The receptor-dependent and non-receptor-dependent mechanism of melatonin activated BK channels in middle cerebral artery myocyte / 中国应用生理学杂志

OBJECTIVE@#To investigate the mechanisms through which myocyte large-conductance Ca-activated K (BK) channels mediate the vasodilation effects of melatonin on cerebral arteries (CAs).@*METHODS@#Middle cerebral arteries (MCA) were obtained from 8-week-old male Wistar rats after anaesthetized. Middle cerebral arterial smooth muscle cells were enzymatically isolated. Whole cell recording mode of patch clamp technique was used to measure the current density of BK channel and voltage-gated potassium (K) channel before and after adding melatonin. Currents density of melatonin on BK channels with melatonin receptor inhibitor 2-phenyl-N-acetyl (luzindole) was recorded using whole cell recording mode and open probability (Po) was recorded using single-channel attached recording mode. The conductance (G) and average open time (To) and off time (Tc) of the BK channel were detected before and after the addition of melatonin in the internal-outward mode.@*RESULTS@#① Melatonin markedly increased the whole-cell BK channel current density but not the voltage-gated potassium (K) channel current density. ② Luzindole (1 μmol/L) greatly suppressed melatonin-induced increase of BK channel current density. ③ The Po of BK channel was significantly increased by melatonin (100 μmol/L) under cell attached recording mode, which was markedly inhibited by luzindole (1 μmol/L). ④ In inside-outside recording mode, melatonin (1 μmol/L, 100 μmol/L) reduced both To and Tc of BK channel, and Tc was reduced much more than To.@*CONCLUSIONS@#Melatonin mediates vasodilation of MCA through the activation of BK channels both melatonin receptor dependent and independent mode.

Involvement of K+ATP and Ca2+ channels in hydrogen sulfide-suppressed ageing of porcine oocytes

BACKGROUND: Hydrogen sulfide has been shown to improve the quality of oocytes destined for in vitro fertilization. Although hydrogen sulfide is capable of modulating ion channel activity in somatic cells, the role of hydrogen sulfide in gametes and embryos remains unknown. Our observations confirmed the hypothesis that the KATP and L-type Ca2+ ion channels play roles in porcine oocyte ageing and revealed a plausible contribution of hydrogen sulfide to the modulation of ion channel activity. RESULTS: We confirmed the benefits of the activation and suppression of the KATP and L-type Ca2+ ion channels, respectively, for the preservation of oocyte quality. CONCLUSIONS: Our experiments identified hydrogen sulfide as promoting the desired ion channel activity, with the capacity to protect porcine oocytes against cell death. Further experiments are needed to determine the exact mechanism of hydrogen sulfide in gametes and embryos.

Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BK(Ca) activator LDD175

Plasma membrane hyperpolarization associated with activation of Ca²⁺-activated K⁺ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pH-sensitive, sperm-specific K⁺ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BK(Ca) activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K⁺ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K⁺ current, and internal alkalinization and Ca²⁺ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K⁺ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca²⁺. In contrast, elevation of intracellular Ca²⁺ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca²⁺-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

Spike Frequency Adaptation in Neurons of the Central Nervous System

Neuronal firing patterns and frequencies determine the nature of encoded information of the neurons. Here we discuss the molecular identity and cellular mechanisms of spike-frequency adaptation in central nervous system (CNS) neurons. Calcium-activated potassium (K(Ca)) channels such as BK(Ca) and SK(Ca) channels have long been known to be important mediators of spike adaptation via generation of a large afterhyperpolarization when neurons are hyper-activated. However, it has been shown that a strong hyperpolarization via these KCa channels would cease action potential generation rather than reducing the frequency of spike generation. In some types of neurons, the strong hyperpolarization is followed by oscillatory activity in these neurons. Recently, spike-frequency adaptation in thalamocortical (TC) and CA1 hippocampal neurons is shown to be mediated by the Ca²⁺-activated Cl- channel (CACC), anoctamin-2 (ANO2). Knockdown of ANO2 in these neurons results in significantly reduced spike-frequency adaptation accompanied by increased number of spikes without shifting the firing mode, which suggests that ANO2 mediates a genuine form of spike adaptation, finely tuning the frequency of spikes in these neurons. Based on the finding of a broad expression of this new class of CACC in the brain, it can be proposed that the ANO2-mediated spike-frequency adaptation may be a general mechanism to control information transmission in the CNS neurons.

