Xiu-Ling Deng

Characterization of ionic currents in human mesenchymal stem cells from bone marrow.

This study characterized functional ion channels in cultured undifferentiated human mesenchymal stem cells (hMSCs) from bone marrow with whole‐cell patch clamp and reverse transcription polymerase chain reaction (RT‐PCR) techniques. Three types of outward currents were found in hMSCs, including a noise‐like rapidly activating outward current inhibited by the large conductance Ca2+‐activated K+ channel (IKCa) blocker iberiotoxin, a transient outward K+ current (Ito) suppressed by 4‐aminopyridine (4‐AP), and a delayed rectifier K+ current (IKDR)‐like ether‐à‐go‐go (eag) K+ channel. In addition, tetrodotoxin‐sensitive sodium current (INa.TTX) and nifedipine‐sensitive L‐type Ca2+ current (ICa.L) were also detected in 29% and 15% hMSCs, respectively. Moreover, RT‐PCR revealed the molecular evidence of high levels of mRNA for the functional ionic currents, including human MaxiK for IKCa, Kv4.2 and Kv1.4 for Ito, heag1 for IKDR, hNE‐Na for INa.TTX, and CACNAIC for ICa.L. These results demonstrate that multiple functional ion channel currents—that is, IKCa, Ito, heag1, INa.TTX, and ICa.L—are expressed in hMSCs from bone marrow.

Ion Channels in Mesenchymal Stem Cells from Rat Bone Marrow

Mesenchymal stem cells (MSCs) from bone marrow are believed to be an ideal cell source for cardiomyoplasty; however, cellular electrophysiology is not understood. The present study was designed to investigate ion channels in undifferentiated rat MSCs. It was found that three types of outward currents were present in rat MSCs, including a small portion of Ca2+‐activated K+ channel (IKCa) sensitive to inhibition by iberiotoxin and/or clotromazole, a delayed rectifier K+ current (IKDR), and a transient outward K+ current (Ito). In addition, tetrodotoxin (TTX)‐sensitive sodium current (INa.TTX) and nifedipine‐sensitive L‐type Ca2+ current (ICa.L) were found in a small population of rat MSCs. Moreover, reverse transcription‐polymerase chain reaction revealed the molecular evidence of mRNA for the functional ionic currents, including Slo and KCNN4 for IKCa; Kv1.4 for Ito; Kv1.2 and Kv2.1 for IKDR; SCN2a1 for INa.TTX; and CCHL2a for ICa.L. These results demonstrate for the first time that multiple functional ion channel currents (i.e., IKCa, Ito, IKDR, INa.TTX, and ICa.L) are present in rat MSCs from bone marrow; however, physiological roles of these ion channels remain to be studied.

Acacetin, a Natural Flavone, Selectively Inhibits Human Atrial Repolarization Potassium Currents and Prevents Atrial Fibrillation in Dogs

Background— The development of atrium-selective antiarrhythmic agents is a current strategy for inhibiting atrial fibrillation (AF). The present study investigated whether the natural flavone acacetin from the traditional Chinese medicine Xuelianhua would be an atrium-selective anti-AF agent. Methods and Results— The effects of acacetin on human atrial ultrarapid delayed rectifier K+ current (IKur) and other cardiac ionic currents were studied with a whole-cell patch technique. Acacetin suppressed IKur and the transient outward K+ current (IC50 3.2 and 9.2 &mgr;mol/L, respectively) and prolonged action potential duration in human atrial myocytes. The compound blocked the acetylcholine-activated K+ current; however, it had no effect on the Na+ current, L-type Ca2+ current, or inward-rectifier K+ current in guinea pig cardiac myocytes. Although acacetin caused a weak reduction in the hERG and hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells, it did not prolong the corrected QT interval in rabbit hearts. In anesthetized dogs, acacetin (5 mg/kg) prolonged the atrial effective refractory period in both the right and left atria 1 to 4 hours after intraduodenal administration without prolongation of the corrected QT interval, whereas sotalol at 5 mg/kg prolonged both the atrial effective refractory period and the corrected QT interval. Acacetin prevented AF induction at doses of 2.5 mg/kg (50%), 5 mg/kg (85.7%), and 10 mg/kg (85.7%). Sotalol 5 mg/kg also prevented AF induction (60%). Conclusions— The present study demonstrates that the natural compound acacetin is an atrium-selective agent that prolongs the atrial effective refractory period without prolonging the corrected QT interval and effectively prevents AF in anesthetized dogs after intraduodenal administration. These results indicate that oral acacetin is a promising atrium-selective agent for the treatment of AF.

