Y-27632 is a selective inhibitor of ROCKs including p160ROCK (Ki: 140 nM) and ROCK2 (IC50: 800 nM).

The inhibited potency of Y-27632 against the ROCK family is 100 times than other kinases including protein kinase C, cAMP-dependent kinase and myosin light chain kinase. Y-27632 prolongs the lag time and delays the appearance of BrdU-labeled cells in a concentration-dependent manner, delays of about 1 and 4 h are noticed in the Swiss 3T3 cells treated with 10 and 100 μM Y-27632, respectively [1]. The application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates [2]. Y-27632 promotes neuronal differentiation of adipose tissue-derived stem cells (ADSCs). Compared to 1.0 and 2.5 μM Y-27632 induced groups, percentages of neuronal-like cells achieved a peak in the 5.0 μM Y-27632 induced group [3]. Y-27632 selectively inhibits smooth-muscle contraction by inhibiting Ca2+ sensitization. We identified the Y-27632 target as a Rho-associated protein kinase, p160ROCK. Y-27632 consistently suppresses Rho-induced [4].

Y-27632 significantly decreased the blood pressure in a dose-dependent manner in spontaneously hypertensive rats: a fall of 50 mm Hg was still observed 7 h after administration of 30 mg/kg of Y-27632. The same dose of this compound also caused a significant and persistent fall in blood pressure in renal hypertensive rats, as well as in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. On the other hand, administration of the same dose of Y-27632 caused only a slight and transient fall in blood pressure in control Wistar rats [4]. Y-27632 (5-10 mg/kg) and fasudil 5-25 (mg/kg) diminished onset of myoclonic jerks, clonic convulsions and tonic hindlimb extensions in mice given pentylenetetrazole [5].

Recombinant ROCK1/2, PKN, or citron kinase is expressed in HeLa cells as Myc-tagged proteins by transfection using Lipofectamine and is precipitated from the cell lysates by the use of 9E10 monoclonal anti-Myc antibody coupled to G protein-Sepharose. Recovered immunocomplexes are incubated with various concentrations of [32P]ATP and 10 mg of histone type 2 as substrates in the absence or presence of various concentrations of either Y-27632 or Y-30141 at 30°C for 30 min in a total volume of 30 μL of the kinase buffer containing 50 mM HEPES-NaOH, pH 7.4, 10 mM MgCl2, 5 mM MnCl2, 0.02% Briji 35, and 2 mM dithiothreitol. PKCa is incubated with 5 μM [32P]ATP and 200 μg/mL histone type 2 as substrates in the absence or presence of various concentrations of either Y-27632 or Y-30141 at 30°C for 10 min in a kinase buffer containing 50 mM Tris-HCl, pH 7.5, 0.5 mM CaCl2, 5 mM magnesium acetate, 25 μg/mL phosphatidylserine, 50 ng/mL 12-O-tetradecanoyl phorbol-13-acetate and 0.001% leupeptin in a total volume of 30 μL. Incubation is terminated by the addition of 10 μL of 43 Laemmli sample buffer. After boiling for 5 min, the mixture is subjected to SDS-polyacrylamide gel electrophoresis on a 16% gel. The gel is stained with Coomassie Brilliant Blue and then dried. The bands corresponding to histone type 2 are excised, and the radioactivity is measured [1].

HeLa cells are plated at a density of 3×10^4 cells per 3.5-cm dish. The cells are cultured in DMEM containing 10% FBS in the presence of 10 mM Thymidine for 16 h. After the cells are washed with DMEM containing 10% FBS, they are cultured for an additional 8 h, and then 40 ng/mL of Nocodazole is added. After 11.5 h of the Nocodazole treatment, various concentrations of Y-27632 (0-300 μM) or vehicle is added and the cells are incubated for another 30 min [1].

A group of animals was injected with a single dose of pentylenetetrazole (PTZ, 65?mg/kg) to investigate if the two Rho-kinase inhibitors, fasudil, and Y-27632, changed the onset of PTZ seizures. Fasudil, Y-27632 or saline was given intraperitoneally 30?min before the PTZ injection. Each mouse was then observed for a 15-min period to measure the onset of the first myoclonic jerk, the onset of the first clonic convulsion and the occurrence of tonic hindlimb extension. Some of the animals died after tonic hindlimb extension, which is an expected outcome of acute PTZ injection. After the observation period, all animals were killed by halothane anesthesia [5]. Seven-week-old male Wistar rats were anesthetized with sodium pentobarbital. A silver clip (0.2 mm in diameter) was placed on the left renal artery in the preparation of the renal hypertensive rats. In the preparation of the DOCA-salt hypertensive rats, the left kidney was removed and a DOCA pellet (50 mg) was implanted subcutaneously. The DOCA rats were then fed an 8% salt diet. Rats from both groups were used after 8 weeks in the experiments, together with a male, 17–22-week old spontaneously hypertensive rats. The average systolic pressure in these groups of hypertensive rats ranged from 209 to 237 mm Hg, and no significant difference was found between groups. Eight-week-old male Wistar rats were used as controls. Their average systolic pressure was 139 mm Hg. Y-27632was administered orally. The systolic blood pressure was measured by the tail cuff method at 1, 3, 5, 7 and 24 h. The rats were prewarmed to 40 8C for 10 min before each measurement. No toxicity was found in rats treated with 30 mg kg?1 of Y-27632 administered per os once per day for 10 days [4].

