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1.
Acta Physiologica Sinica ; (6): 187-195, 2019.
Article in English | WPRIM | ID: wpr-777197

ABSTRACT

Renin-angiotensin system (RAS) is involved in the regulation of vascular smooth muscle cell (VSMC) tension. Angiotensin II (Ang II) as the main effector molecule of RAS can increase the intracellular Ca concentration and cause VSMCs contraction by activating angiotensin II type 1 receptor (AT1R). The large-conductance Ca- and voltage-activated potassium (BK) channel is an essential potassium channel in VSMCs, playing an important role in maintaining membrane potential and intracellular potassium-calcium balance. The BK channel in VSMCs mainly consists of α and β1 subunits. Functional BKα subunits contain voltage-sensors and Ca binding sites. Hence, increase in the membrane potential or intracellular Ca concentration can trigger the opening of the BK channel by mediating transient K outward current in a negative regulatory manner. However, increasing evidence has shown that although Ang II can raise the intracellular Ca concentration, it also inhibits the expression and function of the BK channel by activating the PKC pathway, internalizing AT1R-BKα heterodimer, or dissociating α and β1 subunits. Under some specific conditions, Ang II can also activate the BK channel, but the underlying mechanism remains unknown. In this review, we summarize the potential mechanisms underlying the inhibitory or activating effect of Ang II on the BK channel, hoping that it could provide a theoretical basis for improving intracellular ion imbalance.


Subject(s)
Angiotensin II , Physiology , Calcium , Physiology , Humans , Large-Conductance Calcium-Activated Potassium Channels , Physiology , Muscle, Smooth, Vascular , Cell Biology , Myocytes, Smooth Muscle , Physiology , Renin-Angiotensin System
2.
Acta Physiologica Sinica ; (6): 305-310, 2017.
Article in Chinese | WPRIM | ID: wpr-348270

ABSTRACT

This study was aimed to establish a method to create a stable planar lipid bilayer membranes (PLBMs), in which large conductance calcium-activated potassium channels (BK) were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of N-weathered mixture of phosphatidylcholine and cholesterol at 3:1 ratio. After successful incorporation of BKchannel into PLBMs, single channel characteristics of BKwere studied by patch clamp method. The results showed that i) the single channel conductance of BKwas (206.8 ± 16.9) pS; ii) the activities of BKchannel were voltage dependent; iii) in the bath solution without Ca, there was almost no BKchannel activities regardless of under hyperpolarization or repolarization conditions; iv) under the condition of +40 mV membrane potential, BKchannels were activated in a Caconcentration dependent manner; v) when [Ca] was increased from 1 μmol/L to 100 μmol/L, both the channel open probability and the average open time were increased, and the average close time was decreased from (32.2 ± 2.8) ms to (2.1 ± 1.8) ms; vi) the reverse potential of the reconstituted BKwas -30 mV when [K] was at 40/140 mmol/L (Cis/Trans). These results suggest that the spreading method could serve as a new method for preparing PLBMs and the reconstituted BKinto PLBMs showed similar electrophysiological characteristics to natural BKchannels, so the PLBMs with incorporated BKcan be used in the studies of pharmacology and dynamics of BKchannel.


Subject(s)
Animals , Calcium , Chemistry , Electrophysiological Phenomena , Large-Conductance Calcium-Activated Potassium Channels , Chemistry , Lipid Bilayers , Chemistry , Membrane Potentials , Patch-Clamp Techniques
3.
Chinese Medical Journal ; (24): 200-205, 2016.
Article in English | WPRIM | ID: wpr-310683

