Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca 2ϩ -permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca 2ϩ influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca 2ϩ influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH 2 -terminal kinase, and nuclear factor-B signaling and required de novo protein synthesis. The Ca 2ϩ signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca 2ϩ signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca 2ϩ -signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH 2 , a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own expression.

Microglia are intrinsic immune cells in the brain. In response to neurodegenerative events, excessively activated microglia change their shapes and release various cytokines leading to the pathogenesis of central nervous system (CNS) disease. Because the intracellular mechanisms of this process are still unclear, we have evaluated the functional roles of transient receptor potential vanilloid 4 (TRPV4) channel expressed in the microglia. Robust microglial activation after an injection of lipopolysaccharide (LPS) into the mouse cerebral ventricle was suppressed by concurrent administration of a selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD). When the mechanism was further investigated using cultured rat microglia intrinsically expressing functional TRPV4, release of tumor necrosis factor-α (TNF-α) and expression of galectin-3 were both increased by LPS. These increases were significantly suppressed by cotreatment with 4α-PDD, and the inhibitory effects of 4α-PDD were abolished by knockdown of TRPV4 or TRPV4 antagonists. The amplitude of voltage-dependent K(+) current, which is augmented during microglial activation, was also suppressed by 4α-PDD treatment. Opening of TRPV4 channels with 4α-PDD induced membrane depolarization mainly by increasing Na(+) influx. In addition, mimicking depolarization with a high-K(+) solution suppressed LPS-induced TNF-α release and galectin-3 upregulation. Both depolarizing treatments with 4α-PDD and high-K(+) solution decreased store-operated Ca(2+) influx caused by thapsigargin. These results suggest that depolarization in response to opening of the TRPV4 channel attenuates the driving force for extracellular Ca(2+) and suppresses microglial activation.

A biologically active lipid, sphingosine-1-phosphate (S1P) is highly abundant in blood, and plays an important role in regulating the growth, survival, and migration of many cells. Binding of the endogenous ligand S1P results in activation of various signaling pathways via G protein-coupled receptors, some of which generates Ca mobilization. In astrocytes, S1P is reported to evoke Ca signaling, proliferation, and migration; however, the precise mechanisms underlying such responses in astrocytes remain to be elucidated. Transient receptor potential canonical (TRPC) channels are Ca -permeable cation channels expressed in astrocytes and involved in Ca influx after receptor stimulation. In this study, we investigated the involvement of TRPC channels in S1P-induced cellular responses. In Ca imaging experiments, S1P at 1 μM elicited a transient increase in intracellular Ca in astrocytes, followed by sustained elevation. The sustained Ca response was markedly suppressed by S1P receptor antagonist JTE013, S1P receptor antagonist CAY10444, or non-selective TRPC channel inhibitor Pyr2. Additionally, S1P increased chemokine CXCL1 mRNA expression and release, which were suppressed by TRPC inhibitor, inhibition of Ca mobilization, MAPK pathway inhibitors, or knockdown of the TRPC channel isoform TRPC6. Taken together, these results demonstrate that S1P induces Ca signaling in astrocytes via G -coupled receptors S1P and S1P , followed by Ca influx through TRPC6 that could activate MAPK signaling, which leads to increased secretion of the proinflammatory or neuroprotective chemokine CXCL1.