The Rho-A/Rho-kinase pathway is up-regulated but remains inhibited by cyclic guanosine monophosphate-dependent mechanisms during endotoxemia in small mesenteric arteries

Abstract

Objective: We investigated whether a reduced activity in the Rho-A/Rho-kinase pathway could be involved in the impaired vascular reactivity observed in septic shock.

Design: Ex vivo animal study.

Setting: University research laboratory.

Subjects: Male Wistar rats.

Interventions: Rats received an intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg) either 6 or 24 hours before the onset of our experiments. The effects of Y-27632 (a Rho-kinase inhibitor) were assessed in first-order mesenteric rings taken from these animals using wire myograph. The expression of Rho-A, Rho-kinases I and II, and the total and phosphorylated myosin phosphatase targeting subunit 1 (MYPT1) were assessed by Western blotting.

Measurements and main results: The EC50 to Y-27632 was reduced from 2.10 microM (1.22-3.66 microM) (control) to 0.21 microM (0.09-0.44 microM), and 9.54 (0.82-110.30) nM in LPS-treated groups 6 and 24 hours, respectively. The increased potency of Y-27632 was partially reversed by endothelium removal at both 6 and 24 hours. Incubation of Nomega-nitro-l-arginine methyl ester hydrochloride or 1400W (a nonselective and an inducible nitric oxide synthase inhibitor, respectively) normalized the responses to Y-27632 seen 6 hours after LPS. However, 1400W had no effect, whereas Nomega-nitro-l-arginine methyl ester hydrochloride caused a partial reduction in the enhanced potency of Y-27632 found 24 hours after LPS. The soluble guanylate cyclase inhibitor oxadiazolo[4,3-alpha]quinoxalin-1-one was able to bring the Y-27632 response back to normal both 6 and 24 hours after LPS. Rho-A, Rho-kinase I, Rho-kinase II, and MYPT1 were increased in mesenteric arteries from endotoxemic rats, but the phosphorylated MYPT1 was significantly reduced. However, incubation with oxadiazolo[4,3-alpha]quinoxalin-1-one circumvented the inhibition of MYPT1 phosphorylation found in preparations from LPS-treated animals.

Conclusions: Our findings revealed an impaired Rho-A/Rho-kinase-mediated phosphorylation of MYPT1 in vessels from endotoxemic animals in a cyclic guanosine monophosphate-dependent manner, suggesting that changes in mechanisms involved in calcium sensitization play a pivotal role in cardiovascular changes observed in septic shock.

Figure 1

The vascular relaxation induced by Y-27632 but not by acetylcholine is increased in small mesenteric arteries from LPS-treated rats or after exposure to the nitric oxide donor SNAP. First order mesenteric arterial rings were obtained from animals treated with LPS (10 mg/kg, i.p.) 6 (triangles) or 24 h (squares) before the experiments. Control experiments were performed in preparations obtained from animals treated with saline (0.1 ml/100 g) instead LPS (circles). Concentration response curves to Y-27632 (A and C) or acetylcholine (B and D) were constructed under the maximal contraction elicited by phenylephrine (30 μM). Data presented in panels C and D shown the relaxation obtained in control preparations only, preincubated with SNAP (70 μM for 10 min) 15 min before the exposure to PE and vasodilatory agents. In panel B, triangles from group LPS (6 h) are covered by squares from group LPS (24 h). Results are the mean ± S.E.M. of 6-8 experiments per group. * indicates a p value < 0.05 when compared with the control group (one way ANOVA followed by t-test subjected to Bonferroni’s correction).

Figure 2

Enhanced inhibitory effect of Y-27632 on phenylephrine-induced contraction in small mesenteric arteries from LPS-treated rats. The concentration response curves to phenyleprine (PE) and the maximal contraction induced by KCl (120 mM) are shown in panels A and B, respectively. The following panels reveal the influence of Y-27632 (3 μM, preincubated for 10 min) on PE-induced contraction in vessels from control animals (without LPS administration; C), or animals treated with LPS 6 (C), or 24 h (D) before the experiments. Results are the mean ± S.E.M. of 6 experiments per group. * indicates a p value < 0.05 when compared with the control group (one way ANOVA followed by t-test subjected to Bonferroni’s correction).

Figure 3

Endothelium removal causes a partial reversal of the enhanced relaxation induced by Y-27632 in small mesenteric arteries from LPS-treated animals 6 (B) and 24 h (C) before. The effect of endothelium removal in control rings (from animals not injected with LPS) is shown in A. The dashed lines represent the relaxation induced by Y-27632 in control preparations. Results are the mean ± S.E.M. of 6 experiments per group. * indicates a p value < 0.05 when comparing squares and circles (one way ANOVA followed by t-test subjected to Bonferroni’s correction). E+ indicates endothelium intact and E- endothelium denuded vessels.

