Quercetin inhibits Shc- and phosphatidylinositol 3-kinase-mediated c-Jun N-terminal kinase activation by angiotensin II in cultured rat aortic smooth muscle cells

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) hypertrophy, which results in various cardiovascular diseases. Ang II-induced cellular events have been implicated, in part, in the activation of mitogen-activated protein (MAP) kinases. Although it has been proposed that daily intake of bioflavonoids belonging to polyphenols reduces the incidence of ischemic heart diseases (known as "French paradox"), the precise mechanisms of efficacy have not been elucidated. Thus, we hypothesized that bioflavonoids may affect Ang II-induced MAP kinase activation in cultured rat aortic smooth muscle cells (RASMC). Our findings showed that Ang II stimulated rapid and significant activation of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), and p38 in RASMC. Ang II-induced JNK activation was inhibited by 3,3',4',5,7-pentahydroxyflavone (quercetin), a major bioflavonoid in foods of plant origin, whereas ERK1/2 and p38 activation by Ang II were not affected by quercetin. Ang II caused a rapid tyrosine phosphorylation of Src homology and collagen (Shc), which was inhibited by quercetin. Quercetin also inhibited Ang II-induced Shc.p85 association and subsequent activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in RASMC. Furthermore, LY294002, a PI3-K inhibitor and a quercetin derivative, inhibited Ang II-induced JNK activation as well as Akt phosphorylation. Finally, Ang II-induced [(3)H]leucine incorporation was abolished by both quercetin and LY294002. These findings suggest that the preventing effect of quercetin on Ang II-induced VSMC hypertrophy are attributable, in part, to its inhibitory effect on Shc- and PI3-K-dependent JNK activation in VSMC. Thus, inhibition of JNK by quercetin may imply its usefulness for the treatment of cardiovascular diseases relevant to VSMC growth.     

p38 mitogen-activated protein kinase contributes to angiotensin II-stimulated migration of rat aortic smooth muscle cells

In this study, we clarified the intracellular mechanism of angiotensin II (Ang II) in promoting migration in rat aortic smooth muscle cells (RASMCs). RASMC migration was measured with the Boyden chamber assay, and the result was confirmed with an aortic sprout assay. The activities of kinases were investigated by western blot analysis. Ang II enhanced RASMC migration, which was chemotaxis directed, and induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and heat shock protein 27 (Hsp27). Ang II-enhanced cell migration was inhibited by SB203580 (a p38 MAPK inhibitor) and piceatannol (a spleen tyrosine kinase inhibitor), but only partially by PD98059 (an ERK inhibitor) and PP2 (a Src inhibitor). The Ang II-stimulated phosphorylation of p38 MAPK and Hsp27 in RASMCs was inhibited by piceatannol and SB203580. The phosphorylation of ERK1/2 stimulated by Ang II was suppressed by PD98059, piceatannol, and PP2. Ang II increased the sprout outgrowth from aortic rings and this response was attenuated by pretreatment with SB203580, PD98059, PP2, or piceatannol. These results suggest that p38 MAPK contributes to the regulation of the Ang II-induced chemotactic migration of vascular smooth muscle cells, which is mediated by Hsp27 phosphorylation.     

Mitogen-activated protein kinase contributes to elevated basal tone in aortic smooth muscle from hypertensive rats

The role of mitogen-activated protein kinase (MAPK) in increased basal tone -spontaneous resistance in vascular muscle strips- was clarified in aortic smooth muscle from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The MAPK/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, PD098059 (2'-amino-3'-methoxyflavone), significantly inhibited basal tone in a dose-dependent manner. The basal level of ERK1/2 activation was inhibited by PD098059 and was significantly greater in hypertensive rats than in sham-operated rats. In contrast, inhibition with PD098059 was not observed in sham-operated rats. GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide), an inhibitor of protein kinase C (PKC), decreased both basal tone and ERK1/2 activity in the hypertensive rats. In contrast, Y27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide) and verapamil, inhibitors of Rho kinase and voltage-dependent Ca2+ channels, respectively, significantly inhibited basal tone but not ERK1/2 activity. Thus, basal vascular tone is elevated by the altered activation of MAPK in DOCA-salt hypertensive rats, and this is regulated by PKC, but not by Rho or intracellular Ca2+.     

