THROMBIN RECEPTOR ACTIVATION STIMULATES ASTROCYTE PROLIFERATION AND REVERSAL OF STELLATION BY DISTINCT PATHWAYS - INVOLVEMENT OF TYROSINE PHOSPHORYLATION

Treatment of cultured type-1 astrocytes with thrombin leads to cell pr oliferation and reversal of stellation. The half-maximal concentration s of thrombin required for each response are 500 and 2 pM, respectivel y. To test whether they might be mediated by different receptors, we e xamined the contribution of the G protein-coupled thrombin receptor to these responses in purified rat astrocytes by using the agonist pepti de SFLLRNP. In the absence of added growth factors, SFLLRNP fully mimi cked the effects of thrombin at half-maximal concentrations of 30 mu M for an increase in cell number and DNA synthesis and 100 nM for the r eversal of stellation. The role of protein tyrosine phosphorylation in these events was investigated using antiphosphotyrosine antibodies. T hrombin and SFLLRNP at concentrations at least 10-fold greater than th ose required for half-maximal reversal of stellation but below those r equired for mitogenesis induced an identical pattern of tyrosine phosp horylation 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 a fter similar to 2 min. The specific tyrosine kinase inhibitor herbimyc in A did not affect thrombin- or SFLLRNP-mediated reversal of stellati on at concentrations of up to 1 mu M. In contrast, 1 mu M herbimycin f ully inhibited the ability of thrombin and SFLLRNP to increase cell nu mber and stimulate DNA synthesis. Furthermore, this inhibition by 1 mu M herbimycin A corresponded to inhibition of receptor-induced tyrosin e phosphorylation. Thus, cell proliferation but not reversal of stella tion is dependent on thrombin receptor-activated tyrosine kinase activ ity. These observations support the hypotheses that the thrombin recep tor mediates the actions of thrombin in these cells and that activatio n of the thrombin receptor leads to multiple second messages that stim ulate distinct cellular responses.