Nitric oxide induces dose-dependent Ca2+ transients and causes temporal morphological hyperpolarization in human neutrophils

We exposed adherent neutrophils to the nitric oxide (NO)-radical donors S-n itroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodiu m nitroprusside (SNP) to study the role of NO in morphology and Ca2+ signal ing. Parallel to video imaging of cell morphology and migration in neutroph ils, changes in intracellular free Ca2+ ([Ca2+](i)) were assessed by ratio imaging of Fura-2. NO induced a rapid and persistent morphological hyperpol arization followed by migrational arrest that usually lasted throughout the 10-min experiments. Addition of 0.5-800 mu M SNAP caused concentration-dep endent elevation of [Ca2+](i) with an optimal effect at 50 mu M. This was p robably induced by NO itself, because no change in [Ca2+](i) was observed a lter treatment with NO donor byproducts, i.e. D-penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (greater than or equal to 2 00 eta M) attenuated the Ca2+ response to the soluble chemolactic stimulus formyl-meth ionyl-leucyl-phenylalanine (fMLP), and both NO- and fMLP-induce d Ca2+ transients were abolished at 800 mu M SNAP or more. In kinetic studi es of fluorescently labeled actin cytoskeleton, NO markedly reduced the F-a ctin content and profoundly increased cell area. Immunoblotting to investig ate the formation of nitrotyrosine residues in cells exposed to NO donors d id not imply nitrosylation, nor could we mimic the effects of NO with the c ell permeant form of cGMP, i.e., 8-Br-cGMP. Hence these processes were prob ably not the principal NO targets. In summary, NO donors initially increase d neutrophil morphological alterations, presumably due to an increase in [C a2+](i), and thereafter inhibited such shape changes. Our observations demo nstrate that the effects of NO donors are important for regulation of cellu lar signaling, i.e., Ca2+ homeostasis, and also affect cell migration, e.g. , through Effects on F-actin turnover. Our results are discussed in relatio n to the complex mechanisms that govern basic cell shape changes, required for migration. (C) 2000 Wiley-Liss, Inc.