ABSTRACT

<p><b>BACKGROUND</b>Adenomyosis (AM) has impaired contraction. This study aimed to explore the expression of potassium channels related to contraction in myometrial smooth muscle cells (MSMCs) of AM.</p><p><b>METHODS</b>Uterine tissue samples from 22 patients (cases) with histologically confirmed AM and 12 (controls) with cervical intraepithelial neoplasia were collected for both immunohistochemistry and real-time polymerase chain reaction to detect the expression of large conductance calcium- and voltage-sensitive K + channel (BKCa)-α/β subunits, voltage-gated potassium channel (Kv) 4.2, and Kv4.3. Student's t-test was used to compare the expression.</p><p><b>RESULTS</b>The BKCa-α/β subunits, Kv4.2, and Kv4.3 were located in smooth muscle cells, glandular epithelium, and stromal cells. However, BKCa-β subunit expression in endometrial glands of the controls was weak, and Kv4.3 was almost undetectable in the controls. The expression of BKCa-α messenger RNA (mRNA) (0.62 ± 0.19-fold decrease, P < 0.05) and Kv4.3 mRNA (0.67 ± 0.20-fold decrease, P < 0.05) decreased significantly in the MSMCs of the control group compared with the AM group. However, there were no significant differences in BKCa-β subunit mRNA or Kv4.2 mRNA.</p><p><b>CONCLUSIONS</b>The BKCa-α mRNA and the Kv4.3 mRNA are expressed significantly higher in AM than those in the control group, that might cause the abnormal uterus smooth muscle contractility, change the microcirculation of uterus to accumulate the inflammatory factors, impair the endometrium further, and aggravate the pain.</p>


Subject(s)
Adenomyosis , Metabolism , Adult , Female , Humans , Immunohistochemistry , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Male , Myocytes, Smooth Muscle , Metabolism , Potassium Channels, Voltage-Gated , Metabolism , Real-Time Polymerase Chain Reaction , Shal Potassium Channels , Metabolism , Uterine Contraction , Physiology , Uterine Neoplasms , Metabolism , Uterus , Metabolism
4.
Article in English | WPRIM | ID: wpr-250314

ABSTRACT

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.


Subject(s)
Animals , Aorta , Cell Biology , Metabolism , Physiology , Calcium Signaling , Cells, Cultured , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Male , Muscle, Smooth, Vascular , Metabolism , Physiology , Myocytes, Smooth Muscle , Metabolism , Norepinephrine , Pharmacology , Rats , Rats, Sprague-Dawley , Ryanodine , Pharmacology , Tacrolimus , Pharmacology , Vasoconstriction
5.
Chinese Journal of Burns ; (6): 363-369, 2016.
Article in Chinese | WPRIM | ID: wpr-277068

