Cardiovascular effects of a novel selective Rho kinase inhibitor, 2-(1H-indazole-5-yl)amino-4-methoxy-6-piperazino triazine (DW1865)

Oh, K.S.,Oh, B.K.,Ho Park, C.,Won Seo, H.,Sook Kang, N.,Hyun Lee, J.,Soo Lee, J.,Ho Lee, B. North-Holland ; Elsevier Science Ltd 2013 european journal of pharmacology Vol.702 No.1

The arising critical implications of Rho kinase signaling in cardiovascular diseases have been attracting attention in the pharmacological potential of Rho kinase inhibitors. We identified a novel inhibitor of Rho kinase (2-(1H-indazole-5-yl)amino-4-methoxy-6-piperazino triazine; DW 1865) and characterized its effects in biochemical, cellular, tissue and animal based assays. DW 1865 potently inhibited the kinase activity of both Rho kinase 1 and Rho kinase 2 in vitro, and behaved as an ATP-competitive inhibitor. Interestingly, DW1865 was 10 times more potent in inhibiting Rho kinase activities than fasudil as a selective Rho kinase inhibitor. The activity of DW1865 was shown to be highly selective for Rho kinase in the panel assay of 13 other kinases. In the isolated vascular tissue study, DW1865 exerted vasorelaxation in phenylephrine- or 5-hydroxytriptamine-induced contraction in a concentration-dependent manner manner. In spontaneously hypertensive rats, administration of DW1865 caused a significant and dose-related reduction in blood pressure. Furthermore, DW1865 blocked angiotensin II-induced stress fiber formation and cellular hypertrophy in rat heart-derived H9c2 cells. Taken together, these results suggest that DW1865 is a highly selective and potent Rho kinase inhibitor that will alleviate the pathophysiological actions of Rho kinase such as stress fiber formation, cellular hypertrophy, and hypertension.

Isoflavonoids에 의한 혈관이완효과에 있어 Rho-kinase의 역할

제현동,Je, Hyun-Dong 대한약학회 2006 약학회지 Vol.50 No.4

The aim of present study was to investigate the possible influence of Rho-kinase inhibition on the plant-derived estrogen-like compounds-induced arterial relaxation. Agonist- or depolarization-induced vascular smooth muscle contractions involve the activation of Rho-kinase pathway. However there are no reports addressing the question whether this pathway is involved in genistein-or daidzein-induced vascular relaxation in rat aortae precontracted with phenylephrine or thromboxane $A_2$ mimetic U-46619. We hypothesized that Rho-kinase inhibition plays a role in vascular relaxation evoked by genistein or daidzein in rat aortae. Endothelium-intact and denuded arterial rings from male Sprague-Dawley rats were used and isometric contractions were recorded using a computerized data acquisition system. Genistein concentration-dependently inhibited phenylephrine or thromboxane $A_2-induced$ contraction regardless of endothelial function. Surprisingly, in the agonists-induced contraction, similar results were also observed in aortae treated with daidzein, the inactive congener for protein tyrosine kinase inhibition, suggesting that Rho-kinase might act upstream of tyrosine kinases in phenylephrine-induced contraction. In conclusion, in the agonists-precontracted rat aortae, genistein and daidzein showed similar relaxant response regardless of tyrosine kinase inhibition or endothelial function.

Protein kinases participate in the contraction in response to levobupivacaine in the rat aorta

Shim, H.S.,Ok, S.H.,Lee, S.H.,Kwon, S.C.,Sohn, J.T. North-Holland ; Elsevier Science Ltd 2012 european journal of pharmacology Vol.677 No.1

