S. Regunathan

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.

Agmatine, decarboxylated arginine, is a metabolic product of mammalian cells. Considering the close structural similarity between L-arginine and agmatine, we investigated the interaction of agmatine and nitric oxide synthases (NOSs), which use L-arginine to generate nitric oxide (NO) and citrulline. Brain, macrophages and endothelial cells were respectively used as sources for NOS isoforms I, II and III. Enzyme activity was measured by the production of nitrites or L-citrulline. Agmatine was a competitive NOS inhibitor but not an NO precursor. Ki values were approx. 660 microM (NOS I), 220 microM (NOS II) and 7.5 mM (NOS III). Structurally related polyamines did not inhibit NOS activity. Agmatine, therefore, may be an endogenous regulator of NO production in mammals.

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.

Imidazoline (I) receptors constitute a family of nonadrenergic high-affinity binding sites for clonidine, idazoxan, and allied drugs. One major subclass, the I1 receptors, whose subcellular distribution and signal transduction mechanisms are uncertain, partly mediates the central hypotensive actions of clonidine-like drugs. The I2 receptors, another subclass, are mitochondrial, not G protein coupled, and have diversified functions. Several endogenous ligands for I receptors, collectively termed clonidine-displacing substances (CDSs), have been detected in tissues and serum, but the structure of only one, agmatine (decarboxylated arginine), is known. Agmatine, widely distributed and bioactive, binds, like clonidine, to alpha 2-adrenergic and I receptors of all subclasses. The presence of agmatine and its biosynthetic enzyme in synaptosomes and specific neuronal pathways as well as serum suggests that it may be a novel neurotransmitter/hormone. Another CDS that binds to I receptors and to antibodies to imidazoline drugs has been detected, but its structure is unknown.