Essential role of rho kinase in the ca2+ Sensitization of Prostaglandin F2α‐Induced Contraction of Rabbit Aortae

Ito, Katsuaki; Shimomura, Erika; Iwanaga, Takahiro; Shiraishi, Masayuki; Shindo, Kazutoshi; Nakamura, Junji; Nagumo, Hiromitsu; Seto, Minoru; Sasaki, Yasuharu; Takuwa, Yoh

doi:10.1113/jphysiol.2002.030775

Cited by 83 publications

(68 citation statements)

References 63 publications

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“…The IC 50 values for PKAR3 with HA1077 and H1152P are indeed very close to the corresponding values for Rho-kinase, and to a lesser degree with Y-27632, all with considerably enhanced binding of the inhibitor compared with PKA-C␣. The effect was confirmed by additional measurements of the binding of hydroxyfasudil (HA1100) with PKAR3 (IC 50 1.37 M) and PKA-C␣ (IC 50 6 M), reproducing the ratio of K i values (Rho-kinase 0.56 M, PKA 2.5 M) described in the literature (29). As with the single mutant PKAR(L49I), the IC 50 value for PKAR3 with KT5720 is greatly increased relative to PKA, and binds more weakly relative to Rho-kinase, although to a lesser extent than the single mutation PKAR(L49I).…”

Section: Kinetic Datasupporting

confidence: 48%

“…The Rho-kinase-specific inhibitors fasudil, hydroxyfasudil, and H1152P are isoquinoline sulfonamide derivatives. Relative to fasudil, hydroxyfasudil has an additional hydroxyl group in the position 1 of the isoquinoline ring (29), and H1152P has two additional methyl groups, one at the isoquinoline ring, and the other at the homopiperazine ring that confer higher selectivity and specificity for Rho-kinase. The PKA-specific inhibitor KT5720 is identical to staurosporine in the extended planar portion of the inhibitor but differs especially in its possession of a fatty acid side chain, extending from a furanose instead of a pyranose ring.…”

Section: Mutagenesis Of Pka Residues Toward Rho-kinasementioning

confidence: 99%

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Structural Analysis of Protein Kinase A Mutants with Rho-kinase Inhibitor Specificity

Bonn

Herrero

Breitenlechner

et al. 2006

Journal of Biological Chemistry

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Controlling aberrant kinase-mediated cellular signaling is a major strategy in cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec verify this approach. Specificity of inhibitors for the targeted kinase(s), however, is a crucial factor for therapeutic success. Based on homology modeling, we previously identified four amino acids in the active site of Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid proteins. We have systematically mutated these residues in PKA to their counterparts in Rho-kinase, individually and in combination. Using four Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we measured the inhibitor-binding properties of the mutated proteins and identify the roles of individual residues as specificity determinants. Two combined mutant proteins, containing the combination of mutations T183A and L49I, closely mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain identities form the major determinants of selectivity. An unexpected result of the analysis is the consistent contribution of the individual mutations by simple factors. Crystal structures of the surrogate kinase inhibitor complexes provide a detailed basis for an understanding of these selectivity determinant residues. The ability to obtain kinetic and structural data from these PKA mutants, combined with their Rho-kinase-like selectivity profiles, make them valuable for use as surrogate kinases for structure-based inhibitor design.Phosphorylation via protein kinases is responsible for a large part of cellular signal transduction and is described as a universal regulatory mechanism (1, 2). Perturbation of kinase-mediated signaling pathways results in a number of diseases, including diabetes, cancer, and inflammation (3, 4). Because most protein kinases reside in the cell in an inactive state and are activated by signal transduction processes, many diseases are triggered by overactivation of protein kinases via mutation, overexpression, or malfunctioning cellular inhibition.The human genome encodes some 518 protein kinases (5) that are notably different in how their catalysis is regulated but share a catalytic domain conserved in sequence and structure (6, 7). The latter consists of 250 -300 amino acids, binds substrate and cosubstrate, and catalyzes the phosphorylation reaction.This catalytic domain, together with less conserved surrounding sites, has been the focus of inhibitor design that has exploited differences in kinase structure and pliability to achieve selectivity. Many drugs that target protein kinases are in clinical trials, and some have already been approved, such as the Abl kinase inhibitor Gleevec for therapy against chronic myelogenous leukemia (8) and Tarceva (erlotinib) against nonsmall cell lung cancer (9). The first protein kinase inhibitor that passed the cli...

