In 1994, we isolated agmatine, an amine synthesized from arginine by arginine decarboxylase (ADC), which bound to alpha2 adrenergic and imidazoline receptors of all subclasses, from cow brain. The discovery was a surprise, as neither agmatine nor ADC, prevalent in bacteria, plants and parasites, had been detected in mammals. Following our initial discovery, a number of other studies confirmed the presence of agmatine and ADC in mammalian tissues. Although ADC had been extensively characterized, purified and cloned from bacteria and plants, it was never studied in mammalian systems. The known properties of mammalian (rat brain) ADC clearly distinguishes it from other isoforms and mammalian ornithine decarboxylase, a closely related enzyme. Further studies on the molecular properties, localization and regulation of ADC in mammalian brain is critical in understanding the neurobiology of agmatine. This Proposal will test the hypothesis that mammalian ADC is a novel enzyme with unique primary structure, localized in selective neuronal cells and regulating the production of agmatine, a putative neurotransmitter/neuromodulator. Study 1 will characterize rat liver/brain ADC in parallel with rat liver ODC with respect to subcellular localization, reaction properties, inhibition profiles, and regulation. The proof that mammalian ADC is a novel, and heretofore unrecognized, enzyme will be to establish its primary structure by molecular cloning. We will clone mammalian ADC by two approaches. As several isoforms of non- mammalian ADC have been cloned, mammalian ADC will be cloned by homology screening by PCR to obtain possible partial clones and to use them to screen cDNA libraries to obtain the full length cDNA clone. As an alternate approach, we will purify ADC from rat liver, obtain peptide sequences, and design oligoprobes based on these sequences to be used in PCR based, or cDNA library, screening. Study 2 Will test the hypothesis that ADC may be a glial enzyme, and that its product, agmatine, is released into the extracellular space and taken up into, and stored in, neurons. We will investigate the localization of ADC and agmatine in cultured neuronal cells and whole rat brain by biochemical and immunocytochemical methods to verify this hypothesis. Study 3 will test the hypothesis that activity of ADC is regulated by inflammatory stimuli in glial cells. We will investigate the regulation of the expression of ADC in cultured astrocytes and macrophages by bacterial toxin, LPS, and cytokines. Comparing the regulation of ADC with NOS-2, we will be able to understand how arginine metabolism is regulated in concert with agmatine or nitric oxide synthesis. At the completion of this project, we will be able to demonstrate that mammalian ADC is a novel enzyme with unique sequence, and localization and regulatory properties, thereby providing valuable information and tools to further understand the biological role of agmatine in mammalian brain.
| Wang, Gang; Gorbatyuk, Oleg S; Dayanithi, Govindan et al. (2002) Evidence for endogenous agmatine in hypothalamo-neurohypophysial tract and its modulation on vasopressin release and Ca2+ channels. Brain Res 932:25-36 |
