Essential role of rho kinase in the Ca2+ sensitization of prostaglandin F(2alpha)-induced contraction of rabbit aortae

Abstract

Inhibition of dephosphorylation of the 20 kDa myosin light chain (MLC(20)) is an important mechanism for the Ca(2+)-induced sensitization of vascular smooth muscle contraction. We investigated whether this mechanism operates in prostaglandin F(2alpha) (PGF(2alpha))-induced contraction of rabbit aortic smooth muscle and, if so, whether protein kinase C (PKC) or rho-associated kinase (rho kinase) contribute to the inhibition of dephosphorylation. In normal medium, PGF(2alpha) (10 microM) increased the phosphorylation of MLC(20) and developed tension. The rho-kinase inhibitors fasudil and hydroxyfasudil inhibited these changes, despite having no effect on a phorbol-ester-induced MLC(20) phosphorylation. After treatment with verapamil or chelation of external Ca(2+) with EGTA, PGF(2alpha) increased the MLC(20) phosphorylation and tension without an increase in [Ca(2+)](i), all of which were sensitive to fasudil and hydroxyfasudil. ML-9, a MLC kinase inhibitor, quickly reversed the KCl-induced MLC(20) phosphorylation and contraction to the resting level. However, fractions of PGF(2alpha)-induced contraction and MLC(20) phosphorylation were resistant to ML-9 but were sensitive to fasudil. Ro31-8220 (10 microM), a PKC inhibitor, did not affect the phosphorylation of MLC(20) and the tension caused by PGF(2alpha), thus excluding the possibility of the involvement of PKC in the PGF(2alpha)-induced MLC(20) phosphorylation. PGF(2alpha) increased phosphorylation at Thr654 of the myosin binding subunit (MBS) of myosin phosphatase, which is a target of rho kinase, and fasudil decreased the phosphorylation. These data suggest that the PGF(2alpha)-induced contraction is accompanied by the inhibition of MLC(20) dephosphorylation through rho kinase-induced MBS phosphorylation, leading to Ca(2+) sensitization of contraction. An actin-associated mechanism may also be involved in the PGF(2alpha)-induced sensitization.

Figure 7. Characterization of the antibody pMBS-T654

A, detection of rho kinase-induced phosphorylation of the myosin binding subunit (MBS) by pMBS-T654 in vitro. Recombinant MBS was phosphorylated by rho kinase for 0 min (lanes 1 and 4), 10 min (lanes 2 and 5) or 20 min (lanes 3 and 6) and subjected to SDS-PAGE, followed by Western blotting with pMBS-T654 (upper panel) or with the antibody N-MBS, which recognizes the N-terminus of MBS (lower panel), in the absence (lanes 1–3) or presence (lanes 4–6) of phosphorylated antigen peptide (T654-phosphopeptide). B, PGF_2α-induced phosphorylation at Thr654 of MBS in the rabbit carotid artery. Extracts of arteries before stimulation (lane 1) or 1 (lane 2) and 2 min (lane 3) after the stimulation with PGF_2α (30 µm) were subjected to SDS-PAGE followed by Western blotting with pMBS-T654. C, GTPγS-induced phosphorylation at Thr654 of MBS (MBS-P) and its inhibition by hydroxyfasudil or C3 toxin in permeabilized pig aortic cells. Cells were treated with GTPγS for 10 min. When used, hydroxyfasudil (OH-fasudil, 10 µm) or C3 toxin (1 µg ml⁻¹) was used to pretreat cells for 15 min before application of GTPγS. Photographs: band reacted with pMBS-T654 (upper panel) or N-MBS (lower panel). MBS phosphorylation is expressed as a percentage of the level in the resting state. Data are means ± s.e.m. (n = 4). *P < 0.05.

