherichia coli BL21 (DE3) in a soluble and active form, and recombinant ADC exhibited a maximum specific activity of 0.53 U mg-1 following optimization of culture conditions using an orthogonal array experiment. Up to 14.3 g l−1 of agmatine was obtained from 20 g l−1 of L-arginine in 6 h under optimum conditions (3.5 g l−1 intact cells, 4 mM Mg2+, 30 mM pyridoxal-5′-phosphate (PLP), pH 7, 37 °C). This represents a significant improvement in a method for production of 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.
A method for the determination of endogenous agmatine in rat plasma was developed by isotope dilution-gas chromatography-negative chemical ionization mass spectrometry (GC-NCI/MS). The plasma samples were analyzed after protein precipitation, evaporation, derivatization by hexafluoroacetone (HFAA), and clean-up on a Florisil SPE column. The GC-MS analysis utilized stable isotope d8-agmatine as internal standard. The samples after treatme were tested by negative chemical ionization with selected ion monitoring (SIM) which was set at m/z 492 (molecular ion of agmatine) and m/z 500 (molecular ion of internal standard). The limit of detection (LOD) of agmatine standard solution was 0.005 7 ng/mL. The calibration curve of the agmatine spiked in rat plasma showed a good linear relationship at the range of 1.14-57.0 ng/mL (r = 0.997). The recoveries of agmatine spiked in rat plasma ranged from 92.3% to 109.8%. Inter-day and intra-day precisions were less than 15%. The average concentration level of agmatine in rat plasma was (22 +/- 9) ng/mL, and there was no significant difference between male and female SD rats (p > 0.05). The method is high sensitive and specific, and can be used for the determination of endogenous agmatine in plasma. It provides a strong support for the subsequent research of agmatine.
Agmatine, a cationic polyamine synthesized after decarboxylation of L-arginine by the enzyme arginine decarboxylase, is an endogenous neuromodulator that emerges as a potential agent to manage diverse central nervous system (CNS) disorders. Consistent with its neuromodulatory and neuroprotective properties, there is increasing number of preclinical studies demonstrating the beneficial effects of exogenous agmatine administration on depression, anxiety, hypoxic ischemia, nociception, morphine tolerance, memory, Parkinson`s disease, Alzheimer`s disease, traumatic brain injury related alterations/disorders and epilepsy. The aim of this review is to summarize the knowledge about the effects of agmatine in CNS and point out its potential as new pharmacological treatment for diverse neurological and neurodegenerative diseases. Moreover, some molecular mechanisms underlying the neuroprotective effects of agmatine will be discussed.
Summaryo1.)HPLC quantitation of amines as OPA derivatives has been evaluated and discussed.2.)Stability and characteristics of the C1-C8 aliphatic monoamines, several diamines, including biogenic amines, derivatized with various SH-additives containing OPA reagents of different compositions have been studied from an analytical and theoretical point of view, equally.3.)Stoichiometric studies have been followed as a function of the reaction time by using different SH-additives, varing the molar ratios of OPA/SH-additive from 1/0.5 to 1/50.4.)The composition of derivatives was determined by on-line HPL/MS(ESI) measurements.5.)As a result of an exhaustive derivatization program, performed under strictly the same practical conditions, we obtained comparable results and new knowledge: (i) in the case of the C1-C5 aliphatic amines it has been shown that the use of the OPA/MAP and/or the OPA/NAC=1/50 reagent resulted in two benefits: in an increased stability of the derivatives and in a lower number of species formed, consequently these reagents proved to be proper for their quantitation purposes. (ii) Derivatization studies performed with hexyl, heptyl and ocytl amines revealed that applying the OPA/SH-additive=1/50 reagents, in order to inhibit the formation of the two OPA derivative-containing product, resulted in an additional, transformed OPA derivative: detected and determined by HPLC for the first time, (on the basis of on line HPLC/MS(ESI measurements), these proved to be the two SH-additive-containing OPA derivatives. The proportion of the transformed derivatives can be unambigously influenced by the quality of SH-additive, by the composition of the OPA reagent, i.e., by the molar ratio of the OPA to the SH-additive and by the pH of derivatizations. In terms of side reaction free derivatization the OPA/ET reagents proved to be superb compared to the OPA/MPA one. (iii) In order to improve stability and to increase responses of spermidine and spermine a new principle, the two step derivatization of biogenic amines, has been introduced, applying the OPA/ET/FMOC reagent.
