Background
Rho-associated kinase (ROCK), activated by GTP-linked Rho, phosphorylates targets that are involved in cytoskeletal remodeling, smooth muscle contraction, and neuronal development. H-1152 is a potent, specific, ATP-competitive, and cell permeable inhibitor of ROCK (Ki = 1.6 nM).12 It is a more potent inhibitor of ROCK than either Y-27632 (Ki = 140 nM) or HA-1077 (Ki = 330 nM).2 H-1152 poorly inhibits PKA, PKC, and myosin light chain kinase (Ki = 0.63, 9.27, and 10.1 μM, respectively).2 It has been used to examine the role of ROCK in such diverse processes as stress fiber assembly,3 vasoconstriction,4 as well as spontaneously tonic smooth muscle5 and neurite extension.6
1.Sasaki, Y., Suzuki, M., and Hidaka, H.The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathwayPharmacol. Ther.93225-232(2002)
2.Ikenoya, M., Hidaka, H., Hosoya, T., et al.Inhibition of Rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitorJ. Neurochem.819-16(2002)
3.Davies, S.L., Gibbons, C.E., Vizard, T., et al.Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acidsAm. J. Physiol. Cell Physiol.290C1543-C1551(2006)
4.Johnson, R.P., El-Yazbi, A.F., Takeya, K., et al.Ca2+ sensitization via phosphorylation of myosin phosphatase targeting subunit at threonine-855 by Rho kinase contributes to the arterial myogenic responseJ. Physiol.587(11)2537-2553(2009)
5.Rattan, S., and Patel, C.A.Selectivity of Rho kinase (ROCK) inhibitors in the spontaneously tonic smooth muscleAm. J. Physiol. Gastrointest. Liver Physiol.294(3)G687-G693(2008)
6.Fuentes, E.O., Leemhuis, J., Stark, G.B., et al.Rho kinase inhibitors Y27632 and H1152 augment neurite extension in the presence of cultured Schwann cellsJ. Brachial Plex. Peripher. Nerve Inj.3(19)(2008)
Protocol
Kinase experiment: | Inhibitors (including H-1152) are added at the indicated concentrations to 50 μL of the assay mixture 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 40 μM S6-peptide, various concentrations of [γ-32P]ATP and purified Rho-kinase. The reactions are started by the addition of [γ-32P]ATP and carried out at 30°C for 5 min. The Michaelis-Menten equation is used to calculate the Michaelis constant (Km) and maximal velocity (Vmax) of Rho-kinase. Data are further analyzed with secondary plot to calculate the inhibitory constant (Ki)[2]. |
Cell experiment: | Briefly, cells are routinely plated on poly-d-lysine/laminin coated 96 well plates or in 16 well glass culture slides. Control medium contained Dulbecco's modified Eagles medium/Hams F12(1:1) (DMEM/F12), 2 mM l-glutamine, N2 mix (1:100 dilution), 0.63 mL of 45% glucose for each 100 mL of DMEM/F12, neurotrophin 3 (NT3; final concentration, 8 ng/mL), BDNF (final concentration 8 ng/mL), and 10% fetal bovine serum heat inactivated before use. LIF cultures contain control medium+LIF (50 ng/mL). BMP4 cultures contain control medium+bone morphogenetic protein 4 (BMP4; 25 ng/mL). Total volume of culture is 110 μL. ROCK inhibitor H-1152 is diluted in water and added in an additional 10 μL to cultures 24 h after plating. Water is added to controls. Eighteen hours after the addition of inhibitor, cultures are fixed in 4% paraformaldehyde (1 h at room temperature for peroxidase-linked labeling and 20 min at room temperature for fluorescence labeling). For ArrayScan/Cellomics automated analysis: Cells are plated in a total volume of 50 μL on 384 well plastic plates previously coated with poly-d-lysine/laminin, and cultured in the same medium[3]. |
参考文献: [1]. Tamura M, et al. Development of specific Rho-kinase inhibitors and their clinical application. Biochim Biophys Acta. 2005 Dec 30;1754(1-2):245-52. Epub 2005 Sep 12.
[2]. Ikenoya M, et al. Inhibition of rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor. J Neurochem. 2002 Apr;81(1):9-16.
[3]. Lie M, et al. Accelerated neurite growth from spiral ganglion neurons exposed to the Rho kinase inhibitor H-1152. Neuroscience. 2010 Aug 25;169(2):855-62. |
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