Endothelin-induced protein tyrosine phosphorylation of cultured astrocytes: its relationship to cytoskeletal actin organization.
Endothelins (ETs) promote cytoskeletal actin reorganization of cultured astrocytes (Koyama and Baba, Neuroscience 61:1007-1016, 1994; Koyama and Baba, Glia 16:342-350, 1996). In this study, we examined the signal transduction involved in that activity of ETs. Immunoblot analysis with an anti-phosphotyrosine antibody showed that ET-3 (1 nM) increased tyrosine phosphorylation of 120 Kda and 70 Kda astrocytic proteins. The tyrosine phosphorylations of both proteins reached a maximum at 1 nM ET-3. In morphological examinations, ET-3 (1 nM) induced stress fibers, an organized F-actin structure, and focal adhesions in 0.5 mM dibutyryl cAMP (DBcAMP)-treated astrocytes within 30 min. Immunochemical staining of phosphotyrosine revealed that the newly formed focal adhesions possessed phosphotyrosine immunoreactivity. Phorbol 12-myristate 13 acetate (PMA, 100 nM), bradykinin (1 microM), angiotensin II (100 nM), and A23187 (5 microM) did not induce astrocytic stress fibers and had no obvious effects on tyrosine phosphorylation of 120 Kda and 70 Kda proteins. Tyrosine phosphorylation of astrocytic 120 Kda and 70 Kda proteins was stimulated by 1 mM sodium orthovanadate (VO4(3-)), a protein tyrosine phosphatase inhibitor. VO4(3-) promoted reorganization of stress fibers and focal adhesions in DBcAMP-treated astrocytes. Neither chelation of intra- and extracellular Ca2+ nor pre-treatment with pertussis toxin (PTX) affected the ET-induced tyrosine phosphorylation and stress fiber formation in cultured astrocytes. These results suggest a relationship between cytoskeletal actin reorganization and the tyrosine phosphorylation of astrocytic proteins by ETs.
Koyama
Y
Y
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Japan.
Baba
A
A
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology*; D019332:Endothelin-1 / Q000378:metabolism* / Q000494:pharmacology; D019334:Endothelin-3 / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D037342:Pertussis Toxin; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D014443:Tyrosine / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000199:Actins; D019332:Endothelin-1; D019334:Endothelin-3; D011506:Proteins; D010566:Virulence Factors, Bordetella; D019000:Phosphotyrosine; D014443:Tyrosine; D037342:Pertussis Toxin; D002118:Calcium