Summary A literature overview is given of HPLC methods currently in use to determine amino acids (AAs) and amines (As) as theiro-phthaldialdyde (OPA)/2-mercaptoethanol (MCE) derivatives. This is followed by an exhaustive derivatization study performed
using the classical proteinogen AAs, C1–C4 aliphatic As, several polyamines (PAs) and the OPA/MCE reagent. Model studies have been carried out as a function of reaction
time and reagent composition. The primary importance of reagent composition (mole ratios of OPA to SH-group additive) is repeatedly
proven. Stabilities of the OPA/MCE derivatives have been compared with corresponding OPA/3-mercaptopropionic acid (MPA) and/or
OPA/N-acetyl-L-cysteine (NAC) compounds, obtained under the same conditions.
Crucial factors proven to influence the stability of OPA derivatives of AAs and As are discussed in detail: (i) preparation
and storage conditions of the OPA reagents, (ii) special behavior of the currently believed to be particularly unstable, AAs
and As associated with their original molecular structure, e.g. glycine, β-alanine, γ-amino butyric acid (GABA), histidine,
ornithine and lysine, several As, as well as (iii) the mole ratios of the OPA/SH-additive/AA and/or A.
Summaryo1.Based on stoichiometric and mass spectrometric studies of the OPA derivatives of AAs the nightmare of their instability has been solved.2.It was confirmed, for the first time, that the common characteristic of the believed to be less stable OPA derivatives is associated with their initial molecular structure: they do contain the -NH2-CH2-R moiety and all of them furnish more than one OPA derivative.3.In the light of the common structure of the multiple OPA derivatives providing compounds a plausible reaction pathway has been described. According to the proposed mechanism hydrogen atoms of the -CH2- group, neighbor to the primary amino group, play the key role in the reaction with an additional molecule of OPA: resulting in consecutive OPA-derivatives transformed from the initially obtained species.4.As analytical consequences, the author of this review, based on recent experience, takes the responsibility to state: OPA derivatives of AAs are very much more stable than they are generally believed to be, considering that some of them are providing more than one reaction product (For the time being the successively formed products have not been taken into account). Consequently, in the HPLC analysis of AAs the following requirements are to be followed: (i) the molar ratios of the OPA/SH-group additive/AA should be close to 20/60/1, (ii) the reagents, saved in the refrigerator at ≤4°C, should be at least 90 min and maximum 9 days old, (iii) the reaction time between the OPA reagent and the AAs should be at least 7 min, (iv) the OPA derivatives should not be acidified before loading them onto the chromatographic column, (v) the HPLC elution procedure should be proper for the separation and quantitation of all components formed, including the more than one OPA derivative providing AAs, (vi) last but not least, in order to be able to get reliable AA contents, in particular when working in the low pM level, the blank value of the reagent should be carried out at least every day and the impurity values are to be substracted from the coeluting AA derivative.
We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.
Plasma agmatine concentrations are elevated significantly in depressed patients compared to healthy controls. Treatment with the antidepressant bupropion normalized plasma agmatine levels. Correlational evidence is presented that a change in plasma agmatine levels may lead to similar changes in platelet I1 imidazoline receptors.
Agmatine, an amine and organic cation, is an endogenous ligand at α2-adrenergic and imidazoline (I-) receptors, to which it binds with high affinity. In addition, agmatine has properties of an endogenous neurotransmitter. Thus, agmatine (a) is locally synthesized in brain by a specific enzyme, arginine decarboxylase; (b) is stored in a large number of neurons with selective distribution in the CNS; (c) is associated with small vesicles in axon terminals that, at least in hippocampus, make synaptic asymmetric (excitatory) synapses on pyramidal cells; (d) is released from synaptosomes in a Ca2+-dependent manner; (e) can be enzymatically degraded by agmatinase in synaptosomes; (f) can be inactivated by selective reuptake; (g) blocks the ligand-gated NMDA receptor channel at sites distinct from ligand-binding and polyamine sites; and (h) has systemic actions when administered intraventricularly. Additionally, (i) agmatine is a precursor of brain putrescine and, hence, of higher polyamines, and (j) it competitively inhibits the activity of all isozymes of nitric oxide synthase. Agmatine meets most criteria to establish it as a novel neurotransmitter/neuromodulator in the CNS. However, agmatine differs from forms of clonidine displacing system with respect to distribution, bioactivity, and capacity to interact with antibodies raised to imidazoline-like drugs. Thus, there are multiple endogenous ligands of the imidazoline receptors, one of which is agmatine.