Inhibition of adrenergic human prostate smooth muscle contraction by the inhibitors of c-Jun N-terminal kinase, SP600125 and BI-78D3

BACKGROUND AND PURPOSE α(1) -Adrenoceptor-induced contraction of prostate smooth muscle is mediated by calcium- and Rho kinase-dependent mechanisms. In addition, other mechanisms, such as activation of c-jun N-terminal kinase (JNK) may be involved. Here, we investigated whether JNK participates in α(1)-adrenoceptor-induced contraction of human prostate smooth muscle. EXPERIMENTAL APPROACH Prostate tissue was obtained from patients undergoing radical prostatectomy. Effects of the JNK inhibitors SP600125 (50 μM) and BI-78D3 (30 μM) on contractions induced by phenylephrine, noradrenaline and electric field stimulation (EFS) were studied in myographic measurements. JNK activation by noradrenaline (30 μM) and phenylephrine (10 μM), and the effects of JNK inhibitors of c-Jun phosphorylation were assessed by Western blot analyses with phospho-specific antibodies. Expression of JNK was studied by immunohistochemistry and fluorescence double staining. KEY RESULTS The JNK inhibitors SP600125 and BI-78D3 reduced phenylephrine- and noradrenaline-induced contractions of human prostate strips. In addition, SP600125 reduced EFS-induced contraction of prostate strips. Stimulation of prostate tissue with noradrenaline or phenylephrine in vitro resulted in activation of JNK. Incubation of prostate tissue with SP600125 or BI-78D3 reduced the phosphorylation state of c-Jun. Immunohistochemical staining demonstrated the expression of JNK in smooth muscle cells of human prostate tissue. Fluorescence staining showed that α(1A)-adrenoceptors and JNK are expressed in the same cells. CONCLUSIONS AND IMPLICATIONS Activation of JNK is involved in α(1)-adrenoceptor-induced prostate smooth muscle contraction. Models of α(1)-adrenoceptor-mediated prostate smooth muscle contraction should include this JNK-dependent mechanism.     

Endothelin-1 induces contraction via a Syk-mediated p38 mitogen-activated protein kinase pathway in rat aortic smooth muscle

Although spleen tyrosine kinase (Syk) has crucial roles in various cells, its function on vascular smooth muscle contraction has not been determined. In the present study, we performed experiments to determine if Syk contributes to the endothelin-1 (ET-1)-mediated contraction in rat aortic smooth muscle. ET-1-induced contraction of aortic strips was inhibited by piceatannol, PD98059, and SB203580, inhibitors of Syk, extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK), respectively. Piceatannol also attenuated high K(+)-induced contraction. ET-1 dose-dependently enhanced the activity of Syk and this was inhibited by piceatannol in both rat aortic strip and rat aortic smooth muscle cells. The phosphorylation of p38 MAPK and heat shock protein 27 (HSP27), but not that of ERK1/2, in response to ET-1 was inhibited by both piceatannol and SB203580. These results suggest that Syk may play an important role in the regulation of aortic smooth muscle contraction induced by ET-1, which may be mediated by the p38 MAPK/HSP27 signaling pathway.     

c-Abl kinase regulates curcumin-induced cell death through activation of c-Jun N-terminal kinase

Curcumin, a natural phenolic compound found in turmeric (Curcuma longa) exhibits anticancer properties, attributed to its antiproliferative and apoptosis-inducing activity. The ubiquitously expressed nonreceptor tyrosine kinase c-Abl regulates stress responses induced by oxidative agents such as ionizing radiation and H2O2. In this study, we show that c-Abl is an important component of the cell death response activated by curcumin and that Abl mediates this response partly through activation of c-Jun N-terminal kinase (JNK). Therefore, inhibition of Abl by STI571 [imatinib (Gleevec)] treatment or down-regulation of Abl expression through Abl-specific short-hairpin RNA (shRNA) diminished cell death induction and JNK activation. Highlighting the interdependent nature of the Abl and JNK signaling in the curcumin-induced cell death response, a JNK inhibitor [anthra(1,9-cd)pyrazol-6(2H)-one-1,9-pyrazoloanthrone (SP600125)] caused very little cell death inhibition in STI571-pretreated cells and in Abl shRNA-expressing cells. Moreover, treatment with Abl and JNK inhibitor alone or together caused similar levels of cell death inhibition. Although p53 induction in response to curcumin treatment is dependent on Abl, we found that Abl-->p53 signaling is not necessary for curcumin-induced cell death. Taken together, the results demonstrate the differential roles played by Abl-->p53 and Abl-->JNK signaling events in modulating the cell death response to curcumin.     