Pharmacological study of the mechanisms involved in the vasodilator effect produced by the acute application of triiodothyronine to rat aortic rings

A relationship between thyroid hormones and the cardiovascular system has been well established in the literature. The present in vitro study aimed to investigate the mechanisms involved in the vasodilator effect produced by the acute application of 10-8–10-4 M triiodothyronine (T3) to isolated rat aortic rings. Thoracic aortic rings from 80 adult male Wistar rats were isolated and mounted in tissue chambers filled with Krebs-Henseleit bicarbonate buffer in order to analyze the influence of endothelial tissue, inhibitors and blockers on the vascular effect produced by T3. T3 induced a vasorelaxant response in phenylephrine-precontracted rat aortic rings at higher concentrations (10-4.5–10-4.0 M). This outcome was unaffected by 3.1×10-7 M glibenclamide, 10-3 M 4-aminopyridine (4-AP), 10-5 M indomethacin, or 10-5 M cycloheximide. Contrarily, vasorelaxant responses to T3 were significantly (P<0.05) attenuated by endothelium removal or the application of 10-6 M atropine, 10-5 M L-NG-nitroarginine methyl ester (L-NAME), 10-7 M 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10-6 M (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i](1,6)benzodiazocine-10-carboxylic acid, methyl ester KT 5823, 10-2 M tetraethylammonium (TEA), or 10-7 M apamin plus 10-7 M charybdotoxin. The results suggest the involvement of endothelial mechanisms in the vasodilator effect produced by the acute in vitro application of T3 to rat aortic rings. Possible mechanisms include the stimulation of muscarinic receptors, activation of the NO-cGMP-PKG pathway, and opening of Ca2+-activated K+ channels.

Relaxation mechanism of smooth muscle cells and its relationship with penile erection / 中华男科学杂志

The contractile and diastolic function of smooth muscle cells (SMCs) is closely related to penile erection and erectile dysfunction (ED). In addition to nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), sulfur dioxide (SO2), estrogen receptor (ER), P2Y receptor, perivascular tissue (PVT), and calcium activated potassium channel (Kca) are found to be involved in the relaxation of SMCs. This review updates the mechanisms of the relaxation of SMCs and its relationship with ED.

Effects of hydrogen peroxide on voltage-dependent K+ currents in human cardiac fibroblasts through protein kinase pathways

Human cardiac fibroblasts (HCFs) have various voltage-dependent K+ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide (H2O2) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether H2O2 could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of H2O2 stimulated Ca2+-activated K+ (K(Ca)) currents but not delayed rectifier K+ or transient outward K+ currents, all of which are VDKCs. H2O2-stimulated K(Ca) currents were blocked by iberiotoxin (IbTX, a large conductance K(Ca) blocker). The H2O2-stimulating effect on large-conductance K(Ca) (BK(Ca)) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated BK(Ca) currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the H2O2-stimulating effect on BK(Ca) currents. Using RT-PCR and western blot analysis, three subtypes of K(Ca) channels were detected in HCFs: BK(Ca) channels, small-conductance K(Ca) (SK(Ca)) channels, and intermediate-conductance K(Ca) (IK(Ca)) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to H2O2, but IbTX decreased H2O2-induced apoptosis. These data suggest that among the VDKCs of HCFs, H2O2 only enhances BK(Ca) currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through BK(Ca) channels.

