This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.
Osteogenesis ; Core Binding Factor Alpha 1 Subunit/pharmacology* ; Large-Conductance Calcium-Activated Potassium Channels/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Calcium/metabolism* ; Cell Differentiation ; RNA, Messenger/metabolism* ; Cell Proliferation ; Osteoblasts

The present study attempted to test a novel hypothesis that Ca(2+) sparks play an important role in arterial relaxation induced by tacrolimus. Recorded with confocal laser scanning microscopy, tacrolimus (10 µmol/L) increased the frequency of Ca(2+) sparks, which could be reversed by ryanodine (10 µmol/L). Electrophysiological experiments revealed that tacrolimus (10 µmol/L) increased the large-conductance Ca(2+)-activated K(+) currents (BKCa) in rat aortic vascular smooth muscle cells (AVSMCs), which could be blocked by ryanodine (10 µmol/L). Furthermore, tacrolimus (10 and 50 µmol/L) reduced the contractile force induced by norepinephrine (NE) or KCl in aortic vascular smooth muscle in a concentration-dependent manner, which could be also significantly attenuated by iberiotoxin (100 nmol/L) and ryanodine (10 µmol/L) respectively. In conclusion, tacrolimus could indirectly activate BKCa currents by increasing Ca(2+) sparks released from ryanodine receptors, which inhibited the NE- or KCl-induced contraction in rat aorta.
Animals ; Aorta ; cytology ; metabolism ; physiology ; Calcium Signaling ; Cells, Cultured ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Male ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; physiology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ryanodine ; pharmacology ; Tacrolimus ; pharmacology ; Vasoconstriction

The present study was aimed to investigate the effect of hydrogen peroxide (H₂O₂, oxygen free radical donator) on the current of large-conductance calcium-activated potassium channels (BK(Ca) channels) in isolated outer hair cells of old guinea pig cochlea, and to explore the underlying mechanism. Outer hair cells of old guinea pig cochlea were acutely enzyme-isolated, and currents were recorded by whole-cell patch clamp. The results showed that, rapid activation and non-deactivation electric currents with a string of large amplitude were recorded. Activation voltage of the current was above -40 - -30 mV. The amplitude of current was increased continuously with the rising of membrane potential. The current showed characteristics of outward rectification without "rundown" phenomenon. IbTX (100 nmol/L) could completely block the activity of channel, which confirmed BK(Ca) channel's current. BK(Ca) current amplitude and peak current density increased with the increment of H₂O₂ concentration (1, 2, 4 μmol/L), showing concentration-dependent activation by H₂O₂. Our results suggest that oxygen free radical/BK(Ca) pathway may be able to adjust the balance of intracellular calcium in outer hair cells.
Animals ; Calcium ; metabolism ; Cochlea ; cytology ; Guinea Pigs ; Hair Cells, Vestibular ; drug effects ; Hydrogen Peroxide ; pharmacology ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Membrane Potentials

The aim of present study was to explore the vasodilatation mechanism of angiotensin II (AngII) at the molecular level by investigating the effect of AngII on large-conductance Ca²⁺-activated potassium channels (BK(Ca)) in human mesenteric artery smooth muscle cells. The effect of AngII on BK(Ca) was observed by using patch clamp single channel recording technique and amphotericin-perforated whole-cell recording technique. AngII type 1 receptor (AT₁R) and AngII type 2 receptor (AT₂R) mRNA expression in human mesenteric artery was detected by RT-PCR. In cell-attached patch (Vm = +40 mV), AngII (100 nmol/L) had no significant effect on BK(Ca). After pretreatment with Valsartan (a specific inhibitor of AT₁R, 10 μmol/L), 25, 100 and 250 nmol/L AngII stimulated BK(Ca) activity significantly in a dose response manner. After pretreatment of Valsartan, AngII (100 nmol/L) enhanced BK(Ca) open probability (NP(O)) from 0.010 ± 0.003 to 0.039 ± 0.015, decreased the mean close time (T(C)) of BK(Ca) markedly from (2 729.5 ± 808.6) ms to (487.7 ± 182.5) ms (n = 11, P < 0.05) , but AngII had no significant influences on the amplitude (Amp) and the mean open time (T(O)) of BK(Ca). Further PD123,319 (a specific inhibitor of AT₂R) treatment prevented the stimulatory effect of AngII: PD123,319 decreased the NP(O) of BK(Ca) from 0.016 ± 0.003 to 0.004 ± 0.001 (n = 5, P < 0.05), but had no significant influences on Amp, T(O) and T(C) of BK(Ca). In addition, after pretreatment with Valsartan and PD123,319, AngII (100 nmol/L) had no significant effect on BK(Ca). In the amphotericin-perforated whole-cell patch-clamp configuration, after pretreatment with Valsartan, the current density of BK(Ca) at the voltage of -60 - +30 mV had no significant changes before and after adding 100 nmol/L AngII, but the current density of BK(Ca) at the voltage of +40 mV, +50 mV and +60 mV increased significantly after adding 100 nmol/L AngII, from (9.03 ± 2.23) pA/pF, (12.88 ± 2.55) pA/pF and (17.26 ± 2.84) pA/pF to (12.47 ± 2.22) pA/pF, (18.71 ± 2.51) pA/pF and (27.21 ± 3.12) pA/pF (n = 6, P < 0.05), respectively. Using RT-PCR, the AT₁R mRNA and AT₂R mRNA from isolated human mesenteric artery were detected. So we can draw a conclusion, AngII can stimulate BK(Ca) activity in human mesenteric artery smooth muscle cells after pretreatment with Valsartan, which is possibly mediated by AT₂R.
Angiotensin II ; pharmacology ; Humans ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Mesenteric Arteries ; cytology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Patch-Clamp Techniques ; Receptor, Angiotensin, Type 1 ; metabolism ; Receptor, Angiotensin, Type 2 ; metabolism ; Tetrazoles ; pharmacology ; Valine ; analogs & derivatives ; pharmacology ; Valsartan ; Vasodilation

