Expression and functional role of the RhoA/Rho-kinase pathway in rat coeliac artery

Cleber E. Teixeira, Liming Jin, Zhekang Ying, Trenis Palmer, Fernanda Priviero, R Clinton Webb

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

1. Rho-kinase (ROK) stimulation represents a key step in the maintenance of agonist-induced contraction, an effect counteracted by nitric oxide (NO) released from the endothelium. The aim of the present study was to characterize the involvement of ROK in smooth muscle contraction of the rat coeliac artery using functional and expression studies. 2. Rings of rat coeliac artery were mounted in 5 mL myographs containing warmed and oxygenated Krebs' solution. Rings were connected to isometric transducers and data were recorded in a PowerLab system (ADInstruments, Colorado Springs, CO, USA). After a 60 min equilibration period, preparations were precontracted with phenylephrine (1 μmol/L). Endothelial integrity was assessed by treating the vessels with acetylcholine (1 μmol/L). Expression of ROKα, ROKβ and RhoA was analysed using western blot, whereas Rho guanine nucleotide exchange factors (RhoGEF) were measured at the mRNA level. 3. The addition of Y-27632 (0.01-30 μmol/L) caused sustained relaxation of rings contracted with phenylephrine (PE; 1 μmol/L), with intact or denuded endothelium (pEC50 = 6.38 ± 0.03 and 5.65 ± 0.02, respectively). NG-Nitro-l- arginine methyl ester (100 μmol/L) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin- 1-one (10 μmol/L), but not indomethacin (10 μmol/L), caused marked rightward shifts of the concentration-response curves to Y-27632. The contractile response to KCl (80 mmol/L) was significantly reduced by Y-27632, with a maximal inhibition of 57 ± 6%. Nifedipine (0.1-100 nmol/L) fully blocked KCl-evoked contractions, but only marginally affected those in response to PE (27 ± 2% maximal inhibition). At 1 μmol/L, Y-27632 also significantly enhanced relaxations to sodium nitroprusside (SNP; 0.0001-1 μmol/L). 4. At 1 μmol/L, SNP (but not 1 μmol/L Y-27632) significantly elevated the cGMP content above basal levels. Coincubation with SNP and Y-27632 increased cGMP levels, but the results were not significantly different from those in the presence of SNP alone. 5. Western blot analysis revealed the protein expression of RhoA, ROKα and ROKβ. The PDZ-RhoGEF, p115RhoGEF and leukaemia-associated RhoGEF (LARG) mRNA expression in coeliac artery was visualized by electrophoresis on agarose gels. 6. The results clearly demonstrate a role for the RhoA/ROK signalling pathway in the regulation of rat coeliac artery smooth muscle contraction. The findings of the present study suggest that endogenous nitric oxide-induced relaxation is mediated, in part, by inhibition of RhoA/ROK signalling in this tissue.

Original languageEnglish (US)
Pages (from-to)817-824
Number of pages8
JournalClinical and Experimental Pharmacology and Physiology
Volume32
Issue number10
DOIs
StatePublished - Oct 1 2005

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Celiac Artery
rho-Associated Kinases
Rho Guanine Nucleotide Exchange Factors
Single Nucleotide Polymorphism
Phenylephrine
Muscle Contraction
Endothelium
Smooth Muscle
Nitric Oxide
rhoA GTP-Binding Protein
Western Blotting
Messenger RNA
Agar Gel Electrophoresis
Nitroprusside
Carbon Monoxide
Nifedipine
Transducers
Indomethacin
Acetylcholine
Y 27632

Keywords

  • Coeliac artery
  • Nitric oxide
  • Rho guanine nucleotide exchange factors
  • Rho-kinase
  • RhoA

ASJC Scopus subject areas

  • Physiology
  • Pharmacology (medical)
  • Pharmacology, Toxicology and Pharmaceutics(all)

Cite this

Expression and functional role of the RhoA/Rho-kinase pathway in rat coeliac artery. / Teixeira, Cleber E.; Jin, Liming; Ying, Zhekang; Palmer, Trenis; Priviero, Fernanda; Webb, R Clinton.

In: Clinical and Experimental Pharmacology and Physiology, Vol. 32, No. 10, 01.10.2005, p. 817-824.

Research output: Contribution to journalArticle

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T1 - Expression and functional role of the RhoA/Rho-kinase pathway in rat coeliac artery

AU - Teixeira, Cleber E.

