Soundararajan Regunathan

Is agmatine a novel neurotransmitter in brain

Recent evidence suggests that agmatine, which is an intermediate in polyamine biosynthesis, might be an important neurotransmitter in mammals. Agmatine is synthesized in the brain, stored in synaptic vesicles in regionally selective neurons, accumulated by uptake, released by depolarization, and inactivated by agmatinase. Agmatine binds to alpha2-adrenoceptors and imidazoline binding sites, and blocks NMDA receptor channels and other ligand-gated cationic channels. Furthermore, agmatine inhibits nitric oxide synthase, and induces the release of some peptide hormones. As a result of its ability to inhibit both hyperalgesia and tolerance to, and withdrawal from, morphine, and its neuroprotective activity, agmatine has potential as a treatment of chronic pain, addictive states and brain injury.

Agmatine, the bacterial amine, is widely distributed 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.

Regional localization of agmatine in the rat brain: an immunocytochemical study.

The distribution of agmatine (decarboxylated arginine) was mapped in the central nervous system (CNS) in the rat. Agmatine-like immunoreactivity was identified by light microscopy, exclusively in the cytoplasm of neuronal perikarya. Immunoreactive neurons were present in the cerebral cortex, predominantly within laminae VI and V and, to a lesser extent, III and mainly in retrosplenial, cingulate, primary somatosensory and auditory cortices, and the subiculum. In the lower brainstem, immunoreactivity was selectively localized to visceral relay nuclei: the nucleus tractus solitarii and pontine parabrachial complex, and periventricular areas including the laterodorsal nucleus, locus coeruleus and dorsal raphe. In the midbrain, immunolabeled cells were concentrated in the ventral tegmental area and periaqueductal gray. In the forebrain, subcortical neurons were labeled predominantly in the preoptic area, amygdala, septum, bed nucleus of the stria terminalis, midline thalamus, and the hypothalamus. Ultrastructural analysis of layer V of the somatosensory cortex demonstrated agmatine-immunoreactivity in neurons, primarily in large dense-core vesicles located in the cytoplasm. Agmatine immunoreactivity was also affiliated with endoplasmic reticulum and the plasmalemma. Cortical neurons and the subiculum were labeled in animals not administered the axonal transport inhibitor, colchicine; thus, may normally contain higher concentrations of the amine than other brain regions. The central distribution of agmatine is consistent with the hypothesis that the amine may be a novel neurotransmitter of neurons involved in behavioral and visceral control.

Agmatinase activity in rat brain: a metabolic pathway for the degradation of agmatine.

Abstract: Agmatinase, the enzyme that hydrolyzes agmatine to form putrescine and urea in lower organisms, was found in rat brain. Agmatinase activity was maximal at pH 8–8.5 and had an apparent Km of 5.3 ± 0.99 mM and a Vmax of 530 ± 116 nmol/mg of protein/h. After subcellular fractionation, most of the enzyme activity was localized in the mitochondrial matrix (333 ± 5 nmol/mg of protein/h), where it was enriched compared with the whole‐brain homogenate (7.6–11.8 nmol/mg of protein/h). Within the CNS, the highest activity was found in hypothalamus, a region rich in imidazoline receptors, and the lowest in striatum and cortex. It is interesting that other agmatine‐related molecules such as arginine decarboxylase, which synthesizes agmatine, and I2 imidazoline receptors, for which agmatine is an endogenous ligand, are also located in mitochondria. The results show the existence of rat brain agmatinase, mainly located in mitochondria, indicating possible degradation of agmatine by hydrolysis at its sites of action.

Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase.

Abstract: We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylornithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatines degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.

Agmatine: An Endogenous Ligand at Imidazoline Receptors Is a Novel Neurotransmittera

ABSTRACT: 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.

Agmatine (decarboxylated arginine) is synthesized and stored in astrocytes

We investigated whether astrocytes store and synthesize agmatine (decarboxylated arginine), an endogenous ligand for imidazoline and α2adrenergic receptors, in brain. Agmatine, detected chemically and immunocytochemically, is contained in cultured astrocytes and C6 glioma cells (8.5 ± 1.4 and 1.8 ± 0.6nmol mg−1protein, respectively). Glial membranes express activity for arginine decarboxylase (ADC), the biosynthetic enzyme for agmatine (astrocytes 85.4 ± 9.2; C6 cells 18.2 ± 3.12 nmolh−1mg−1protein). Lipopolysaccharide, an inducer of glial nitric oxide synthase (iNOS), significantly reduced (C6) or did not affect (astrocytes) ADC activity. Inferferon-γ, not affecting iNOS, elevated ADC activity in both cell types. Astrocytes are a site of synthesis and storage of agmatine. ADC and iNOS enzymes synthesizing distinct bioactive products from L-arginine, may be reciprocally regulated.

