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Research ArticleArticle

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

Yong Xu and Teresa L. Krukoff
Molecular Pharmacology July 2007, 72 (1) 112-120; DOI: https://doi.org/10.1124/mol.106.033761
Yong Xu
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Teresa L. Krukoff
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Abstract

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.

Footnotes

  • This work was supported by the Heart and Stroke Foundation of Alberta/Northwest Territories/Nunavut. Y.X. is the recipient of a studentship from the Heart and Stroke Foundation of Canada.

  • ABBREVIATIONS: ADM, adrenomedullin; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; Ca2+-CaM, Ca2+-calmodulin complex; CaMKII, calmodulin-dependent kinase II; CRLR, calcitonin receptor-like receptor; MEM, minimum essential medium; NMDA, N-methyl-d-aspartate; NOS, nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; NOSIP, nitric-oxide synthase interaction protein; PVN, paraventricular nucleus; RAMP-2/3, receptor-activity-modifying protein 2 or 3; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; FK506, tacrolimus; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein; PKA, protein kinase A; OA, okadaic acid; CsA, cyclosporin A; PBS, phosphate-buffered saline; vinyl-l-NIO, N5-(1-imino-3-butenyl)-ornithine; PP1, protein phosphatase 1.

    • Received January 1, 2007.
    • Accepted April 18, 2007.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 72 (1)
Molecular Pharmacology
Vol. 72, Issue 1
1 Jul 2007
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Research ArticleArticle

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

Yong Xu and Teresa L. Krukoff
Molecular Pharmacology July 1, 2007, 72 (1) 112-120; DOI: https://doi.org/10.1124/mol.106.033761

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Research ArticleArticle

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

Yong Xu and Teresa L. Krukoff
Molecular Pharmacology July 1, 2007, 72 (1) 112-120; DOI: https://doi.org/10.1124/mol.106.033761
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