Li-Mei Zhao

K Ca 3.1 channels mediate the increase of cell migration and proliferation by advanced glycation endproducts in cultured rat vascular smooth muscle cells

The mechanisms underlying the involvement of advanced glycation endproducts (AGEs) in diabetic atherosclerosis are not fully understood. The present study was designed to investigate whether intermediate-conductance Ca2+-activated K+ channels (KCa3.1 channels) are involved in migration and proliferation induced by AGEs in cultured rat vascular smooth muscle cells (VSMCs) using approaches of whole-cell patch voltage-clamp, cell proliferation and migration assay, and western blot analysis. It was found that the current density and protein level of KCa3.1 channels were enhanced in cells incubated with AGE-BSA (bovine serum albumin), and the effects were reversed by co-incubation of AGE-BSA with anti-RAGE (anti-receptors of AGEs) antibody. The ERK1/2 inhibitors PD98059 and U0126, the P38-MAPK inhibitors SB203580 and SB202190, or the PI3K inhibitors LY294002 and wortmannin countered the KCa3.1 channel expression by AGE-BSA. In addition, AGE-BAS increased cell migration and proliferation, and the effects were fully reversed with anti-RAGE antibody, the KCa3.1 channel blocker TRAM-34, or KCa3.1 small interfering RNA. These results demonstrate for the first time that AGEs-induced increase of migration and proliferation is related to the upregulation of KCa3.1 channels in rat VMSCs, and the intracellular signals ERK1/2, P38-MAPK and PI3K are involved in the regulation of KCa3.1 channel expression.

Angiotensin II upregulates KCa3.1 channels and stimulates cell proliferation in rat cardiac fibroblasts

The proliferation of cardiac fibroblasts is implicated in the pathogenesis of myocardial remodeling and fibrosis. Intermediate-conductance calcium-activated K⁺ channels (K(Ca)3.1 channels) have important roles in cell proliferation. However, it is unknown whether angiotensin II (Ang II), a potent profibrotic molecule, would regulate K(Ca)3.1 channels in cardiac fibroblasts and participate in cell proliferation. In the present study, we investigated whether K(Ca)3.1 channels were regulated by Ang II, and how the channel activity mediated cell proliferation in cultured adult rat cardiac fibroblasts using electrophysiology and biochemical approaches. It was found that mRNA, protein, and current density of K(Ca)3.1 channels were greatly enhanced in cultured cardiac fibroblasts treated with 1 μM Ang II, and the effects were countered by the angiotensin type 1 receptor (AT₁R) blocker losartan, the p38-MAPK inhibitor SB203580, the ERK1/2 inhibitor PD98059, and the PI3K/Akt inhibitor LY294002. Ang II stimulated cell proliferation and the effect was antagonized by the K(Ca)3.1 blocker TRAM-34 and siRNA targeting K(Ca)3.1. In addition, Ang II-induced increase of K(Ca)3.1 expression was attenuated by transfection of activator protein-1 (AP-1) decoy oligodeoxynucleotides. These results demonstrate for the first time that Ang II stimulates cell proliferation mediated by upregulating K(Ca)3.1 channels via interacting with the AT₁R and activating AP-1 complex through ERK1/2, p38-MAPK and PI3K/Akt signaling pathways in cultured adult rat cardiac fibroblasts.

Insulin-mediated upregulation of KCa3.1 channels promotes cell migration and proliferation in rat vascular smooth muscle

