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.

Electron paramagnetic resonance study on free radical scavenging and/or generating activity of dopamine-4-O-sulfate.

The free radical scavenging and/or generating activity of dopamine-4-O-sulfate was examined and compared with that of dopamine. In humans, dopamine mostly exists in two isomeric forms of sulfate ester conjugates as metabolites; i.e., dopamine-3-O-sulfate and dopamine-4-O-sulfate in the circulation. Dopamine is generally believed to be oxidized by molecular oxygen or another reactive oxygen species under physiological conditions, to form oxidized dopamine derivatives that are cytotoxic. However, it is not known whether dopamine conjugates are generated on interaction with reactive oxygen species or not. In the present study, we measured the susceptibility to oxidization of dopamine-4-O-sulfate by using electron paramagnetic resonance (EPR) spectroscopy and optical absorption spectrometry. Dopamine was easily oxidized and dopamine-derived radicals appeared, whereas dopamine-4-O-sulfate was not oxidized under physiological conditions. Furthermore, dopamine-4-O-sulfate did not react with a strong oxidizing agent, sodium periodate. These results suggest that dopamine-4-O-sulfate has resistance against autoxidation, and seems to be a stable metabolite of dopamine.

Antioxidants inhibit JNK and p38 MAPK activation but not ERK 1/2 activation by angiotensin II in rat aortic smooth muscle cells.

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) hypertrophy, which results in several cardiovascular diseases. Ang II-induced cellular events have been mediated, in part, by reactive oxygen species (ROS) which also involve activation of mitogen-activated protein (MAP) kinases. Although it has been proposed that the therapeutic administration of antioxidants is useful for vascular diseases, the precise mechanisms which regulate ROS-sensitive signaling events have not been well characterized. Thus, we hypothesized that antioxidants may affect ROS-mediated MAP kinases activation induced by Ang II. The present findings showed that Ang II stimulated rapid and significant activation of ERK 1/2, JNK and p38 MAPK in cultured rat aortic smooth muscle cells (RASMC). Ang II-induced ERK 1/2 activation was not affected by all antioxidants examined, whereas JNK was sensitive to all antioxidants. In contrast, p38 MAPK activation was inhibited by DPI and ascorbic acid concentration-dependently, but by NAC only at high concentration. DETC and Trolox C had no effects on p38 MAPK activation by Ang II. We further examined the effects of antioxidants on Ang II-induced increases in oxygen consumption as an index of ROS generation in RASMC. DPI strongly inhibited Ang II-induced increases in oxygen consumption. DETC also inhibited Ang II-induced oxygen consumption, whereas ascorbic acid markedly augmented it. These findings suggest that the inhibitory effects of antioxidants on MAP kinases activation in VSMC are attributable, in part, to their modulating effects on ROS generation by Ang II in VSMC. Thus, inhibition of MAP kinases by antioxidants may imply their usefulness for relief of cardiovascular diseases.

Effect of endothelin-1(1-31) on p38 mitogen-activated protein kinase in cultured human mesangial cells.

It was reported that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs(1-31). In this study, we investigated the effect of ET-1(1-31) on p38 mitogen-activated protein kinase (p38-MAPK) activity in human mesangial cells (HMCs). By measuring the kinase activity, we demonstrated that ET-1 (1-31) activated the p38-MAPK dose-dependently (10(-9) M to 10(-7) M), which was inhibited by SB203580. The p38-MAPK activation induced by ET-1(1-31) peaked at 10 minutes. BQ123 almost abolished ET-1(1-31)-induced p38-MAPK activation, whereas BQ788 failed to inhibit it. These findings suggest that the stimulatory effect of ET-1(1-31) on p38-MAPK activation is mediated through ET(A) or ET(A)-like receptor. In conclusion, ET-1(1-31) induced increase in p38-MAPK activation in cultured HMCs.

Effect of endothelin-1(1-31) on intracellular free calcium in cultured human mesangial cells.

We found that human chymase selectively produces 31-amino-acid length endothelins (1-31) (ETs(1-31)). We investigated the effect of synthetic ET-1(1-31) on intracellular free Ca2+ concentration ([Ca2+]i) in cultured human mesangial cells. ET-1(1-31) increased [Ca2+]i in a concentration-dependent manner to a similar extent as ET-1. The ET-1 (1-31)-induced [Ca2+]i increase was not influenced by removal of extracellular Ca2+ but was inhibited by thapsigargin. ET-1(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon. It was inhibited by BQ123, but not by BQ788. These results suggest that ET-1(1-31) by itself exhibits bioactive properties probably through endothelin ET(A) or ET(A)-like receptors. Since human chymase has been reported to exist in the kidney, ET-1(1-31) may be a candidate substance for mesangium-relevant diseases.

Comparison of the effects of endothelin-1, -2 and -3 (1-31) on changes in [Ca2+]i in human coronary artery smooth muscle cells.

We have previously found that human chymase selectively cleaves big endothelins (ETs) at the Tyr31-Gly32 bond to produce 31-amino-acid endothelins, ETs (1-31). In the present study, we investigated the effects of ETs (1-31) on changes in intracellular free Ca2+ ([Ca2+]i) in cultured human coronary artery smooth muscle cells (HCASMCs) using confocal laser microscopy. ETs (1-31) increased [Ca2+]i in a concentration-dependent manner. Phosphoramidon did not inhibit the increases in [Ca2+]i caused by ETs (1-31). The [Ca2+]i increases induced by ETs (1-31) were compared to those of ETs (1-21) and big ETs. ET-1 (1-21) was about 10-times more potent than big ET-1 or ET-1 (1-31), whereas big ET-2 was 10-times less potent than ET-2 (1-21) or ET-2 (1-31). ETs (1-31) may induce [Ca2+]i increase through ET(A)-type or ET(A)-type-like receptors. The 10(-12) M ET (1-31)-induced increases in [Ca2+]i were not affected by removal of extracellular Ca2+, but were inhibited by thapsigargin. These results suggested that ET-1, -2 and -3 (1-31) showed similar potencies in increasing [Ca2+]i and mechanisms of ET (1-31)-induced increases in [Ca2+]i may be similar among the three ETs (1-31).