17Beta-Estradiol Inhibits Calcium-Activated Potassium Channel Expressions in Rat Whole Bladder / 대한배뇨장애요실금학회지

PURPOSE: To investigate the effect of estrogen on the expression of calcium-activated potassium (KCa) channels in an overactive bladder rat model. To this end, mRNA and protein levels of KCa channel subtypes in the bladder of ovariectomized rats were measured by reverse transcription polymerase chain reaction and western blotting, respectively. METHODS: Ten-week-old female Sprague-Dawley rats were divided randomly into 3 groups: sham-operated control group (n=11), ovariectomy group (n=11), and the group treated with estrogen after ovariectomy (n=12). Rats in the last group were subcutaneously injected with 17β-estradiol (50 µg/kg) every other day for 2 weeks, whereas rats in the other 2 groups received vehicle (soybean oil) alone. Two weeks after treatment, the whole bladder was excised for mRNA and protein measurements. RESULTS: Protein levels of the large-conductance KCa (BK) channels in the ovariectomy group were 1.5 folds higher than those in the sham-operated control group. However, the protein levels of the other KCa channel subtypes did not change significantly upon bilateral ovariectomy. Treatment with 17β-estradiol after ovariectomy restored BK channel protein levels to the control value. In contrast, BK channel mRNA levels were not significantly affected by either ovariectomy alone or 17β-estradiol treatment. The small-conductance KCa type 3 channel (SK3) mRNA and protein levels decreased to 75% of control levels upon 17β-estradiol treatment. CONCLUSIONS: These results suggest that 17β-estradiol may influence urinary bladder function by modulating BK and SK3 channel expression.

Isoliquiritigenin relaxes the cerebral basilar artery by enhancing BKCa current in spontaneously hypertensive rat: role of sGC/cGMP / 生理学报

The purpose of the present study is to investigate the effect of isoliquiritigenin (ISL) on the cerebral basilar artery in spontaneously hypertensive rats (SHR). The change of SHR systolic pressure was measured by tail artery pressure measurement instrument before and after ISL intervention. After perfusion with 1 × 10(-5) mol/L phenylephrine (PE), 1 × 10(-5) mol/L PE + 1 × 10(-4) mol/L ISL and 1 × 10(-5) mol/L PE, the diameter of the cerebral basilar artery separated from SHR was measured by pressure myograph. The current of large-conductance calcium-activated potassium (BKCa) channel of SHR single vascular smooth muscle cell (VSMC) was recorded by whole-cell patch-clamp technique and the cGMP levels of basilar artery was evaluated by ELISA. The results showed that 1) after intervention with ISL for 14 days, the systolic pressure of SHR was decreased from (218.3 ± 1.6) mmHg to (119.2 ± 1.9) mmHg (P < 0.01), but there was no difference in systolic pressure between ISL-treated SHR and Wistar-Kyoto (WKY) rat; 2) 1 × 10(-4) mol/L ISL relaxed the SHR cerebral basilar artery (P < 0.01); 3) ISL significantly increased the outward current density of VSMC from SHR cerebral basilar artery (P < 0.01, n = 6), and the effect could be reversed by 1 × 10(-3) mol/L TEA (a BKCa channel inhibitor), but 3 × 10(-4) mol/L 4-AP (a Kv channel inhibitor) had no effect on the enhanced current density induced by ISL in VSMC; 4) 1 × 10(-5) mol/L Methylene blue (a sGC inhibitor) significantly inhibited the ISL-enhanced current density in VSMC (P < 0.05, n = 6); 5) ISL significantly increased the cGMP level of SHR basilar artery (P < 0.05, n = 6). The results suggest that the role of the ISL in relaxing the SHR cerebral basilar artery may be related to its effect in enhancing BKCa current by increasing the levels of cGMP in the VSMC.

