Prevention of the wortmannin-induced inhibition of phosphoinositide 3-kinase by sulfhydryl reducing agents.

The effects of the sulfhydryl reducing agents 2-mercaptoethanol and dithiothreitol on wortmannin-induced inhibition of phosphoinositide 3-kinase (PI3K) were studied in order to examine whether the sulfhydryl reducing agents directly affect the wortmannin inhibition of PI3K. These reducing agents are commonly used to stabilize enzyme structures by maintaining protein sulfhydryl groups in the reduced state. Preincubation of wortmannin with millimolar levels of 2-mercaptoethanol, a sulfhydryl derivative of ethanol, markedly prevented subsequent wortmannin-induced inhibition of PI3K. In contrast, ethanol, 2-mercaptoethanol lacking sulfhydryl group, and 2-(methylthio)ethanol, a methyl derivative of the sulfhydryl group of 2-mercaptoethanol, had little effect on the wortmannin-induced inhibition of PI3K, which suggests that the prevention of wortmannin-induced inhibition by 2-mercaptoethanol occurs through the sulfhydryl group of this agent. Moreover, dithiothreitol, a second sulfhydryl reducing agent, also markedly prevented wortmannin-induced inhibition of PI3K. These results indicate that the wortmannin-induced inhibition of PI3K is markedly prevented by millimolar concentrations of sulfhydryl reducing agents such as 2-mercaptoethanol and dithiothreitol in the medium, presumably by the binding of wortmannin to the agents.

Olmesartan inhibits angiotensin II-Induced migration of vascular smooth muscle cells through Src and mitogen-activated protein kinase pathways.

Clinical studies have shown that angiotensin-receptor blockers (ARBs) reduce the risk of cardiovascular diseases in hypertensive patients. It is assumed that the reduction of the risk by ARBs may be attributed in part to the inhibition of angiotensin II (AII)-induced vascular smooth muscle cell (VSMC) migration associated with atherosclerosis. However, the effect of ARBs on AII-induced changes in intracellular signaling and resultant cell migration has not been well established. Here, we investigated the effect of olmesartan, an ARB, on AII-induced extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) activation and rat aortic smooth muscle cell (RASMC) migration. Olmesartan inhibited AII-induced ERK1/2 and JNK activation at lower concentrations (10 nM). On the other hand, PP2, a Src tyrosine kinase inhibitor, also inhibited AII-induced ERK1/2 and JNK activation, but its effect on ERK1/2 was less pronounced than that of olmesartan. Olmesartan, U0126 (an ERK1/2 inhibitor), SP600125 (a JNK inhibitor), and PP2 potently inhibited AII-induced RASMC migration. From these findings, it was inferred that angiotensin-receptor blockade by olmesartan results in the inhibition of AII-induced activation of Src, ERK1/2, and JNK in RASMC. Olmesartan may be a potent inhibitor of AII-induced VSMC migration, which may be involved in the progression of atherosclerosis.