To detect the phosphorylation of potassium channel protein (Kv1.2 and Kv1.5) in rat cardiac muscle cells accurately, we applied the combined method of immunoprecipitation and Western blot in this study. Compared with using Western blot alone, the combination of immunoprecipitation and Western blot displayed high sensitivity to detect the activation of potassium channel proteins. Because of its simplicity, quickness and reproducibility, we find that this method was promising for detecting the phosphorylation of Kv1.2 and Kv1.5 proteins or other potassium channel proteins in rat cardiac muscle cells.
Animals ; Blotting, Western ; methods ; Cell Membrane ; metabolism ; Female ; Immunoprecipitation ; methods ; Kv1.2 Potassium Channel ; analysis ; genetics ; metabolism ; Kv1.5 Potassium Channel ; analysis ; genetics ; metabolism ; Male ; Myocytes, Cardiac ; cytology ; metabolism ; Phosphorylation ; Rats ; Rats, Sprague-Dawley

To investigate the role of potassium channels in the pathogenesis of airway hyperresponsiveness induced by cigarette smoking, the alteration in expression of large-conductance calcium-activated potassium channel (BKca) and voltage-dependent delayed rectifier potassium channel (Kv1.5) in bronchial smooth muscle cells were investigated in chronic cigarette smoking rats. Airway responsiveness was determined, hematoxylin and eosin staining, immuno-histochemistry, in-situ hybridization and western blot techniques were used. The results showed: (1) Chronic cigarette smoking down-regulated the protein synthesis and mRNA expression of BKca and Kv1.5 in bronchial and bronchiolar smooth muscles. (2) BKca decreased more markedly than Kv1.5 in bronchi, but there was no difference between them in bronchioli. (3) No changes in the expression of these two potassium channel proteins were found in extracted cell membrane protein from lung tissue. The results suggest that chronic cigarette smoking can down-regulate the levels of BKca and Kv1.5 in rat bronchial smooth muscle cells in vivo, which might contribute to the mechanism of airway hyperresponsiveness induced by cigarette smoking.
Animals ; Bronchi ; metabolism ; Cells, Cultured ; Kv1.5 Potassium Channel ; Male ; Muscle, Smooth ; cytology ; metabolism ; Potassium Channels, Calcium-Activated ; biosynthesis ; genetics ; Potassium Channels, Voltage-Gated ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Smoking ; adverse effects

BACKGROUNDPotassium (K+) channels are important in regulating cell membrane potential and excitability. Although bronchial myocytes from asthmatic rats show a significant reduction in voltage-dependent delayed rectifier potassium channel (Kv) current density and higher excitability, the activity and expression of Kv in human bronchial smooth muscle cells (HBSMCs) have never been studied. The objective of this study was to investigate the effect of passive sensitization by asthmatic serum on the activity of Kv and the expression of Kv isoform Kv1.5 in HBSMCs.

METHODSHBSMCs were randomly divided into two groups: control group (containing 10% serum from nonatopic individuals) and sensitized group (containing 10% asthmatic serum), then cultured for 24 hours. Whole-cell patch clamp, immunofluorescence staining, reverse transcription-polymerase chain reaction and Western blot techniques were used to study the effect of passive sensitization on the activity of Kv and the expression of Kv1.5 in HBSMCs.

RESULTSThe membrane potential in passively sensitized HBSMCs was significantly depolarized to -(26.7 +/- 5.2) mV compared with -(41.3 +/- 6.4) mV in the control group (P < 0.01). Passive sensitization caused a significant inhibition of Kv currents in HBSMCs, resulting in a downward shift in the current-voltage (I-V) relationship curve. At +50 mV, the peak Kv current density of passively sensitized HBSMCs was significantly decreased from (54.6 +/- 8.7) picoamperes per picofarad (pA/pF) to (32.1 +/- 7.1) pA/pF (P < 0.01). The expression level of Kv1.5 mRNA in passively sensitized HBSMCs was significantly lower than that in the control group (0.76 +/- 0.07 vs 1.04 +/- 0.13, P < 0.05). The expression of Kv1.5 protein of passively sensitized HBSMCs was also significantly reduced compared to that from the control group (984 +/- 168 vs 2200 +/- 380, P < 0.05).

