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

J. Eduardo Da Silva-Santos, Chin Wei Chiao, Romulo Leite, R Clinton Webb

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)1716-1723
Number of pages8
JournalCritical care medicine
Volume37
Issue number5
DOIs
StatePublished - Jan 1 2009

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rho-Associated Kinases
Myosin-Light-Chain Phosphatase
Mesenteric Arteries
Endotoxemia
Cyclic GMP
Quinoxalines
Septic Shock
Phosphorylation
Nitric Oxide Synthase Type II
Intraperitoneal Injections
Endothelium
Blood Vessels
Lipopolysaccharides
Y 27632
Wistar Rats
Western Blotting
Calcium
Research

Keywords

  • Guanylate cyclase
  • Hypotension
  • Myosin phosphatase
  • Nitric oxide
  • Septic shock
  • Vascular hyporesponsiveness

ASJC Scopus subject areas

  • Critical Care and Intensive Care Medicine

Cite this

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; Chiao, Chin Wei; Leite, Romulo; Webb, R Clinton.

In: Critical care medicine, Vol. 37, No. 5, 01.01.2009, p. 1716-1723.

Research output: Contribution to journalArticle

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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 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.",
keywords = "Guanylate cyclase, Hypotension, Myosin phosphatase, Nitric oxide, Septic shock, Vascular hyporesponsiveness",
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AU - Chiao, Chin Wei

AU - Leite, Romulo

AU - Webb, R Clinton

PY - 2009/1/1

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N2 - 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.

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KW - Hypotension

KW - Myosin phosphatase

KW - Nitric oxide

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KW - Vascular hyporesponsiveness

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