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

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Abstract
SUMMARY: 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.

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