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 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 microM 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 approximately 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 microM. In contrast, 1 microM herbimycin fully inhibited the ability of thrombin and SFLLRNP to increase cell number and stimulate DNA synthesis. Furthermore, this inhibition by 1 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Grabham
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Department of Microbiology and Molecular Genetics, University of California, Irvine 92717.
Cunningham
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D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects; D016227:Benzoquinones; D002455:Cell Division / Q000187:drug effects; D047029:Lactams, Macrocyclic; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011809:Quinones / Q000494:pharmacology; D051381:Rats; D018179:Receptors, Thrombin / Q000378:metabolism*; D017828:Rifabutin / Q000031:analogs & derivatives; D013917:Thrombin / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D010446:Peptide Fragments; D011809:Quinones; D018179:Receptors, Thrombin; C086309:thrombin receptor peptide SFLLRNP; D017828:Rifabutin; D014443:Tyrosine; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D013917:Thrombin