A high performance liquid chromatography (HPLC) method is described for the determination of agmatine, an endogenous neuromodulator. The method involves pre-column derivatization of the sample with a fluorescent tagging reagent, 7-fluoro-4-nitrobenzoxadiazole (NBD-F). The resulting agmatine derivative is stable and can be readily extracted into ethyl acetate at pH 8.5. The extraction enhances the quantification of low level agmatine because it eliminates chromatographic peaks caused by endogenous amino acids. The HPLC separation is carried out on a C8 reversed phase column and completed in less than 10 min. With laser-induced fluorescence (LIF) detection, the detection limit is 5×10−9 M agmatine. Method precision (coefficient of variation) is 5% for agmatine in human plasma at the sub-μM level. This method has been validated by determination of agmatine in biological samples including human plasma and rat brain and stomach tissues.
A microchip electrophoresis method with laser induced fluorescence detection was developed for the detection of agmatine (Agm)
and octopamine (Oct). The fluorescent derivatization reagent, fluorescein isothiocyanate was used for precolumn derivatization
of Agm and Oct. The sodium dodecyl sulfate (SDS) micelles was employed as pseudostationary phase for the separation of Agm
and Oct with other endogenous compounds exist in biological samples. Some parameters including buffer concentration, buffer
pH, SDS concentration and separation voltage were investigated in detail. Under the optimum conditions, the separation and
determination of Agm and Oct was performed within 40s. The calibration curves were linear for both Agm and Oct over the concentration
range of 1.0×10−7 to 4.0×10−5M and 1.5×10−7 to 4.5×10−5M, respectively. The detection limits of Agm and Oct (S/N=3) are 5.0×10−8 and 8.0×10−8M, respectively. These values make the method very suitable for the determination of Agm and Oct in rat brain tissue and
human plasma as demonstrated in this paper.
The polyamine agmatine modulates a variety of behavioral effects including the abuse-related effects of opioids and has been proposed as a potential medication candidate for the treatment of opioid abuse. However, little is known of the effects of agmatine on the abuse-related effects of other drugs of abuse. This study examined the effects of agmatine on the rewarding effects of methamphetamine in rats using a conditioned place preference paradigm. Methamphetamine (0.1-1.0mg/kg) dose-dependently increased the time spent in methamphetamine-paired side (place preference). Agmatine, at doses that did not produce place preference or aversion (10-32mg/kg), significantly decreased the development of methamphetamine-induced place preference when agmatine was administered in combination with methamphetamine during place conditioning. Agmatine also significantly decreased the expression of methamphetamine-induced place preference when an acute injection of agmatine was given immediately before test session. These doses of agmatine do not alter the motor activity in rats, suggesting that the observed attenuation of methamphetamine-induced place preference was not due to general behavioral disruption. Together, these data suggests that agmatine attenuates the rewarding effects of methamphetamine and may be able to modulate the abuse liability of methamphetamine.
Agmatine is a putative neurotransmitter in the brain. Current analytical techniques do not allow the detection of agmatine in extracellular fluid, making it difficult to study its physiological role. However, a new method for in vivo monitoring agmatine in the brain was developed. Capillary zone electrophoresis and laser induced fluorescence detection (CZE-LIFD) was used to measure nanomolar concentrations of agmatine in submicroliter sample volumes. This analytical technique proved to detect 0.49 attomole of agmatine improving the sensitivity of previous analytical techniques. On the other hand, the hippocampus is a brain region well known for having a population of agmatine containing neurons. Therefore, intracerebral microdialysis was performed in the hippocampus and agmatine was extracted from the extracellular environment. Detectable amounts of agmatine were found in dialysates from probes located in the hippocampus but not from the probes located in the lateral ventricle. Furthermore, extracellular agmatine was calcium and impulse dependent and depolarization of hippocampal neurons increased extracellular agmatine concentration. The methods reported here are sensitive enough to study the physiological role of brain agmatine in freely moving animals.