CONCLUSIONSThe activity and expression of Kv were all decreased in HBSMCs passively sensitized by asthmatic serum compared with nonsensitized cells. These changes might be involved in the mechanisms of formation and development of asthma.

Asthma ; blood ; Bronchi ; metabolism ; Cells, Cultured ; Female ; Fluorescent Antibody Technique ; Humans ; Immunization ; Kv1.5 Potassium Channel ; Male ; Myocytes, Smooth Muscle ; metabolism ; Potassium Channels, Voltage-Gated ; analysis ; genetics ; RNA, Messenger ; analysis

Large-conductance calcium-activated potassium channel (BK(Ca)) and voltage-gated potassium channel Kv1.5 play an important role in the pathogenesis of bronchial hyperresponsiveness (BHR). It is known that cigarette smoke can induce BHR, however, the role of BK(Ca) and Kv1.5 expression in it remains to be further elucidated. The purpose of the present study was to investigate the direct effects of cigarette smoke extract (CSE) on BK(Ca) and Kv1.5 expression, and the role of protein kinase C (PKC) isoforms activation in primary cultured rat bronchial smooth muscle cells (BSMCs). Primarily cultured rat BSMCs were treated with 5% CSE, the expression and translocation of PKC isoforms were measured by Western blot, and the mRNA and protein levels of BK(Ca) and Kv1.5 alpha-subunits were determined by semi-quantitative RT-PCR and Western blot, respectively. The results showed that 5% CSE induced the translocation of PKCepsilon, PKCeta, PKCtheta from soluble fraction to particulate fraction, and reduced mRNA and protein expressions of BK(Ca) and Kv1.5 alpha-subunits. The decreased expression of potassium channels was partly restored by PKC inhibitor, BIM or Goe6983. In summary, CSE may activate PKC isoforms epsilon, eta, theta, thereby down-regulate the expressions of BK(Ca) and Kv1.5 in BSMCs.
Animals ; Bronchi ; cytology ; Cells, Cultured ; Kv1.5 Potassium Channel ; metabolism ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; enzymology ; Protein Isoforms ; metabolism ; Protein Kinase C ; metabolism ; Rats ; Smoke ; adverse effects ; Tobacco

AIMTo study the mRNA expression changes in the brain of rats after middle cerebral artery occlusion.

METHODSMiddle cerebral artery occlusion was used to induce ischemia in rat brain. The mRNA expression of voltage-dependent potassium channel subtypes, including Kv1.4, Kv1.5, Kv2.1 and Kv4.2, were detected in rat hippocampus and cortex by RT-PCR.

RESULTSMiddle cerebral artery occlusion induced a significant neurological injury in rats. After ischemia 2 h, the mRNA of Kv1.4, Kv2.1 and Kv4.2 in hippocampus increased by 50%, 67% and 90% , respectively. And the mRNA of Kv1.4 and Kv4.2 maintained at a high level in hippocampus after ischemia 24 h. In cortex, the mRNA level of all the four subtypes were not changed significantly after ischemia 2 h, but the mRNA of Kv2.1 and Kv4.2 increased by 70% and 62% after ischemia 24 h, respectively.

CONCLUSIONThe mRNA expression levels of voltage-dependent potassium channels were up-regulated in rat hippocampus and cortex after middle cerebral artery occlusion.

Animals ; Brain ; metabolism ; Infarction, Middle Cerebral Artery ; metabolism ; Kv1.4 Potassium Channel ; biosynthesis ; genetics ; Kv1.5 Potassium Channel ; biosynthesis ; genetics ; Male ; Potassium Channels, Voltage-Gated ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Shab Potassium Channels ; biosynthesis ; genetics ; Shal Potassium Channels ; biosynthesis ; genetics ; Up-Regulation