ABSTRACT

<p><b>OBJECTIVE</b>To study the changes in the expression of large-conductance calcium-activated potassium (BKCa) channels in dorsal root ganglion (DRG) neurons after electrical injury in rats' sciatic nerves and its influence on sensory conduction function.</p><p><b>METHODS</b>One-hundred and thirty-six adult SD rats were divided into normal control group, sham electrical injury group, and 75, 100, 125 V electrical injury groups according to the random number table, with 8 rats in normal control group and 32 rats in each of the rest 4 groups. Rats in normal control group were routinely fed without any treatment. Blunt dissection of the sciatic nerves of left hind leg of rats was performed in sham electrical injury group, while sciatic nerves of left hind leg of rats in electrical injury groups were electrically injured with corresponding voltage. Eight rats of normal control group fed for one week, and 8 rats from each of the rest four groups on post injury day (PID) 3 and in post injury week (PIW) 1, 2, 3 respectively were collected to detect the paw withdrawal mechanical threshold (PWMT). In addition, rats of 100 V electrical injury group in PIW 1 were collected and intrathecally injected with NS1619 after former PWMT detection, and PWMT was detected per 30 minutes within three hours post injection. The rats in each group at each time point were sacrificed after PWMT detection. The DRG of L4 to L6 segments of spinal cord was sampled to observe the BKCa channels distribution with immunohistochemical staining and to detect the protein and mRNA expressions of BKCa channels with Western blotting and reverse transcription-polymerase chain reaction respectively. Data were processed with one-way analysis of variance, analysis of variance of factorial design, and SNK test.</p><p><b>RESULTS</b>(1) The PWMT values of rats in 75 and 100 V electrical injury groups on PID 3 and in PIW 1, 2, 3 were (5.8±0.6), (5.0±0.8), (4.2±0.3), (5.9±1.1) g; (5.3±1.3), (5.9±2.0), (4.5±2.7), (4.3±1.3) g, respectively, which were significantly lower than the value (s) in normal control group [(11.2±2.0) g] and sham electrical injury group [respectively (11.3±2.1), (12.0±2.0), (11.1±1.6), (10.3±2.1) g, with P values below 0.05]. The PWMT values of rats in 125 V electrical injury group decreased obviously on PID 3 and in PIW 1 [(6.1±1.6) and (5.7±1.7) g] as compared with the value (s) in normal control group and sham electrical injury group, and they were obviously increased in PIW 2 and 3 [(26.7±3.3) and (21.7±3.4) g] as compared with the value (s) of the rest 4 groups (with P values below 0.05). The PWMT of 100 V electrical injury group in PIW 1 firstly increased and then decreased within three hours post injection, which increased significantly at post injection minutes 30, 60, 90, 120 as compared with that before intervention [respectively (8.5±0.8), (9.7±1.2), (11.0±1.5), (8.6±0.8) g, with P values below 0.05]. (2) The positive expression of BKCa channels in large amount was observed in the cytoplasm and cytomembrane of neurons on the DRG of rats in normal control group and sham electrical injury group at each time point. The positive expression of BKCa channels in the cytoplasm and cytomembrane of neurons on the DRG of rats decreased over time in electrical injury groups, which was most obvious in 125 V electrical injury group. (3) There were no statistically significant differences in the protein expression of BKCa channels in DRG of rats among the five groups on PID 3 (with P values above 0.05). Compared with those in normal control group (0.477±0.027, 0.521±0.034, 0.475±0.022) and sham electrical injury group (0.511±0.025, 0.489±0.025, 0.483±0.032) in PIW 1, 2, 3, the protein expressions of BKCa channels in DRG of rats in 75, 100, 125 V electrical injury groups were decreased significantly (0.274±0.026, 0.202±0.019, 0.285±0.033; 0.253±0.022, 0.233±0.024, 0.203±0.017; 0.092±0.017, 0.095±0.021, 0.087±0.016, with P values below 0.05). The protein expressions of BKCa channels in DRG of rats in 125 V electrical injury group in PIW 1, 2, 3 were obviously lower than those in 75 and 100 V electrical injury groups (with P values below 0.05). (4) The mRNA expression levels of BKCa channels in DRG of rats in 75, 100, 125 V electrical injury groups on PID 3 and in PIW 1, 2, 3 were 0.326±0.021, 0.238±0.019, 0.291±0.022, 0.364±0.018; 0.264±0.020, 0.293±0.017, 0.243±0.023, 0.295±0.021; 0.134±0.023, 0.089±0.017, 0.074±0.018, 0.087±0.020, respectively, significantly decreased as compared with the level (s) in normal control group (0.581±0.051) and sham electrical injury group (0.603±0.045, 0.586±0.032, 0.614±0.045, 0.572±0.038), with P values below 0.05. The mRNA expression levels of BKCa channels in DRG of rats in 125 V electrical injury group at each time point were lower than those in 75 and 100 V electrical injury groups (with P values below 0.05).</p><p><b>CONCLUSIONS</b>The electrical injury in sciatic nerves results in reduction of the BKCa channels expression in rat's DRG of corresponding spinal segments, which plays a role in the pathological process of sensory conduction dysfunction.</p>


Subject(s)
Animals , Blotting, Western , Electricity , Ganglia, Spinal , Metabolism , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Random Allocation , Rats , Rats, Sprague-Dawley , Sciatic Nerve , Wounds and Injuries
6.
Article in Chinese | WPRIM | ID: wpr-254953