Figure 4

The non-selective nitric oxide synthase inhibitor L-NAME blocks at 6 h (B) and partially reduces at 24 h (C) the enhanced relaxation induced by Y-27632 in small mesenteric arteries from LPS-treated animals. The effect of L-NAME in control rings (from animals not injected with LPS) is shown in A. The dashed lines represent the relaxation induced by Y-27632 in control preparations. L-NAME was pre-incubated for 40 min. Results are the mean ± S.E.M. of 6 to 8 experiments per group. * indicates a p value < 0.05 when comparing squares and circles (one way ANOVA followed by t-test subjected to Bonferroni’s correction).

Figure 5

The selective inhibition of iNOS by 1400W abolishes at 6 h (B) but not at 24 h (C) the enhanced relaxation induced by Y-27632 in small mesenteric arteries from LPS-treated animals. Incubation of 1400W was done for 40 min. Panel A shows the absence of influence of 1400W in control preparations (from animals not injected with LPS). The dashed lines represent the relaxation induced by Y-27632 in control preparations. Results are the mean ± S.E.M. of 5 to 6 experiments per group. * indicates a p value < 0.05 when comparing squares and circles (one way ANOVA followed by t-test subjected to Bonferroni’s correction).

Figure 6

Inhibition of soluble guanylate cyclase counteracts the enhanced relaxation induced by Y-27632 in small mesenteric arteries from LPS-treated animals both 6 (B) and 24 h (C) before. ODQ was incubated for 10 min. Panel A shows the influence of ODQ in control preparations (from animals not injected with LPS). The dashed lines represent the relaxation induced by Y-27632 in control preparations. Results are the mean ± S.E.M. of 8 to 9 experiments per group. * indicates a p value < 0.05 when comparing squares and circles (analyses of variance followed by t-test subjected to Bonferroni’s correction).

Figure 7

Representative western blots and corresponding densitometric analyses (relative to β-actin) of iNOS (A) and soluble guanylate cyclase β1 subunit (B) in small mesenteric arteries from control (indicated by “C”) and rats treated with LPS 6 and 24 h before. The data shown in graphs are the mean ± S.E.M. of 3 animals per group. * indicates a p value < 0.05 when comparing with the control group (one way ANOVA followed by t-test subjected to Bonferroni’s correction).

Figure 8

Increased protein levels of Rho-A/Rho-kinase components in the mesenteric bed of endotoxemic rats. Panels show representative western blots and corresponding densitometric analyses (relative to β-actin) of Rho-A (A), ROCK-I (B), and ROCK-II (C) in small mesenteric arteries from control rats (indicated by “C”) and rats treated with LPS 6 and 24 h before. The data shown in graphs are the mean ± S.E.M. of 3 animals per group. * indicates a p value < 0.05 when comparing with the control group; # indicates a p values < 0.05 when compared with LPS 6 h group (one way ANOVA followed by t-test subjected to Bonferroni’s correction).

Figure 9

Increased protein levels of MYPT1 and reduced phosphorilated-MYPT1 in vessels of endotoxemic rats. Densitometric analyses (relative to β-actin) of total MYPT1 (A) and phosphorilated-MYPT1 (B) found in small mesenteric arteries from control rats (indicated by “C”) and rats treated with LPS 6 and 24 h before. The data shown in graphs are the mean ± S.E.M. of 4 animals per group. * indicates a p value < 0.05 when comparing with the control group (one way ANOVA followed by t-test subjected to Bonferroni’s correction).

Figure 10

Inhibition of the soluble guanylate cyclase by ODQ reestablishes the phosphorilation of MYPT1 in vessels from endotoxemic animals. Panels show representative western blots and corresponding densitometric analyses (relative to β-actin) of phospho-MYPT1 in the whole mesenteric arterial bed (except the superior artery) from LPS-treated animals (6 or 24 h before, as indicated) or control animals (“C”). After removal from animals the vessels were kept in warmed nutritive solution and were incubated with ODQ (10 μM for 10 min) or vehicle (DMSO) followed by phenylephrine (30 μM for 5 min). After these treatments the vessels were immediately frozen in liquid nitrogen for western blot analyses. The data shown are the mean ± S.E.M. of 4 animals per group. # indicates a p value < 0.05 when comparing with the control group and * comparing with the respective time in tissue not incubated with ODQ (one way ANOVA followed by t-test subjected to Bonferroni’s correction).

Figure 11

Schematic representation showing the main steps in the Rho-A/Rho-kinase pathway in normal vessels (left box) and the changes described in this study regarding the up-regulation of Rho-A/Rho-kinase components and the failure of this pathway, by cGMP-dependent mechanisms, to phosphorylate and inhibit the myosin phosphatase in endotoxemic vessels (right box).