Figure 1. Effects of fasudil and hydroxyfasudil on the prostaglandin F_2α (PGF_2α)- or 12-deoxyphorbol 13-isobutyrate (DPB)-induced contraction and 20 kDa myosin light chain (MLC₂₀) phosphorylation in rabbit aortae

Fasudil (▪) or hydroxyfasudil (OH-fasudil, (Δ) at 10 µm was added 20 min after the application of 10 µm PGF_2α (A) or 1 µm DPB (B). Left panels, a change in tension after the addition of fasudil or hydroxyfasudil (○, agonist alone). Tension is expressed as a percentage of contraction just before the addition of fasudil or hydroxyfasudil. In the right panels, MLC₂₀ phosphorylation (MLC-P) was observed 20 min after the addition of PGF_2α or DPB (just before the addition of fasudil or hydroxyfasudil (a) or 50 min after the addition of PGF_2α or DPB (b), and is expressed as a percentage of phosphorylated MLC₂₀ of total MLC₂₀. MLC₂₀ phosphorylation in the resting state was 8–9 % in each case (data not shown). Since PGF_2α caused diphosphorylation of MLC₂₀, monophosphorylation (MLC-P1, open columns) and diphosphorylation (MLC-P2, black columns) are shown (A, right panel). Data are presented as the mean ± s.e.m. (n = 5–6). * Significantly different from the time-matched control (P < 0.05).

Figure 2. Changes in[Ca²⁺]_i, tension and MLC₂₀ phosphorylation due to PGF_2α under normal conditions or conditions where an increase in [Ca2+]_i was inhibited

A, examples of changes in[Ca²⁺]_i in normal physiological saline solution (PSS), in the presence of verapamil (10 µm) or EGTA (4 mm). For[Ca²⁺]_i and tension, 100 % represents the change induced by 65.4 mm KCl, which was obtained before the application of PGF_2α. Verapamil or EGTA was applied 10 min before the addition of PGF_2α (10 µm). B, summarized data of the PGF_2α-induced contraction and MLC₂₀ phosphorylation in the normal medium or in the presence of verapamil and fasudil or hydroxyfasudil (OH-fasudil). Developed tension is expressed as a percentage of the maximum contraction to 65.4 mm KCl. C, summarized data of the PGF_2α-induced contraction and MLC₂₀ phosphorylation in the normal medium or in the presence of EGTA and fasudil or OH-fasudil. In B and C, MLC₂₀ phosphorylation (MLC-P) was measured at 5 min after the addition of PGF_2α (10 µm). When used, fasudil (10 µm) or hydroxyfasudil (OH-fasudil, 10 µm) was added 30 min before PGF_2α. The protocol in B and C was the same as that shown in A, but the experiments were performed in a different series. MLC₂₀ phosphorylation under the resting condition in normal PSS was 8.9 ± 2.4 % (data not shown). Verap: verapamil.

Figure 3. Effects of fasudil, hydroxyfasudil or ONO-RS-082 on MLC₂₀ phosphorylation and tension maintained in Ca²⁺-free, EGTA solution in the presence of PGF_2α

A, change in tension. After PGF_2α (10 µm)-induced contraction had reached a peak, the external medium was changed from normal PSS to Ca²⁺ free, 1 mm EGTA solution (5 min in the figure; ○, PGF_2α alone). When used, fasudil (δ, 10 µm) or hydroxyfasudil (□, 10 µm) was added at the time when the external Ca²⁺ was omitted (the left panel). ONO-RS-082 (•, 5 µm, the right panel) was applied 15 min before the addition of PGF_2α. Data are expressed as a percentage of the maximal contraction to PGF_2α in normal medium (n = 6-7). B, MLC₂₀ phosphorylation (MLC-P) measured in the resting state (0 min), just before switching the medium to Ca²⁺ free, EGTA solution (5 min), 5, 10 and 15 min after Ca²⁺ removal (10, 15 and 20 min after the addition of PGF_2α). Open columns: controls; hatched columns: fasudil-treated muscles; filled columns: hydroxyfasudil-treated muscles; grey column: ONO-RS-082-treated muscles. * Significantly different from the level in the absence of fasudil or hydroxyfasudil (P < 0.05); § significantly different from the resting level (P < 0.05).

