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Effects of rho-kinase inhibitors on contractile synergism involving a prostanoid EP3 agonist in rat femoral artery We have recently shown that prostaglandin E2 and sulprostone, acting as prostanoid EP3 receptor agonists, weakly contract rat femoral artery, whilst exhibiting pronounced synergism with strong contractile agents (phenylephrine, U-46619, K+) (Jones et al., 2005). We now report the effects of two specific Rho-kinase inhibitors, H-1152 (Sasaki et al., 2002) and Y-27632 (Uehata et al., 1997), on these interactions. Rho-kinase has been previously implicated in endothelin-1 / phenylephrine synergism in rat corpus cavernosum (Wingard et al., 2003). Femoral arteries were removed from male Sprague-Dawley rats (250 - 300 g). Ring preparations were set up in a wire myograph system in Krebs-Henseleit solution gassed with 95% O2 / 5% CO2 at 37 oC and containing 1 μM indomethacin. The main protocol involved the following sequence: TP antagonist GR-32191 (1 μM) / NOS inhibitor L-NAME (100 μM), Rho-kinase inhibitor or vehicle, priming with phenylephrine or K+, cumulative addition of sulprostone (0.1 - 144 nM). Responses were normalized to contraction induced by 40 mM K+. H-1152 (0.01 - 4.44 μM) abolished or almost abolished established contractions to 1 μM phenylephrine, 30 nM sulprostone primed with 100 nM phenylephrine, and 10 nM U-46619, but only reduced 40 mM K+ responses by 65 - 80%. pIC50 values (± se mean, n = 6) were 6.97 ± 0.12, 6.70 ± 0.18, 6.84 ± 0.06 and 6.75 ± 0.06 respectively (all P >0.05 relative to phenylephrine value, 1-factor ANOVA). Y-27632 (0.1 - 44.4 μM) had a similar profile; the corresponding pIC50 values (n = 5) were 6.51 ± 0.10, 6.22 ± 0.13, 5.85 ± 0.07 and 5.83 ± 0.09. The phenylephrine pIC50 was significantly greater than the U-46619 and K+ values (P <0.001), possibly due to slightly larger established contractions to the latter agents. Based on these findings, preparations were treated with vehicle and either 100, 300 or 1000 nM H-1152 (3 sets of experiments, each n = 6) before cumulative addition of sulprostone with and without 100 nM phenylephrine priming. H-1152 itself slightly reduced the resting tension, which was most obvious at 1000 nM (-2.3 ± 0.9%, n = 6), and markedly inhibited responses to phenylephrine alone. Under phenylephrine priming, H-1152 progressively reduced the pEC50 and maximum of sulprostone, with the vector passing through the mid-points of the fitted sigmoids having a gradient of -37%.log unit-1 (co-ordinates, -9.33,78%; -7.79,20%). The reverse vector for sulprostone under variable phenylephrine priming had a similar magnitude (-39%.log unit-1; -7.28,14%; -9.49,100%). Considerable synergism between phenylephrine and sulprostone was still evident in the presence of 1000 nM H-1152. In a less comprehensive series of experiments (sulprostone alone curve not obtained, n = 4), Y-27632 (1 and 10 μM) suppressed 20 mM K+-primed sulprostone curves. The vector gradient was -62%.log unit-1 (co-ordinates, -9.59,104%; -8.57,40%), similar to the gradient for sulprostone under variable K+ priming (-55%.log unit-1; -7.95,16%; -9.65,110%). A probable explanation of these data is that Rho-kinase activation is an integral step in the transduction pathways of both the strong receptor agonists and K+ (see Sakurada et al., 2003); inhibition of priming responses by H-1152 or Y-27632 would consequentially suppress enhanced sulprostone responses. From the inhibition profiles obtained, it would be unwise to infer that the EP3 receptor directly transduces through a Rho-kinase step.
Jones, R.L. et al. (2005). Proceedings of British Pharmacological Society, Cambridge, in press. |
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