Levobupivacaine is a long-acting amide local anesthetic that intrinsically produces vasoconstriction both in vivo and in vitro. Levobupivacaine increases intracellular calcium concentrations ([Ca<SUP>2+</SUP>]<SUB>i</SUB>) in vascular smooth muscle cells. The goals of this in vitro study were to investigate whether levobupivacaine-induced contraction is associated with increased Ca<SUP>2+</SUP> sensitivity and to identify the protein kinases involved in mediating contraction in response to levobupivacaine in isolated rat aortic smooth muscle. The effect of levobupivacaine and potassium chloride (KCl) on the [Ca<SUP>2+</SUP>]<SUB>i</SUB> and tension was measured simultaneously with acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative levobupivacaine concentration-response curves were generated in the presence or absence of the following antagonists: GF 109203X; Y-27632; genistein; SP600125; PD 98059; and SB 203580. Levobupivacaine-induced protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and c-Jun NH<SUB>2</SUB>-terminal kinase (JNK) phosphorylation and Rho-kinase (ROCK-2) membrane translocation were detected in rat aortic vascular smooth muscle cells using Western blotting. The slope of the [Ca<SUP>2+</SUP>]<SUB>i</SUB>-tension curve for levobupivacaine was higher than that for KCl. Y-27632, GF 109203X, and SP600125 attenuated levobupivacaine-induced contraction in a concentration-dependent manner. Genistein, PD 98059, and SB 203580 attenuated levobupivacaine-induced contraction. Pretreatment with GF 109203X and Y-27632 inhibited levobupivacaine-induced PKC phosphorylation and Rho-kinase (ROCK-2) membrane translocation, respectively. Pretreatment with SP600125 or PD 98059 attenuated the levobupivacaine-induced phosphorylation of JNK and ERK, respectively. These results indicate that levobupivacaine-induced contraction involving an increase in myofilament Ca<SUP>2+</SUP> sensitivity involves the primary activation of Rho-kinase-, PKC-, and JNK-mediated pathways of rat aortic smooth muscle.

스트레스에 의한 백서 음경조직의 Rho-kinase 및 Nitric Oxide Synthase의 발현 변화

김규현,윤하나 대한남성과학회 2008 The World Journal of Men's Health Vol.26 No.3

Purpose: The autonomic nervous system (ANS) and stress hormones are important mediators in the stress response. The ANS controls the tone of corpus cavernosal smooth muscle. Clinically, it is not uncommon to develop erectile dysfunction as the sequelae of acute or chronic stress, with or without loss of libido. In this study, we investigated the effect of stress on the expression of mediators of penile corpus cavernosal smooth muscle relaxation and contraction. Materials and methods: Forty-eight male Sprague-Dawley rats were used, with 8 rats in each group. They were divided into six groups; stress+10, stress+20, and stress+30, which were named after their duration (days) of exposure, and a control group for each stress group. The stress group was maintained under a scheduled stress condition, while the control group was provided a comfortable general environment. Penile tissues were sampled and expressions of Rho-kinase, inducible nitric oxide synthase (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) were measured and analyzed by Western blotting. Results: Penile Rho-kinase expression was significantly increased in the stress groups compared with the control group (p<0.05), with a greater increase during longer stress. Expression of iNOS was increased in stress+30 group (p<0.05). Expression of eNOS, nNOS, and Rho-kinase were increased in stress+20, stress+30 group (p<0.05). Conclusions: These results suggest that stress may alter penile corpus cavernosal smooth muscle relaxation by affecting mediators.

Involvement of α_1B-adrenoceptors and Rho kinase in contractions of rat aorta and mouse spleen

Hadeel A. Alsufyani,James R. Docherty The Korean Society of Pharmacology 2023 The Korean Journal of Physiology & Pharmacology Vol.27 No.4

