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Abstract

We previously reported that protein kinase C (PKC)-delta was initially translocated from the cytosol to the membrane fraction (on day 4), followed by PKC-alpha, with the progression of cerebral vasospasm after subarachnoid hemorrhage (SAH) on day 7. Rho/Rho-kinase pathways have also been proposed to be involved in the vasospasm. Thus we investigated the interactive role of Rho-kinase and PKC in the development of cerebral vasospasm after SAH. The cerebral vasospasm was produced using a 'two-hemorrhage' canine model. The animals were treated with Y-27632, a Rho-kinase inhibitor, and rottlerin, a PKC-delta inhibitor, both injected into the cisterna magna. Y-27632 inhibited the vasospasm, 20-kDa myosin light chain (MLC20) phosphorylation, and PKC-delta translocation after the second injection of autologous blood on day 4. In contrast, Y-27632 did not affect the vasospasm on day 7. Rottlerin also inhibited the vasospasm on day 4, but had no effect on MLC20 phosphorylation and RhoA translocation. The vasospasm was accompanied with the phosphorylation of caldesmon (CaD), an actin-linked regulatory protein, which was strongly attenuated by Y-27632 and rottlerin. The application of PKC-delta to skinned strips of isolated canine basilar arteries caused a contraction and an increase in CaD phosphorylation. The development of cerebral vasospasm after SAH (on day 4) is caused by at least two mechanisms: one involves MLC20 phosphorylation mediated by the inhibition of MLC20 phosphatase by Rho-kinase, and the other CaD phosphorylation mediated by the activation of PKC-delta by Rho-kinase, which results in the alleviation of the inhibition by CaD of myosin Mg2+-ATPase activity.

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Specificity and mechanism of action of some commonly used protein kinase inhibitors.

S. P. Davies, H. Reddy, M Caivano … (2000)

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

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Regulation of Myosin Phosphatase by Rho and Rho-Associated Kinase (Rho-Kinase)

A Iwamatsu, K Okawa, M Nakafuku … (1996)

The small guanosine triphosphatase Rho is implicated in myosin light chain (MLC) phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in nonmuscle cells. The guanosine triphosphate (GTP)-bound, active form of RhoA (GTP.RhoA) specifically interacted with the myosin-binding subunit (MBS) of myosin phosphatase, which regulates the extent of phosphorylation of MLC. Rho-associated kinase (Rho-kinase), which is activated by GTP.RhoA, phosphorylated MBS and consequently inactivated myosin phosphatase. Overexpression of RhoA or activated RhoA in NIH 3T3 cells increased phosphorylation of MBS and MLC. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase.

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Functional roles of the rho/rho kinase pathway and protein kinase C in the regulation of cerebrovascular constriction mediated by hemoglobin: relevance to subarachnoid hemorrhage and vasospasm.

Grant Wickman, Bozena Vollrath, C. X. Lan (2003)

Although there is evidence that the Rho/Rho kinase pathway and protein kinase C (PKC) are involved in the development of cerebral vasospasm, the mechanism by which subarachnoid hemorrhage (SAH) activates these pathways is unclear. A large body of evidence points to oxyhemoglobin (OxyHb) as a major causative component of blood clot responsible for vasospasm. Therefore, the present studies were conducted to explore whether the Rho/Rho kinase and PKC may be involved in a sustained vasoconstriction induced by OxyHb in cerebral arteries. OxyHb evoked sustained vasoconstriction in the endothelium-denuded rabbit basilar arteries, which was reversed by the selective inhibitors of Rho kinase, Y-27632, and HA-1077, with the IC50 values of 0.26+/-0.02 and 0.74+/-0.1 micromol/L, respectively. In quiescent cerebrovascular smooth muscle (CVSM) cells, OxyHb induced Rho translocation, as assessed by immunoblotting, with a time course, which paralleled the contractile action of OxyHb. Rho translocation was also observed in intact arteries stimulated with OxyHb for 24 hours (219%) and 48 hours (160%). The increase in Rho translocation was fully inhibited by GGTI-297, an inhibitor of Rho prenylation. OxyHb also caused significant translocation of both PKCalpha and PKCepsilon (P<0.01), which was maximal at the time corresponding to maximal tension developed in response to OxyHb. Ro-32-0432, an inhibitor of PKC, attenuated vasoconstriction mediated by OxyHb in basilar artery. These results show, for the first time, that OxyHb-mediated signaling in CVSM utilizes the Rho/Rho kinase and PKC-based mechanisms.

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PubMed ID:: 15637442

DOI:: 10.1159/000083093

ScienceOpen disciplines: Chemistry

Keywords: Acetophenones,pharmacology,Amides,Animals,Benzopyrans,Calmodulin-Binding Proteins,metabolism,Disease Models, Animal,Dogs,Female,In Vitro Techniques,Intracellular Signaling Peptides and Proteins,Male,Myosin Light Chains,Phosphorylation,Protein Kinase C,physiology,Protein Kinase C-delta,Protein Transport,Protein-Serine-Threonine Kinases,Pyridines,Subarachnoid Hemorrhage,complications,Vasoconstriction,drug effects,Vasospasm, Intracranial,etiology,rho-Associated Kinases

Interactive role of protein kinase C-delta with rho-kinase in the development of cerebral vasospasm in a canine two-hemorrhage model.

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Karger: Cardiovascular System

Most cited references 14

Specificity and mechanism of action of some commonly used protein kinase inhibitors.

Regulation of Myosin Phosphatase by Rho and Rho-Associated Kinase (Rho-Kinase)

Functional roles of the rho/rho kinase pathway and protein kinase C in the regulation of cerebrovascular constriction mediated by hemoglobin: relevance to subarachnoid hemorrhage and vasospasm.

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