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.

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

“…First, the elimination of ADM-stimulated increases in [Ca 2ϩ ] i with H-89 or Rp-cAMP suggests that PKA mediates ADM-stimulated NO production by stimulating accumulation of intracellular Ca 2ϩ . Likewise, this PKA-Ca 2ϩ -NO cascade has been reported in urothelial cells (Birder et al, 2002), enterocytes (Canani et al, 2003), and neuroblastoma cells (Xu and Krukoff, 2005). Alternatively but not exclusively, PKA may stimulate nNOS activation by activating protein phosphatases to remove the inhibitory serine 847 phosphorylation from nNOS.…”

“…Likewise, the NO-cGMP cascade mediates ADM's hypertensive effects in the rostral ventrolateral medulla, an autonomic center in the brainstem that participates in maintaining sympathetic tone (Xu and Krukoff, 2004a). Finally, we demonstrated a direct stimulatory effect of ADM on NO production in SK-N-SH human neuroblastoma cells in vitro (Xu and Krukoff, 2005). However, the mechanisms by which ADM stimulates NO production in primary neurons remain to be identified.…”

“…We believe that this response can be attributed to the inhibition of CaMKII evoked by Ca 2ϩ chelation, given that CaMKII is involved in the constitutive phosphorylation of nNOS (Komeima et al, 2000). The cAMP-PKA pathway is coupled to ADM receptors in various types of cells (Kitamura et al, 1993;Shimekake et al, 1995), including neural cells (Xu and Krukoff, 2005). In addition, PKA mediates nNOS activation induced by diverse stimuli, such as electrical field stimulation (Ferrer et al, 2004) and application of endothelin (Jaureguiberry et al, 2004) or ␤-adrenoceptor agonists (Birder et al, 2002).…”

“…As described previously (Xu and Krukoff, 2005), cells (grown at a density of 10 5 / well in 96-well culture plates) were treated with 0.5 mM 1-methyl-3-isobutylxanthine (a cyclic nucleotide phosphodiesterase inhibitor; Sigma-Aldrich) for 30 min to prevent breakdown of accumulated cAMP, followed by treatment with vehicle, 1 or 10 nM ADM, for 5 min. Intracellular cAMP levels were measured with a commercial enzyme immunoassay kit according to the manufacturer's instructions (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).…”

Calcitonin gene-related peptide- and adrenomedullin-induced facilitation of calcium current by different signal pathways in nucleus tractus solitarius

“…ADM facilitates VDCCs involving MAPK in the NTS. cAMP-PKA pathways are coupled to ADM receptors in various types of cells (Kitamura, et al, 1993;Shimekake, et al, 1995;Ross and Yallampalli, 2006), including neural cells (Xu and Krukoff, 2005).…”