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Adrenomedullin Stimulates Nitric Oxide Production from Primary Rat Hypothalamic Neurons: Roles of Calcium and Phosphatases

Department of Cell Biology and Center for Neuroscience, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada

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.

Address correspondence to: Dr. Teresa L. Krukoff, Department of Cell Biology, Center for Neuroscience, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7. E-mail: teresa.krukoff{at}ualberta.ca

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				A. Geambasu and T. L. Krukoff Adrenomedullin acts in the lateral parabrachial nucleus to increase arterial blood pressure through mechanisms mediated by glutamate and nitric oxide Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2008; 295(1): R38 - R44. [Abstract] [Full Text] [PDF]