scholarly journals A Method for Measuring Rho Kinase Activity in Tissues and Cells

2008 ◽  
pp. 181-189 ◽  
Author(s):  
Ping‐Yen Liu ◽  
James K. Liao
Keyword(s):  
Kinase Activity ◽  
Rho Kinase

Effect of Early Normotension with Olmesartan on Rho-kinase Activity in Hypertensive Patients

2019 ◽  
Vol 18 (1) ◽  
pp. 87-91 ◽  
Author(s):  
Claudio Cantin ◽  
Jorge E. Jalil ◽  
Juan F. Bulnes ◽  
Ulises Novoa ◽  
Paul MacNab ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Kinase Activity ◽  
Clinical Evidence ◽  
Mononuclear Cells ◽  
Treatment Initiation ◽  
Rho Kinase ◽  
Angiotensin Ii Receptor ◽  
Peripheral Blood Mononuclear ◽  
Hypertensive Patients ◽  
Rock Activity

Background: Angiotensin II is a potent activator of the Rho-kinase (ROCK) pathway, through which it exerts some of its adverse vasoconstrictor effects. Clinical evidence on the effects of blocking the angiotensin II receptor 1 on ROCK activity in hypertensive patients is scarce. Objective: To demonstrate that ROCK activity in peripheral blood mononuclear cells (PMBCs) in patients with essential hypertension is reduced earlier than previously observed, along with blood pressure (BP) lowering on treatment with olmesartan. Methods: Prospective pilot open study; 17 hypertensive patients were treated with progressive olmesartan doses starting with 20 mg qd. BP was measured at 3, 6 and 9 weeks after treatment initiation. If treatment failed to normalize BP after 3 weeks, olmesartan dose was increased to 40 mg qd, and if still hypertensive after 6 weeks, 12.5 mg of hydrochlorothiazide qd was added. ROCK activity was measured at baseline and 9 weeks after treatment as myosin phosphatase target subunit 1 phosphorylation (MYPT1-p/T ratio) in PBMC. Results: Mean baseline BP was 162 ± 4.9/101 ± 2.4 mmHg. After 9 weeks of treatment, both systolic and diastolic BP were reduced by 41 and 22 mmHg, respectively (p<0.05). Mean pretreatment MYPT1- p/T ratio in PMBCs was significantly reduced by 80% after 9 weeks with olmesartan (p<0.01). Conclusion: Normotension achieved after 9 weeks in 82% of the patients treated with olmesartan was associated with a significant reduction of ROCK activity in PBMC.

scholarly journals Pro-Nerve Growth Factor Induces Activation of RhoA Kinase and Neuronal Cell Death

2019 ◽  
Vol 9 (8) ◽  
pp. 204 ◽  
Author(s):  
Marina Sycheva ◽  
Jake Sustarich ◽  
Yuxian Zhang ◽  
Vaithinathan Selvaraju ◽  
Thangiah Geetha ◽  
...  
Keyword(s):  
Cell Death ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Neuronal Cell Death ◽  
Rho Kinase ◽  
Neuronal Cell ◽  
Nerve Growth ◽  
Rhoa Kinase

We have previously shown that the expression of pro-nerve growth factor (proNGF) was significantly increased, nerve growth factor (NGF) level was decreased, and the expression of p75NTR was enhanced in Alzheimer’s disease (AD) hippocampal samples. NGF regulates cell survival and differentiation by binding TrkA and p75NTR receptors. ProNGF is the precursor form of NGF, binds to p75NTR, and induces cell apoptosis. The objective of this study is to determine whether the increased p75NTR expression in AD is due to the accumulation of proNGF and Rho kinase activation. PC12 cells were stimulated with either proNGF or NGF. Pull-down assay was carried out to determine the RhoA kinase activity. We found the expression of p75NTR was enhanced by proNGF compared to NGF. The proNGF stimulation also increased the RhoA kinase activity leading to apoptosis. The expression of active RhoA kinase was found to be increased in human AD hippocampus compared to control. The addition of RhoA kinase inhibitor Y27632 not only blocked the RhoA kinase activity but also reduced the expression of p75NTR receptor and inhibited the activation of JNK and MAPK induced by proNGF. This suggests that overexpression of proNGF in AD enhances p75NTR expression and activation of RhoA, leading to neuronal cell death.

