Involvement of Rho-kinase in contraction of guinea-pig aorta induced by prostanoid EP3 receptor agonists

Abstract

1. The mechanism of contraction of guinea-pig isolated aorta induced by the prostanoid EP(3) receptor agonist sulprostone (0.1-300 nM) has been investigated. In 60% of the experiments, the sulprostone log concentration-response curve (maximum=15-40% of 100 nM U-46619 response; low-responders) was unaffected by the removal of extracellular Ca(2+), blockade of L-type Ca(2+) channels with nifedipine and depletion of internal Ca(2+) stores. In the remaining preparations (35-65% of 100 nM U-46619 response; high-responders), contractions to higher sulprostone concentrations showed a nifedipine-sensitive component, which was enhanced by charybdotoxin. 2. In Ca(2+)-free Krebs solution, established contractions to 300 nM sulprostone were abolished by the Rho-kinase inhibitors H-1152, Y-27632 and HA-1077 (IC(50) values=190, 770 and 2030 nM). The PKA/Rho-kinase inhibitor H-89 (10 nM-10 micro M) caused enhancement progressing to inhibition. The selective PKC inhibitor Ro 32-0432 (3 micro M) had no effect, while staurosporine, recently shown to be a potent Rho-kinase inhibitor, abolished sulprostone responses (IC(50) approximately 47 nM), but its action was slow. The MAP kinase inhibitors SB 202190, SB 203580 and PD 80958 produced little inhibition. 3. In normal Krebs solution, H-1152 and Y-27632 abolished established contractions to 300 nM sulprostone and 1 micro M phenylephrine, and partially inhibited 10 micro M phenylephrine and 50 mM K(+) responses. 4. The results are discussed in relation to the reported potencies of the protein kinase inhibitors in enzyme assays. Activation of the Rho-kinase pathway appears to be a primary mechanism of contraction induced by EP(3) receptor agonists in guinea-pig aorta.

Figure 1

Characteristics of sulprostone-induced contraction of guinea-pig aorta in normal Krebs solution, (a), (b) and (c) Low/high-responder status defined by lower/higher responses to sulprostone coupled with insensitivity/sensitivity to nifedipine (n=5 in panel c). An experimental record of the effect of nifedipine on K⁺-induced contraction is also shown. (d) Effect of nifedipine on log concentration–response curves for phenylephrine and U-46619 (n=6). (e) Experimental record showing enhancement of sulprostone-induced contraction by CTX in preparations from the same guinea-pig. (f) Effect of nifedipine on the log concentration – response curve for sulprostone enhanced by CTX. Data were analyzed by repeated-measures two-factor ANOVA: panels (c), (d) and (f), *P<0.05, **P<0.01, ***P<0.001 for comparisons of control and corresponding nifedipine or CTX data, P>0.05 for the remaining comparisons; panel (d), ns indicates P>0.05 for comparison of pre- and post-nifedipine data; panel (f), all ns indicates P>0.05 for the three contrasts within the box. In panels (b) and (c), contraction is expressed as a percentage of the 100 nM U-46619 response. Values are means±s.e.m.

Figure 2

Effects of Ca²⁺ modulation on agonist-induced contraction of high-responder guinea-pig aorta preparations. (a) Log concentration–response curves for sulprostone under control, Ca²⁺-free and Ca²⁺ store depletion (phenylephrine, Phe) conditions (n=12). The control response to 10 μM phenylephrine and the corresponding third response during the Ca²⁺ store depletion (Phe) procedure are also shown (n=12). (b) Log concentration–response curves for U-46619 (n=4) and phenylephrine (n=9) under control and Ca²⁺-free conditions. (c) Experimental tracing showing the Ca²⁺ store depletion (Phe) protocol and a subsequent response to sulprostone. (d) Log concentration–response curves for sulprostone under control (same as panel a), CPA treatment, Ca²⁺ store depletion (CPA), and Ca²⁺ store depletion (CPA and Phe) conditions (n=12). ***P<0.001 for comparsion with control data (repeated-measures two-factor ANOVA). Values are means±s.e.m.

Figure 3

Effects of protein kinase inhibitors on established contractile responses of guinea-pig aorta in Ca²⁺-free Krebs solution (mixture of low- and high-responders). (a) Experimental tracings showing the effect of Ro 32-0432 on a PMA response, Ro 32-0432 followed by Y-27632 on a phenylephrine response, and staurosporine on a sulprostone response. (b) Log concentration–inhibition curves: tone induced by 300 nM sulprostone (n=5–7). For staurosporine, each preparation was exposed to a single concentration of staurosporine for 60 min; the broken line indicates that steady-state inhibition was not achieved. Values are means±s.e.m.

Figure 4

Effects of Rho-kinase inhibitors on established responses of guinea-pig aorta preparations (mostly high-responders) in normal Krebs solution: (a) H-1152 and (b) Y-27632 vs phenylephrine (n=4 and 6), sulprostone (n=4 and 6) and K⁺ (n=10 and 6). Values are means±s.e.m.

Figure 5

Effects of cumulative addition of protein kinase inhibitors on K⁺ (50 mM)-induced contraction of guinea-pig aorta preparations (mostly low responders) suppressed by addition of 10 μM nifedipine. (a) H-1152 followed by Ro 32-0432, (b) Y-27632 followed by Ro 32-0432, (c) Ro 32-0432 followed by H-1152, (d) vehicle controls for panel c (all n=4). **P<0.01, ***P<0.001 compared to control value (=100%); ns P>0.05, ^††P<0.01 for comparison indicated by parenthesis (repeated-measures one-factor ANOVA performed on raw data for each panel). Values are means±s.e.m.

Figure 6

Guinea-pig aorta in normal Krebs solution: effects of Y-27632 on log concentration–response curves for (a) U-46619, phenylephrine and sulprostone (low-responder preparations, n=4), (b) sulprostone in the presence of CTX, (c) phenylephrine in the presence of CTX, (d) K⁺ (all high-responder preparations, n=4). Statistical analysis by repeated-measures two-factor ANOVA: ns P>0.05, *P<0.05, **P<0.01, ***P<0.001 for comparison of pre- and post-nifedipine data. Values are means±s.e.m.