129830-38-2

C14H23Cl2N3O

320.26

Y-27632 2HCl;反式-4-[(R)-1-氨基乙基]-N-(4-吡啶基)环己烷甲酰胺二盐酸盐;Y-27632 dihydrochloride

[1]Uehata M, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997 Oct 30;389(6654):990-4. [2]Inan S, et al. Antiepileptic effects of two Rho-kinase inhibitors, Y-27632 and fasudil, in mice. Br J Pharmacol. 2008 Sep;155(1):44-51. [3]Gong-Bo Fu, Wei-Jian Huang, Min Zeng, Xu Zhou, Hong-Ping Wu, Chang-Cheng Liu, Han Wu, Jun Weng, Hong-Dan Zhang, Yong-Chao Cai, Charles Ashton, Min Ding, Dan Tang, Bao-Hua Zhang, Yi Gao, Wei-Feng Yu, Bo Zhai, Zhi-Ying He, Hong-Yang Wang, and He-Xin Yan . Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens [J]. Cell Research. 2019 Jan;29(1):8-22. [4]Chen X, Lin J, Hu T, et al. Galectin‐3 exacerbates ox‐LDL‐mediated endothelial injury by inducing inflammation via integrin β1‐RhoA‐JNK signaling activation[J]. Journal of cellular physiology. 2019 Jul;234(7):10990-11000. [5]Watanabe K, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 2007 Jun;25(6):681-6. [6]Schenke M, Schjeide B M, Püschel G P, et al. Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays[J]. Toxins. 2020, 12(5): 276. [7]Li Z, Luo W, Fang S, et al. Prostacyclin facilitates vascular smooth muscle cell phenotypic transformation via activating TP receptors when IP receptors are deficient[J]. Acta Physiologica. 2020: e13555. [8]Zhu Y, Wang L, Yu H, et al. In situ generation of human brain organoids on a micropillar array[J]. Lab on a Chip. 2017 Aug 22;17(17):2941-2950. [9]Xue ZW, et al. Rho-associated coiled kinase inhibitor Y-27632 promotes neuronal-like differentiation of adult human adipose tissue-derived stem cells.Chin Med J (Engl). 2012 Sep;125(18):3332-5. [10]Ishizaki T, et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 2000 May;57(5):976-83.

[1]Fu G B, Huang W J, Zeng M, et al. Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens. Cell Research. 2019, 29(1): 8-22 [2]Zhu Y, Wang L, Yu H, et al. In situ generation of human brain organoids on a micropillar array. Lab on A Chip. 2017 Aug 22;17(17):2941-2950 [3]Wen L, Miao Y, Fan Z, et al. Establishment of an Efficient Primary Culture System for Human Hair Follicle Stem Cells Using the Rho-Associated Protein Kinase Inhibitor Y-27632. Frontiers in Cell and Developmental Biology. 2021 Mar 5;9:632882. doi: 10.3389/fcell.2021.632882. eCollection 2021. [4]Li Z, Luo W, Fang S, et al. Prostacyclin facilitates vascular smooth muscle cell phenotypic transformation via activating TP receptors when IP receptors are deficient. Acta Physiologica. 2020: e13555 [5]Chen X, Lin J, Hu T, et al. Galectin‐3 exacerbates ox‐LDL‐mediated endothelial injury by inducing inflammation via integrin β1‐RhoA‐JNK signaling activation. Journal of Cellular Physiology. 2019 Jul;234(7):10990-11000 [6]Schenke M, Schjeide B M, Püschel G P, et al. Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays. Toxins. 2020, 12(5): 276 [7]Zhu Y, Wang L, Yin F, et al. A hollow fiber system for simple generation of human brain organoids. Integrative Biology. 2017 Sep 18;9(9):774-781 [8]Wang C, Li Y, Jia L, et al. CD276 expression enables squamous cell carcinoma stem cells to evade immune surveillance. Cell Stem Cell. 2021 Sep 2;28(9):1597-1613.e7. doi: 10.1016/j.stem.2021.04.011. Epub 2021 May 3.

DMSO:199.8 mM
H2O:43.7 mM
Powder: -20°C for 3 years
In solvent: -80°C for 2 years