ABSTRACT

<p><b>OBJECTIVE</b>To observe the effect of β₃adrenoceptors (β₃-AR) activation on rat thoracic aorta smooth muscle contractility and the possible related mechanism.</p><p><b>METHODS</b>The endothelium removed thoracic aorta was pre-contracted with 30 mmol/L KCl physiological saline solution (PSS). Then the tension of the thoracic aorta was recorded in presence of BRL37344 (BRL) to determine the action of β₃-AR. The tension of the thoracic aorta was also recorded in the presence of Propranolol (PRA), SR59230A (SR), L-NNA, H-89 and Iberiotoxin (IBTX) respectively to reveal the underling mechanism of β₃-AR activation on rat vascular smooth muscle. Immunohistochemistry was adopted to confirm the existence and the distribution of β₃-AR in rat thoracic aorta.</p><p><b>RESULTS</b>The results showed that: (1) The thoracic aorta was relaxed by β₃-AR activation, with a relaxation percentage of (10.59 ± 0.79). (2) β₃-AR was expressed in both endothelial and smooth muscle layer in thoracic aorta sections of rats. (3) PRA did not block the effect of BRL on the thoracic aorta. The relaxation actions of BRL could be antagonized by pre-incubating the thoracic aorta with SR. (4) L-NNA (a NOS inhibitor) and H-89 (a PKA inhibitor) reversed the relaxation effect of BRL on vascular smooth muscle. (5) The effect of BRL was decreased after application of Ibriotoxin (IBTX), a large conductance calcium dependent potassium channel blocker.</p><p><b>CONCLUSION</b>The results confirmed that activation of β₃-AR led to relaxation of thoracic aorta smooth muscle. The relaxation action of β₃-AR on smooth muscle of rat thoracic aorta was related to activation of NOS and PKA signaling pathway. Large conductance Ca²⁺-K⁺ channels were involved in the relaxation action of β₃-AR activation on rat thoracic aorta smooth muscle.</p>


Subject(s)
Animals , Aorta, Thoracic , Physiology , In Vitro Techniques , Isoquinolines , Large-Conductance Calcium-Activated Potassium Channels , Physiology , Muscle Contraction , Muscle Relaxation , Muscle, Smooth, Vascular , Physiology , Nitroarginine , Peptides , Propanolamines , Propranolol , Rats , Receptors, Adrenergic, beta-3 , Physiology , Signal Transduction , Sulfonamides
7.
Article in English | WPRIM | ID: wpr-188411

ABSTRACT

PURPOSE: Familial hypokalemic periodic paralysis (HOKPP) is an autosomal dominant channelopathy characterized by episodic attacks of muscle weakness and hypokalemia. Mutations in the calcium channel gene, CACNA1S, or the sodium channel gene, SCN4A, have been found to be responsible for HOKPP; however, the mechanism that causes hypokalemia remains to be determined. The aim of this study was to improve the understanding of this mechanism by investigating the expression of calcium-activated potassium (KCa) channel genes in HOKPP patients. METHODS: We measured the intracellular calcium concentration with fura-2-acetoxymethyl ester in skeletal muscle cells of HOKPP patients and healthy individuals. We examined the mRNA and protein expression of KCa channel genes (KCNMA1, KCNN1, KCNN2, KCNN3, and KCNN4) in both cell types. RESULTS: Patient cells exhibited higher cytosolic calcium levels than normal cells. Quantitative reverse transcription polymerase chain reaction analysis showed that the mRNA levels of the KCa channel genes did not significantly differ between patient and normal cells. However, western blot analysis showed that protein levels of the KCNMA1 gene, which encodes KCa1.1 channels (also called big potassium channels), were significantly lower in the membrane fraction and higher in the cytosolic fraction of patient cells than normal cells. When patient cells were exposed to 50 mM potassium buffer, which was used to induce depolarization, the altered subcellular distribution of BK channels remained unchanged. CONCLUSION: These findings suggest a novel mechanism for the development of hypokalemia and paralysis in HOKPP and demonstrate a connection between disease-associated mutations in calcium/sodium channels and pathogenic changes in nonmutant potassium channels.


Subject(s)
Blotting, Western , Calcium , Calcium Channels , Channelopathies , Cytosol , Humans , Hypokalemia , Hypokalemic Periodic Paralysis , Large-Conductance Calcium-Activated Potassium Channels , Membranes , Muscle Weakness , Muscle, Skeletal , Paralysis , Polymerase Chain Reaction , Potassium , Potassium Channels , Potassium Channels, Calcium-Activated , Reverse Transcription , RNA, Messenger , Sodium Channels
8.
Acta Physiologica Sinica ; (6): 302-306, 2014.
Article in Chinese | WPRIM | ID: wpr-297488

ABSTRACT

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.


