title: Adrenomedullin stimulates nitric oxide release from SK-N-SH human neuroblastoma cells by modulating intracellular calcium mobilization.
abstract: We used SK-N-SH human neuroblastoma cells to test the hypothesis that adrenomedullin (ADM), a multifunctional neuropeptide, stimulates nitric oxide (NO) release by modulating intracellular free calcium concentration ([Ca2+]i) in neuron-like cells. We used a nitrite assay to demonstrate that ADM (10 pM to 100 nM) stimulated NO release from the cells, with a maximal response observed with 1 nM at 30 min. This response was blocked by 1 nM ADM(22-52), an ADM receptor antagonist or 2 microM vinyl-L-NIO, a neuronal NO synthase inhibitor. In addition, 5 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular calcium chelator, eliminated the ADM-induced NO release. Similar results were observed when the cells were incubated in calcium-free medium or when L-type calcium channels were inhibited with 5 microM nifedipine or 10 microM nitrendipine. Depletion of calcium stores in the endoplasmic reticulum (ER) with 1 microM cyclopiazonic acid or 150 nM thapsigargin, or inhibition of ryanodine-sensitive receptors in the ER with 10 microM ryanodine attenuated the ADM-induced NO release. NO responses to ADM were mimicked by 1 mM dibutyryl cAMP, a cAMP analog, and were abrogated by 5 microM H-89, a protein kinase A inhibitor. Furthermore, Fluo-4 fluorescence-activated cell sorter analysis showed that ADM (1 nM) significantly increased [Ca2+]i at 30 min. This response was blocked by nifedipine (5 microM) or H-89 (5 microM) and was reduced by ryanodine (10 microM). These results suggest that ADM stimulates calcium influx through L-type calcium channels and ryanodine-sensitive calcium release from the ER, probably via cAMP-protein kinase A-dependent mechanisms. These elevations in [Ca2+)]i cause activation of neuronal NO synthase and NO release.
authors.affiliation: Department of Cell Biology and Center for Neuroscience, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Yong
authors.initials: Y
authors.lastname: Xu
authors.forename: Teresa L
authors.initials: TL
authors.lastname: Krukoff
mesh_terms: D053607:Adrenomedullin; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D003593:Cytoplasm / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D009569:Nitric Oxide / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D058265:Receptors, Adrenomedullin; D018000:Receptors, Peptide / Q000235:genetics; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000502:physiology; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D020746:Calcium Channels, L-Type; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D010455:Peptides; D012333:RNA, Messenger; D058265:Receptors, Adrenomedullin; D018000:Receptors, Peptide; D019837:Ryanodine Receptor Calcium Release Channel; C104782:adrenomedullin (22-52); C058636:calcitonin gene-related peptide (8-37); C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D053607:Adrenomedullin; D009569:Nitric Oxide; D004533:Egtazic Acid; D019284:Thapsigargin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015740:Calcitonin Gene-Related Peptide; D002118:Calcium