α₁-adrenoceptors link via the G-protein Gq/G₁₁ to both Ca²⁺ entry and release from stores, but may also activate Rho kinase, which causes calcium sensitization. This study aimed to identify the subtype(s) of α₁-adrenoceptor involved in Rho kinase-mediated responses in both rat aorta and mouse spleen, tissues in which contractions involve multiple subtypes of α₁-adrenoceptor. Tissues were contracted with cumulative concentrations of noradrenaline (NA) in 0.5 log unit increments, before and in the presence of an antagonist or vehicle. Contractions produced by NA in rat aorta are entirely α₁-adrenoceptor mediated as they are competitively blocked by prazosin. The α_1A-adrenoceptor antagonist RS100329 had low potency in rat aorta. The α_1D-adrenoceptor antagonist BMY7378 antagonized contractions in rat aorta in a biphasic manner: low concentrations blocking α_1D-adrenoceptors and high concentrations blocking α_1B-adrenoceptors. The Rho kinase inhibitor fasudil (10 µM) significantly reduced aortic contractions in terms of maximum response, suggesting inhibition of α_1B-adrenoceptor mediated responses. In the mouse spleen, a tissue in which all 3 subtypes of α₁-adrenoceptor are involved in contractions to NA, fasudil (3 µM) significantly reduced both early and late components to the NA contraction, the early component involving α_1B- and α_1D-adrenoceptors, and the late component involving α_1B- and α_1A-adrenoceptors. This suggests that fasudil inhibits α_1B-adrenoceptor mediated responses. It is concluded that α_1D- and α_1B-adrenoceptors interact in rat aorta and α_1D-, α_1A- and α_1B-adrenoceptors interact in the mouse spleen to produce contractions and these interactions suggest that one of the receptors preferentially activates Rho kinase, most likely the α_1B-adrenoceptor.

Rho-Associated Kinase 2 Polymorphism in Patients With Vasospastic Angina

유상용,김정욱,정상식,신대희,Jinkun Jang,이창근,Seung-Jea Tahk,신준한,최소연,윤명호 대한심장학회 2012 Korean Circulation Journal Vol.42 No.6

Background and Objectives: Recent studies indicate that in response to vasoconstrictor stimuli, the small GTPase RhoA and its down-stream effector, Rho-associated kinase 2 (ROCK)/Rho-kinase, are associated with hypercontraction of the vascular smooth muscle of coro-nary arteries through augmentation of myosin light chain phosphorylation and Ca 2+ sensitization. Expression of ROCK/Rho-kinase mRNA was significantly increased and up-regulated in the spastic coronary artery in a porcine model, and a specific inhibitor of ROCK/Rho-ki-nase inhibited coronary artery spasm in humans. We therefore explored the role of ROCK2 polymorphisms in the pathogenesis of vaso-spastic angina (VA). Subjects and Methods: We studied 106 patients with VA who exhibited spontaneous or provoked coronary spasm during coronary angio-graphy and compared the prevalence of ROCK2 polymorphisms between this group of patients with VA and controls whose angiograms were normal, and in whom the ergonovine test did not cause spasm (n=107). Five single nucleotide polymorphisms (SNPs) of the ROCK2gene were selected. SNPs were genotyped by high-resolution melting. Linkage disequilibrium and haplotype analyses were performed us-ing the SHEsis program. Results: The prevalence of genotypes of the 5 interesting SNPs in patients with VA was not different from that in the control group. In haplo-type analysis, the haplotype G-T-C-T-G (in order of rs978906, rs2271621, rs2230774, rs1515210, and rs3771106) was significantly associ-ated with a decreased risk of VA (p=0.007). Conclusion: The haplotype G-T-C-T-G in the ROCK2 gene had a protective effect against VA, suggesting the involvement of ROCK2 in VA pathogenesis.

Rho-Kinase as a Therapeutic Target for Nonalcoholic Fatty Liver Diseases

Sousa-Lima Inês,Kim Hyun Jeong,John Jones,김영범 대한당뇨병학회 2021 Diabetes and Metabolism Journal Vol.45 No.5