scholarly journals Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway

2013 ◽  
Vol 304 (5) ◽  
pp. G527-G535 ◽  
Author(s):  
Senthilkumar Rajagopal ◽  
Divya P. Kumar ◽  
Sunila Mahavadi ◽  
Sayak Bhattacharya ◽  
Ruizhe Zhou ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Kinase Activity ◽  
Muscle Relaxation ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Receptor Activation ◽  
Gastric Smooth Muscle ◽  
Selective Ligand ◽  
Gastric Muscle ◽  
Kinase Pathway

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5−/− mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2′-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

scholarly journals Effects of Tolvaptan on Oxidative Stress in ADPKD: A Molecular Biological Approach

2022 ◽  
Vol 11 (2) ◽  
pp. 402
Author(s):  
Matteo Rigato ◽  
Gianni Carraro ◽  
Irene Cirella ◽  
Silvia Dian ◽  
Valentina Di Di Vico ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Renal Deterioration ◽  
Deterioration Rate ◽  
Function Loss ◽  
State Marker ◽  
Molecular Biological Approach

Autosomal dominant polycystic disease (ADPKD) is the most frequent monogenic kidney disease. It causes progressive renal failure, endothelial dysfunction, and hypertension, all of which are strictly linked to oxidative stress (OxSt). Treatment with tolvaptan is known to slow the renal deterioration rate, but not all the molecular mechanisms involved in this effect are well-established. We evaluated the OxSt state in untreated ADPKD patients compared to that in tolvaptan-treated ADPKD patients and healthy subjects. OxSt was assessed in nine patients for each group in terms of mononuclear cell p22phox protein expression, NADPH oxidase key subunit, MYPT-1 phosphorylation state, marker of Rho kinase activity (Western blot) and heme oxygenase (HO)-1, induced and protective against OxSt (ELISA). p22phox protein expression was higher in untreated ADPKD patients compared to treated patients and controls: 1.42 ± 0.11 vs. 0.86 ± 0.15 d.u., p = 0.015, vs. 0.53 ± 0.11 d.u., p < 0.001, respectively. The same was observed for phosphorylated MYPT-1: 0.96 ± 0.28 vs. 0.68 ± 0.09 d.u., p = 0.013 and vs. 0.47 ± 0.13 d.u., p < 0.001, respectively, while the HO-1 expression of untreated patients was significantly lower compared to that of treated patients and controls: 5.33 ± 3.34 vs. 2.08 ± 0.79 ng/mL, p = 0.012, vs. 1.97 ± 1.22 ng/mL, p = 0.012, respectively. Tolvaptan-treated ADPKD patients have reduced OxSt levels compared to untreated patients. This effect may contribute to the slowing of renal function loss observed with tolvaptan treatment.

Abstract MP09: Vascular Rho-kinase Activation by Inflammatory Signaling Mediates Blood Pressure Increase in Overweight

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Cristiane Aoqui Aoqui ◽  
Stefan Chmielewski ◽  
Uwe Heemann ◽  
Marcus Baumann
Keyword(s):  
Blood Pressure ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Normal Weight ◽  
Mesenteric Arteries ◽  
Pressure Increase ◽  
Tnf Alpha ◽  
Kinase Activation ◽  
Blood Pressure Increase ◽  
Tlr4 Ligand