Subject(s)
Animals , Calcium , Metabolism , Cochlea , Cell Biology , Guinea Pigs , Hair Cells, Vestibular , Hydrogen Peroxide , Pharmacology , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Membrane Potentials
9.
Chinese Medical Journal ; (24): 1775-1781, 2014.
Article in English | WPRIM | ID: wpr-248113

ABSTRACT

<p><b>OBJECTIVE</b>This review focuses on the role of the large conductance calcium-activated potassium (BKCa) channels in diabetic vascular complications.</p><p><b>DATA SOURCES</b>Relevant articles published in English or Chinese from 1981 to present were selected from PubMed. The search terms were "BKCa channels" and "diabetes". Important references from selected articles were also retrieved.</p><p><b>STUDY SELECTION</b>Articles regarding the role of BKCa channels in diabetic vascular complications and relevant mechanisms were selected.</p><p><b>RESULTS</b>The BKCa channels are abundantly expressed in vascular smooth cells and play an important role in regulation of vascular tone. Multiple studies indicated that the expression and function of BKCa channels are altered by different mechanisms in diabetic vascular diseases such as coronary arterial disease, cerebral arterial disease, and diabetic retinopathy.</p><p><b>CONCLUSION</b>BKCa channels may play an important role in diabetic vascular complications and may be an effective therapeutic target for relieving and reducing the burden of diabetic vascular complications.</p>


Subject(s)
Cerebral Arterial Diseases , Metabolism , Coronary Artery Disease , Diabetic Angiopathies , Metabolism , Diabetic Retinopathy , Metabolism , Humans , Large-Conductance Calcium-Activated Potassium Channels , Metabolism
10.
Acta Physiologica Sinica ; (6): 39-46, 2013.
Article in Chinese | WPRIM | ID: wpr-333137

ABSTRACT

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.


Subject(s)
Angiotensin II , Pharmacology , Humans , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Mesenteric Arteries , Cell Biology , Muscle, Smooth, Vascular , Cell Biology , Myocytes, Smooth Muscle , Metabolism , Patch-Clamp Techniques , Receptor, Angiotensin, Type 1 , Metabolism , Receptor, Angiotensin, Type 2 , Metabolism , Tetrazoles , Pharmacology , Valine , Pharmacology , Valsartan , Vasodilation
11.
Chinese Journal of Cardiology ; (12): 770-774, 2012.
Article in Chinese | WPRIM | ID: wpr-326423

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the changes of open probability (Po) of large conductance Ca(2+)-activated K(+) channel (BK channel) in diabetic coronary smooth muscle cells and elucidate the underlying cellular electrophysiology mechanisms of coronary dysfunction.</p><p><b>METHODS</b>Rat coronary smooth muscle cells were isolated from control group and diabetic group. BK single channel currents were recorded by patch clamp technique in inside-out configuration. Open probabilities were calculated and compared between two groups. After exposure to DHS-1, a specific BK channel activator, Po at 0.2 and 1 µmol/L free Ca(2+) were compared between control and diabetic groups.</p><p><b>RESULTS</b>In the presence of 0.2 µmol/L free Ca(2+), the Po at baseline was significantly lower in diabetic rats than in control rats (0.0032 ± 0.0012 vs. 0.095 ± 0.036, P < 0.05). Cytoplasmic application of DSH-1 significantly increased the Po to 0.335 ± 0.096 (P < 0.05 vs. baseline) in control rats, whereas DSH-1 had no effect in diabetic rats (Po = 0.022 ± 0.018, P > 0.05 vs. baseline). In the presence of 1 µmol/L free Ca(2+), the Po at baseline was also significantly lower in diabetic rats than in control rats (0.210 ± 0.055 vs. 0.458 ± 0.077, P < 0.05). Cytoplasmic application of DHS-1 further robustly enhanced Po to 0.823 ± 0.019 (P < 0.05 vs. baseline) in control rats and to 0.446 ± 0.098 in diabetic rats (P < 0.05 vs. baseline of diabetic rats; P < 0.05 vs. control rats with DHS-1).</p><p><b>CONCLUSION</b>The decrease of Po of BK single channel in coronary smooth muscle cells may be a potential cause for coronary dysfunction in diabetic rats.</p>


Subject(s)
Animals , Coronary Vessels , Metabolism , Diabetes Mellitus, Experimental , Metabolism , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Male , Muscle, Smooth, Vascular , Cell Biology , Myocytes, Smooth Muscle , Metabolism , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley
12.
Article in Chinese | WPRIM | ID: wpr-329955