Nonalcoholic fatty liver disease (NAFLD) is a major public health problem and the most common form of chronic liver disease, affecting 25% of the global population. Although NAFLD is closely linked with obesity, insulin resistance, and type 2 diabetes mellitus, knowledge on its pathogenesis remains incomplete. Emerging data have underscored the importance of Rho-kinase (Rho-associated coiled-coil-containing kinase [ROCK]) action in the maintenance of normal hepatic lipid homeostasis. In particular, pharmacological blockade of ROCK in hepatocytes or hepatic stellate cells prevents the progression of liver diseases such as NAFLD and fibrosis. Moreover, mice lacking hepatic ROCK1 are protected against obesity-induced fatty liver diseases by suppressing hepatic de novo lipogenesis. Here we review the roles of ROCK as an indispensable regulator of obesity-induced fatty liver disease and highlight the key cellular pathway governing hepatic lipid accumulation, with focus on de novo lipogenesis and its impact on therapeutic potential. Consequently, a comprehensive understanding of the metabolic milieu linking to liver dysfunction triggered by ROCK activation may help identify new targets for treating fatty liver diseases such as NAFLD.

Dexmedetomidine-Induced Contraction Involves CPI-17 Phosphorylation in Isolated Rat Aortas

Ok, Seong-Ho,Kwon, Seong-Chun,Baik, Jiseok,Hong, Jeong-Min,Oh, Jiah,Han, Jeong Yeol,Sohn, Ju-Tae MDPI AG 2016 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.17 No.10

<P>Dexmedetomidine, a highly selective α-2 adrenoceptor agonist, produces vasoconstriction, which leads to transiently increased blood pressure. The goal of this study was to investigate specific protein kinases and the associated cellular signal pathways responsible for the increased calcium sensitization induced by dexmedetomidine in isolated rat aortas, with a particular focus on phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17). The effect of Y-27632 and chelerythrine on the dexmedetomidine-induced intracellular calcium concentration ([Ca<SUP>2+</SUP>]<SUB>i</SUB>) and tension were assessed using fura-2-loaded aortic strips. The effects of rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride on the dexmedetomidine-induced phosphorylation of CPI-17 or of the 20-kDa regulatory light chain of myosin (MLC<SUB>20</SUB>) were investigated in rat aortic vascular smooth muscle cells. The effects of rauwolscine, Y-27632, and chelerythrine on the membrane translocation of Rho-kinase and protein kinase C (PKC) phosphorylation induced by dexmedetomidine were assessed. Y-27632 and chelerythrine each reduced the slopes of the [Ca<SUP>2+</SUP>]<SUB>i</SUB>-tension curves of dexmedetomidine-induced contraction, and Y-27632 more strongly reduced these slopes than did chelerythrine. Rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride attenuated the dexmedetomidine-induced phosphorylation of CPI-17 and MLC<SUB>20</SUB>. Taken together, these results suggest that dexmedetomidine-induced contraction involves calcium sensitization, which appears to be mediated by CPI-17 phosphorylation via Rho-kinase or PKC.</P>

Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway

Inji Baek,Su Bun Jeon,Min-Ji Song,Enyue Yang,Uy Dong Sohn,In Kyeom Kim 대한생리학회-대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.3

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300ՌM) or vehicle. We determined the phosphorylation level of the myosin light chain (MLC₂₀), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of MLC₂₀ phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619- or NaF-induced phosphorylation of MYPT1^Thr855 and CPI17^Thr38, the downstream effectors of Rho-kinase. In regards to the Ca²⁺-free solution, flavone inhibited the phosphorylation of MYPT1^Thr855 and CPI17^Thr38, as well as vascular contractions induced by U46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.

Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway

Baek, In-Ji,Jeon, Su-Bun,Song, Min-Ji,Yang, Enyue,Sohn, Uy-Dong,Kim, In-Kyeom The Korean Society of Pharmacology 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.3

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 $({\mu}M$) or vehicle. We determined the phosphorylation level of the myosin light chain ($MLC_{20}$), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of $MLC_{20}$ phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619 or NaF-induced phosphorylation of $MYPT1^{Thr855}$ and $CPI17^{Thr38}$, the downstream effectors of Rho-kinase. In regards to the $Ca^{2+}$-free solution, flavone inhibited the phosphorylation of $MYPT1^{Thr855}$ and $CPI17^{Thr38}$, as well as vascular contractions induced by U 46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.