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Adrenomedullin Stimulates Nitric Oxide Release from SK-N-SH Human Neuroblastoma Cells by Modulating Intracellular Calcium Mobilization

Department of Cell Biology and Center for Neuroscience, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

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

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 µM vinyl-L-NIO, a neuronal NO synthase inhibitor. In addition, 5 µM 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 µM nifedipine or 10 µM nitrendipine. Depletion of calcium stores in the endoplasmic reticulum (ER) with 1 µM cyclopiazonic acid or 150 nM thapsigargin, or inhibition of ryanodine-sensitive receptors in the ER with 10 µM 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 µM 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 µM) or H-89 (5 µM) and was reduced by ryanodine (10 µM). 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.

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