The detailed molecular mechanisms underlying pathogenesis of various vascular diseases such as atherosclerosis are not fully understood in type-2 diabetes. The present study was designed to investigate whether insulin regulates K(Ca)3.1 channels and participates in vasculopathy in type-2 diabetes. A rat model with experimental insulin-resistant type-2 diabetes was used for detecting pathological changes in the aorta wall, and cultured vascular smooth muscle cells (VSMCs) were employed to investigate the regulation of K(Ca)3.1 channels by insulin and roles of K(Ca)3.1 channels in cell migration and proliferation using molecular biology and electrophysiology. Early pathological changes were observed and expression of K(Ca)3.1 channels increased in the aorta wall of the type 2 diabetic rats. K(Ca)3.1 channel mRNA, protein levels and current density were greatly enhanced in cultured VSMCs treated with insulin, and the effects were countered in the cells treated with the ERK1/2 inhibitor PD98059, but not the p38-MAPK inhibitor SB203580. In addition, insulin stimulated cell migration and proliferation in cultured VSMCs, and the effects were fully reversed in the cells treated with the K(Ca)3.1 blocker TRAM-34 or PD98059, but not SB203580. These results demonstrate the novel information that insulin increases expression of K(Ca)3.1 channels by stimulating ERK1/2 phosphorylation thereby promoting migration and proliferation of VSMCs, which likely play at least a partial role in the development of vasculopathy in type-2 diabetes.

Daidzein relaxes rat cerebral basilar artery via activation of large-conductance Ca2+-activated K+ channels in vascular smooth muscle cells

Daidzein, a phytoestrogen, has been reported to produce vasodilation via inhibition of Ca(2+) inflow. However, the involvement of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in the effect of daidzein is debated. Therefore, the present study was designed to investigate the effect of daidzein on the rat cerebral basilar artery and the underlying molecular mechanisms. Isolated cerebral basilar artery rings and single vascular smooth muscle cells (VSMCs) were used for vascular reactivity and electrophysiology measurements, to investigate the effect of daidzein on BK(Ca) channels in cerebral basilar artery smooth muscle. In addition, the human BK(Ca) channel alpha-subunit gene (hslo) was transfected into HEK293 cells, to directly assess whether daidzein activates BK(Ca) channels. The results showed that daidzein produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Paxilline, a selective BK(Ca) channel blocker, significantly inhibited the daidzein-induced vasodilation, whereas NS1619, a selective BK(Ca) channel opener, enhanced the vasodilation. In the whole-cell configuration, daidzein increased noisy oscillation currents in cerebral basilar artery VSMCs in a concentration-dependent manner, and washout of daidzein or blockade of BK(Ca) channels with paxilline fully reversed the increase. However, daidzein did not substantially affect hSlo currents in HEK293 cells when applied to the outside of the cell membrane. In conclusion, these results indicate that the activation of BK(Ca) channels in VSMCs at least partly contributes to the daidzein-induced vasodilation of the rat cerebral basilar artery. The beta1-subunit of BK(Ca) channels plays a critical role in the activation of BK(Ca) currents by daidzein.

Equol is Neuroprotective During Focal Cerebral Ischemia and Reperfusion that Involves p-Src and gp91 phox

Both of gp91(phox) (an isoform of nicotinamide adenine dinucleotide phosphate reduced oxidases) and Src (a nonreceptor protein tyrosine kinase) are abundantly expressed in the brain and play a prominent role in mediating ischemic alteration in neurons. The inhibitory strategy of them is believed to be the promising treatment of stroke. The present study was designed to investigate the effect of equol (0.625-2.5 mg·kg(-1), i.g. for 3 days), a predominant active metabolite of daidzein, on neuroprotection against cerebral ischemia/reperfusion injury in rats and the underlying mechanisms. We found that equol decreased the mortality, neurological deficit, brain histological damage, infarct volume, serum lactate dehydrogenase activity and malondialdehyde content in a dose-dependent manner in rats with 2-h middle cerebral artery occlusion, followed by 22-h reperfusion. Western blot analysis revealed that protein levels of gp91(phox) and phosphorylated Src-Tyr416 (p-Src) in ischemic cerebral cortex were increased in rats treated with vehicle, which was reversed in animals treated with equol. In rat pheochromocytoma cell line (PC12) with hypoxia/reoxygenation injury, silencing of gp91(phox) with specific siRNA did not affect the increase of p-Src level by hypoxia/reoxygenation injury and the inhibition of p-Src level by equol, while silencing of Src suppressed the upregulation of gp91(phox) by hypoxia/reoxygenation injury and enhanced the inhibitory effect of equol on gp91(phox) expression. These results demonstrate that equol confers a neuroprotection in rats via inhibiting the activation of Src and upregulation of gp91(phox) induced by focal cerebral ischemia/reperfusion, and Src may play a partial role in regulating gp91(phox) expression of neurons.