Protein kinase C-mediated intracellular alkalinization in rat and rabbit aortic smooth muscle cells

The influence of protein kinase C (C-kinase) activation on intracellular pH (pHi) of cultured rat (RASM) and rabbit (RBASM) aortic smooth muscle cells was studied by employing a pH-sensitive fluorescent-dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The known C-kinase activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) and mezerine as well as the agonist angiotensin II each caused an intracellular alkalinization of approximately 0.1-0.15 pH units in RASM and RBASM cells grown in serum-free conditions. TPA-induced alkalinization was sensitive to the Na+/H+ exchange blockers amiloride and 5-N-ethylisopropyl-amiloride (EIPA). These results suggest that protein kinase C activation leads to intracellular alkalinization in vascular smooth muscle cells and the increase in pHi might play an important role in receptor-coupled arterial contraction.     

蛋白酪氨酸磷酸化参与调控主动脉平滑肌细胞容积调节性氯通道电流

目的　探讨蛋白酪氨酸磷酸化在胚胎鼠主动脉平滑肌细胞系A10细胞容积调节性氯通道电流中的调控作用。方法　膜片钳全细胞记录技术。结果　①在A10细胞 ,低渗溶液激活一外向整流电流 ,该电流在正电位 (≥ 6 0mV)时缓慢失活。其反转电位为 (- 1 5± 3 4 )mV ,接近Cl-平衡电位 (Ecl=0mV)。降低胞外Cl-浓度其反转电位随之变化。高渗溶液可抑制该电流。②DIDS(10 0 μmol·L-1)电压依赖性抑制容积调节性氯通道电流 ,在 +80mV和 - 80mV的抑制率分别为 5 3 7%± 6 2 %和 2 9 8%± 4 9%。③酪氨酸激酶抑制剂genistein (30 μmol·L-1)抑制该电流 ,在 +80mV和 - 80mV对其抑制率分别为6 2 3%± 11 3%和 6 6 9%± 10 5 % ,其抑制作用无电压依赖性。④酪氨酸磷酸酶抑制剂sodiumorthovanadate(Na3 VO4,5 0 0 μmol·L-1)增强该电流 ,其作用无电压依赖性 ,在 +80mV和 - 80mV电流幅度分别增加了 4 4 8%±14 5 %和 4 7 1%± 13 6 %。结论　A10细胞上存在有容积调节性氯通道。蛋白酪氨酸磷酸化参与调节A10细胞容积调节性氯电流的激活     

Inhibition of c-Jun N-terminal kinase 1, but not c-Jun N-terminal kinase 2, suppresses apoptosis induced by ischemia/reoxygenation in rat cardiac myocytes

In the present study, rat cardiac myocytes were used as an in vitro ischemia/reperfusion injury model to delineate the role of c-Jun N-terminal kinase (JNK) 1 and JNK2 isoforms in ischemia/reoxygenation-induced apoptosis using an antisense approach. Exposure of rat cardiac myocytes to ischemia did not induce apoptosis as detected by staining with either acridine orange/ethidium bromide or annexin-V-fluorescein/propidium iodide. In contrast, a time-dependent increase in the number of apoptotic cells was noted after reoxygenation of ischemic myocytes, whereas the level of necrotic cells remained unaltered. Reoxygenation, but not ischemia alone, also caused a time-dependent increase in JNK activation that preceded apoptosis induction. Treatment of cardiac myocytes with antisense (AS) oligonucleotides that specifically targeted either JNK1 or JNK2 significantly reduced both mRNA and protein expression of the target isoform but had no effect on the expression of the alternate isoform. Pretreatment of cardiac myocytes with JNK1 AS, but not JNK2 AS, resulted in almost complete attenuation of reoxygenation-induced apoptosis. Furthermore, control oligonucleotides for JNK1 AS or JNK2 AS had no effect on JNK mRNA or protein expression or reoxygenation-induced apoptosis, indicating a sequence-specific mode of action. Additional studies revealed that apoptosis induced by other JNK-activating stimuli, including ceramide, heat shock, and UV irradiation, was partly suppressed after treatment with JNK1 AS but not JNK2 AS. These findings demonstrate that the JNK1 isoform plays a preferential role in apoptosis induced by ischemia/reoxygenation as well as diverse JNK-activating cellular stresses.     