The biophysiology of the amino acid l-arginine has been a field of active research. Agmatine, which is a metabolite of l-arginine, is known to participate in many biophysical reactions, including those in the cardiovascular system. We sought to investigate plasma agmatine levels in human subjects as a potential biomarker for the metabolic syndrome.
Agmatine concentration was measured in plasma from 322 elderly participants in the Ansan Geriatric study. The metabolic syndrome was defined according to an Asian modified version of criteria established in the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. We observed that the metabolic syndrome was associated with low levels of plasma agmatine concentration.
The mean plasma agmatine level in the metabolic syndrome group was lower than that in the non-metabolic syndrome group (79.42 ng/mL vs. 82.44 ng/mL, P = 0.024). Agmatine remained significant within the regression model after adjustment for different covariates (adjusted odds ratio, 0.962; 95% confidence interval, 0.933-0.993).
We concluded that plasma agmatine levels were lower in subjects with the metabolic syndrome than in those without the metabolic syndrome.
Agmatine-cannabinoid interactions are supported by the close association between cannabinoid CB(1) receptors and agmatine immunoreactive neurons and evidence that shared brain mechanisms underlie the pharmacological effects of agmatine and cannabinoid agonists. In the present study, we used the hot-plate assay of thermal nociception to determine if agmatine alters cannabinoid action through activation of imidazoline sites and/or alpha(2)-adrenoceptors. WIN 55212-2 (1, 2 or 3 mg/kg, i.p.) or CP55,940 (1, 2 or 3 mg/kg, i.p.) administration increased hot-plate response latency. Agmatine (50 or 100 mg/kg, i.p.) was ineffective. Administration of agmatine (50 mg/kg, i.p.) with WIN 55212-2 (1, 2 or 3 mg/kg, i.p.) or CP55,940 (1, 2 or 3 mg/kg, i.p.) produced response-latency enhancement. Regression analysis indicated that agmatine increased the potency of WIN 55212-2 and CP55,940 by 3- and 4.4-fold, respectively, indicating synergy for both drug interactions. Idazoxan, a mixed imidazoline site/alpha(2)-adrenoceptor antagonist, but not yohimbine (5 mg/kg, i.p.), a selective alphia(2)-adrenoceptor antagonist, blocked response-latency enhancement produced by a combination of WIN 55212-2 (2 mg/kg) and agmatine. Response-latency enhancement produced by WIN 55212-2 (2 mg/kg) was blocked by SR 141716A (5 mg/kg, i.p.), a cannabinoid CB(1) receptor antagonist; attenuated by idazoxan (2 and 5 mg/kg); and not affected by yohimbine (5 mg/kg). These results demonstrate a synergistic interaction between agmatine and cannabinoid agonists and suggest that agmatine administration enhances cannabinoid action in vivo.
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.
Two routes of putrescine biosynthesis exist in Escherichia coli both emanating from the latter portion of the arginine-biosynthetic pathway. Isotope competition experiments indicate that both arginine and ornithine can give rise to putrescine. The formation of putrescine from ornithine is independent of the conversion of the latter to arginine.
The decarboxylation of ornithine to yield putrescine is catalyzed by cell-free preparations. This enzyme has been termed “biosynthetic” ornithine decarboxylase to distinguish it from the inducible degradative ornithine decarboxylase, from which it differs in many properties.
Cell-free extracts also carry out the conversion of arginine to putrescine. In this pathway, the arginine carboxyl group is first released as CO2, and the amidine group is then removed as urea. The arginine decarboxylase has optimal activity at pH 8 and an absolute magnesium ion requirement. Both of these characteristics distinguish it from the catabolic arginine decarboxylase. Agmatine ureohydrolase, the enzyme catalyzing the second step of this pathway, is also present in cell-free extracts.
Arginine biosynthesis in E. coli involves a branched pathway, the branch point to putrescine and arginine occurring at ornithine. The existence of two pathways of putrescine biosynthesis is interpreted in relation to control mechanisms of the arginine biosynthetic pathway.
