Endothelin-1 Induced the Reactivation of Primary Rat Ocular Astrocytes




Ma, Hai-Ying
Park, Yong
Wang, Junming
Yorio, Thomas


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Purposes: Astrocytes play a crucial role in cell survival and axon function of retinal ganglion cells (RGCs) by providing the structural support to neurons, secreting neurotrophic factors to regulate apoptosis and maintenance of the extracellular milieu. Endothelin-1(ET-1) and its receptors are found to be involved in the etiology of glaucoma. However, ET-mediated reactivation of astrocytes affecting RGC survival is still not fully understood. This study aimed to investigating the mechanisms by which ET-1 promotes the reactivation of primary rat ocular astrocytes. Methods: The primary astrocytes were isolated from retinas and optic nerve of rats. Immunostaining of glial fibrillary acid protein (GFAP), RNA binding protein with multiple splicing (RBPMS) and alpha smooth muscle actin (α-SMA) was performed on the cultured primary astrocytes to identify the purity of cells. The cultured primary astrocytes were treated with 100nM endothelin-1 for 24 hours followed the protein detection using Western Blot. ET-1-mediated influx of calcium was monitored in astrocytes using Fura-2 AM calcium imaging. Results: GFAP was uniformly stained on the primary astrocytes, and no staining of RBPMS and α-SMA was identified, whereas the staining of α-SMA was identified in NIH3T3 fibroblast cells. The treatment of ET-1 and ET-3 induced the upregulation of GFAP, neural cell adhesion molecule (NCAM), c-Jun, c-Jun N-terminal kinase (JNK) and Ki67 (a protein marker of cell proliferation). Administration of SP600125, an inhibitor of JNK, attenuated the increased GFAP induced by ET-1 in astrocytes. However, BQ788, an antagonist of ETB receptor, didn’t inhibit ET-1-mediated upregulation of GFAP. In addition, ET-1 triggered augment of intracellular calcium in the primary astrocytes, whereas the application of verapamil, an L-type calcium channel blocker, inhibited the influx of calcium. Conclusions: The hallmark of reactive astrocytes, GFAP, is tightly regulated in astrocytes. An increase in protein levels of GFAP and Ki67 induced by ET-1 reflected the reactivation of astrocytes. Meanwhile, other proteins were also found to be upregulated, such as NCAM, c-Jun and JNK. In addition, intracellular of calcium was also promoted with ET-1 treatment. Taken together, the results suggest that calcium-mediated signaling and JNK/c-Jun pathway are involved in reactivation of astrocytes. This reactivation could lead to dysfunction in the optic nerve and affect RGC survival.