ABSTRACT

<p><b>OBJECTIVE</b>The present study was to explore signaling mechanisms underlying nicotine-induced inhibition of large-conductance calcium-activated potassium channels (BK(Ca)).</p><p><b>METHODS</b>8 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.</p><p><b>RESULTS</b>CPT-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.</p><p><b>CONCLUSION</b>This phenomenon may serve as a physiological mechanism that nicotine inhibits BK(Ca) channel activity to increase via cAMP/PKA-dependent pathway.</p>


Subject(s)
Animals , Arteries , Cell Biology , Metabolism , Coronary Vessels , Cell Biology , Metabolism , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Male , Myocytes, Smooth Muscle , Metabolism , Nicotine , Pharmacology , Patch-Clamp Techniques , Rats , Rats, Wistar , Signal Transduction
13.
Acta Physiologica Sinica ; (6): 121-128, 2012.
Article in Chinese | WPRIM | ID: wpr-335933

ABSTRACT

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).


Subject(s)
Aged , Estradiol , Pharmacology , Female , Humans , Hypertension , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Physiology , Mesenteric Arteries , Metabolism , Physiology , Middle Aged , Muscle, Smooth, Vascular , Cell Biology , Metabolism , Physiology , Patch-Clamp Techniques , Postmenopause , Physiology , Receptors, Estrogen
14.
Acta Physiologica Sinica ; (6): 355-364, 2012.
Article in English | WPRIM | ID: wpr-333191

ABSTRACT

The large-conductance calcium-activated potassium (BK) channels distributed in both excitable and non-excitable cells are key participants in a variety of physiological functions. By employing numerous high-affinity natural toxins originated from scorpion venoms the pharmacological and structural characteristics of these channels tend to be approached. A 37-residue short-chain peptide, named as martentoxin, arising from the venom of the East-Asian scorpion (Buthus martensi Karsch) has been investigated with a comparatively higher preference for BK channels over other voltage-gated potassium (Kv) channels. Up to now, since the specific drug tool probing for clarifying structure-function of BK channel subtypes and related pathology remain scarce, it is of importance to illuminate the underlying mechanism of molecular interaction between martentoxin and BK channels. As for it, the current review will address the recent progress on the studies of pharmacological characterizations and molecular determinants of martentoxin targeting on BK channels.


Subject(s)
Amino Acid Sequence , Humans , Large-Conductance Calcium-Activated Potassium Channels , Ligands , Peptides , Chemistry , Scorpion Venoms , Chemistry
15.
Chinese Journal of Cardiology ; (12): 157-160, 2012.
Article in Chinese | WPRIM | ID: wpr-275084

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the role of C-type natriuretic peptide receptor (NPR-C) and large-conductance calcium-activated potassium channels (BK(Ca)) in brain natriuretic peptide (BNP) induced porcine coronary artery dilation.</p><p><b>METHODS</b>Porcine coronary artery rings were obtained and treated with BNP (10(-6) mol/L), BNP + NPR-C antagonist cANF4-28 (10(-6) mol/L) and BNP + BK(Ca) blocker tetraethylammonium (TEA, 1 mmol/L). The vascular tone experiments were observed on 10 vessel segments. BK(Ca) current density was measured by the whole-cell patch clamp technique.</p><p><b>RESULTS</b>The maximum diastolic rate was similar between BNP group (68.51% ± 11.50%) and cANF4-28 + BNP group (65.67% ± 11.90%, P > 0.05) while significantly reduced in TEA + BNP group (28.87% ± 4.55%, all P < 0.05). When the holding potential was set at +60 mV, the BK(Ca) current density of BNP group was (78.48 ± 5.86) pA/pF, which was significantly higher than control group [(53.84 ± 4.55) pA/pF, P < 0.05], which was equally reduced in the TEA group and TEA + BNP group [(28.80 ± 2.76) pA/pF and (30.60 ± 3.88) pA/pF respectively, all P < 0.05 vs. control group].</p><p><b>CONCLUSION</b>BNP could relax the porcine coronary arterial smooth muscles by increasing BK(Ca) current, and this effect is not mediated by NPR-C.</p>