Effects of clonidine and other imidazole-receptor binding agents on second messenger systems and calcium influx in bovine adrenal chromaffin cells

Clonidine and related imidazoline compounds bind to alpha 2-adrenergic as well as to newly described non-adrenergic imidazole/imidazoline receptors in brain and peripheral tissues. The present study was undertaken to identify the signal transduction mechanism coupled to this new class of receptors (imidazole receptors) using bovine adrenal chromaffin cells. Clonidine did not modify the basal or forskolin-stimulated production of cyclic AMP (cAMP), suggesting the absence of functionally active alpha 2-adrenergic receptors in adrenal chromaffin cells. Clonidine also failed to modify the basal and GTP gamma S- or carbachol-stimulated increase in phosphoinositide hydrolysis. However, clonidine increased significantly the production of cyclic GMP (cGMP) as well as the uptake of 45Ca2+. The cGMP response to clonidine was slower (peak at 15 min) and smaller (only about 50% over control) than the response to acetylcholine and was not shared by other agents that bind to imidazole receptors. In contrast, all agents that bind to imidazole receptors increased the influx of 45Ca2+ into chromaffin cells. It is concluded that (a) alpha 2-adrenergic and imidazole receptors are functionally distinct and linked to different signal transduction mechanisms; (b) the classical G-protein coupled soluble second messenger systems are not coupled to imidazole receptors; (c) clonidine may increase cGMP by a non-receptor-mediated intracellular action; and (d) imidazole receptors may regulate intracellular calcium levels through an ion regulating system that may be different from calcium channels.

Immunocytochemical localization of an imidazoline receptor protein in the central nervous system

Imidazoline (I) receptors have been implicated in the regulation of arterial blood pressure and behavior although their distribution in the central nervous system (CNS) remains in question. Presumptive I- receptor sites were detected in the rat central nervous system with a polyclonal antibody to an imidazoline receptor protein (IRP) with binding characteristics of the native receptor. IRP-like immunoreactivity (LI) was detected in neurons and glia by light and electron microscopy. Spinal cord: processes were heavily labeled in superficial laminae I and II of the dorsal horn, lateral-cervical and -spinal nuclei and sympathetic cell column. Medulla: label was concentrated in the area postrema, rostral, subpostremal and central subnuclei of nucleus tractus solitarii, spinal trigeminal nucleus caudalis, and inferior olivary subnuclei. Visceromotor neurons in the dorsal vagal and ambigual nuclei were surrounded by high concentrations of immunoreactive processes. In reticular formation, label was light, though predominant in the intermediate reticular zone and ventrolateral medulla. Pons: label was detected in the neuropil of the periventricular gray, concentrated in the dorsal- and external-lateral subnuclei of lateral parabrachial nucleus, and present intracellularly in the mesencephalic trigeminal nucleus. Midbrain: IRP-LI was most heavily concentrated in the interpeduncular nucleus, nuclei interfascicularis and rostral-linearis, the subcommissural organ, central gray, and in glia surrounding the cerebral aqueduct. Diencephalon: high densities were detected in the medial habenular nucleus, nucleus paraventricularis thalami, other midline-intralaminar thalamic nuclei, the supramammillary and mediobasal hypothalamic nuclei. In the median eminence, immunolabeled processes were restricted to the lamina interna and lateral subependymal zone. Telencephalon: IRP-LI was concentrated in the central amygdaloid nucleus, bed nucleus of stria terminalis and globus pallidus, followed by moderate labeling of the medial amygdaloid nucleus, amygdalostriatal zone and caudoputamen, the hilus of the dentate gyrus, and stratum lacunosum-moleculare of field CA1 of Ammons horn. The subfornical organ and organum vasculosum lamina terminalis were filled with diffuse granular immunoreactivity. Ultrastructural studies identified IRP-LI within glia and neurons including presynaptic processes. I-receptor(s) localize to a highly restricted network of neurons in the CNS and circumventricular regions lying outside of the blood-brain barrier. Putative imidazoline receptors have a unique distribution pattern, show partial overlap with alpha 2 adrenoreceptors and are heavily represented in sensory processing centers and the visceral nervous system.

Uptake of Agmatine into Rat Brain Synaptosomes: Possible Role of Cation Channels

Abstract: Agmatine (decarboxylated arginine), an endogenous ligand for imidazoline receptors, has been identified in brain where it is synthesized from arginine by arginine decarboxylase. Here we report a mechanism for the transport of agmatine into rat brain synaptosomes. The uptake of agmatine was energy‐ and temperature‐dependent and saturable with a Km of 18.83 ± 3.31 mM and a Vmax of 4.78 ± 0.67 nmol/mg of protein/min. Treatment with ouabain (Na+,K+‐ATPase inhibitor) or removal of extracellular Na+ did not attenuate the uptake rate. Agmatine transport was not inhibited by amino acids, polyamines, or monoamines, indicating that the uptake is not mediated by any amino acid, polyamine, or monoamine carriers. When we examined the effects of some ion‐channel agents on agmatine uptake, only Ca2+‐channel blockers inhibited the uptake, whereas a reduction in extracellular Ca2+ increased it. In addition, some imidazoline drugs, such as idazoxan and phentolamine, were strong noncompetitive inhibitors of agmatine uptake. Thus, a selective, Na+‐independent uptake system for agmatine exists in brain and may be important in regulating the extracellular concentration of agmatine.