Nonadrenergic imidazoline receptors (I receptors) mediate the central antihypertensive effects of clonidine. Agmatine, an arginine metabolite that is synthesized within bovine brain and exhibits clonidine-displacing substance (CDS) activity, might be the endogenous I receptor neurotransmitter. The authors compared the affinity of agmatine versus the potency of endogenous bovine hypothalamic CDS, at bovine adrenomedullary I1 receptors and at human clonidine-binding sites: human alpha-2A, alpha-2B and alpha-2C expressed on transfected cell lines, I1 sites on human platelet plasma membranes and I2b (amiloride-insensitive) sites on human platelet intracellular membranes. The alpha-2 and I1 sites were labeled with [125I]p-iodoclonidine and the I2b sites were labeled with [3H]-idazoxan. Agmatine displayed preferential affinity for I1 receptors, with both high (H) and low (L) affinity components; the rank order was I1(human)(H) > I1(bovine)(H) > I2b = I1(human)(L) = I1(bovine) (L) = alpha-2A = alpha-2B = alpha-2C. By comparison, hypothalamic CDS competition curves modeled to a single site for all receptors, i.e., I1(bovine) > I2b = alpha-2C > I1(human) = alpha-2A = alpha-2B. Thus, agmatine alone cannot explain the rank order of potency of hypothalamic CDS. Moreover, I1 sites on human platelets differ from I1 sites on bovine adrenomedullary cells with respect to CDS potency but not agmatine affinity. These results do not rule out agmatine as an I1 transmitter but suggest that other I-receptor ligands might exist within endogenous CDS.
The literature on alpha 2-adrenoceptors in depression is replete with seemingly contradictory findings, including reports of both hypersensitive and hyposensitive alterations. Now, with the discovery of nonadrenergic imidazoline receptors (I receptors) and an endogenous I receptor ligand (agmatine), new light is being shed on this controversy. Specifically, those studies that had utilized allegedly "alpha 2-selective" imidazoline radioligands, i.e., 3H-clonidine, could be reinterpreted in terms of increased I receptors in depression. Although the molecular identity of the I1 binding site remains unknown, an I2 receptive site has been reported to be encoded by monoamine oxidase genes (both MAO-A and MAO-B), suggesting a novel explanation for the antidepressant efficacy of idazoxan, a prototypic I2 ligand. Platelet I1 binding sites are also reported to be elevated in patients with unipolar depression and are lowered by antidepressant treatments. Furthermore, clonidine challenge and animal studies of the behavioral effects of imidazolines may be reinterpreted to support a role for I1 sites in the central control of behavior. A hypothesis for depletion of brain clonidine-displacing substance (CDS) in depression is presented. Lowered concentrations of CDS could account for an elevation of I receptors, via compensatory upregulation. Our model offers an explanation for a number of previously discrepant observations as well as testable hypotheses for the study of imidazoline receptors in depression.
An improved method is described for determining picogram quantities of 3-methoxy-4-hydroxyphenylglycol (MHPG) in plasma of humans and of other species. The method makes use of gas-liquid chromatography and electron capture detection. Low level nonlinearity of detector response was corrected by operating the detector in the pulsed rather then the customary steady state mode. Detector overloading was prevented by heat coagulation of plasma proteins and subsequent ultrafiltration. Sensitivity was significantly enhanced by utilizing a derivatizing agent carrying a higher number of electrophores. Baseline conditions are described and control values for plasma MHPG of human volunteers, Rhesus monkeys and rats are presented.
The major cholinergic innervation of the rat cerebral cortex arises from the nucleus basalis in the basal forebrain. Introduction of the excitotoxins kainate or ibotenate into the nucleus basalis by stereotaxic injection results in degeneration of the cholinergic cells. We have investigated the effect of this excitotoxic action on ornithine decarboxylase (ODC) activity and cholinergic responsiveness in the cerebral cortex. A massive and rapid induction of ODC activity was seen in ipsilateral cortex after injection of excitotoxin. A maximal increase in ODC activity of 268 times the control value was seen in ipsilateral cerebral cortex 8 h after lesioning. Thereafter, ODC activity declined but remained significantly greater than control levels for 32 h. Pretreatment of animals with the irreversible ODC inhibitor difluoromethylornithine prevented the induction of ODC by kainate. Tissue content of the ODC product putrescine showed a marked increase in cerebral cortex ipsilateral to the lesion, increasing sevenfold at 24 h, the maximal concentration reached. After 24 h, the level of putrescine decreased but remained significantly elevated above control values for 5 days. Levels of the polyamines spermidine and spermine were unaffected by lesioning. Increases on ODC activity of much smaller magnitude were also seen in brain regions not directly innervated from the ipsilateral nucleus basalis. However, the response in ipsilateral cortex was found to be dependent on an intact projection from nucleus basalis to cortex. The induction of ODC was shown to be prevented by treatment of rats with MK-801, a result indicating the involvement of N-methyl-D-aspartate (NMDA) receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Tritiated agmatine has been used by others in ion flux methods to measure nicotinic receptor function in neurones. However, as shown here, agmatine blocks nicotinic receptor function in both the chick retina and the rat superior cervical ganglion at high concentrations.