Subject(s)
Animals , Coronary Vessels , Physiology , Large-Conductance Calcium-Activated Potassium Channels , Physiology , Natriuretic Peptide, Brain , Pharmacology , Patch-Clamp Techniques , Receptors, Atrial Natriuretic Factor , Physiology , Swine
16.
Chinese Journal of Cardiology ; (12): 421-426, 2012.
Article in Chinese | WPRIM | ID: wpr-275031

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and voltage-dependent K(+) (K(V)) channels in rat coronary artery smooth muscle cells (CASMCs), and evaluate the vasorelaxation mechanisms of DHA.</p><p><b>METHODS</b>BK(Ca) and K(V) currents in individual CASMC were recorded by patch-clamp technique in whole-cell configuration. Effects of DHA at various concentrations (0, 10, 20, 40, 60 and 80 µmol/L) on BK(Ca) and K(V) channels were observed.</p><p><b>RESULTS</b>(1) DHA enhanced IBK(Ca) and BK(Ca) tail currents in a concentration-dependent manner while did not affect the stably activated curves of IBK(Ca). IBK(Ca) current densities were (68.2 ± 22.8), (72.4 ± 24.5), (120.4 ± 37.9), (237.5 ± 53.2), (323.6 ± 74.8) and (370.6 ± 88.2)pA/pF respectively (P < 0.05, n = 30) with the addition of 0, 10, 20, 40, 60 and 80 µmol/L DHA concentration, and half-effect concentration (EC(50)) of DHA was (36.22 ± 2.17)µmol/L. (2) IK(V) and K(V) tail currents were gradually reduced, stably activated curves of IK(V) were shift to the right, and stably inactivated curves were shifted to the left in the presence of DHA. IK(V) current densities were (43.9 ± 2.3), (43.8 ± 2.3), (42.9 ± 2.0), (32.3 ± 1.9), (11.7 ± 1.5) and (9.6 ± 1.2)pA/pF respectively(P < 0.05, n = 30)post treatment with 0, 10, 20, 40, 60 and 80 µmol/L DHA under manding potential equal to +50 mV, and EC(50) of DHA was (44.19 ± 0.63)µmol/L.</p><p><b>CONCLUSION</b>DHA can activate BK(Ca) channels and block K(V) channels in rat CASMCs, the combined effects on BK(Ca) and K(V) channels lead to the vasodilation effects of DHA on vascular smooth muscle cells.</p>


Subject(s)
Animals , Coronary Vessels , Cell Biology , Metabolism , Docosahexaenoic Acids , Pharmacology , Female , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Male , Myocytes, Smooth Muscle , Metabolism , Patch-Clamp Techniques , Potassium Channels, Calcium-Activated , Metabolism , Rats , Rats, Sprague-Dawley
17.
Article in Chinese | WPRIM | ID: wpr-268951

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the role of K(Ca)3.1 channel in the proliferation and migration of rat vascular smooth muscle cells of the proliferative phenotype.</p><p><b>METHODS</b>Rat 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Upregulated expression of K(Ca)3.1 channel can promote the proliferation and migration of rat VSMCs of the proliferative phenotype.</p>


Subject(s)
Animals , Cell Movement , Cell Proliferation , Cells, Cultured , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Muscle, Smooth, Vascular , Cell Biology , Metabolism , Myocytes, Smooth Muscle , Cell Biology , Metabolism , Potassium Channel Blockers , Pharmacology , Pyrazoles , Pharmacology , Rats , Rats, Sprague-Dawley
18.
Chinese Medical Journal ; (24): 2548-2555, 2012.
Article in English | WPRIM | ID: wpr-283724