Cell cycle-dependent expression of potassium channels and cell proliferation in rat mesenchymal stem cells from bone marrow

Abstract.  Objective: Recently, our team has demonstrated that voltage‐gated delayed rectifier K+ current (IKDR) and Ca2+‐activated K+ current (IKCa) are present in rat bone marrow‐derived mesenchymal stem cells; however, little is known of their physiological roles. The present study was designed to investigate whether functional expression of IKDR and IKCa would change with cell cycle progression, and whether they could regulate proliferation in undifferentiated rat mesenchymal stem cells (MSCs). Materials and Methods: Membrane potentials and ionic currents were recorded using whole‐cell patch clamp technique, cell cycling was analysed by flow cytometry, cell proliferation was assayed with DNA incorporation method and the related genes were down‐regulated by RNA interference (RNAi) and examined using RT‐PCR. Results: It was found that membrane potential hyperpolarized, and cell size increased during the cell cycle. In addition, IKDR decreased, while IKCa increased during progress from G1 to S phase. RT‐PCR revealed that the mRNA levels of Kv1.2 and Kv2.1 (likely responsible for IKDR) reduced, whereas the mRNA level of KCa3.1 (responsible for intermediate‐conductance IKCa) increased with the cell cycle progression. Down‐regulation of Kv1.2, Kv2.1 or KCa3.1 with the specific RNAi, targeted to corresponding gene inhibited proliferation of rat MSCs. Conclusion: These results demonstrate that membrane potential, IKDR and IKCa channels change with cell cycle progression and corresponding alteration of gene expression. IKDR and intermediate‐conductance IKCa play an important role in maintaining membrane potential and they participate in modulation of proliferation in rat MSCs.

Experimental diabetes mellitus down-regulates large-conductance Ca2+-activated K+ channels in cerebral artery smooth muscle and alters functional conductance.

Cerebral vascular dysfunction and associated vascular complications often develop over time in type-2 diabetes, but the underlying mechanisms are not wholly understood. The aim of the present study was to investigate whether large-conductance Ca(2+)-activated K(+) (BKCa) channels in cerebral artery smooth muscle cells (CASMCs) were impaired in experimental model of type-2 diabetes, and the changes could account for cerebral vascular complication in type-2 diabetes. Sprague-Dawley rats were fed with high fat and glucose diet for 8 weeks and then injected with streptozotocin (STZ/30 mg/kg i.p.). Three months after injection of STZ, the alterations of BKCa channels were assessed by using multi-myograph system, patch-clamp, RT-PCR and Western blot. Our results show that the model is characterized by insulin resistance, hyperglycaemia, hyperlipidemia and moderate hypertension, which resembles the clinical manifestation of patients with typre-2 diabetes. Inhibition of BKCa channels with 1 mM tetraethylammonium (TEA) or 1 microM paxilline (PAX) causes smaller constriction in type-2 diabetic cerebral basilar arteries than control arteries. The contractile efficacy of 5-Hydroxytryptamine (5-HT) is substantially reduced by TEA or PAX pretreatment in control > diabetic basilar artery rings. The response to 5-HT in diabetic basilar artery rings is higher than that of control artery rings after activation of BKCa channels with NS1619. The whole-cell K(+) currents are significantly decreased in type-2 diabetic CASMCs compared to control, and the sensitivity of BKCa channels to voltage, the specific inhibitor and opener are all diminished in diabetic CASMCs. The expression of BKCa channel beta1, but not alpha-subunits is markedly reduced at both of mRNA and protein levels in endothelial-denudated cerebral arteries. In conclusion, type-2 diabetes downregulates BKCa channel beta1-subunits in CASMCs, resulting in reduced activity of BKCa channel, increased vascular tone and blood pressure, thereby contributing to cerebral vascular complication in type-2 diabetes.

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.

Modulation of human cardiac transient outward potassium current by EGFR tyrosine kinase and Src-family kinases

AIMS The human cardiac transient outward K(+) current I(to) (encoded by Kv4.3 or KCND3) plays an important role in phase 1 rapid repolarization of cardiac action potentials in the heart. However, modulation of I(to) by intracellular signal transduction is not fully understood. The present study was therefore designed to determine whether/how human atrial I(to) and hKv4.3 channels stably expressed in HEK 293 cells are regulated by protein tyrosine kinases (PTKs). METHODS AND RESULTS Whole-cell patch voltage-clamp, immunoprecipitation, western blotting, and site-directed mutagenesis approaches were employed in the present study. We found that human atrial I(to) was inhibited by the broad-spectrum PTK inhibitor genistein, the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556, and the Src-family kinases inhibitor PP2. The inhibitory effect was countered by the protein tyrosine phosphatase inhibitor orthovanadate. In HEK 293 cells stably expressing human KCND3, genistein, AG556, and PP2 significantly reduced the hKv4.3 current, and the reduction was antagonized by orthovanadate. Interestingly, orthovanadate also reversed the reduced tyrosine phosphorylation level of hKv4.3 channels by genistein, AG556, or PP2. Mutagenesis revealed that the hKv4.3 mutant Y136F lost the inhibitory response to AG556, while Y108F lost response to PP2. The double-mutant Y108F-Y136F hKv4.3 channels showed no response to either AG556 or PP2. CONCLUSION Our results demonstrate that human atrial I(to) and cloned hKv4.3 channels are modulated by EGFR kinase via phosphorylation of the Y136 residue and by Src-family kinases via phosphorylation of the Y108 residue; tyrosine phosphorylation of the channel may be involved in regulating cardiac electrophysiology.