doi: 10.1042/BJ20050237.
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
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- PMID:15823093
- PMCID: PMC1180727
- DOI: 10.1042/BJ20050237
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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
David P Wilson et al. Biochem J..
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Abstract
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.
Figures
(A) A typical U-46619-induced contractile response of rat caudal arterial smooth muscle. De-endothelialized rat caudal arterial smooth muscle helical strips were contracted with 87 mM KCl (K+) and relaxed in H-T solution. Subsequent addition of a submaximal concentration of U-46619 (0.1 μM) elicited a slow, sustained contractile response. Washout of the agonist resulted in slow relaxation. (B,C) The rate of contraction in response to a maximal concentration (1 μM) of U-46619 (B) was significantly lower than that due to K+ depolarization (C). (D) U-46619 (1 μM)-mediated contraction was blocked by pre-treatment with the TP receptor antagonist SQ-29548 (1 μM). The viability of the preparation after treatment with SQ-29548 and U-46619 was verified by a normal contractile response to K+ stimulation. (n=4).
(A) In the presence of extracellular Ca2+ (H-T solution), U-46619 (0.1 μM) elicited a typical contractile response, as seen in Figure 1(A). In the absence of extracellular Ca2+ (5 min exposure to H-T solution containing 2 mM EGTA), U-46619 (0.1 μM) failed to induce contraction. Following 60 min exposure to EGTA-containing solution, U-46619 again failed to elicit a contractile response. Replenishment of extracellular Ca2+ in the presence of U-46619 resulted in an immediate contractile response. (n=4). (B) In control experiments, caffeine (20 mM) elicited a contractile response in the presence of extracellular Ca2+ and following removal of extracellular Ca2+, but not following depletion of Ca2+ stores (n=4). (C) Following K+ (87 mM)- and U-46619 (0.1 μM)-induced contractions, tissue was exposed to EGTA and CPA (10 μM) prior to addition of caffeine (20 mM) for 5 min in the continued presence of EGTA and CPA. After prolonged exposure to EGTA-containing solution in the continued presence of CPA, addition of U-46619 (0.1 μM) had no effect until extracellular Ca2+ was provided (n=3). Similar responses were observed when a higher concentration of U-46619 (1 μM) was used.
De-endothelialized smooth muscle strips were stimulated with U-46619 (1 μM) (A) or 87 mM KCl (B). Once steady-state tension development was complete, increasing concentrations of nicardipine were added, and relaxation of the tissue was monitored [n=3–8 (A);n=4 (B)].
(A) De-endothelialized smooth muscle strips were untreated, stimulated with U-46619 (1 μM) for 2, 5 or 10 min, with U-46619 (1 μM) in the presence of SQ-29548 (1 μM) for 10 min, or with U-46619 (1 μM) in the presence of Y-27632 (10 μM) for 10 min. Phosphorylated and unphosphorylated LC20 were separated by urea/glycerol gel electrophoresis, detected by Western blotting with anti-LC20 and quantified by scanning densitometry (n=7). Asterisks indicate statistically significant differences from control (P<0.05). (B,C) Following treatment with KCl, de-endothelialized rat caudal arterial smooth muscle strips were pre-treated without (B) or with (C) ML-7 (30 μM) prior to addition of U-46619 (1 μM) (n=3).
(A) Following a control contraction–relaxation cycle, α-toxin-permeabilized, Ca2+-ionophore (A23187)-treated, de-endothelialized smooth-muscle strips were exposed to threshold [Ca2+] (pCa 6.25). Addition of GTP (10 μM) elicited a small contractile response. Subsequent addition of U-46619 (1 μM) induced a further increase in force. The contraction was fully reversed by Y-27632 (10 μM) treatment. (B) Following a control contraction–relaxation cycle, α-toxin-permeabilized tissue was transferred to sub-threshold [Ca2+] (pCa 6.5). Addition of GTP[S] (10 μM) resulted in a robust Ca2+-sensitization response (n=4). (C) Cumulative data showing the Ca2+ sensitivity of contraction of α-toxin-permeabilized strips in the absence and presence of GTP (10 μM) and U-46619 (1 μM).
