Rho-kinase inhibitors prevent agonist-induced vasospasm in human internal mammary artery

Abstract

1. Vasospasm of arterial conduits used for coronary artery surgery is an important cause of graft failure and is likely to result partly from raised levels of vasoconstrictor substances such as thromboxane A(2) and endothelin-1. Our aim was to find pharmacological agents that could prevent agonist-induced vasospasm. 2. Isometric tension was recorded from discarded segments of human left internal mammary artery (LIMA). Submaximal contraction evoked by the thromboxane A(2) mimetic U46619 (10 nM) was not inhibited by a blocker of store- and receptor-operated Ca(2+) channels (30 microM SKF96365) in the presence of diltiazem. Furthermore, contractions to < or =1 nM U46619 were preserved when extracellular Ca(2+) was reduced from 2.5 mM to 60 nM. Thus, sustained U46619-evoked contraction occurred without Ca(2+) influx. 3., We hypothesized that contraction might occur via Rho-kinase-mediated Ca(2+)-sensitization of myofilaments. Inhibitors of Rho-kinase (Y27632 and HA1077) were profound relaxants. If contraction was pre-evoked by 10 nM U46619, Y27632 and HA1077 caused full relaxation with EC(50)s of 1.67+/-0.22 microM and 3.58+/-0.35 microM respectively. Y27632 was also effective if applied before U46619, but was less potent. 4. Y27632 abolished contraction evoked by endothelin-1 and significantly reduced resting tone in the absence of a vasoconstrictor. 5. Rho-kinase-mediated Ca(2+)-sensitization appears to be a major mechanism of vasoconstriction in human LIMA. Rho-kinase inhibitors may have an important role in preventing vasospasm in arterial grafts used for coronary artery surgery.

Figure 1

Role of Ca²⁺ entry in contraction evoked by U46619 in human LIMA. (A) Concentration-response curves for U46619 in control conditions and following preincubation with 30 μM SKF96365. EC₅₀ values for U46619 were 3.30±0.41 nM (control, n=4) and 4.76±1.01 nM (SKF96365, n=4, P>0.05). (B) Typical original experimental traces showing contraction evoked by U46619 in one vessel segment bathed in Krebs solution (2.5 mM Ca²⁺) and another in low-Ca²⁺ Krebs solution (60 nM free Ca²⁺). (C) Summary of experiments as shown in (B). Data points are mean±s.e.mean normalized tension (tension (g)/wet weight of the vessel (g)) (n=6 for both groups). Experiments were performed in the presence of 10 μM diltiazem. The smooth curves in this and all other figures are fitted Hill equations.

Figure 2

Concentration-response curve for HA1077-induced relaxation (EC₅₀=3.58±0.35 μM, n=6) of LIMA pre-contracted with 10 nM U46619. Data points are mean±s.e.mean for tension as a percentage of that evoked by U46619. Experiments were performed in the presence of 10 μM diltiazem.

Figure 3

Effect of Y27632 on contraction pre-induced by U46619. (A) The upper (larger) experimental record is representative of contraction induced by 10 nM U46619 followed by relaxation in response to Y27632. The lower trace on the same plot is the effect of 10 μM Y27632 on another vessel segment in the absence of U46619. Contraction is expressed as a percentage of that evoked by 80 mM K⁺. (B) Mean±s.e.mean Y27632-induced relaxation of U46619-evoked tone in endothelium-intact vessels given as a percentage 80 mM K⁺-evoked contraction (EC₅₀ 1.67±0.22 μM, n=6). (C) Effect of Y27632 (1 and 10 μM) and GF 109203X on baseline tension in the absence of U46619 (n=4 in each group). Data points are mean±s.e.mean as a percentage of the maximum contractile responses to 80 mM K⁺ (*P=0.044, ^**P=0.0048). Experiments were performed in the presence of 10 μM diltiazem.

Figure 4

Effect of Y27632 applied before contraction with U46619. (A) Mean±s.e.mean contraction to U46619 as a percentage of contraction evoked by 80 mM K⁺. Data were collected in the absence of Y27632 and following preincubation with either 1 μM or 10 μM Y27632. EC₅₀ values in control conditions and in the presence of 1 μM Y27632 were not significantly different (5.94±0.74 nM and 7.37±1.11 nM respectively, n=4 for each). Preincubation with 10 μM Y27632 changed the EC₅₀ to 18.69±5.39 nM (P=0.046, n=4). (B,C) Use of a double concentration-response protocol to detect an effect of 1 μM Y27632. (B) Control experiments (n=5) showing no effect on maximum contraction to U46619 (100% vs 90.8±6.3%) or the EC₅₀ (8.43±1.55 nM vs 9.03±2.00 nM) between the first and second concentration-response curves. (C) Preincubation with 1 μM Y27632 attenuated the contractile response to low concentrations of U46619 (1 – 10 nM) but not to higher concentrations. Y27632 increased the EC₅₀ for U46619 from 4.81±0.56 nM to 8.93±0.69 nM (P=0.0017, n=5). Experiments were performed in the presence of 10 μM diltiazem. (*P<0.05, ^**P<0.01, #P<0.005, ##P<0.001, +P<0.0001).

Figure 5

Comparison of the effectiveness of Y27632 applied before or after U46619. Data points are mean±s.e.mean responses as a percentage of the maximum response to 10 nM U46619 in the absence of Y27632.

Figure 6

Concentration-response curve for Y27632-induced relaxation (EC₅₀=0.53±0.11 μM, n=5) of LIMA pre-contracted with 10 nM endothelin-1. Data points are mean±s.e.mean for tension as a percentage of that evoked by endothelin-1. Experiments were performed in the presence of 10 μM diltiazem.