• 약학회지
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• 제50권4호
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• pp.293-299
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• 2006

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.

• Molecules and Cells
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• 제27권2호
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• pp.191-198
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• 2009

The present study was undertaken to determine whether treatment with genistein, the plant-derived estrogen-like compound influences agonist-induced vascular smooth muscle contraction and, if so, to investigate related mechanisms. The measurement of isometric contractions using a computerized data acquisition system was combined with molecular experiments. Genistein completely inhibited KCl-, phorbol ester-, phenylephrine-, fluoride- and thromboxane $A_2$-induced contractions. An inactive analogue, daidzein, completely inhibited only fluoride-induced contraction regardless of endothelial function, suggesting some difference between the mechanisms of RhoA/Rho-kinase activators such as fluoride and thromboxane $A_2$. Furthermore, genistein and daidzein each significantly decreased phosphorylation of MYPT1 at Thr855 had been induced by a thromboxane $A_2$ mimetic. Interestingly, iberiotoxin, a blocker of large-conductance calcium-activated potassium channels, did not inhibit the relaxation response to genistein or daidzein in denuded aortic rings precontracted with fluoride. In conclusion, genistein or daidzein elicit similar relaxing responses in fluoride-induced contractions, regardless of tyrosine kinase inhibition or endothelial function, and the relaxation caused by genistein or daidzein was not antagonized by large conductance $K_{Ca}$-channel inhibitors in the denuded muscle. This suggests that the RhoA/Rho-kinase pathway rather than $K^+$- channels are involved in the genistein-induced vasodilation. In addition, based on molecular and physiological results, only one vasoconstrictor fluoride seems to be a full RhoA/Rho-kinase activator; the others are partial activators.

• The Korean Journal of Physiology and Pharmacology
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• 제13권3호
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• pp.201-207
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• 2009

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.

• 약학회지
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• 제58권4호
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• pp.223-228
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• 2014

The present study was undertaken to investigate the influence of sulforaphane on vascular smooth muscle contractility and to determine the mechanism involved. We hypothesized that sulforaphane, the primary ingredient of broccoli of cruciferous vegetables, plays a role in vascular relaxation through inhibition of Rho-kinase in rat aortae. Intact of denuded arterial rings from male Sprague-Dawley rats were used and isometric tensions were recorded using a computerized data acquisition system. Interestingly, sulforaphane significantly inhibited fluoride, phorbol ester or thromboxane $A_2$ mimetic-induced contraction in denuded muscles suggesting that additional pathways different from endothelial nitric oxide synthesis such as inhibition of Rho-kinase or MEK might be involved in the vasorelaxation. Furthermore, sulforaphane inhibited thromboxane $A_2$-induced increases in pERK1/2 levels suggesting the mechanism including inhibition of thromboxane $A_2$-induced increases in ERK1/2 phosphorylation. This study provides evidence that sulforaphane induces vascular relaxation through inhibition of Rho-kinase or MEK in rat aortae.

• 약학회지
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• 제57권5호
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• pp.376-381
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• 2013

The present study was undertaken to investigate the influence of curcumin on vascular smooth muscle contractility and to determine the mechanism involved. We hypothesized that curcumin, the primary ingredient of Curcuma longa, plays a role in vascular relaxation through inhibition of Rho-kinase in rat aortae. Denuded arterial rings from male Sprague-Dawley rats were used and isometric tensions were recorded using a computerized data acquisition system. Interestingly, curcumin inhibited fluoride-induced contraction but didn't inhibit phorbol ester-induced contraction suggesting that additional pathways different from endothelial nitric oxide synthesis might be involved in the vasorelaxation. Furthermore, curcumin significantly inhibited fluoride-induced increases in pMYPT1 levels. On the other hand, it didn't significantly inhibit phorbol ester-induced increases in pERK1/2 levels suggesting the mechanism involving inhibition of fluoride-induced MYPT1 phosphorylation. This study provides evidence that curcumin induces vascular relaxation through inhibition of Rho-kinase in rat aortae.

• The Korean Journal of Physiology and Pharmacology
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• 제20권1호
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• pp.69-74
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• 2016

The present study was undertaken to investigate the influence of cardamonin on vascular smooth muscle contractility and to determine the mechanism(s) involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Cardamonin significantly relaxed fluoride-, phenylephrine-, and phorbol ester-induced vascular contractions, suggesting that it has an anti-hypertensive effect on agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, cardamonin significantly inhibited the fluoride-induced increase in pMYPT1 level and phenylephrine-induced increase in pERK1/2 level, suggesting inhibition of Rho-kinase and MEK activity and subsequent phosphorylation of MYPT1 and ERK1/2. This study provides evidence that the relaxing effect of cardamonin on agonist-induced vascular contraction regardless of endothelial function involves inhibition of Rho-kinase and MEK activity.