ABSTRACT

<p><b>BACKGROUND</b>Diabetes mellitus is associated with coronary dysfunction, contributing to a 2- to 4-fold increase in the risk of coronary heart diseases. The mechanisms by which diabetes induces vasculopathy involve endothelial-dependent and -independent vascular dysfunction in both type 1 and type 2 diabetes mellitus. The purpose of this study is to determine the role of vascular large conductance Ca(2+)-activated K(+) (BK) channel activities in coronary dysfunction in streptozotocin-induced diabetic rats.</p><p><b>METHODS</b>Using videomicroscopy, immunoblotting, fluorescent assay and patch clamp techniques, we investigated the coronary BK channel activities and BK channel-mediated coronary vasoreactivity in streptozotocin-induced diabetic rats.</p><p><b>RESULTS</b>BK currents (defined as the iberiotoxin-sensitive K(+) component) contribute (65 ± 4)% of the total K(+) currents in freshly isolated coronary smooth muscle cells and > 50% of the contraction of the inner diameter of coronary arteries from normal rats. However, BK current density is remarkably reduced in coronary smooth muscle cells of streptozotocin-induced diabetic rats, leading to an increase in coronary artery tension. BK channel activity in response to free Ca(2+) is impaired in diabetic rats. Moreover, cytoplasmic application of DHS-1 (a specific BK channel b(1) subunit activator) robustly enhanced the open probability of BK channels in coronary smooth muscle cells of normal rats. In diabetic rats, the DHS-1 effect was diminished in the presence of 200 nmol/L Ca(2+) and was significantly attenuated in the presence of high free calcium concentration, i.e., 1 mmol/L Ca(2+). Immunoblotting experiments confirmed that there was a 2-fold decrease in BK-b(1) protein expression in diabetic vessels, without altering the BK channel α-subunit expression. Although the cytosolic Ca(2+) concentration of coronary arterial smooth muscle cells was increased from (103 ± 23) nmol/L (n = 5) of control rats to (193 ± 22) nmol/L (n = 6, P < 0.05) of STZ-induced diabetic rats, reduced BK-b(1) expression made these channels less sensitive to intracellular Ca(2+), which in turn led to enhanced smooth muscle contraction.</p><p><b>CONCLUSIONS</b>Our results indicated that BK channels are the key determinant of coronary arterial tone. Impaired BK channel function in diabetes mellitus is associated with down-regulation of BK-b(1) expression and reduction of the b(1)-mediated BK channel activation in diabetic vessels.</p>


Subject(s)
Animals , Blotting, Western , Coronary Vessels , Metabolism , Diabetes Mellitus, Experimental , Metabolism , Diabetes Mellitus, Type 1 , Metabolism , Electrophysiology , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Male , Muscle, Smooth, Vascular , Metabolism , Rats , Rats, Sprague-Dawley
19.
Protein & Cell ; (12): 883-892, 2012.
Article in English | WPRIM | ID: wpr-757860

ABSTRACT

Large-conductance Ca²⁺-activated K⁺ channels (BK channels) constitute an key physiological link between cellular Ca²⁺ signaling and electrical signaling at the plasma membrane. Thus these channels are critical to the control of action potential firing and neurotransmitter release in several types of neurons, as well as the dynamic control of smooth muscle tone in resistance arteries, airway, and bladder. Recent advances in our understanding of K⁺ channel structure and function have led to new insight toward the molecular mechanisms of opening and closing (gating) of these channels. Here we will focus on mechanisms of BK channel gating by Ca²⁺, transmembrane voltage, and auxiliary subunit proteins.


Subject(s)
Animals , Calcium Signaling , Cytoplasm , Metabolism , Electric Conductivity , Electrophysiological Phenomena , Humans , Ion Channel Gating , Large-Conductance Calcium-Activated Potassium Channels , Chemistry , Metabolism , Protein Subunits , Chemistry , Metabolism
20.
Chinese Journal of Cardiology ; (12): 348-352, 2011.
Article in Chinese | WPRIM | ID: wpr-272247

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the mechanism of enhanced large conductance calcium-activated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA).</p><p><b>METHODS</b>Coronary 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.</p><p><b>RESULTS</b>BK 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.</p><p><b>CONCLUSION</b>DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.</p>


Subject(s)
Animals , Coronary Vessels , Cell Biology , Metabolism , Cytochrome P-450 Enzyme Inhibitors , Docosahexaenoic Acids , Pharmacology , Fatty Acids, Unsaturated , Pharmacology , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Muscle, Smooth, Vascular , Metabolism , Myocytes, Smooth Muscle , Metabolism , Proadifen , Pharmacology , Rats , Rats, Sprague-Dawley
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