Inhibition of c-Jun N-terminal kinase sensitizes tumor cells to flavonoid-induced apoptosis through down-regulation of JunD

Reduction of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun NH2-terminal kinase (JNK) has been implicated in stress-induced apoptosis. However, many studies also emphasize the role of JNK on cell survival, although its mechanisms are not completely understood. Previously, we found that inhibition of JNK activity promotes flavonoid-mediated apoptosis of human osteosarcoma cells. We thus determined whether inhibition of JNK sensitizes tumor cells to a bioflavonoid-induced apoptosis, and whether this effect of JNK is a general effect. As the results, quercetin and genistein as well as a flavonoid fraction induced apoptosis of tumor cells, which was further accelerated by specific JNK inhibitor, SP600125 or by small interfering RNA specific to JNK1/2. This effect was specific to types of cells because it was further apparent in tumorigenic cell lines. Inhibition of JNK by SP600125 also reduced flavonoid-stimulated nuclear induction of JunD which was known to have protective role in apoptosis, whereas JNK inhibition alone had little effect on apoptosis. The flavonoid-induced apoptosis of tumor cells was significantly enhanced by transfecting them with antisense JunD oligonucleotides. These results suggest that inhibition of JNK facilitates flavonoid-induced apoptosis through down-regulation of JunD, which is further sensitive to tumor cells. Therefore, combination with a specific JNK inhibitor further enhances the anti-cancer and chemopreventive potential of bio-flavonoids.     

Resveratrol suppresses angiotensin II-induced Akt/protein kinase B and p70 S6 kinase phosphorylation and subsequent hypertrophy in rat aortic smooth muscle cells

Resveratrol (RV), a polyphenolic substance found in grape skin, is proposed to account in part for the protective effect of red wine in the cardiovascular system. Angiotensin II (Ang II)-induced hypertrophy of vascular smooth muscle cells (VSMCs) is a pivotal step in the development of cardiovascular disease. The aims of this study were to test the hypothesis that RV may alter Ang II-mediated hypertrophic VSMC growth and to identify the putative underlying signaling pathways. We show that RV indeed potently inhibits Ang II-induced [(3)H]leucine incorporation in a concentration-dependent manner (50 microM RV, 71% inhibition). Western blot analysis reveals that phosphorylation of Akt/protein kinase B (PKB) and to a lesser extent the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) 1/2, both essentially involved in Ang II-mediated hypertrophy, is dose dependently reduced by RV. Consistent with these results, we show that RV attenuates phosphorylation of the p70 ribosomal protein S6 kinase (p70(S6K)), a kinase downstream of the ERK 1/2 as well as the Akt pathway, that is implicated in Ang II-induced protein synthesis. Upstream of Akt/PKB RV seems to mediate its antihypertrophic effect by inhibiting phosphorylation of the phosphatidylinositol 3-kinase (PI(3)K) rather than by activating phosphatases. In summary, we demonstrate for the first time that RV inhibits Ang II-induced VSMC hypertrophy, possibly by interfering mainly with the PI(3)K/Akt and p70(S6K) but also with the ERK 1/2 signaling pathway. Thus, this study delivers important new insight in the molecular pathways that may contribute to the proposed beneficial effects of RV in cardiovascular disease.     

Rho-kinase inhibitors decrease TGF-beta-stimulated VEGF synthesis through stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts

We have previously reported that transforming growth factor-beta (TGF-beta) stimulates the synthesis of vascular endothelial growth factor (VEGF) through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In order to investigate whether Rho-kinase is involved in the TGF-beta-stimulated VEGF synthesis in these cells we examined the effects of Rho-kinase inhibitors on the VEGF synthesis. TGF-beta time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1) which is a well known substrate of Rho-kinase. Y27632 and fasudil, Rho-kinase inhibitors, significantly reduced the TGF-beta-stimulated VEGF synthesis as well as the MYPT-1 phosphorylation. Y27632 and fasudil failed to affect the TGF-beta-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or Smad2. On the contrary, Y27632 as well as fasudil markedly suppressed the TGF-beta-induced phosphorylation of SAPK/JNK. Taken together, our results strongly suggest that Rho-kinase regulates TGF-beta-stimulated VEGF synthesis via SAPK/JNK activation in osteoblasts.     

