Bradykinin (BK) is produced and acts at the site of injury and inflammation. In the CNS, migration of microglia toward the lesion site plays an important role pathologically. In the present study, we investigated the effect of BK on microglial migration. Increased motility of cultured microglia was mimicked by B 1 receptor agonists and markedly inhibited by a B 1 antagonist , but not by a B 2 receptor antagonist. BK induced chemotaxis in microglia isolated from wild-type and B 2 -knock-out mice but not from B 1 -knock-out mice. BK-induced motility was not blocked by pertussis toxin but was blocked by chelating intracellular Ca 2ϩ or by low extracellular Ca 2ϩ , implying that Ca 2ϩ influx is prerequisite. Blocking the reverse mode of Na ϩ /Ca 2ϩ exchanger (NCX) completely inhibited BK-induced migration. The involvement of NCX was further confirmed by using NCX ϩ/Ϫ mice; B 1 -agonist-induced motility and chemotaxis was decreased compared with that in NCX ϩ/ϩ mice. Activation of NCX seemed to be dependent on protein kinase C and phosphoinositide 3-kinase, and resultant activation of intermediate-conductance (IK-type) Ca 2ϩ -dependent K ϩ currents (I K(Ca) ) was activated. Despite these effects, BK did not activate microglia, as judged from OX6 staining. Using in vivo lesion models and pharmacological injection to the brain, it was shown that microglial accumulation around the lesion was also dependent on B 1 receptors and I K(Ca) . These observations support the view that BK functions as a chemoattractant by using the distinct signal pathways in the brain and, thus, attracts microglia to the lesion site in vivo.

L-tri-iodothyronine (3, 3', 5-triiodothyronine; T3) is an active form of the thyroid hormone (TH) essential for the development and function of the CNS. Though nongenomic effect of TH, its plasma membrane-bound receptor, and its signaling has been identified, precise function in each cell type of the CNS remained to be investigated. Clearance of cell debris and apoptotic cells by microglia phagocytosis is a critical step for the restoration of damaged neuron-glia networks. Here we report nongenomic effects of T3 on microglial functions. Exposure to T3 increased migration, membrane ruffling and phagocytosis of primary cultured mouse microglia. Injection of T3 together with stab wound attracted more microglia to the lesion site in vivo. Blocking TH transporters and receptors (TRs) or TRα-knock-out (KO) suppressed T3-induced microglial migration and morphological change. The T3-induced microglial migration or membrane ruffling was attenuated by inhibiting Gi /o -protein as well as NO synthase, and subsequent signaling such as phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). Inhibitors for Na(+) /K(+) -ATPase, reverse mode of Na(+) /Ca(2+) exchanger (NCX), and small-conductance Ca(2+) -dependent K(+) (SK) channel also attenuated microglial migration or phagocytosis. Interestingly, T3-induced microglial migration, but not phagocytosis, was dependent on GABAA and GABAB receptors, though GABA itself did not affect migratory aptitude. Our results demonstrate that T3 modulates multiple functional responses of microglia via multiple complex mechanisms, which may contribute to physiological and/or pathophysiological functions of the CNS.

Microglia express AMPA (α-amino-hydroxy-5-methyl-isoxazole-4-propionate)-type of glutamate (Glu) receptors (AMPAR), which are highly Ca(2+) impermeable due to the expression of GluA2. However, the functional importance of AMPAR in microglia remains to be investigated, especially under pathological conditions. As low expression of GluA2 was reported in some neurodegenerative diseases, GluA2(-/-) mice were used to show the functional change of microglial AMPARs in response to Glu or kainate (KA). Here we found that Glu-induced currents in the presence of 100 μM cyclothiazide, an inhibitor of AMPAR desensitization, showed time-dependent decrease after activation of microglia with lipopolysaccharide (LPS) in GluA2(+/+) microglia, but not in GluA2(-/-) microglia. Upon activation of microglia, expression level of GluA2 subunits significantly increased, while expression of GluA1, A3 and A4 subunits on membrane surface significantly decreased. These results suggest that nearly homomeric GluA2 subunits were the main reason for low conductance of AMPAR in activated microglia. Increased expression of GluA2 in microglia was also detected partially in brain slices from LPS-injected mice. Cultured microglia from GluA2(-/-) mice showed higher Ca(2+) -permeability, consequently inducing significant increase in the release of proinflammatory cytokine, such as TNF-α. The conditioning medium from KA-treated GluA2(-/-) microglia had more neurotoxic effect on wild type cultured neurons than that from KA-treated GluA2(+/+) microglia. These results suggest that membrane translocation of GluA2-containing AMPARs in activated microglia has functional importance and thus, dysfunction or decreased expression of GluA2 may accelerate Glu neurotoxicity via excess release of proinflammatory cytokines from microglia.

J. Neurochem. (2011) 117, 61–70. Abstract Galanin (GAL) is a neuropeptide which is up‐regulated following neuronal axotomy or inflammation. One subtype of GAL receptor (GalR2) is reported to be expressed in the brain’s immune cell population, microglia. In the present study, we investigated the effect of GAL on microglial migration and compared the mechanism with that of bradykinin (BK). GAL significantly increased the migration of rat cultured microglia at 0.1 pM. The GAL‐induced signal cascade was partly similar to that induced by BK. It was not dependent on Gi/o protein but involved activation of protein kinase C, phosphoinositide 3‐kinase and Ca2+‐dependent K+ channels. However, reverse‐mode activation of the Na+/Ca2+‐exchanger 1 was not involved in GAL‐induced microglial migration, unlike BK‐induced migration. Likewise, nominally‐free extracellular Ca2+ inhibited BK‐induced migration but not GAL‐induced migration. An inositol‐1,4,5‐triphosphate receptor antagonist significantly inhibited GAL‐induced migration. GAL‐induced Ca2+ signaling did not induce nitric oxide synthase expression, but up‐regulated class II major histocompatibility complex expression. These results indicate that activation of inositol‐1,4,5‐triphosphate receptor and increase in intracellular Ca2+ are important for GAL‐induced migration and immunoreactivity in microglia. The differences in down‐stream signal transduction induced by GAL and BK suggest that GAL and BK may control distinct microglial functions under pathological conditions.

Microglia, the immune cells of the central nervous system (CNS), are busy and vigilant housekeepers in the adult brain. The main candidate as a chemoattractant for microglia at damaged site is adenosine triphosphate (ATP). However, many other substances can induce immediate change of microglia. Some neuropeptides such as angiotensin II, bradykinin (BK), endothelin, galanin (GAL), and neurotensin are also chemoattractants for microglia. Among them, BK increased microglial migration via B 1 receptor with different mechanism from that of ATP. BK-induced migration was controlled by a G i/o protein-independent pathway, while ATP-induced migration was via a G i/o protein-dependent and also a mitogen-activated protein kinase (MAPK) / extracellular signal-regulated kinase (ERK)-dependent pathway. On the other hand, GAL is reported to have a similar signal cascade as that of BK, though only part of the signaling was similar to that of BK-induced migration. For example, BK activates reverse-mode Na + /Ca 2+ exchange allowing extracllular Ca 2+ influx, while GAL induces intracellular Ca 2+ mobilization via increasing inositol-1,4,5-trisphosphate. In addition, GAL activates MAPK/ERK-dependent signaling but BK did not. These results suggest that chemoattractants for immune cells in the brain including ATP and each peptide may have distinct role under both physiological and pathophysiological conditions.

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