ABSTRACT-Agmatine was about as potent as aminoguanidine to inhibit the activity of the inducible form of nitric oxide synthase (iNOS) in isolated rat aorta. Like aminoguanidine, agmatine was devoid of significant activity on the constitutive form of NOS. Agmatine inhibited the conversion of [3H]L-arginine in [3H]L-citrulline in partially purified iNOS from macrophages (IC50=262±39.9 pM). Thus, our data suggest that agmatine may act as endogenous inhibitor of iNOS.Keywords: Agmatine, Nitric oxide, Inducible enzyme L-Arginine is a nutritionally semiessential amino acid involved in a variety of physiological processes (1). Arginine plays a role in protein and creatine biosynthesis and, as an intermediate in the urea cycle, is converted to ornithine by arginase. However, other enzymes that catalyze the conversion of arginine have been identified. In this respect, nitric oxide synthase (NOS) produces the potent vasodilator nitric oxide (NO) (2, 3), and arginine decarboxylase converts arginine to agmatine (4). This latter enzyme, orginally observed in bacteria, has been recently found in mammalian brain (5). In addition, agmatine was identified as an endogenous clonidine-displacing substance (5). Thus, it was hypothesized that arginine may have an antihypertensive effect through NO and agmatine formation by NOS and arginine decarboxylase, respectively (6). However, Pinthong et al. (7) showed that despite recognition at a2-adrenoceptor binding sites, agmatine failed to produce functional a2-adrenoceptor activity not only in the isolated guinea pig ileum but also in other functional models of a2-adrenoceptors. In this respect, agmatine failed to mimic the effect of clonidine in isolated rat vas deferens or isolated porcine palmar lateral vein. In addition, in rat cerebral cortex, agmatine produced a concentrationdependent inhibition of 3H-clonidine, but failed, unlike UK-14304, to inhibit forskolin-stimulated cyclic AMP. Thus collectively, the results indicate that agmatine may not posses sufficient a2-adrenoceptor agonist properties to influence cardiovascular function as recently observed (8). Although a possible role of agmatine as a substrate for NOS has been ruled out (9), other guanidines such as aminoguanidine may act against NO formation (10).Thus, the aim of this study was to test the possible interaction of agmatine with NO synthesis. Thoracic aortae were excised from male Sprague Dawley rats (270-360g;Charles River, Paris, France). Rings, 2-mm-wide, were suspended (2 g tension) in organ baths containing 20 ml of Krebs-Henseleit solution (for composition see below) at 371C and gassed with 95% 02 /5% CO2. After a 1-hr rest period, contractile responses were measured using force-displacement transducers connected to a data collection system (IOS; Dei Leirre, Mitry Mory, France). Physiological solution was composed of: 118 mM NaCl, 4.7 mM KC1, 2.5 mM CaC12, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3 and 11 mM glucose.In a first series of experiments, rings possessing a functional endothelium were precontracted wit...

ABSTRACT

Formation of the lipid peroxidation product 8-epi-prostaglandin2alpha (8-epi-PGF2alpha) a bioactive marker of oxidative stress, was quantified in in vitro and in vivo models of neuronal death. In culture media of primary rat cortical neurones exposed to hypoxia followed by reoxygenation, a 3.7-fold increase of 8-epi-PGF2alpha concentration was observed in comparison to control cells. In rats submitted to 2h middle cerebral artery occlusion followed by a 22h reperfusion period, a 27-fold increase of 8-epi-PGF2alpha was observed in the ischaemic hemisphere compared with the corresponding hemisphere of sham-operated rats. Treatment with the neuroprotective agent BN 80933 significantly reduced both 8-epi-PGF2alpha elevations in vitro and in vivo. These data suggest that 8-epi-PGF2alpha elevations might reflect the damaging free radical overproduction and subsequent lipid peroxidation during neuronal injury induced by hypoxia and ischaemia. Inhibition of 8-epi-PGF2alpha elevations participates to the neuroprotective effects of BN 80933.

Cell death is a common feature observed in neurodegenerative disorders, and is often associated with calpain activation and overproduction of reactive oxygen species (ROS). This study investigated the use of calpain inhibitors and antioxidants in combination to protect cells against necrosis. Maitotoxin (MTX), which induces a massive influx of calcium, was used to provoke neuronal cell death. This toxin increased, in a concentration-dependent manner, both calpain activity and ROS formation. Calpain inhibitors or antioxidants inhibited MTX-induced necrosis only marginally (below 20%), whereas their association protected against cell death by 40-66% in a synergistic manner. BN 82204, which possesses both calpain-cathepsin L inhibitory and antioxidant properties, and its acetylated pro-drug BN 82270, totally protected cells at 100 lM. The pro-drug BN 82270, which had better cell penetration, was twice as effective as the active principle BN 82204 in protecting glioma C6 or neuroblastoma SHSY5Y cells against death. These results suggest the potential therapeutic relevance of using a single molecule with multiple activities (cysteine protease inhibitor/antioxidant), and warrant further in vivo investigations in models of neuronal disorders.