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

da Silva-Santos, J Eduardo PhD; Chiao, Chin-Wei PhD; Leite, Romulo PhD; Webb, R Clinton PhD

doi: 10.1097/CCM.0b013e31819efb43
Laboratory Investigations

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 μM (1.22–3.66 μM) (control) to 0.21 μM (0.09–0.44 μM), 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 Nω-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 Nω-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.

From the Department of Physiology (JEdS-S, RL, C-WC, RCW), Medical College of Georgia, Augusta, GA; and Institute of Biological Sciences (JEdS-S), Universidade Federal do Pará, Belém, PA, Brazil.

Supported, in part, by grants from National Institute of Health (HL-71138 and HL-74167), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES—Brazil).

This study was performed at the Department of Physiology, Medical College of Georgia, GA.

The authors have not disclosed any potential conflicts of interest.

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© 2009 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins