Stellation

Abstract: Treatment of cultured type-1 astrocytes with thrombin leads to cell proliferation and reversal of stellation. The half-maximal concentrations of thrombin required for each response are 500 and 2 pM, respectively. To test whether they might be mediated by different receptors, we examined the contribution of the G protein-coupled thrombin receptor to these responses in purified rat astrocytes by using the agonist peptide SFLLRNP. In the absence of added growth factors, SFLLRNP fully mimicked the effects of thrombin at half-maximal concentrations of 30 µM for an increase in cell number and DNA synthesis and 100 nM for the reversal of stellation. The role of protein tyrosine phosphorylation in these events was investigated using antiphosphotyrosine antibodies. Thrombin and SFLLRNP at concentrations at least 10-fold greater than those required for half-maximal reversal of stellation but below those required for mitogenesis induced an identical pattern of tyrosine phosphorylation on several proteins of 55–65, 106, 110–115, and 120–130 kDa. The response was rapid (<1 min) and transient with a peak response after ∼2 min. The specific tyrosine kinase inhibitor herbimycin A did not affect thrombin- or SFLLRNP-mediated reversal of stellation at concentrations of up to 1 µM. In contrast, 1 µM herbimycin fully inhibited the ability of thrombin and SFLLRNP to increase cell number and stimulate DNA synthesis. Furthermore, this inhibition by 1 µM herbimycin A corresponded to inhibition of receptor-induced tyrosine phosphorylation. Thus, cell proliferation but not reversal of stellation is dependent on thrombin receptor-activated tyrosine kinase activity. These observations support the hypotheses that the thrombin receptor mediates the actions of thrombin in these cells and that activation of the thrombin receptor leads to multiple second messages that stimulate distinct cellular responses.

Abstract: A number of factors appear to be involved in the proliferative and hypertrophic processes which characterize reactive astrocytosis. We have investigated the possibility that ATP, an agent that is released by injured cells following tissue destruction, may be one such factor. For this purpose, we utilized primary cultures of astrocytes derived from cerebral cortices of neonatal rats to study the effect of extracellular ATP on properties associated with astrogliosis. Light microscopic studies disclosed marked stellation of astrocytes after 30-60 min of exposure to 100 microM-1 mM ATP. In addition, the content of the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), was increased 35-40% following 60-min exposure to ATP; this effect persisted for 1-3 days of exposure to 100 microM ATP. [3H]Thymidine incorporation increased progressively from 1-3 days; a 3.6-fold increase in DNA synthesis was observed following 3 days of exposure to 1 mM ATP, suggesting stimulation of cellular proliferation. These findings show that high micromolar to low millimolar concentrations of extracellular ATP reproduce several features associated with reactive gliosis and suggest that extracellular ATP may be involved in the activation of astrocytes following CNS injury.

Abstract: Astrocytes are typically star shaped cells playing diverse roles in the function of the nervous system. In astrocyte cultures established from the cerebral hemispheres of newborn rats, the cells have generally a polygonal fibroblast-like morphology, but acquire a stellate shape upon serum removal. When the serine protease thrombin or the bioactive lipid lysophosphatidic acid is added, the stellate cells revert to the flat morphology. Here we show that the effect of these agents is mediated via activation of the small GTP-binding protein Rho. Neither thrombin nor lysophosphatidic acid induced spreading of astrocytes microinjected with C3 transferase, an exoenzyme which ADP-ribosylates and thereby inactivates Rho. In contrast, the response of cells injected with a dominant negative form of Rac was unaffected. In addition, the injection of active Rho into stellate astrocytes mimicked the effect of thrombin and lysophosphatidic acid and an injection of C3 into flat cells grown in serum induced stellation. The conversion from a stellate to a spread morphology upon activation of Rho resulted in the formation of stress fibers and focal adhesions which most probably are key events in establishing and stabilizing the altered cytoarchitecture. These results suggest that Rho plays a crucial role in determining the shape of astrocytes and thereby may modulate their interaction with neurons in vivo.