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Section: Kinetic Datasupporting

confidence: 48%

Section: Mutagenesis Of Pka Residues Toward Rho-kinasementioning

confidence: 99%

Structural Analysis of Protein Kinase A Mutants with Rho-kinase Inhibitor Specificity

Bonn

Herrero

Breitenlechner

et al. 2006

Journal of Biological Chemistry

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“…The phosphorylation of MYPT1 at T696 has been shown to increase in response to PGF2α in a manner dependent on the presence of rho-kinase in the rabbit aorta (Ito et al, 2003). On the other hand, the relative contribution of MYPT1 and CPI-17 to the regulation of the MLCP activity has been studied in the tissues expressing both MYPT1 and CPI-17 (Kitazawa et al, 2003;Niiro et al, 2003).…”

Section: Q2 Relative Contribution Of Mypt1 and Cpi-17 To The Regulatmentioning

confidence: 99%

Regulation of myosin phosphorylation and myofilament Ca2+ sensitivity in vascular smooth muscle

Hirano

Kobayashi

2004

J. Smooth Muscle Res.

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The Ca 2+ -dependent, reversible phosphorylation of the 20 kDa regulatory myosin light chain (MLC) plays a primary role in regulating the contraction of smooth muscle. However, it is well known that the Ca 2+ signal is not the only factor which regulates such contraction, however, the alteration of the Ca 2+ sensitivity in the contractile apparatus is also known to play an important role. The degree of MLC phosphorylation is determined by the balance of the activity between phosphorylation and dephosphorylation. Either the Ca 2+ -independent activation of MLC phosphorylation or the inhibition of MLC dephosphorylation causes a greater MLC phosphorylation for a given level of Ca 2+ signal and thereby potentiates the myofilament Ca 2+ sensitivity. The smooth muscle myosin light chain phosphatase (MLCP) consisting of three subunits was first isolated and cloned in the early '90s. The intensive investigation thereafter has uncovered the biochemical basis for regulating the activity of MLCP. The regulation of the MLCP activity is now considered to play a critical role in regulating the myofilament Ca 2+ sensitivity. There are three major mechanisms in the regulation of MLCP; (1) the phosphorylation of a 110 kDa regulatory subunit of MLCP (2) the conformational change of the trimeric structure, and (3) the inhibition by a smooth muscle specific inhibitor protein, CPI-17. Furthermore, some kinases have been found to phosphorylate the MLC and activate the contraction of smooth muscle in a Ca 2+ -independent manner. Numerous protein kin ases hav e been fou nd to be involved in the reg ulation of MLC phosphorylation, and rho-kinase is one of the most frequently investigated kinases. The smooth muscle physiology is now asked to integrate the current understanding of the biochemical mechanisms and to clarify which kinases and/or proteins in the contractile apparatus play a physiological role in regulating the myofilament Ca 2+ sensitivity and how such extracellular contractile stimulation modulates these mechanisms.

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“…Ozagrel is a thromboxane A 2 synthase inhibitor, and ameliorates vascular contraction and platelet aggregation. Fasudil is a Rho-kinase inhibitor 7,16) and does not have direct anticoagulant and antiplatelet effects, but upregulates endothelial nitric oxide synthase activity in endothelial cells 21) and prevents the occurrence of endothelial injury, such as platelet aggregation. 15) Fasudil relaxes the cerebral arterial strips contracted by various vasoconstrictive substances including thromboxane A 2 .…”

Section: Efficacymentioning

confidence: 99%

“…The phosphorylated MBS was detected with the anti phospho-MBS antibody. 7) MBS phosphorylation produced by the addition of a reaction mixture was defined as 100%.…”

Section: Drug Interactionmentioning

confidence: 99%

Safety and Efficacy of Fasudil Monotherapy and Fasudil-Ozagrel Combination Therapy in Patients With Subarachnoid Hemorrhage: Sub-analysis of the Post-marketing Surveillance Study

Suzuki

Shibuya

Satoh

et al. 2008

Neurol. Med. Chir.(Tokyo)

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Sub-analysis of the fasudil post-marketing surveillance study compared the safety and efficacy of fasudil plus ozagrel to fasudil only. A total of 3690 patients received fasudil and 1138 received fasudil plus ozagrel between 1995 and 2000. The occurrence of adverse events, occurrence of low density areas associated with vasospasm on computed tomography, absence of symptomatic vasospasm, and poor clinical outcomes associated with vasospasm were compared between the fasudil and fasudil plus ozagrel groups. The pharmacokinetics of fasudil were assessed in 5 patients with subarachnoid hemorrhage. The drug interaction between fasudil and ozagrel was pharmacologically investigated in vitro and in vivo. The occurrence of adverse events and clinical outcomes were similar between the two groups. The occurrences of symptomatic vasospasm and low density areas were lower in the fasudil group than in the fasudil plus ozagrel group. The average trough value (8-hour value) of the fasudil active metabolite, hydroxyfasudil, was 50 nM. Fasudil showed no pharmacological interaction with ozagrel. The combination of fasudil plus ozagrel was well tolerated, but did not result in better efficacy than fasudil only.

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Essential role of rho kinase in the ca²⁺ Sensitization of Prostaglandin F_2α‐Induced Contraction of Rabbit Aortae

Cited by 83 publications

References 63 publications

Structural Analysis of Protein Kinase A Mutants with Rho-kinase Inhibitor Specificity

Structural Analysis of Protein Kinase A Mutants with Rho-kinase Inhibitor Specificity

Regulation of myosin phosphorylation and myofilament Ca<sup>2+</sup> sensitivity in vascular smooth muscle

Safety and Efficacy of Fasudil Monotherapy and Fasudil-Ozagrel Combination Therapy in Patients With Subarachnoid Hemorrhage: Sub-analysis of the Post-marketing Surveillance Study

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