Cyclic ADP‐ribose is a second messenger in the lipopolysaccharide‐stimulated activation of murine N9 microglial cell line

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Abstract
Lipopolysaccharide, the main component of the cell wall of Gram‐negative bacteria, is known to activate microglial cells following its interaction with the CD14/Toll‐like receptor complex (TLR‐4). The activation pathway triggered by lipopolysaccharide in microglia involves enhanced basal levels of intracellular calcium ([Ca2+]i) and terminates with increased generation of cytokines/chemokines and nitric oxide. Here we demonstrate that in lipopolysaccharide‐stimulated murine N9 microglial cells, cyclic ADP‐ribose, a universal and potent Ca2+ mobiliser generated from NAD+ by ADP‐ribosyl cyclases (ADPRC), behaves as a second messenger in the cell activation pathway. Lipopolysaccharide induced phosphorylation, mediated by multiple protein kinases, of the mammalian ADPRC CD38, which resulted in significantly enhanced ADPRC activity and in a 1.7‐fold increase in the concentration of intracellular cyclic ADP‐ribose. This event was paralleled by doubling of the basal [Ca2+]i levels, which was largely prevented by the cyclic ADP‐ribose antagonists 8‐Br‐cyclic ADP‐ribose and ryanodine (by 75% and 88%, respectively). Both antagonists inhibited, although incompletely, functional events downstream of the lipopolysaccharide‐induced microglia‐activating pathway, i.e. expression of inducible nitric oxide synthase, overproduction and release of nitric oxide and of tumor necrosis factor α. The identification of cyclic ADP‐ribose as a key signal metabolite in the complex cascade of events triggered by lipopolysaccharide and eventually leading to enhanced generation of pro‐inflammatory molecules may suggest a new therapeutic target for treatment of neurodegenerative diseases related to microglia activation.