The purpose of the present study was to investigate the effects of different concentrations of ethanol on action potential (AP) in the isolated rat myocardium and the possible mechanism of electric-physiological changes. Standard microelectrode technique was used to record AP in isolated rat myocardium, and whole cell patch clamp technique was used to record the human Kv1.5 (hKv1.5) channel currents in HEK293 cells. The effects of different concentrations of ethanol (6.25, 12.5, 25.0, 50.0, 100.0 and 200.0 mmol/L) on AP parameters in rat atrium and papillary and Kv1.5 channel currents in HEK293 cells were analyzed. The results showed that in isolated atrium, action potential amplitude (APA), action potential duration (APD), action potential duration of 50% repolarization (APD(50)) and action potential duration of 90% repolarization (APD(90)) were not affected by 6.25 and 12.5 mmol/L ethanol, while APD, APD(50) and APD(90) were prolonged significantly by 25.0-200.0 mmol/L ethanol (P < 0.05 or P < 0.01), and APA was reduced with 100.0 and 200.0 mmol/L ethanol (P < 0.05 or P < 0.01). In isolated papillary, APA, APD, APD(50) and APD(90) were not affected by 6.25-25.0 mmol/L ethanol, while APD, APD(50) and APD(90) were prolonged significantly with 50.0-200.0 mmol/L ethanol (P < 0.05 or P < 0.01), and APA was reduced with 200.0 mmol/L ethanol (P < 0.05). The Kv1.5 channel currents were inhibited by ethanol in a concentration dependent manner in HEK293 cells. These findings suggest that 6.25 and 12.5 mmol/L ethanol produce no effects on AP parameters, and 50.0-200.0 mmol/L ethanol prolong APD significantly in isolated rat atrium and papillary. The prolonged effect on APD in isolated myocardium may be due to the inhibition of the Kv1.5 channel currents.
Action Potentials ; drug effects ; Animals ; Dose-Response Relationship, Drug ; Ethanol ; pharmacology ; HEK293 Cells ; Humans ; Kv1.5 Potassium Channel ; antagonists & inhibitors ; drug effects ; Male ; Myocardium ; metabolism ; Myocytes, Cardiac ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley

PURPOSE: Pulmonary Kv channels are thought to play a crucial role in the regulation of cell proliferation and apoptosis. Previous studies have shown that fluoxetine upregulated the expression of Kv1.5 and prevented pulmonary arterial hypertension in monocrotaline-induced or hypoxia-induced rats and mice. The current study was designed to test how fluoxetine regulates Kv1.5 channels, subsequently promoting apoptosis in human PASMCs cultured in vitro. MATERIALS AND METHODS: Human PASMCs were incubated with low-serum DMEM, ET-1, and fluoxetine with and without ET-1 separately for 72 h. Then the proliferation, apoptosis, and expression of TRPC1 and Kv1.5 were detected. RESULTS: In the ET-1 induced group, the upregulation of TRPC1 and down regulation of Kv1.5 enhanced proliferation and anti-apoptosis, which was reversed when treated with fluoxetine. The decreased expression of TRPC1 increased the expression of Kv1.5, subsequently inhibiting proliferation while promoting apoptosis. CONCLUSION: The results from the present study suggested that fluoxetine protects against big endothelin-1 induced anti-apoptosis and rescues Kv1.5 channels in human pulmonary arterial smooth muscle cells, potentially by decreasing intracellular concentrations of Ca2+.
Apoptosis/drug effects/genetics ; Blotting, Western ; Cell Proliferation/drug effects ; Cells, Cultured ; Endothelin-1/*pharmacology ; Flow Cytometry ; Fluoxetine/*pharmacology ; Humans ; Kv1.5 Potassium Channel/genetics/*metabolism ; Muscle, Smooth, Vascular/*cytology/drug effects ; Pulmonary Artery/*cytology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the effects of puerarin on proliferation, apoptosis and Kv1.5 gene expression of rat pulmonary artery smooth muscle cells (PASMCs) induced by hypoxia.

METHODSThe rat PASMCs were divided into 5 groups: control group, hypoxia group, hypoxia plus puerarin (1 × 10(-5) mol/L) group, hypoxia plus puerarin (1 × 10(-4) mol/L) group and hypoxia plus puerarin (1 × 10(-3) mol/L) group, and cultured at 37°C for 24 h. The proliferation of rat PASMCs was detected by CCK-8 assay and flow cytometry, the activity of caspase-3 was measured with spectrophotometric method, Kv1.5 protein was detected by western blot, Kv1.5 mRNA was detected by real-time PCR.