Equol increases cerebral blood flow in rats via activation of large-conductance Ca(2+)-activated K(+) channels in vascular smooth muscle cells.

The present study was designed to investigate the effect of equol on cerebral blood flow and the underlying molecular mechanisms. The regional cerebral blood flow in parietal lobe of rats was measured by using a laser Doppler flowmetry. Isolated cerebral basilar artery and mesenteric artery rings from rats were used for vascular reactivity measurement with a multi wire myography system. Outward K(+) current in smooth muscle cells of cerebral basilar artery, large-conductance Ca(2+)-activated K(+) (BK) channel current in BK-HEK 293 cells stably expressing both human α (hSlo)- and β1-subunits, and hSlo channel current in hSlo-HEK 293 cells expressing only the α-subunit of BK channels were recorded with whole cell patch-clamp technique. The results showed that equol significantly increased regional cerebral blood flow in rats, and produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Both paxilline and iberiotoxin, two selective BK channel blockers, significantly inhibited equol-induced vasodilation in cerebral arteries. Outward K(+) currents in smooth muscle cells of cerebral basilar artery were increased by equol and fully reversed by washout or blockade of BK channels with iberiotoxin. Equol remarkably enhanced human BK current in BK-HEK 293 cells, but not hSlo current in hSlo-HEK 293 cells, and the increase was completely abolished by co-application of paxilline. Our findings provide the first information that equol selectively stimulates BK channel current by acting on its β1 subunit, which may in turn contribute to the equol-mediated vasodilation and cerebral blood flow increase.

Metformin Restores Intermediate-Conductance Calcium-Activated K+ Channel– and Small-Conductance Calcium-Activated K+ Channel–Mediated Vasodilatation Impaired by Advanced Glycation End Products in Rat Mesenteric Artery

The present study was designed to investigate the effect of metformin on the impairment of intermediate-conductance and small-conductance Ca2+-activated potassium channels (IKCa and SKCa)–mediated relaxation in diabetes and the underlying mechanism. The endothelial vasodilatation function of mesenteric arteries was assessed with the use of wire myography. Expression levels of IKCa and SKCa and phosphorylated Thr172 of AMP-activated protein kinase (AMPK) were measured using Western blot technology. The channel activity was observed using a whole-cell patch voltage clamp. Reactive oxygen species (ROS) were measured using dihydroethidium and 2′,7′-dichlorofluorescein diacetate. Metformin restored the impairment of IKCa- and SKCa-mediated vasodilatation in mesenteric arteries from streptozotocin-induced type 2 diabetic rats and that from normal rats incubated with advanced glycation end products (AGEs) for 3 hours. In cultured human umbilical vein endothelial cells (HUVECs), 1 μM metformin reversed AGE-induced increase of ROS and attenuated AGE- and H2O2- induced downregulation of IKCa and SKCa after long-term incubation (>24 hours). Short-term treatment (3 hours) with 1 μM metformin reversed the decrease of IKCa and SKCa currents induced by AGE incubation for 3 hours without changing the channel expression or the AMPK activation in HUVECs. These results are the first to demonstrate that metformin restored IKCa- and SKCa-mediated vasodilatation impaired by AGEs in rat mesenteric artery, in which the upregulation of channel activity and protein expression is likely involved.