In intact chick retina, agmatine 1 m m decreases dimethylphenylpiperazinium (DMPP)‐induced depolarizations measured in the optic nerve by approximately 70%, while having little effect on responses induced by glutamate analogues. DMPP dose‐response curves are reduced in a manner consistent with a non‐competitive effect of agmatine, and agmatine at 1 m m does not prevent binding of ¹²⁵ I‐labelled neuronal bungarotoxin, a snake venom neurotoxin that competitively binds and blocks functional nicotinic receptors in chick retinal homogenates.
Agmatine (10 μ m ) substantially blocks both DMPP‐induced depolarizations of rat superior cervical ganglion and synaptic transmission through the ganglion. Others have established that [ ³ H]‐agmatine will pass through nicotinic receptor channels in the rat ganglion. These data suggest that agmatine acts both as a cation and as a weak channel blocker at neuronal nicotinic receptors.
Escherichia coli K-12 mutants that carry deletions in their genes for ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (speC), arginine decarboxylase (L-arginine carboxy-lyase, EC 4.1.1.19) (speA), and agmatine ureohydrolase (agmatinase or agmatine amidinohydrolase, EC 3.5.3.11) (speB) can still synthesize very small amounts of putrescine and spermidine. The putrescine concentration in these mutants was found to be 1/2500th that in spe+ cells. The pathway of putrescine synthesis appears to be through the biodegradative arginine decarboxylase, which converts arginine to agmatine, in combination with a low agmatine ureohydrolase activity--1/2000th that in spe+ strains. These results suggest that even such low levels of polyamines permit a low level of protein synthesis. Evidence is presented that the polyamine requirement for the growth of the polyamine-dependent speAB, speC deletion mutants, which are also streptomycin resistant, is not due to a decreased ability to synthesize polyamines.
A method is presented for the pre-column derivatization of agmatine, arginine, citrulline or ornithine with o-phthalaldehyde-2-mercaptoethanol, and subsequent separation of the derivatives by reversed-phase liquid chromatography. Fluorescent response is linear from 10 to 150 pmol of injected analyte and detection limits range from 28 to 100 fmol. Response factors relative to the internal standard, homocysteic acid, were 1.16 (agmatine and arginine), 1.03 (citrulline) and 0.34 (ornithine). The applicability of the method to the measurement of arginase, arginine deaminase, arginine decarboxylase and other enzyme activities in bacterial extracts was examined.
To check whether the central hypotensive effect of alpha adrenergic agonists is linked with the stimulation of alpha-2 receptors, such drugs were administered directly to the nucleus reticularis lateralis, which is an important site for the hypotensive action of clonidine. These experiments were carried out by microinjections (0.5 microliter on each side) in normotensive cats anesthetized with pentobarbital. alpha-Methylnorepinephrine, a selective alpha-2 agonist (0.1-10 micrograms/kg) had no hypotensive effect in this region, whereas potent alpha-1 agonists such as cirazoline (0.01-1 micrograms/kg) and ST 587 (1-10 micrograms/kg), like clonidine, produced dose-dependent hypotensive effects. Our results suggest that alpha-2 selective catecholamines are not active in the nucleus reticularis lateralis region, whereas imidazolines induce a hypotensive effect whatever their affinity for one subtype of alpha adrenoceptors. Therefore, there may be some form of structure-activity relationship which would indicate the existence, in this particular region of the medulla oblongata, of sites preferring the imidazoline structure.