RESULTSThe cell viability and proportion of synthesis phase in control group were 0.940 ± 0.045 and 9.67% ± 1.28%, which were significantly lower than those (1.296 ± 0.034 and 18.19% ± 1.19%) in hypoxia group (P < 0.05). The Caspase-3 activity, Kv 1.5 protein and Kv 1.5 mRNA in control group were 0.1073 ± 0.0113, 0.886 ± 0.038 and 0.0377 ± 0.0031, which were significantly higher than those (0.0664 ± 0.0049, 0.602 ± 0.064 and 0.0108 ± 0.0014) in hypoxia group (P < 0.05). As compared with hypoxia group, the cell viability and proportion of synthesis phase in 3 hypoxia plus puerarin groups significantly decreased, and the Caspase-3 activity, Kv 1.5 protein and Kv 1.5 mRNA in 3 hypoxia plus puerarin groups significantly enhanced (P < 0.05).

CONCLUSIONPuerarin could decrease the proliferation and increase the apoptosis induced by hypoxia in rat PASMCs, and the up-regulated expression of Kv1.5 gene may be the mechanism of puerarin effects.

Animals ; Apoptosis ; drug effects ; Cell Hypoxia ; Cell Proliferation ; drug effects ; Cells, Cultured ; Isoflavones ; pharmacology ; Kv1.5 Potassium Channel ; metabolism ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; Pulmonary Artery ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of exercise stress on chronic cigarette smoking induced downregulation of large conductance calcium-activated potassium channel (BKca) and voltage-dependent delayed rectifier potassium channel (Kv1.5) expression in pulmonary arterial smooth muscle cells of rats.

METHODSRats were divided into three groups: the normal control group, the smoking control group and the smoking + exercise group. The plasma cortisol level, the potassium channel expression and the pathological changes in lung tissue were determined with HE staining, the immunohistochemistry and the in-situ hybridization.

RESULTS(1) In the smoking + exercise group, the plasma cortisol level was determined immediately after exercise [(1528.7 +/- 469.7) ng/L] and was higher than that determined before exercise [(672.4 +/- 235.7) ng/L] (P < 0.01); (2) The HE staining showed that the chronic pulmonary inflammatory response in the smoking control group was severe while it was mild in the smoking + exercise group; (3) The mRNA and protein expression (OD value) of BKca in the smoking control group (mRNA: 0.2206 +/- 0.0415 for big artery and 0.3935 +/- 0.1378 for small artery; protein: 0.2634 +/- 0.1219 for big artery and 0.0995 +/- 0.0851 for small artery) were less than those in the normal control group. The mRNA expression of BKca in the smoking + exercise group (OD value) (0.5022 +/- 0.1134 for big artery and 0.6408 +/- 0.2135 for small artery) was higher than that in the smoking control group; (4) The mRNA and protein expression of Kv1.5 in the smoking control group (OD value) (mRNA: 0.9354 +/- 0.3290 for big artery and 0.5012 +/- 0.1170 for small artery; protein: 1.1112 +/- 0.3310 for big artery and 0.4736 +/- 0.1250 for small artery) were less than those in the normal control group. The protein expression of Kv1.5 in the smoking + exercise group (0.7445 +/- 0.2690) in small artery was higher than that in the smoking control group.

CONCLUSIONProper exercise stress can decrease inhibition effect of the chronic smoking on the expression of potassium channel BKca and Kv1.5, which perhaps partly results from exercise induced increase of cortisol secretion.

Animals ; Down-Regulation ; Hydrocortisone ; blood ; Kv1.5 Potassium Channel ; biosynthesis ; genetics ; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits ; Male ; Movement ; physiology ; Muscle, Smooth, Vascular ; metabolism ; Potassium Channels ; biosynthesis ; genetics ; Pulmonary Artery ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Smoking ; adverse effects

AIMTo observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE.

METHODSIn the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia.

RESULTSBlocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs.

CONCLUSIONThe results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.

Animals ; Cell Hypoxia ; Cells, Cultured ; Hydroxyeicosatetraenoic Acids ; pharmacology ; Hypoxia ; physiopathology ; Kv1.5 Potassium Channel ; biosynthesis ; genetics ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Potassium Channels, Voltage-Gated ; antagonists & inhibitors ; Pulmonary Artery ; physiopathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Shab Potassium Channels ; biosynthesis ; genetics ; Vasoconstriction ; drug effects