• 약학회지
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• 제58권5호
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• pp.337-342
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• 2014

The present study was undertaken to investigate the influence of diosgenin on vascular smooth muscle contractility and to determine the mechanism involved. We hypothesized that diosgenin, the primary ingredient of Dioscorea villosa, plays a role in vascular relaxation through inhibition of Rho-kinase in rat aortae. Denuded arterial rings from male Sprague-Dawley rats were used and isometric tensions were recorded using a computerized data acquisition system. Interestingly, diosgenin inhibited fluoride-induced contraction but didn't inhibit phorbol ester-induced contraction suggesting that additional pathways different from endothelial nitric oxide synthesis such as inhibition of Rho-kinase might be involved in the vasorelaxation. Furthermore, diosgenin didn't inhibit thromboxane $A_2$-induced increases in pERK1/2 levels suggesting the mechanism excluding inhibition of thromboxane $A_2$-induced increases in ERK1/2 phosphorylation. This study provides evidence that diosgenin induces vascular relaxation through inhibition of Rho-kinase in rat aortae.

• The Korean Journal of Physiology and Pharmacology
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• 제27권4호
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• pp.325-331
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• 2023

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.

• 생명과학회지
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• 제13권6호
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• pp.903-910
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• 2003

Phosphipase D(PLD)는 호중구의 활성에서 중요한 신호전달 인자로 작용한다. 본 연구에서는 호중구에서 PLD의 활성화에 대한 nitric oxide(NO)와 cGMP의 영향을 조사하였다. 세포 내 NO의 생성을 증가시키는 물질인 sodium nitroprusside (SNP)를 단독으로 처리하였을 때 SNP를 처리하지 않은 세포에 비교하여 PLD 활성은 0.5 mM 농도에서 2배 이상 증가하였다. 세포 내 cAMP의 농도를 증가시키는 물질인 dibutyryl-cAMP를 처리하였을 때 formyl-Met-Leu-Phe(fMLP)에 의한 PLD활성은 억제되었으나 cGMP를 증가시키는 물질인 8-bromo-cGMP(300 $\mu$M)를 단독으로나 fMLP와 같이 처리하였을 때 PLD의 활성은 큰 영향이 없었다. NO에 의한 PLD의 활성은 cGMP-의존형 인산화 효소인 protein kinase G(PKG)의 억제제인 KT 5823에 의하여 억제되지 않았는데 이러한 결과는 PKG 이외의 경로를 통하여 일어남을 제시한다. NO를 처리한 호중구에서 p38 mitogen activated protein kinase(MAPK)가 활성화되어 인산화된 p38 MAPK가 Western blot에서 증가되었다. NO에 의한 p38 MAPK의 인산화는 KT 5823에 의하여 억제되지 않았고 PLD 억제제인 n-butanol에 의하여도 영향을 받지 않았다. PLD 활성의 인자인 RhoA는 fMLP나 phorbol myristate acetate(PMA)의 자극에 의하여 세포질로부터 세포막으로 전이가 되었으나 cGMP의 전처리에 의하여 fMLP에 의한 RhoA의 전이는 억제되었으나 PMA에 의한 전이는 영향을 받지 않았다. 이들 결과들은 호중구 내 증가된 cGMP가 RhoA를 억제하였으나 세포 내 증가된 NO는 cGMP 이외의 인자를 통하여 PLD의 활성화를 일으킨다는 것을 제시하고 있다.

• 약학회지
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• 제55권2호
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• pp.138-144
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• 2011

The aim of present study was to investigate the possible influence and related mechanism of resveratrol on U-46619 (high concentration)-induced vasoconstriction. Agonist-induced vascular smooth muscle contractions involve the activation of thick or thin filament pathway. However, there are no reports addressing the question whether this pathway is involved in resveratrol-induced relaxation in rat aortae contracted with high U-46619. We hypothesized that MEK or Rho-kinase inhibition plays a role in vascular relaxation evoked by resveratrol in rat aortae. Endothelium-denuded arterial rings from male Sprague-Dawley rats were used and isometric contractions were recorded using a computerized data acquisition system. Resveratrol fully inhibited U-46619 in low concentration-induced contraction regardless of endothelial function. However, resveratrol partially decreased U-46619 in high concentration-induced contraction regardless of endothelial function. Interestingly, only in U-46619 (high concentration)-induced contraction, no significant decrease was observed in phospho-ERK1/2 levels and slight decrease in phospho-MYPT1 levels suggesting that additional pathways different from them or endothelial nitric oxide synthesis might be involved in the vasorelaxation. In conclusion, in high U-46619-contracted rat aortae, resveratrol showed relaxation response regardless of endothelial function significantly but slightly decreasing MYPT1 phosphorylation rather than ERK1/2 phosphorylation.