Figure 4. Effects of the protein kinase C (PKC) inhibitor Ro31-8220 on phosphorylation of MLC₂₀ at Ser19 and contraction induced by PGF_2α and DPB

A, the effects of Ro31-8220 on the PGF_2α-induced inhibition of Ser19 dephosphorylation and relaxation. The protocol was the same as in Fig. 3. When the external medium was changed from normal PSS to Ca²⁺-free, 1 mm EGTA solution, Ro31-8220 (5 µm) was simultaneously added to one group (filled columns) but not to the control group (open columns). B, the effects on the DPB-induced inhibition of Ser19 dephosphorylation and relaxation. After 65.4 mm KCl caused a maximal contraction (5 min), the external medium was switched to Ca²⁺ free, 1 mm EGTA solution (5.4 mm K⁺) containing 1 µm DPB. When present, Ro31-8220 (5 µm) was added simultaneously with DPB. Data were taken 15 min after the omission of external Ca²⁺. The left panels represent a relative value of the 65.4 mm KCl-induced contraction, which was obtained before PGF_2α. The right panels are Ser19 phosphorylation expressed as a value relative to the phosphorylation in the resting state. Data are mean ± s.e.m. (n = 4). * Significantly different from DPB alone (P < 0.05, N.S. not significant).

Figure 5. Sensitivity of KCl- and PGF_2α-induced contraction and MLC₂₀ phosphorylation to ML-9 and fasudil

After KCl (65.4 mm, left)- or PGF_2α (10 µm, right)-induced contraction reached a respective peak (5 min), ML-9 (100 or 200 µm, respectively) was applied. Fasudil (10 µm) was added 15 min later. A, a change in tension in fasudil-treated (•) or untreated muscles (○). The ordinate represents the relative tension of maximum contraction to KCl or PGF_2α, respectively. B, MLC₂₀ phosphorylation (MLC-P) measured in the protocol shown in A. Data were sampled just before the addition of ML-9 (open columns, corresponding to a in A) and 30 min after the addition of ML-9 for KCl-induced contraction or 45 min after addition of ML-9 for PGF_2α-induced contraction (corresponding to b in A). Hatched columns: in the absence of fasudil, filled columns: in the presence of fasudil. * Significantly different from the level before the addition of ML-9 (P < 0.05), § significantly different from the level in the absence of fasudil (P < 0.05n = 5–8).

Figure 6. Differential effects of hydroxyfasudil and ML-9 on the calyculin A-induced contraction and MLC₂₀ phosphorylation

Open columns: tension expressed as a percentage of the maximum contraction due to 65.4 mm KCl, which was obtained before the addition of calyculin A, filled columns: MLC-P. At 30 min after the application of calyculin A (300 nm), hydroxyfasudil or ML-9 at each concentration was added. * Significantly different from the level in the absence of ML-9 (P < 0.05).

Figure 8. PGF_2α-induced phosphorylation of the MBS of phosphatase, and its inhibition by fasudil

A, time-course of the PGF_2α (10 µm)-induced contraction in the absence (○) or presence (•) of fasudil (10 µm). When present, fasudil was applied 15 min before addition of PGF_2α. B, (photographs) example of Western blots with the pMBS-T654 antibody or the N-MBS antibody. Samples were taken before the addition of PGF_2α (resting) and 1 min after PGF_2α. Bar graph, quantified phosphorylation of MBS (MBS-P) in the resting state, 1 or 15 min after the addition of PGF_2α in the absence (open columns) or presence (filled columns) of fasudil. The ratio of the density of a band with the pMBS-T654 antibody to that with the N-MBS antibody in the resting state is referred as 1. Data are presented as the means ± s.e.m. of four experiments. * Significantly different from the resting level (P < 0.05); § significantly different from PGF_2α alone (P < 0.05).