Metformin improves IKCa-mediated endothelial dilative dysfunction of arteriole in diabetic rats

Methods Diabetic rat model was induced by a single intraperitoneal injection of 30 mg/kg STZ after high fat and glucose diet for 8 weeks. Animals whose blood glucose > 11.1 mmol/L were included in diabetic and metformin group. Agematched animals fed with standard chow and injected with citric acid buffer were served as control. Four weeks after STZ injection, rats in three groups were fed with normal diet for additional 8 weeks. After that, fasting blood was drawn and third-order mesenteric arteries were separated. Hemoglobin A1c (HbA1c) was measured with an automatic analyzer. The changes of Achand NS309 (opener of IKCa and small conductance Ca -activated K channel, SKCa) -induced vasodilatation mediated by IKCa in mesentery arterioles of each group and mesentery arterioles of normal rats incubated with 200 μg/mL AGE-BSA (200 μg/mL BSA as control) for 3 hours were measured by multi-myograph system. The effect of metformin on AGEBSA (200 μg/mL) and H2O2 (100 μmol/L) induced changes of IKCa mRNA and protein expression in cultured human umbilical vein endothelial cells (HUVECs) were detected by RT-PCR and Western blot. The level of malondialdehyde (MDA) and the activity of Cu-Zn superoxide dismutase (Cu-Zn SOD) in cellular supernatant were determined by colorimetric method. Results Increased HbA1c level and reduceded endotheliumdependent dilative response mediated by IKCa in mesentery arterioles were observed in diabetic rats, and metformin treatment (300 mg/kg/day by gavage) restored the adverse condition. The vasodilatation mediated by IKCa was also impaired in 200 μg/mL AGE-BSA-incubated mesentery arterioles. AGE-BSA at 200 μg/mL concentration and H2O2 (100 μmol/L) significantly decreased the mRNA and protein expression of IKCa. AGE-BSA also increased the production of MDA and inhibited Cu-Zn SOD activity in HUVECs. Metformin of 10 mol/L and 10 mol/L reversed those effects.

The role and mechanism of K Ca 3.1 channels in human monocyte migration induced by palmitic acid

Abstract Monocyte migration into diseased tissues contributes to the pathogenesis of diseases. Intermediate‐conductance Ca2+‐activated K+ (KCa3.1) channels play an important role in cell migration. However, the role of KCa3.1 channels in mediating monocyte migration induced by palmitic acid (PA) is still unclear. Using cultured THP‐1 cells and peripheral blood mononuclear cells from healthy subjects, we investigated the role and signaling mechanisms of KCa3.1 channels in mediating the migration induced by PA. Using methods of Western blotting analysis, RNA interference, cell migration assay and ELISA, we found that PA‐treated monocytes exhibited increment of the protein levels of KCa3.1 channel and monocyte chemoattractant protein‐1 (MCP‐1), and the effects were reversed by co‐incubation of PA with anti‐TLR2/4 antibodies or by specific inhibitors of p38‐MAPK, or NF‐&kgr;B. In addition, PA increased monocyte migration, which was abolished by a specific KCa3.1 channel blocker, TRAM‐34, or KCa3.1 small interfering RNA (siRNA). The expression and secretion of MCP‐1 induced by PA was also similarly prevented by TRAM‐34 and KCa3.1 siRNA. These results demonstrate for the first time that PA upregulates KCa3.1 channels through TLR2/4, p38‐MAPK and NF‐&kgr;B pathway to promote the expression of MCP‐1, and then induce the trans‐endothelial migration of monocytes. Graphical abstract Figure. No Caption available. HighlightsPalmitic acid upregulates KCa3.1 channels in human monocytes.TLR2/4, p38‐MAPK and NF‐&kgr;B pathway is involved in the effect of palmitic acid.The migration of monocytes induced by palmitic acid is mediated by KCa3.1 channels.KCa3.1 channels contribute to palmitic acid‐induced MCP‐1 production in monocytes.