Y-27632 2HCl

ROCK1

320, 26

Male Wistar rats with spontaneous or induced hypertension

Dissolved in DMSO, and diluted in saline

Y-27632 2HCl inhibits ROCK-II while displaying little activity against PKC, cAMP-dependent protein kinase and myosin light-chain kinase (MLCK) with Ki of 26 uM, 25 uM and > 250 uM, respectively, as well as PKA activated by another Rho-family GTPase member, Cdc42. Y-27632 2HCl inhibits smooth-muscle contraction induces by various agonists including phenylephrine, histamine, acetylcholine, serotonin, endothelin, and thromboxane with IC50 of 0.3-1 uM, by selectively inhibiting Ca2+ sensitization. Y-27632 2HCl suppresses Rho-induced, p160ROCK-mediated formation of stress fibres in cultured cells. [1] Y-27632 2HCl treatment blocks both Rho-mediated activation of actomyosin and LPA-stimulated invasive activity of MM1 cells in a concentration-dependent manner. [2] Y-27632 2HCl treatment is not only sufficient to initiate formation of exuberant axonal processes but also facilitates axonal maturation during the very early stages of axonogenesis, while largely sparing axon elongation. [3] In human embryonic stem (hES) cells, Y-27632 2HCl treatment at 10 uM markedly diminishes dissociation-induced apoptosis even in serum-free suspension (SFEB) culture, increases cloning efficiency (from ca.1% to ca.27%), facilitates subcloning after gene transfer, and enables SFEB-cultured hES cells to survive and differentiate into Bf1+ cortical and basal telencephalic progenitors. [4]

Phosphorylation reactions, The p160ROCK is expressed in COS cells as tagged full-length proteins, and immunoprecipitated by the use of anti-tag antibodies. The p160ROCK (30 ng) is incubated with 40 uM [gamma-32P]ATP (3.3 Ci/mmol) and with 3 ug of either histone (HF2A), dephosphorylated casein or MBP in the presence of various concentrations of Y-27632 2HCl at 30 C in a total volume of 31 uL. A 7 uL aliquot is taken at 0, 5, 10, and 20 minutes, mixed with an equal volume of 2, Laemmli sample buffer, and applied to SDS-PAGE. The gel is stained with Commassie Blue, dried and subjected to analysis by a Bioimage Analyzer BAS2000. The Y-27632 2HCl concentration required to inhibit p160ROCK activity by 50% (IC50 value) is obtained. Ki value is calculated according to the equation, Ki = IC50/(1 + S/Km), where S and Km represent concentrations of and Km value for ATP.

Y-27632 2HCl is a selective ROCK1 and ROCK2 inhibitor with a Ki of 140 nM and 300nM in a cell-free assay, exhibits >200-fold selectivity over other kinases, including PKC, cAMP-dependent protein kinase, MLCK and PAK.