Pseudomonas aeruginosa is known to break down arginine by the arginine deiminase pathway. An additional pathway has now been found whereby arginine is converted to putrescine with agmatine and N-carbamoylputrescine as intermediates. The following enzyme activities belonging to this pathway were detected in crude extracts: arginine decarboxylase (EC 4.1.1.19), which catalyses the release of CO2 from arginine to give agmatine; agmatine deiminase (EC 3.5.3.12), which degrades agmatine to N-carbamoylputrescine; and N-carbamoylputrescine amidinohydrolase (EC 3.5.3.-), which then removes the ureido group of carbamoylputrescine. In crude extracts, arginine decarboxylase activity was stimulated by pyridoxal phosphate, Mg2+ and by the products of the catabolic pathway, putrescine and spermidine. Growth of P. aeruginosa on arginine as the sole carbon and nitrogen source markedly increased the activity of arginine decarboxylase. Agmatine and N-carbamoylputrescine induced the synthesis of agmatine deiminase and N-carbamoylputrescine hydrolase. Addition of succinate or citrate to medium containing arginine or agmatine led to repression of the enzymes involved in the arginine decarboxylase pathway. Moreover, the repression of agmatine deiminase and N-carbamoylputrescine hydrolase was further increased when P. aeruginosa was grown in media with agmatine plus glutamine or agmatine plus succinate and ammonia. This suggests that the expression of the agmatine pathway may be regulated by carbon catabolite repression as well as nitrogen catabolite repression.
Agmatine, a clonidine displacing substance and imidazoline receptor agonist, was recently isolated from bovine brain and shown to be present in the rat hypothalamus. Since clonidine can stimulate the release of pituitary luteinizing hormone (LH), we tested the hypothesis that agmatine may similarly act in the rat to stimulate the hypothalamic luteinizing hormone-releasing hormone (LHRH)-pituitary LH axis. Administration of agmatine intracerebroventricularly rapidly augmented the release of LH in a dose-related fashion in ovariectomized, ovarian steroid-primed rats. Additionally, agmatine enhanced the in vitro efflux of LH releasing hormone from the median eminence-arcuate nucleus of the hypothalami of rats similarly pretreated with steroids. These studies imply that the endogenous imidazoline receptor agonist, agmatine, may serve as an excitatory neurotransmitter/neuromodulator in the hypothalamic control of LH release and we suggest that the previously reported excitatory effects of clonidine on LH release may be attributed to stimulation by clonidine of imidazoline receptors.
The present experiments tested the actions of a putative endogenous imidazoline receptor agonist, agmatine, on gastric secretion and on experimental gastric mucosal injury in rats. Agmatine, given i.p. (0.5-20.0 mg/kg) or i.c.v. (0.5-2.5 micrograms), augmented basal gastric acid secretion in conscious rats to a maximum of 40% when given i.p. and 44% when given i.c.v. Agmatine also potentiated total secretory volume as well as gastric acid and pepsin output in pylorus-ligated rats. When administered before exposure to stress, agmatine significantly decreased gastric glandular mucus levels and exacerbated stress-induced gastric mucosal injury. These results are in contrast to our data showing that an exogenous agonist of I1-imidazoline receptors, moxonidine, is a potent antisecretory and gastroprotective agent. A precise physiological role for agmatine in blood pressure regulation and in gastrointestinal function awaits clarification. However, it is possible that agmatine functions as an "inverse agonist" at central imidazoline receptors, resulting in hypertension, augmented gastric secretion and exacerbated gastric mucosal injury.
In summary, classic CDS appears bioactive at some a 2-adrenergic and I-receptor sites where it appears to act as an agonist. It is expressed in serum and CSF wherein in pathological states it may be elevated. However, absence of a structure for classic CDS and of receptor-selective antagonists for I-receptors limits the certainty of whether the biological responses represent the action of a single molecule and to what extent the responses relate to actions at I-receptors. Also, the fact that agmatine may also interact with the same receptor makes it uncertain how much co-purified agmatine may distort computations.
I-receptors can be localized immunocytochemically in rat nervous system with polyclonal antibodies to an IRBP. I-receptors are cytoplasmic and detected in neuronal perikarya, processes, and glia. Labeled neuronal perikarya in the CNS are uncommon and localized to the mesencephalic trigeminal nucleus. I-receptors are heavily represented in primary sensory systems including: somatosensory systems (spinal and trigeminal) and visceral afferent systems (NTS), in central networks subserving autonomic regulation, neuroendocrine control and emotional behaviors, in circumventricular (neurohaemal) organs and in nonneuronal cells including astrocytes with regional densities paralleling neuronal innervation. The distributions of I-receptors and alpha 2-adrenergic receptors partially differ. I-receptors in the CNS appear to relate broadly to the visceral brain and its afferent inputs, particularly pain. Its functions may relate to regulation of integrative behaviors related to stress.
