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 ADM22–52, 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.
Gab1, SHP2, and Protein Kinase A Are Crucial for the Activation of the Endothelial NO Synthase by Fluid Shear Stress