Background: Overweight is associated with a high prevalence of hypertension. The mechanisms linking overweight to blood pressure increase remain unclear. We hypothesized that vascular Rho-kinase activation contributes to blood pressure increase in overweight by involving TNF-α and TLR4. Methods: C57/BL6 mice fed a high-fat diet for 2 weeks were used to induce overweight associated blood pressure increase. Additional treatment in overweight and normal weight mice contained Rho-kinase inhibitors (fasudil and pravastatin; n=7/all groups), etanercept and TNFR1 and TLR4 null-mice. Microvascular studies were performed in a wire myograph and arterial blood pressure measured with a carotid catheter. Rho-kinase activity was determined in small mesenteric arteries of all groups. Inflammatory ligands such as TNF-alpha and free fatty acids were determined. Effects of TNF-alpha and TLR4 ligand LPS and palmitic acid on Rho-kinase activity and were determined ex vivo in mesenteric arteries and in in vivo. Results: Overweight mice had higher blood pressure (Delta: 9±2 mmHg) and vasoconstriction as normal weight mice. Small mesenteric arteries of overweight mice had a 50% higher Rho-kinase activity as normal weight mice. Ex vivo treatment with the Rho-kinase inhibitor Y-27632 reversed vasoconstriction of mesenteric arteries of overweight mice to constriction level in normal weight mice. In vivo treatment with the Rho-kinase inhibitor fasudil or pravastatin along with high-fat diet abolished the overweight associated blood pressure increase and enhanced vasoconstriction. TNF-alpha and TLR4 ligand free fatty acid were enhanced in overweight mice. TNF-alpha and TLR4 ligand LPS and palmitic acid increased Rho-kinase activity in mesenteric arteries. Use of etenercept, TNFR1 and TLR4 null-mice in the overweight model prevented blood pressure increase, vasoconstriction and Rho-kinase activation. All described effects were independent of adiposity. Conclusion: These results indicated that in diet-induced overweight vascular Rho-kinase activation is a key element of increased blood pressure and vasoconstriction. Potential activator of Rho-kinase are mediated by inflammatory factors including TLR4 ligands and TNF-alpha.

scholarly journals RHO KINASE ACTIVITY, CONNEXIN40 AND ATRIAL FIBRILLATION

2019 ◽  
Vol 37 ◽  
pp. e54-e55
Author(s):  
G. Bertoldi ◽  
E. Pagnin ◽  
V. Ravarotto ◽  
F. Nalesso ◽  
B. Rossi ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Kinase Activity ◽  
Rho Kinase

scholarly journals RhoA/Rho Kinase Blocks Muscle Differentiation via Serine Phosphorylation of Insulin Receptor Substrate-1 and -2

2007 ◽  
Vol 21 (9) ◽  
pp. 2282-2293 ◽  
Author(s):  
Min Jin Lim ◽  
Kyu Jin Choi ◽  
Yan Ding ◽  
Jin Hwan Kim ◽  
Bum Shik Kim ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Muscle Differentiation ◽  
Serine Phosphorylation ◽  
Fibroblast Growth Factor 2 ◽  
Insulin Receptor Substrate ◽  
Fibroblast Growth ◽  
Proliferation Medium

Abstract Although the RhoA/Rho kinase (RhoA/ROK) pathway has been extensively investigated, its roles and downstream signaling pathways are still not well understood in myogenic processes. Therefore, we examined the effects of RhoA/ROK on myogenic processes and their signaling molecules using H9c2 and C2C12 cells. Increases in RhoA/ROK activities and serine phosphorylation levels of insulin receptor substrate (IRS)-1 (Ser307 and Ser636/639) and IRS-2 were found in proliferating myoblasts, whereas IRS-1/2 tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity increased during the differentiation process. ROK strongly bound to IRS-1/2 in proliferation medium but dissociated from them in differentiation medium (DM). ROK inactivation by a ROK inhibitor, Y27632, or a dominant-negative ROK, decreased IRS-1/2 serine phosphorylation with increases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity, which led to muscle differentiation even in proliferation medium. Inhibition of ROK also enhanced differentiation in DM. ROK activation by a constitutive active ROK blocked muscle differentiation with the increased IRS-1/2 serine phosphorylation, followed by decreases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity in DM. Interestingly, fibroblast growth factor-2 added to DM also blocked muscle differentiation through RhoA/ROK activation. Fibroblast growth factor-2 blockage of muscle differentiation was reversed by Y27632. Collectively, these results suggest that the RhoA/ROK pathway blocks muscle differentiation by phosphorylating IRS proteins at serine residues, resulting in the decreased IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity. The absence of the inhibitory effects of RhoA/ROK in DM due to low concentrations of myogenic inhibitory growth factors seems to allow IRS-1/2 tyrosine phosphorylation, which stimulates muscle differentiation via transducing normal myogenic signaling.

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