Endothelins increase tyrosine phosphorylation of astrocytic focal adhesion kinase and paxillin accompanied by their association with cytoskeletal components.
Astrocytic endothelin receptors are involved in the appearance of activated astrocytes upon injury of the brain [Ishikawa N. et al. (1997) Eur. J. Neurosci. 9, 895-901; Koyama Y. et al. (1999) Glia 26, 268-271]. To clarify signal transduction triggered by endothelin receptors, we examined the effects of endothelins on protein tyrosine phosphorylation in cultured rat astrocytes. Endothelin-1 (1 nM) increased tyrosine phosphorylation of focal adhesion kinase and paxillin. The tyrosine phosphorylation was also induced by endothelin-1 (1 nM) and Ala(1,3,11,15)-endothelin-1 (10nM), an endothelin-B receptor agonist. BQ788 (100 nM), an endothelin-B receptor antagonist, inhibited the effects of endothelin-3. Orthovanadate (VO(4)(3-)), a tyrosine phosphatase inhibitor, but not bradykinin (1 microM), angiotensin II (100 nM), A23187 (5 microM) and phorbol 12-myristate 13-acetate (100 nM), increased tyrosine phosphorylation of focal adhesion kinase and paxillin. The tyrosine phosphorylation by endothelin-3 was not prevented by pertussis toxin, Ca(2+) chelation, protein kinase C inhibitors (calphostin C and staurosporine) or wortmannin. Immunocytochemical staining showed that endothelin-3 and VO(4)(3-) induced redistribution of focal adhesion kinase and paxillin to focal adhesions concomitant with stress fiber formation in dibutyryl cyclic-AMP-treated astrocytes. Treatment with endothelin-3 and VO(4)(3-) increased focal adhesion kinase and paxillin associated with astrocytic cytoskeletal fraction. In the presence of cytochalasin B, an actin disrupting agent, endothelin-3 and VO(4)(3-) did not phosphorylate focal adhesion kinase and paxillin. Application of cytochalasin B after treatment with endothelin-3 and VO(4)(3-) stimulated dephosphorylation of focal adhesion kinase and paxillin. These results suggest that the associations of focal adhesion kinase and paxillin with cytoskeletal components are required in the endothelin-induced tyrosine phosphorylation of the astrocytic proteins.
Koyama
Y
Y
Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Yamada-Oka 1-6, Suita 565-0871, Japan.
Yoshioka
Y
Y
Hashimoto
H
H
Matsuda
T
T
Baba
A
A
D000730:Androstadienes / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D001920:Bradykinin / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002614:Chelating Agents / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000187:drug effects* / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1 / Q000378:metabolism / Q000494:pharmacology; D019334:Endothelin-3 / Q000378:metabolism / Q000494:pharmacology; D016232:Endothelins / Q000378:metabolism* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051419:Paxillin; D037342:Pertussis Toxin; D010750:Phosphoproteins / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017466:Receptors, Endothelin / Q000819:agonists; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000187:drug effects* / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D002614:Chelating Agents; D003598:Cytoskeletal Proteins; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D019334:Endothelin-3; D016232:Endothelins; D004791:Enzyme Inhibitors; D051419:Paxillin; D010750:Phosphoproteins; C496063:Pxn protein, rat; D017466:Receptors, Endothelin; D010566:Virulence Factors, Bordetella; D000804:Angiotensin II; D003571:Cytochalasin B; D014638:Vanadates; D014443:Tyrosine; D003994:Bucladesine; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D000077191:Wortmannin