RT Journal Article
SR Electronic
T1 Adrenomedullin Stimulates Nitric Oxide Production from Primary Rat Hypothalamic Neurons: Roles of Calcium and Phosphatases
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 112
OP 120
DO 10.1124/mol.106.033761
VO 72
IS 1
A1 Yong Xu
A1 Teresa L. Krukoff
YR 2007
UL http://molpharm.aspetjournals.org/content/72/1/112.abstract
AB Adrenomedullin (ADM) in the brain plays important roles in the maintenance of homeostasis. Although in vivo evidence has suggested that nitric oxide (NO) mediates ADM's effects in the brain, mechanisms for ADM stimulation of NO production in neurons have not been identified. In the present study, primary hypothalamic neurons were used to characterize ADM-induced NO production and to study the underlying mechanisms. Using Calcium Orange/4-amino-5-methylamino-2′,7′-difluorofluorescein fluorescence live cell imaging, we found that ADM (1 or 10 nM, 5 min) significantly elevated [Ca2+]i and NO production in a concentration-dependent manner. Ca2+ and NO responses induced by 10 nM ADM were abolished by pretreatment with 50 μM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), an intracellular Ca2+ chelator, or protein kinase A (PKA) inhibitors 5 μM N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and 50 μM Rp-cAMP. Furthermore, the ADM-induced NO production was significantly attenuated by a protein phosphatase 1/2A inhibitor, okadaic acid (OA; 0.1 μM), or calcineurin inhibitors, tacrolimus (FK506) (1 μM) and cyclosporin A (CsA; 0.1 μM). Using Western blotting, we found that ADM significantly decreased phosphorylation of neuronal nitric-oxide synthase (nNOS) at serine 847. This dephosphorylation was inhibited by 0.1 μM OA, 1 μM FK506, 0.1 μM CsA, or 5 μM H-89, and attenuated by 50 μM BAPTA-AM. These results suggest that, in hypothalamic neurons, ADM elevates [Ca2+]i via PKA-associated mechanisms. The PKA/Ca2+ cascade leads to protein phosphatase (PP) 1/PP2A- and calcineurin-mediated dephosphorylation of nNOS. We hypothesize that the Ca2+ increase and nNOS dephosphorylation contribute to activation of nNOS and production of NO in hypothalamic neurons. The American Society for Pharmacology and Experimental Therapeutics