OBJECTIVEThe present study was to explore signaling mechanisms underlying nicotine-induced inhibition of large-conductance calcium-activated potassium channels (BK(Ca)).

METHODS8 week male Wistar rats were divided randomly into saline group and nicotine group and received respectively injection with saline or nicotine (Sigma, Shanghai, China) at 2 mg/(kg x d) for 21 days. Coronary vascular smooth muscle cells were dissociated enzymatically. Dissociated smooth muscle cells were interfered with CPT-cAMP (100 micromol/L) or forskolin (10 micromol/L). The signal channel open dwell-time (To), close dwell-time (Tc) and open probability (Po) were recorded.

RESULTSCPT-cAMP or forskolin significantly prolonged To, shorten Tc and increased Po in saline group (P < 0.01). But in nicotine group To, Tc and Po did not been changed.

CONCLUSIONThis phenomenon may serve as a physiological mechanism that nicotine inhibits BK(Ca) channel activity to increase via cAMP/PKA-dependent pathway.

Animals ; Arteries ; cytology ; drug effects ; metabolism ; Coronary Vessels ; cytology ; drug effects ; metabolism ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Male ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Nicotine ; pharmacology ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Signal Transduction

The aim of the present study was to study the effect of β-estradiol (β-E(2)) on the large-conductance Ca(2+)-activated potassium (BK(Ca)) channel in mesenteric artery smooth muscle cells (SMCs). The mesenteric arteries were obtained from post-menopause female patients with abdominal surgery, and the SMCs were isolated from the arteries using an enzymatic disassociation. According to the sources, the SMCs were divided into non-hypertension (NH) and essential hypertension (EH) groups. Single channel patch clamp technique was used to investigate the effect of β-E(2) and ICI 182780 (a specific blocker of estrogen receptor) on BK(Ca) in the SMCs. The results showed the opening of BK(Ca) in the SMCs was voltage and calcium dependent, and could be blocked by IbTX. β-E(2) (100 μmol/L) significantly increased open probability (Po) of BK(Ca) in both NH and EH groups. After β-E(2) treatment, NH group showed higher Po of BK(Ca) compared with EH group. ICI 182780 could inhibit the activating effect of β-E(2) on BK(Ca) in no matter NH or EH groups. These results suggest β-E(2) activates BK(Ca) in mesenteric artery SMCs from post-menopause women via estrogen receptor, but hypertension may decline the activating effect of β-E(2) on BK(Ca).
Aged ; Estradiol ; analogs & derivatives ; pharmacology ; Female ; Humans ; Hypertension ; physiopathology ; Large-Conductance Calcium-Activated Potassium Channels ; agonists ; metabolism ; physiology ; Mesenteric Arteries ; metabolism ; physiology ; Middle Aged ; Muscle, Smooth, Vascular ; cytology ; metabolism ; physiology ; Patch-Clamp Techniques ; Postmenopause ; physiology ; Receptors, Estrogen ; antagonists & inhibitors

OBJECTIVETo investigate the role of K(Ca)3.1 channel in the proliferation and migration of rat vascular smooth muscle cells of the proliferative phenotype.

METHODSRat vascular smooth muscle cells (VSMCs) were cultured with tissue adhesion method. The morphological characteristics of the fist and ninth passages of VSMCs were observed with light and electron microscopy and immunocytochemistry. The expressions of K(Ca)3.1 channel mRNA and protein in the cells were detected using RT-PCR and immunocytochemistry, respectively. MTT and transwell assay were employed to assess the effect of the K(Ca)3.1 channel blocker TRAM-34 on the proliferation and migration of VSMCs.