AU - Jin, Liming

AU - Ying, Zhekang

AU - Palmer, Trenis

AU - Priviero, Fernanda

AU - Webb, R Clinton

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N2 - 1. Rho-kinase (ROK) stimulation represents a key step in the maintenance of agonist-induced contraction, an effect counteracted by nitric oxide (NO) released from the endothelium. The aim of the present study was to characterize the involvement of ROK in smooth muscle contraction of the rat coeliac artery using functional and expression studies. 2. Rings of rat coeliac artery were mounted in 5 mL myographs containing warmed and oxygenated Krebs' solution. Rings were connected to isometric transducers and data were recorded in a PowerLab system (ADInstruments, Colorado Springs, CO, USA). After a 60 min equilibration period, preparations were precontracted with phenylephrine (1 μmol/L). Endothelial integrity was assessed by treating the vessels with acetylcholine (1 μmol/L). Expression of ROKα, ROKβ and RhoA was analysed using western blot, whereas Rho guanine nucleotide exchange factors (RhoGEF) were measured at the mRNA level. 3. The addition of Y-27632 (0.01-30 μmol/L) caused sustained relaxation of rings contracted with phenylephrine (PE; 1 μmol/L), with intact or denuded endothelium (pEC50 = 6.38 ± 0.03 and 5.65 ± 0.02, respectively). NG-Nitro-l- arginine methyl ester (100 μmol/L) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin- 1-one (10 μmol/L), but not indomethacin (10 μmol/L), caused marked rightward shifts of the concentration-response curves to Y-27632. The contractile response to KCl (80 mmol/L) was significantly reduced by Y-27632, with a maximal inhibition of 57 ± 6%. Nifedipine (0.1-100 nmol/L) fully blocked KCl-evoked contractions, but only marginally affected those in response to PE (27 ± 2% maximal inhibition). At 1 μmol/L, Y-27632 also significantly enhanced relaxations to sodium nitroprusside (SNP; 0.0001-1 μmol/L). 4. At 1 μmol/L, SNP (but not 1 μmol/L Y-27632) significantly elevated the cGMP content above basal levels. Coincubation with SNP and Y-27632 increased cGMP levels, but the results were not significantly different from those in the presence of SNP alone. 5. Western blot analysis revealed the protein expression of RhoA, ROKα and ROKβ. The PDZ-RhoGEF, p115RhoGEF and leukaemia-associated RhoGEF (LARG) mRNA expression in coeliac artery was visualized by electrophoresis on agarose gels. 6. The results clearly demonstrate a role for the RhoA/ROK signalling pathway in the regulation of rat coeliac artery smooth muscle contraction. The findings of the present study suggest that endogenous nitric oxide-induced relaxation is mediated, in part, by inhibition of RhoA/ROK signalling in this tissue.

AB - 1. Rho-kinase (ROK) stimulation represents a key step in the maintenance of agonist-induced contraction, an effect counteracted by nitric oxide (NO) released from the endothelium. The aim of the present study was to characterize the involvement of ROK in smooth muscle contraction of the rat coeliac artery using functional and expression studies. 2. Rings of rat coeliac artery were mounted in 5 mL myographs containing warmed and oxygenated Krebs' solution. Rings were connected to isometric transducers and data were recorded in a PowerLab system (ADInstruments, Colorado Springs, CO, USA). After a 60 min equilibration period, preparations were precontracted with phenylephrine (1 μmol/L). Endothelial integrity was assessed by treating the vessels with acetylcholine (1 μmol/L). Expression of ROKα, ROKβ and RhoA was analysed using western blot, whereas Rho guanine nucleotide exchange factors (RhoGEF) were measured at the mRNA level. 3. The addition of Y-27632 (0.01-30 μmol/L) caused sustained relaxation of rings contracted with phenylephrine (PE; 1 μmol/L), with intact or denuded endothelium (pEC50 = 6.38 ± 0.03 and 5.65 ± 0.02, respectively). NG-Nitro-l- arginine methyl ester (100 μmol/L) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin- 1-one (10 μmol/L), but not indomethacin (10 μmol/L), caused marked rightward shifts of the concentration-response curves to Y-27632. The contractile response to KCl (80 mmol/L) was significantly reduced by Y-27632, with a maximal inhibition of 57 ± 6%. Nifedipine (0.1-100 nmol/L) fully blocked KCl-evoked contractions, but only marginally affected those in response to PE (27 ± 2% maximal inhibition). At 1 μmol/L, Y-27632 also significantly enhanced relaxations to sodium nitroprusside (SNP; 0.0001-1 μmol/L). 4. At 1 μmol/L, SNP (but not 1 μmol/L Y-27632) significantly elevated the cGMP content above basal levels. Coincubation with SNP and Y-27632 increased cGMP levels, but the results were not significantly different from those in the presence of SNP alone. 5. Western blot analysis revealed the protein expression of RhoA, ROKα and ROKβ. The PDZ-RhoGEF, p115RhoGEF and leukaemia-associated RhoGEF (LARG) mRNA expression in coeliac artery was visualized by electrophoresis on agarose gels. 6. The results clearly demonstrate a role for the RhoA/ROK signalling pathway in the regulation of rat coeliac artery smooth muscle contraction. The findings of the present study suggest that endogenous nitric oxide-induced relaxation is mediated, in part, by inhibition of RhoA/ROK signalling in this tissue.

KW - Coeliac artery

KW - Nitric oxide

KW - Rho guanine nucleotide exchange factors

KW - Rho-kinase

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