Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC30
Macrophage apoptosis and loss of motility is induced by asymmetric dimethylarginine
B. Ahmetaj1, J. Leiper2, N. Freestone1, F. Arrigoni1
1. School of Pharmacy and Chemistry, Kingston University, Penryhn Road, Surrey KT1 2EE, United Kingdom. 2. MRC Clinical Sciences Centre, Imperial College, Hammersmith Hospital, DuCane Road, London W12 0NN, United Kingdom.
Asymmetrically methylated forms of arginine (asymmetric dimethylarginine, (ADMA) and L-N-monomethylarginine, (L-NMMA)) are competitive inhibitors of all three isoforms of nitric oxide synthase (NOS), the enzyme responsible for the conversion of L-arginine to NO 1. Nitric oxide (NO) is a critical signalling molecule that amongst other effects can deplete macrophages in atherosclerotic plaques 2. Additionally, the ADMA/NO pathway is a key regulator of endothelial cell motility 3, although its role in monocyte and macrophage motility remains unclear. U937 cells, a human leukemic monocyte lymphoma cell line, were differentiated into macrophages using 20-50ng/ml phorbel ester (PMA). To test cell proliferation Interferon-γ (10U/mol) and ADMA (10μM, 50μM, and 100μM) were added either singly or in combination (24 h before) and incubated with the tetrazolium salt [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] for 3 h. The absorbance at 570nm was measured. Nitrite levels were measured by incubating the supernatant of treated cells with griess reagents [sulfanilamide and N-1-naphthylethylenediamine dihydrochloride] for 30 min and measuring the absorbance at 540nm. Here we show that in U937 differentiated monocytes, cell proliferation decreases significantly (P<0.05; n=3) with ADMA (50μM, 100μM) concomitantly with decreased NO production, suggesting that ADMA may induce a reduction in NO release or NOS activity and cell proliferation as a result of cell apoptosis. Spontaneous migration of differentiated monocytes was also digitally recorded by microscopy with a time interval of 10 min for a period of 12 h. ADMA (1μM, 10μM and 100μM) was shown to significantly reduce motility and directional travel dose dependently by approximately 0.3μm/min (P<0.05; n=6). Symmetric dimethylarginine (SDMA), which does not inhibit NOS, had variable dose dependant effects and reduced motility and directional travel at pathophysiological concentrations (100μM). Subsequent immunostaining of f-actin using an Alexa Fluor 488® phalloidin demonstrated an increase in stress fibre and focal adhesion formation following incubation with ADMA (100 μM). This implicates ADMA as a critical regulator of monocyte/macrophage motility and highlights the possible therapeutic benefits of pharmacological tools to promote NO production and/or increase ADMA removal from the body.
Where applicable, experiments conform with Society ethical requirements