title: Pressure-dependent contribution of Rho kinase-mediated calcium sensitization in serotonin-evoked vasoconstriction of rat cerebral arteries.
abstract: Our understanding of the cellular signalling mechanisms contributing to agonist-induced constriction is almost exclusively based on the study of conduit arteries. Resistance arteries/arterioles have received less attention as standard biochemical approaches lack the necessary sensitivity to permit quantification of phosphoprotein levels in these small vessels. Here, we have employed a novel, highly sensitive Western blotting method to assess: (1) the contribution of Ca(2+) sensitization mediated by phosphorylation of myosin light chain phosphatase targeting subunit 1 (MYPT1) and the 17 kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17) to serotonin (5-HT)-induced constriction of rat middle cerebral arteries, and (2) whether there is any interplay between pressure-induced myogenic and agonist-induced mechanisms of vasoconstriction. Arterial diameter and levels of MYPT1 (T697 and T855), CPI-17 and 20 kDa myosin light chain subunit (LC(20)) phosphorylation were determined following treatment with 5-HT (1 micromol l(1)) at 10 or 60 mmHg in the absence and presence of H1152 or GF109203X to suppress the activity of Rho-associated kinase (ROK) and protein kinase C (PKC), respectively. Although H1152 and GF109203X suppressed 5-HT-induced constriction and reduced phospho-LC(20) content at 10 mmHg, we failed to detect any increase in MYPT1 or CPI-17 phosphorylation. In contrast, an increase in MYPT1-T697 and MYPT1-T855 phosphorylation, but not phospho-CPI-17 content, was apparent at 60 mmHg following exposure to 5-HT, and the phosphorylation of both MYPT1 sites was sensitive to H1152 inhibition of ROK. The involvement of MYPT1 phosphorylation in the response to 5-HT at 60 mmHg was not dependent on force generation per se, as inhibition of cross-bridge cycling with blebbistatin (10 micromol l(1)) did not affect phosphoprotein content. Taken together, the data indicate that Ca(2+) sensitization owing to ROK-mediated phosphorylation of MYPT1 contributes to 5-HT-evoked vasoconstriction only in the presence of pressure-induced myogenic activation. These findings provide novel evidence of an interplay between myogenic- and agonist-induced vasoconstriction in cerebral resistance arteries.
authors.affiliation: The Smooth Muscle Research Group, Department of Physiology and Pharmacology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1.
authors.forename: Ahmed F
authors.initials: AF
authors.lastname: El-Yazbi
authors.forename: Rosalyn P
authors.initials: RP
authors.lastname: Johnson
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Walsh
authors.forename: Kosuke
authors.initials: K
authors.lastname: Takeya
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
authors.forename: William C
authors.initials: WC
authors.lastname: Cole
mesh_terms: D000818:Animals; D001794:Blood Pressure / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology*; D002536:Cerebral Arteries / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D008297:Male; D009124:Muscle Proteins / Q000502:physiology; D009213:Myography; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D011312:Pressure; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054645:Protein Phosphatase 1 / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000494:pharmacology*; D014661:Vasoconstriction / Q000187:drug effects*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D009124:Muscle Proteins; D010750:Phosphoproteins; C491519:Ppp1r14a protein, rat; D047428:Protein Kinase Inhibitors; D012701:Serotonin; D054460:rho-Associated Kinases; D011493:Protein Kinase C; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D002118:Calcium