Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2+ entry and Ca2+ sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855, but not Thr-697.
The signal transduction pathway whereby the TxA2 (thromboxane A2) mimetic U-46619 activates vascular smooth muscle contraction was investigated in de-endothelialized rat caudal artery. U-46619-evoked contraction was inhibited by the TP receptor (TxA2 receptor) antagonist SQ-29548, the ROK (Rho-associated kinase) inhibitors Y-27632 and H-1152, the MLCK (myosin light-chain kinase) inhibitors ML-7, ML-9 and wortmannin, the voltagegated Ca2+-channel blocker nicardipine, and removal of extracellular Ca2+; the protein kinase C inhibitor GF109203x had no effect. U-46619 elicited Ca2+ sensitization in a-toxin-permeabilized tissue. U-46619 induced activation of the small GTPase RhoA, consistent with the involvement of ROK. Two downstream targets of ROK were investigated: CPI-17 [protein kinase C-potentiated inhibitory protein for PP1 (protein phosphatase type 1) of 17 kDa], a myosin light-chain phosphatase inhibitor, was not phosphorylated at the functional site (Thr-38); phosphorylation of MYPT1 (myosin-targeting subunit of myosin light-chain phosphatase) was significantly increased at Thr-855, but not Thr-697. U-46619-evoked contraction correlated with phosphorylation of the 20 kDa light chains of myosin. We conclude that: (i) U-46619 induces contraction via activation of the Ca2+/calmodulin/MLCK pathway and of the RhoA/ROK pathway; (ii) Thr-855 of MYPT1 is phosphorylated by ROK at rest and in response to U-46619 stimulation; (iii) Thr-697 of MYPT1 is phosphorylated by a kinase other than ROK under resting conditions, and is not increased in response to U-46619 treatment; and (iv) neither ROK nor protein kinase C phosphorylates CPI-17 in this vascular smooth muscle in response to U-46619.
Wilson
David P
DP
Smooth Muscle Research Group and Department of Biochemistry & Molecular Biology, University of Calgary Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1.
Susnjar
Marija
M
Kiss
Eniko
E
Sutherland
Cindy
C
Walsh
Michael P
MP
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001158:Arteries / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type; D020013:Calcium Signaling; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D013928:Thromboxane A2 / Q000037:antagonists & inhibitors / Q000502:physiology*; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology*; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D054460:rho-Associated Kinases
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; C491519:Ppp1r14a protein, rat; D014662:Vasoconstrictor Agents; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D002118:Calcium