酪氨酸激酶抑制剂对cyclopiazoni cacid引起的大鼠主动脉血管环收缩效应的影响

目的　探讨酪氨酸激酶在Ca2 +池操纵性Ca2 +内流中的作用。方法　记录大鼠胸主动脉环收缩反应。结果　①不同剂量酪氨酸激酶抑制剂 genistein (1～ 10 0 μmol·L-1)和tyrphostin 2 5 (Tyr 2 5 ,1～ 30 μmol·L-1)均以浓度依赖性抑制cyclopiazonicacid (CPA)引起的平滑肌收缩平台期。Tyr 2 5的最大作用浓度为 10 μmol·L-1,抑制率为 42 %±11%。② 10 μmol·L-1Tyr 2 5和 1μmol·L-1nifedipine(Nif)对CPA引起电压依赖性Ca2 +通道 (VDCC)开放过程的抑制作用存在部分交叉。③ 1μmol·L-1Nif预处理阻断VDCC作用后 ,10 μmol·L-1Tyr 2 5只能部分阻断CPA引起的Ca2 +池操纵性Ca2 +通道 (SOCC)开放过程 ,再加入 6 0 μmol·L-1SK&F96 36 5可完全阻断SOCC的开放过程。结论　CPA引起平滑肌的收缩过程中 ,蛋白质酪氨酸激酶参与了开启VDCC和SOCC的信号转导过程     

Cytochalasin-B and phloretin depress contraction and relaxation of aortic smooth muscle

Cytochalasin-B (20 microM) and phloretin (100 microM) blocked by more than 80% the contractile responses to calcium ions in partially depolarized rabbit aortic strips. Both also blocked, but only by approximately 50%, the responses to noradrenaline and histamine in normal calcium medium. The responses to these agonists in calcium-free EGTA medium were also blocked partially. Cytochalasin-B partially blocked the acetylcholine-induced relaxation of aortic strips precontracted with phenylephrine but not the relaxation due to nitroglycerine or compound A 23187. The relaxation due to isoprenaline was potentiated by cytochalasin B. Since both compounds are known to block hexose transport but share no other known effects, it is suggested that a glycolytic intermediate could be required for the contraction in response to calcium. However, the concentrations of cytochalasin-B required for these effects were somewhat greater than those usually required to block hexose transport.     

Lack of involvement of pertussis toxin-sensitive G-proteins in norepinephrine-induced vasoconstriction of rat aorta smooth muscle

Several studies have shown that stimulation of pertussis toxin (PTX)-sensitive G-proteins amplified alpha-adrenoceptor (alpha-AR) agonist-induced vasoconstriction in small muscular and resistance arteries. The aim of this study was to assess the potential involvement of PTX-sensitive G-proteins in norepinephrine (NE)-induced constriction in a large diameter artery, the rat aorta. PTX (1 microg/mL, 2 hr; 3 microg/mL, 4 hr) did not modify concentration-response curves to NE in endothelium-denuded aortic rings. However, several lines of evidence suggested that aortic smooth muscle cells (SMC) had a PTX-sensitive G-protein pathway. [alpha-(32)P]ADP-ribosylation of G(i/o)-proteins by PTX (3 microg/mL, 4 hr) was demonstrated in situ in the intact aorta without endothelium. alpha(i/o) subunits were identified in vitro by both immunoblotting and ADP-ribosylation experiments in rat aorta SMC membranes. The measurement of G(i/o)-specific GTPase activity evidenced an effective coupling between NE receptors and G(i/o)-proteins, as NE induced an increase in basal G(i/o)-specific GTPase activity (20.7 +/- 2.8 vs 7.2 +/- 2.2 pmol P(i)/mg protein at 5 min; P < 0.05 vs basal). Co-immunoprecipitation revealed the in vitro coupling between alpha(1D)-ARs and G(i)-protein in rat aorta SMC membranes. In conclusion, we identified a PTX-sensitive G(i/o)-protein pathway in rat endothelium-denuded aorta. We showed an effective coupling between NE receptors and G(i)-proteins via alpha(1D)-ARs. Since PTX has no effect on NE-induced vasoconstriction, the PTX-sensitive G(i)-protein pathway does not play a predominant role in NE-induced responses in rat aorta SMC in contrast to small diameter muscular and resistance arteries.     

