Endothelins, a vasoconstrictor peptide family, are present in the brain. The production of brain ETs is in creased in various Crenolanib buy brain disorders. Increases in brain ETs are involved in the pathophysiological responses of nerve tissues. Receptors for ETs are classified as ETA or ETB types. In the brain, high expression of ETB receptors was observed in astrocytes. ETs have been shown to regulate the function of astrocytes through ETB recep tors. In animal brain injury models, ETB antagonists re duced astrocytic proliferation, indicating that ETB receptors are involved in the induction of astrogliosis. Ac tivation of ETB receptors was shown to induce the production of several signaling molecules, such as neuro trophic factors and cytokines, in cultured astrocytes and in the rat brain.

These findings suggest that ETs regu late the pathophysiological response of the damaged brain by modulating the production of astrocytic signaling mol ecules. As for Inhibitors,Modulators,Libraries the production of chemokines in the brain, we previously showed that administration of an ETB agon ist increased CCL2 and CXCL1 production in the adult rat brain. In this study, to clarify the role of ETB re ceptors in astrocytic chemokine production, the effect of Astrocytes were prepared from the cerebra of one to two day old Wistar rats as described previously. The isolated cells were seeded at 1 �� 104 cells cm2 in 75 cm2 culture flasks and grown in minimal essential medium supplemented with 10% fetal calf serum. To re move small process bearing cells, the culture flasks were shaken at 250 rpm overnight, 10 to 14 days after seeding.

The monolayer cells were trypsinized and seeded on six well culture plates. Astro cytes were identified by immunocytochemical observations of glial fibrillary acidic protein, an astrocytic marker protein. At this stage, approximately 95% of cells Inhibitors,Modulators,Libraries showed immunoreactivity for GFAP. Cultured neurons and microglia Inhibitors,Modulators,Libraries were prepared from the rat cerebrum according to previously described methods. Treatment with ETs and the other drugs Before treatment with ETs and other drugs, astrocytes in six Inhibitors,Modulators,Libraries well culture plates were cultured in serum free MEM for 48 hours. ET 1 and Ala1,3,11,15 ET 1 were dissolved in distilled H2O to make stock solutions. ET antagonists and signal transduction inhibitors were dissolved in di methyl sulfoxide.

Treatments of cultured astro cytes with ETs and other drugs were started by addition of the stock solutions to serum Inhibitors,Modulators,Libraries free MEM. As a control for treatments with ET antagonists and signal transduc tion inhibitors, equal volumes of DMSO were included in the medium. ETs on chemokine expression in rat cultured astrocytes was examined. Methods Preparation of rat primary cultured astrocytes All selleckbio experimental protocols conformed to the Guiding Prin ciples for the Care and Use of Animals of the Japanese Pharmacological Society and were approved by the Animal Experiment Committee of Osaka Ohtani University.