title: Endothelin-1 modulates hemoglobin-mediated signaling in cerebrovascular smooth muscle via RhoA/Rho kinase and protein kinase C.
abstract: Endothelin-1 (ET-1) and oxyhemoglobin (OxyHb) have been implicated in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage. However, the contribution of ET-1 to this condition has not been definitely established. In this study, we investigated whether threshold concentration of ET-1 enhances cerebrovascular smooth muscle (CVSM) contraction to OxyHb by activating the RhoA/Rho kinase and protein kinase C (PKC) pathways. CVSM contraction was measured in endothelium-denuded rabbit basilar arteries. Cytosolic and particulate fractions of CVSM cells were examined for RhoA and PKC reactivity with specific antibodies using immunoblotting procedures. ET-1 (0.1 nM) alone did not produce any significant contraction, but it markedly potentiated the magnitude (223% of control) and rate (149% of control) of contraction in response to OxyHb, which was attenuated by the inhibitors of Rho kinase Y-27632 and HA-1077. ET-1-mediated potentiation of the contraction was also inhibited by inhibitors of PKC, Ro-32-0432, and GF-109203X. BQ-123 prevented potentiation of vasoconstriction mediated by ET-1, indicating that the action of ET-1 was mediated by the endothelin type A receptor. Pretreatment with ET-1 significantly enhanced OxyHb-mediated RhoA translocation in CVSM cells and intact basilar arteries. ET-1 also caused potentiation of PKC-epsilon expression in membranes of CVSM cells exposed to OxyHb for 10 and 60 min but did not markedly change the distribution of PKC-alpha. Thus, in CVSM, threshold concentration of ET-1 potentiates contraction induced by OxyHb via RhoA/Rho kinase- and PKC-epsilon-dependent mechanisms. This process may contribute to the pathological contraction of cerebral arteries observed after subarachnoid hemorrhage.
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002560:Cerebrovascular Circulation; D004056:Differential Threshold; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000008:administration & dosage / Q000494:pharmacology / Q000502:physiology*; D005260:Female; D006454:Hemoglobins / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009994:Osmolar Concentration; D010108:Oxyhemoglobins / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology*; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
chemical_list: D019332:Endothelin-1; D006454:Hemoglobins; D047908:Intracellular Signaling Peptides and Proteins; D010108:Oxyhemoglobins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D020742:rhoA GTP-Binding Protein