Corynoxeine isolated from the hook of Uncaria rhynchophylla inhibits rat aortic vascular smooth muscle cell proliferation through the blocking of extracellular signal regulated kinase 1/2 phosphorylation

The proliferation of vascular smooth muscle cells (VSMCs) induced by injury to the intima of arteries is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis. Uncaria rhynchophylla is traditional Chinese herb that has been applied to the treatment of convulsive disorders, such as epilepsy, in China. In the present study, we examined whether corynoxeine exerts inhibitory effects on platelet-derived growth factor (PDGF)-BB-induced rat aortic VSMC proliferation and the possible mechanism of such effects. Pre-treatment of VSMCs with corynoxeine (5-50 microM) for 24 h resulted in significant decreases in cell number without any cytotoxicity; the inhibition percentages were 25.0+/-12.5, 63.0+/-27.5 and 88.0+/-12.5% at 5, 20 and 50 microM, respectively. Also, corynoxeine significantly inhibited the 50 ng/ml PDGF-BB-induced DNA synthesis of VSMCs in a concentration-dependent manner without any cytotoxicity; the inhibitions were 32.8+/-11.0, 51.8+/-8.0 and 76.9+/-7.4% at concentrations of 5, 20 and 50 microM, respectively. Pre-incubation of VSMCs with corynoxeine significantly inhibited PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, whereas corynoxeine had no effects on mitogen-activated protein kinase (MAPK/ERK)-activating kinase 1 and 2 (MEK1/2), Akt, or phospholipase C (PLC)gamma1 activation or on PDGF receptor beta (PDGF-Rbeta) phosphorylation. These results suggest that corynoxeine is a potent ERK1/2 inhibitor of key PDGF-BB-induced VSMC proliferation and may be useful in the prevention and treatment of vascular diseases and restenosis after angioplasty.     

Inhibition of LIM kinase reduces contraction and proliferation in bladder smooth muscle

Overactive bladder (OAB) is the most bothersome symptom in lower urinary tract symptoms (LUTS). Current pharmacologic treatment aims to inhibit detrusor contraction; however, shows unsatisfied efficacy and high discontinuation rate. LIM kinases (LIMKs) promote smooth muscle contraction in the prostate; however, their function in the bladder smooth muscle remains unclear. Here, we studied effects of the LIMK inhibitors on bladder smooth muscle contraction and proliferation both in vitro and in vivo experiments. Bladder expressions of LIMKs are elevated in OAB rat detrusor tissues. Two LIMK inhibitors, SR7826 and LIMKi3, inhibit contraction of human detrusor strip, and cause actin filament breakdown, as well as cell proliferation reduction in cultured human bladder smooth muscle cells (HBSMCs), paralleled by reduced cofilin phosphorylation. Silencing of LIMK1 and LIMK2 in HBSMCs resulted in breakdown of actin filaments and decreased cell proliferation. Treatment with SR7826 or LIMKi3 decreased micturition frequency and bladder detrusor hypertrophy in rats with bladder outlet obstruction. Our study suggests that LIMKs may promote contraction and proliferation in the bladder smooth muscle, which could be inhibited by small molecule LIMK inhibitors. LIMK inhibitors could be a potential therapeutic strategy for OAB- related LUTS.KEY WORDS: LIMK, Cofilin phosphorylation, Overactive bladder (OAB), Lower urinary tract symptoms (LUTS), Bladder smooth muscle contraction     

Hydrogen peroxide-mediated inhibition of lipopolysaccharide-stimulated inhibitory kappa B kinase activity in rat aortic smooth muscle cells