(A) De-endothelialized smooth muscle strips were treated for 5 min with U-46619 (1 μM), the α1-adrenoceptor agonist cirazoline (1 μM) or vehicle, and quick-frozen in liquid N2. (B) α-Toxin-permeabilized, de-endothelialized smooth-muscle strips were treated with GTP[S] (10 μM) or vehicle and quick-frozen in liquid N2. Tissues were homogenized (see the Experimental section), and samples were removed and added to SDS-gel sample buffer for analysis of total RhoA content. Activated (GTP-bound) RhoA was selectively pulled down from the remaining samples by interaction with the Rho-binding domain of Rhotekin coupled to agarose beads. Samples of washed beads (activated RhoA) and supernatants (inactive, GDP-bound RhoA) were treated with SDS-gel sample buffer. Proteins were separated by SDS/PAGE and RhoA was detected by Western blotting with anti-RhoA at 1:1000 dilution (n=2).
Following initial control K+-induced contractions, tissues were either untreated (A) or pre-treated with Y-27632 (10 μM) (B), H-1152 (100 nM) (C) or GF109203x (5 μM) (D) prior to addition of U-46619 (1 μM). (E) Tissue pre-contracted with U-46619 (1 μM) was treated with Y-27632 (10 μM) in the continued presence of U-46619. Control tissues were treated with PdBu (1 μM) without (F) or with (G) pre-treatment with GF-109203x (0.1 μM). (H) Cumulative data (n=8, except for 100 nM GF109203x and H-1552, in which casesn=4).
(A) Determination of the detection limit of anti-[PThr38]-CPI-17 and anti-CPI-17. Different amounts of phosphorylated (P-CPI-17) or unphosphorylated recombinant CPI-17 (unP-CPI-17) were subjected to SDS/PAGE and Western blotting with anti-[PThr38]-CPI-17 (a) or anti-CPI-17 (b). (B) Phosphorylation of CPI-17 in response to PdBu treatment. Tissue samples were quick-frozen at the indicated times following addition of PdBu (1 μM), proteins were separated by SDS/PAGE, and CPI-17 phosphorylation at Thr-38 was assessed by Western blotting with anti-[PThr38]-CPI-17. (C) Lack of phosphorylation of CPI-17 in response to U-46619 treatment. Tissue samples were quick-frozen at selected times following addition of U-46619 (1 μM), proteins were separated by SDS/PAGE and CPI-17 phosphorylation at Thr-38 was assessed by Western blotting with anti-[PThr38]-CPI-17 (a). Blots were subsequently re-probed with anti-CPI-17, which recognizes both phosphorylated and unphosphorylated CPI-17 (b) (n=3).
Analysis of phosphorylation of MYPT1 in response to U-46619 treatment. Tissue samples were quick-frozen at selected times following addition of U-46619 (1 μM), proteins were separated by SDS/PAGE, and MYPT1 phosphorylation at Thr-697 and Thr-855 was assessed by dual labelling of Western blots with anti-[PThr697]-MYPT1 (rabbit polyclonal) and pan-MYPT1 (monoclonal) antibodies (A,B) or anti-[PThr855]-MYPT1 (rabbit polyclonal) and pan-MYPT1 (monoclonal) antibodies (C,D), all at 1:1000 dilution (see the Experimental section). To account for any variations in loading levels, the data are expressed in arbitrary units as the ratio of signal intensities of phosphorylated MYPT1:total MYPT1, the latter being detected by an antibody that recognizes both phosphorylated and unphosphorylated forms of the protein (n=8). Phosphorylated MYPT1 was produced by incubating purified MLCP with ROK in the presence of ATP (MLCP+ROK+ATP). Unphosphorylated MYPT1 was from identical incubations in the absence of ATP (MLCP+ROK).
Abbreviations undefined elsewhere: G12/13 are heterotrimeric G-proteins; RhoA-GDP, inactive form of RhoA; RhoA-GTP, active form of RhoA; ROKi, inactive form of ROK; ROKa, activated form of ROK; CaM, calmodulin.
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