Induction of Prostanoid Biosynthesis by Bacterial Lipopolysaccharide and Isoproterenol in Rat Microglial Cultures

Abstract: We have used purified microglial cultures obtained from neonatal rat cerebral cortex to investigate the ability of microglia to release prostanoids after exposure to bacterial lipopolysaccharide, a classic macrophage activator. Release of prostaglandin E₂, prostaglandin D₂, and thromboxane A₂ was low in basal conditions and increased in a dose- and time-dependent way upon lipopolysaccharide treatment (1–100 ng/ml), by a mechanism requiring de novo protein synthesis. When compared with astrocytes, microglial cells appeared to respond more effectively to lipopolysaccharide, being able to release prostanoids after exposure to a 100-fold lower concentration of lipopolysaccharide. In addition to prostanoids, we also measured the release of leukotriene B₄; although lipopolysaccharide failed to stimulate leukotriene B₄ release by microglial cells, it doubled the basal production in astrocytes. Lipopolysaccharide enhanced the release of preloaded [³H]arachidonic acid from microglial membrane phospholipids by a mechanism inhibited by the protein synthesis inhibitor cycloheximide, which suggests that the increased availability of arachidonic acid contributed to the enhanced prostanoid production. Lipopolysaccharide, however, also stimulated prostanoid synthesis by inducing cyclooxygenase activity, as shown by determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin and by assessing the level of the inducible form of cyclooxygenase by western blot analysis. Among the mechanisms potentially involved in the regulation of microglial prostanoid production, we studied the effect of β-adrenergic receptor activation. The β-agonist isoproterenol was inactive by itself but doubled the effect of lipopolysaccharide. The drug appeared to act mainly through the inducible cyclooxygenase; because it did not stimulate arachidonic acid release, it enhanced the lipopolysaccharide-evoked prostanoid production observed after aspirin pretreatment and induced de novo synthesis of cyclooxygenase detectable by western blot analysis. We suggest that during cerebral inflammatory processes microglia can contribute to the establishment of high prostanoid levels, which can be further elevated by β-adrenergic activation.