1. In rat aortic smooth muscle cells (RASMC), exposure to lipopolysaccharide (LPS) resulted in NF-kappaB-DNA binding, degradation of IkappaB-alpha, -beta and -epsilon and increased activity of both alpha and beta isoforms of inhibitory kappa B kinase (IKK). 2. Expression of dominant-negative (DN)-IKK-alpha, IKK-beta and NF-kappaB-inducing kinase (NIK) abolished LPS-stimulated NF-kappaB reporter activity, suggesting that activation of a NIK/IKK-dependent pathway is indispensable for NF-kappaB activation by LPS in this cell type. 3. The tyrosine phosphatase inhibitor, pervanadate, abolished LPS-stimulated NF-kappaB-DNA-binding activity. However, the effect of pervanadate was shown to be mediated by excess hydrogen peroxide (H(2)O(2)) present in the reaction mix. Preincubation of RASMC with H(2)O(2) inhibited LPS-stimulated IKK kinase activity and downstream NF-kappaB-DNA binding activity. 4. H(2)O(2) also strongly stimulated p38 MAP kinase activity in RASMCs. Effective inhibition of this pathway using SB203580 did not reverse the effects of H(2)O(2) on LPS-stimulated IKK/NF-kappaB signalling. 5. These studies show that hydrogen peroxide-mediated inhibition of LPS-stimulated NF-kappaB activation in RASMC occurs upstream of IKK. The inhibitory effect of H(2)O(2) is not due to tyrosine phosphatase inhibition, it is mediated by H(2)O(2) through a mechanism which is independent of any cross-talk involving MAP kinase homologues.     

Augmentation of alpha1-adrenoceptor-mediated contraction by warming without increased phosphorylation of myosin in rat caudal arterial smooth muscle

We previously reported the relationship between alpha1-adrenoceptor-mediated contraction and phosphorylation of 20-kDa myosin light chain (LC20) in de-endothelialized rat caudal arterial smooth muscle at room temperature (Mita M, Walsh MP. Biochem J. 1997;327:669-674). We now describe the effect of increasing the temperature to 37 degrees C on this relationship. The EC50 value (76.6 +/- 18.2 nM) for cirazoline (alpha1-adrenergic agonist)-induced contraction of the strips at room temperature (23 degrees C) was significantly greater than that (14.5 +/- 1.9 nM) at 37 degrees C. The initial rate of the contraction to a sub-maximal concentration of cirazoline (0.3 microM) was similar at the two temperatures. However, cirazoline-induced maximal force at 37 degrees C was approximately 1.8 times that at room temperature. LC20 phosphorylation in response to cirazoline at room temperature and 37 degrees C closely matched the time courses of contraction, but values were not significantly different at the two temperatures: resting phosphorylation levels were 0.09 +/- 0.04 mol P(i)/mol LC20 at 37 degrees C and 0.22 +/- 0.06 mol P(i)/mol LC20 at room temperature; maximal cirazoline-stimulated LC20 phosphorylation levels were 0.58 +/- 0.08 mol P(i)/mol LC20 at room temperature and 0.49 +/- 0.05 mol P(i)/mol LC20 at 37 degrees C. We conclude, therefore, that the enhanced cirazoline-induced contraction at 37 degrees C is not due to increased LC20 phosphorylation.     

Big mitogen-activated protein kinase 1 protects cultured rat aortic smooth muscle cells from oxidative damage

Oxidative stress is considered a major mediator of arteriosclerosis. In vascular smooth muscle cells, oxidative stress-induced cell death (including apoptosis) is probably related to arterial calcification in arteriosclerosis. Big mitogen-activated protein kinase-1 / extracellular signal-regulated kinase 5 (BMK1/ERK5) is a newly identified member of the mitogen-activated protein kinases family. Like Src tyrosine kinase, BMK1/ERK5 is known to be sensitive to oxidative stress; however, its pathophysiological significance is poorly understood. In this study, we investigated the involvement of BMK1 and Src in H(2)O(2)-induced cell death using cultured rat aortic smooth muscle cells (RASMCs). Cell apoptosis was evaluated by using the TdT-mediated dUTP nick end labeling (TUNEL) method, and BMK1 and Src activities were determined by Western blotting. The main results are as follows: 1) BMK1 and Src were activated by H(2)O(2) in a time- and concentration-dependent manner in RASMCs; 2) BMK1 activation by H(2)O(2) was attenuated both in Src-knockdown RASMCs and in RASMCs pretreated with 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a Src family kinases inhibitor; and 3) H(2)O(2)-induced cell death was increased in BMK1- and Src-knockdown RASMCs as well as in PP2-treated RASMCs. These findings suggested that Src and BMK1 may play defensive and resistive roles against oxidative stress-induced death in RASMCs.