RESULTSThe first and ninth passages of VSMCs showed morphological characteristics of contractile and proliferative phenotypes, respectively. Compared with the first- passage cells, the ninth-passage VSMCs exhibited significantly increased K(Ca)3.1 channel mRNA and protein expressions with enhanced cell proliferation and migration (P<0.01), which was inhibited by the application of TRAM-34 (P<0.01). TRAM-34 produced no obvious effect on the first-passage VSMCs.

CONCLUSIONUpregulated expression of K(Ca)3.1 channel can promote the proliferation and migration of rat VSMCs of the proliferative phenotype.

Animals ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Potassium Channel Blockers ; pharmacology ; Pyrazoles ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the mechanism of enhanced large conductance calcium-activated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA).

METHODSCoronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16, 17-epoxydocosapentaenoic acid (16, 17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration.

RESULTSBK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2 ± 2.7)% of total potassium currents (n = 20). DHA could activate BK channels, and its 50% effective concentration (EC(50)) was (0.23 ± 0.03) µmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16, 17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC(50) was (19.7 ± 2.8) nmol/L.

CONCLUSIONDHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.

Animals ; Coronary Vessels ; cytology ; drug effects ; metabolism ; Cytochrome P-450 Enzyme Inhibitors ; Docosahexaenoic Acids ; pharmacology ; Fatty Acids, Unsaturated ; pharmacology ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Proadifen ; pharmacology ; Rats ; Rats, Sprague-Dawley

To investigate the characteristics of large-conductance calcium-activated potassium channels (BK(Ca)) and the effect of hydrogen peroxide (H2O2) on BK(Ca) in guinea-pig proximal colon smooth muscle cells, single smooth muscle cells of guinea-pig colon were enzymatically isolated in low calcium solution containing papain (3 mg/mL), DTT (2 mg/mL), and bovine serum albumin (BSA, 2 mg/mL). Tissues were incubated at 36 degrees C in enzyme solution for 15 min and were then suspended in enzyme-free low calcium solution. Inside-out single channel recording technique was used to record BK(Ca) current. The intracellular (bath) and microelectrode solution both contained symmetrical high potassium. The BK(Ca) in guinea-pig colon smooth muscle cell possesses: 1) voltage-dependence, 2) high selectivity for potassium ion, 3) large conductance (223.7 pS+/-9.2 pS), 4) dependence of [Ca(2+)](i). Intracellular application of H2O2 decreased the open probability (P(o)) of BK(Ca) at low concentration (conductance. The effects of H2O2 were reversed by reducing agent dithiothreitol (DTT). Similarly, cysteine specific oxidizing agent, DTNB, also increased or decreased P(o) of BK(Ca) and DTT partially reversed the effect of DTNB. It is thus suggested that H2O2 and DTNB may modulate P(o) of BK(Ca) via the oxidation of cysteine residue.
Animals ; Colon ; cytology ; Guinea Pigs ; Hydrogen Peroxide ; pharmacology ; Large-Conductance Calcium-Activated Potassium Channels ; physiology ; Myocytes, Smooth Muscle ; physiology ; Oxidation-Reduction

The goal of the present study was to investigate the effects of quercetin-filled phosphatidylcholine liposomes (PCL-Q) on the currents carried by large conductance Ca(2+)-dependent K(+) channels (BK(Ca)) in rat thoracic aorta following non-fatal whole-body ionizing irradiation. Using patch-clamp technique, it is found that the outward K(+) currents of isolated smooth muscle cells (SMCs) stimulated by depolarizing voltage steps were sensitive to BK(Ca) inhibitor, paxilline, and this kind of outward K(+) currents in SMCs from irradiated animals demonstrated a significant decrease in amplitude. Radiation-induced BK(Ca) suppression was evident 9 days post-irradiation and progressively increased over 30 days of experimental period. Thus, the vasorelaxing force of these SMCs may be diminished following irradiation. PCL-Q effectively restored BK(Ca) function in post-irradiated SMCs. It is noteworthy that the constituents of PCL-Q, i.e., free quercetin (Q) and "empty" liposomes (PCL), being taken separately, showed a decreased ability to recover BK(Ca) function as compared with combined composition. These results suggest that PCL-Q is able to regain normal function of BK(Ca) following irradiation. The protective effects of PCL-Q can be explained by its antioxidant and membrane repairing properties as well as its ability to inhibit protein kinase C activity. Thus, the lipid encapsulation of flavonoid, PCL-Q, appears to be a potential medication in the case of ionizing irradiation accident, and for the patients with neoplasm who have to receive external radiotherapy as well.
Animals ; Aorta, Thoracic ; drug effects ; radiation effects ; Large-Conductance Calcium-Activated Potassium Channels ; physiology ; Liposomes ; chemistry ; Myocytes, Smooth Muscle ; physiology ; Patch-Clamp Techniques ; Phosphatidylcholines ; chemistry ; Quercetin ; pharmacology ; Radiation, Ionizing ; Rats