Noradrenaline reduces the ATP-stimulated phosphorylation of p38 MAP kinase via β-adrenergic receptors–cAMP–protein kinase A-dependent mechanism in cultured rat spinal microglia

To elucidate the involvement of the noradrenergic system in the regulation of spinal microglial activity, we examined the effects of noradrenaline (NA) on the phosphorylation of three MAP kinases (extracellular signal-regulated kinase (ERK), p38, or c-Jun N-terminal kinase (JNK)) stimulated by ATP in rat cultured spinal microglia using Western blotting. ATP (100 μM) quickly induced the phosphorylation of three MAP kinases and MKK3/6, which are upstream kinases of p38. Under these conditions, NA inhibited only the ATP-stimulated phosphorylation of p38 in a time (30–60 min)- and dose (10–100 μM)-dependent manner, but did not affect those of ERK, JNK, or MKK3/6. The inhibitory action of NA was completely reversed by pretreatment with propranolol, an antagonist for β-adrenoceptors, or both atenolol and ICI118551, selective antagonists for β1 and β2, respectively. Treatment with dibutyryl cAMP or the selective activator of PKA mimicked the inhibitory effect of NA. Furthermore, treatment with KT5720, an inhibitor of protein kinase A, completely blocked the action of NA. These data suggest that NA could control the activation of p38 through the β1/2-adrenergic pathways, which include the production of cAMP and the activation of PKA. Simultaneously, we found that NA also markedly inhibited the ATP-induced increase in the expression of tumor necrosis factor (TNF)-α mRNA through β-adrenergic pathways. Furthermore, preincubation with either actinomycin D or cyclohexamide, general inhibitors of transcription or protein synthesis, respectively, almost completely blocked the inhibitory action of NA on the ATP-stimulated phosphorylation of p38. These results suggest that de novo synthesis of certain factors by NA through β-adrenoceptors would participate in the modulation of p38 activity. Thus, the inhibitory system via β1/2-adrenergic pathways in spinal microglia appears to have an important role in the modulation of microglial functions through the downregulation of p38 activity.