Effect of adrenomedullin on antioxidant enzymes activity in cerebellar vermis of rats

A Israel, L Figueira, MR Garrido. Universidad Central de Venezuela, Laboratory of Neuropeptides, Venezuela

Human adrenomedullin (AM) is a 52-amino acid peptide involved in cardiovascular control. AM has two specific receptors formed by the calcitonin-receptor-like receptor (CLRL) and receptor activity-modifying protein (RAMP) 2 or 3. These are known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the calcitonin gene-1 related peptide receptor (CGRP1), which is composed of CRLR and RAMP1 (1,2). In brain, AM and their receptors are expressed in several localized regions, including the cerebellum (3,4). It has been reported in vascular tissue that AM signals through either the elevation of intracellular cAMP or stimulation of endothelial nitric oxide synthase (NOS) activity (5). Little is known about AM receptor signaling pathways in cerebellum. Therefore, we assessed the effect of AM on NOS activity, cGMP production and on the activity of three antioxidant enzymes: catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), in rat cerebellum vermis. In addition, we evaluated the possible regulatory action of angiotensin II (ANG) on the latter AM action. For this purpose, male Sprague Dawley rats (200-220 g) were sacrificed, the cerebellar vermis was dissected under stereomicroscopic control and the tissue was stimulated in vitro with AM (2x10-7 M) for 5 min, in the presence or absence of: ANG (2x10-7 M), AM22 -50 or CGRP8 -37 (1x10-6 M) for 10 min. After homogenization and centrifugation, CAT, GPx and SOD activity was determined spectrophotometrically. NOS activity was assayed by monitoring the conversion of radiolabelled [3H]-L-arginine to [3H]-L-citruline and cGMP was determined by radioimmunoassay. Results were expressed as the means ± S.E.M of N=12 per experimental group. Statistical significance was assessed by one-way ANOVA and the Bonferroni test. P values <0.05 were considered statistically significant. Our findings indicate that in cerebellum, AM significantly increased NOS activity (+67±5%, p<0.01) and cGMP production (+68±6%, p<0.01) and these effects were blocked by AM22-50, suggesting that AM receptor mediate AM-induced cGMP formation and NO production. Stimulation of cerebellar vermis with ANG increased (+93.3±8; +60±7 and +23±3 %, respectively, p<0.01) and AM reduced (-70.9±6; -22.9±3 and -15±1.2 %, respectively, p<0.01) CAT, GPx and SOD basal activity. Tissue preincubation with ANG, AM22-50 or CGRP8 - 37 blunted AM-induced decrease of CAT, SOD and GPX activity. These results indicate that ANG exerts a regulatory role on AM-induced decrease of CAT, SOD and GPX activity, and they demonstrated that both CGRP1 and AM receptors are involved in AM-induced effects. Our findings suggest a functional role for AM and their receptors in cerebellum.

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