We investigated the cardiovascular responses in anesthetized ventilated rats to agmatine (decarboxylated arginine), an amine which is an endogenous clonidine-displacing substance (CDS) synthesized in brain. Intracisternal agmatine dose-dependently increased sympathetic nerve activity and arterial pressure (at 400 nmol by 8.7 +/- 2.1 microV and 28.6 +/- 2.7 mmHg, respectively) and blocked arterial baroreflex reflexes. Microinjection of agmatine into the rostral ventrolateral medulla (RVL) had no effect on arterial pressure or sympathetic nerve activity while iontophoresis of agmatine onto defined vasomotor neurons of RVL was also without effect. Agmatine (i.v.) decreased sympathetic nerve activity and arterial pressure probably by blocking the transmission through sympathetic ganglia and by direct dilation of vascular smooth muscles. Despite binding like clonidine to alpha 2-adrenergic receptors and imidazoline (I)-receptors of both classes, agmatine does not replicate the central or peripheral actions of clonidine. The results suggest that earlier cardiovascular actions of partially purified CDS were either attributable to contaminating molecules and/or that CDS may be a family of molecules.
Agmatine, a newly identified amine in mammalian brain, is an endogenous ligand for imidazoline and alpha 2-adrenergic receptors. We sought to develop a polyclonal antibody to agmatine suitable for immunocytochemistry. Agmatine was conjugated to keyhole limpet hemocyanin and injected into rabbits. The polyclonal antiserum so generated dose-dependently recognized the agmatine conjugate but not carrier protein by dot blot. Its reaction with the conjugate was selectively antagonized by agmatine but not related compounds. The antiserum, but not pre-immune or pre-adsorbed antiserum, selectively stained cultured adrenal chromaffin cells. Our results indicate that agmatine immunoreactivity is contained in a sub-population of adrenal chromaffin cells and, thus, these antibodies are useful for immunocytochemical localization of the amine in mammalian tissues.
We sought to determine whether agmatine (decarboxylated arginine), a bacterial product recently discovered for the first time in mammalian brain, was contained in other organs. A method was developed for isolation of agmatine from tissue and detection by RP-HPLC following solid-liquid extraction and derivatization with o-phthalaldehyde and mercaptoethanol. Recovery was about 80% and the limit of fluorometric detection was about 10 pg on column. In male Sprague-Dawley rats agmatine was unevenly and widely distributed in many tissues confirming its presence in mammals. The highest concentration (approximately 71 ng/mg net weight) was found in stomach, with aorta and small intestine next, followed by smaller levels in spleen, adrenal, aorta, and skeletal muscle and brain. Serum concentrations were high. Agmatine in male Long Evans rats of 3, 12, and 24 months of age demonstrated similar but not identical tissue distribution without any effect of aging. Since agmatine binds to alpha 2-adrenergic and imidazoline receptors, is bioactive in a number of tissues, is contained in neurons and is found in serum and tissues, the findings are consistent with a potential role for agmatine as a neurotransmitter and/or hormone. It also raises the possibility that agmatine may, as in bacteria, serve as a polyamine precursor along metabolic pathways previously not detected in mammals.
Clonidine, an antihypertensive drug, binds to alpha 2-adrenergic and imidazoline receptors. The endogenous ligand for imidazoline receptors may be a clonidine-displacing substance, a small molecule isolated from bovine brain. This clonidine-displacing substance was purified and determined by mass spectroscopy to be agmatine (decarboxylated arginine), heretofore not detected in brain. Agmatine binds to alpha 2-adrenergic and imidazoline receptors and stimulates release of catecholamines from adrenal chromaffin cells. Its biosynthetic enzyme, arginine decarboxylase, is present in brain. Agmatine, locally synthesized, is an endogenous agonist at imidazoline receptors, a noncatecholamine ligand at alpha 2-adrenergic receptors and may act as a neurotransmitter.