Characteristics of K+ Outward Currents in the Cochlear Outer Hair Cells of Circling Mice within the First Postnatal Week

K+ outward currents in the outer hair cells (OHCs) of circling mice (homozygous (cir/cir) mice), an animal model for human deafness (DFNB6 type), were investigated using a whole cell patch clamp technique. Littermate heterozygous (+/cir) mice of the same age (postnatal day (P) 0 -P6) were used as controls. Similar slow rising K+ currents were observed in both genotypes, but their biophysical and pharmacological properties were quite different. The values of V(half) for activation were significantly different in the heterozygous (+/cir) and homozygous (cir/cir) mice (-8.1+/-2.2 mV, heterozygous (+/cir) mice (n=7) and -17.2+/-4.2 mV, homozygous (cir/cir) mice (n=5)). The inactivation curve was expressed by a single first order Boltzmann equation in the homozygous (cir/cir) mice, while it was expressed by a sum of two first order Boltzmann equations in the heterozygous (+/cir) mice. The K+ current of homozygous (cir/cir) mice was more sensitive to TEA in the 1 to 10 mM range, while the 4-AP sensitivities were not different between the two genotypes. Removal of external Ca2+ did not affect the K+ currents in either genotype, indicating that the higher sensitivity of K+ current to TEA in the homozygous (cir/cir) mice was not due to an early expression of Ca2+ activated K+ channels. Our results suggest that the K+ outward current of developing homozygous (cir/cir) mice OHCs is different in both biophysical and pharmacological aspects than that of heterozygous (+/cir) mice.

Relation of intermediate-conductance Ca(2+)-activated K(+) channels with ability of proliferation, migration, invasion and IgE secretion of multiple myeloma cells / 中国实验血液学杂志

This study was aimed to investigate the effects of the intermediate-conductance Ca(2+)-activated K(+) (IKCa1) channels on the proliferation, migration, invasion ability and monoclonal immunoglobulin (IgE) secretion of multiple myeloma (MM) cells. Trypan blue exclusion was used to evaluate the impact of clotrimazole (CLO, an inhibitor of the KCa1) on the survival ability of MM cell line U266; transwell chamber and matrigel experiments were used to evaluate the impact of CLO on the ability of the migration and invasion of U266 cells; the influence of CLO on IgE secretion in U266 cells was detected by ELISA. The results showed that small dose of CLO ( ≤ 1.0 µmol/L) could not inhibit the viability of U266 cells. The Transwell and Matrigel invading tests displayed that the cell number moving into lower chamber of transwell decreased after U266 cells treated with small dose of CLO ( ≤ 1.0 µmol/L). After treating the cells with 1.00 µmol/L CLO for 24 h and 48 h, the concentration of IgE in cell supernatant was (4.98 ± 0.39) and (4.38 ± 0.32) ng/ml, while those in control group were (15.41 ± 1.88) and (19.73 ± 2.01) ng/ml, respectively, suggesting significant difference between them(P < 0.05). It is concluded that CLO can decrease the ability of migration and monoclonal immunoglobulin secretion of multiple myeloma cells by blocking the IKCa1, thus this study provides a new think for the targeted therapy of MM.

Role of calcium activated-potassium channels in the injury to rat alveolar macrophages induced by quartz / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of calcium activated-potassium channels (KCa) in the injury to rat alveolar macrophages induced by quartz.</p><p><b>METHODS</b>The experiments were conducted on a rat alveolar macrophage cell line (NR8383) in vitro, where crystal silica (100 üg/ml) and amorphous silica (100 üg/ml) were used as the test substances and the cells without any treatment as negative controls. At first the effects of two kinds of quartz were compared. Then KCa special inhibitors (Paxilline for BK, Tram-34 for IK, Apamin for SK) were added in different doses to the in vitro test system with 100 üg/ml crystal quartz as matrix, to observe the function of such channels. Cell viability, lactate dehydrogenase (LDH), interleukin-1β (IL-1β) and tumor necrosis factor-a (TNF-α) were tested.</p><p><b>RESULTS</b>Comparing to the negative control group, cell viability reduced, LDH leakage, IL-1β and TNF-α release increased significantly in the amorphous quartz group, furthermore, the effects by crystal quartz were much more serious than those by amorphous quartz, with a statistical significance (P < 0.01). Comparing to the crystal quartz group, IK blockers (Tram-34) led to increase in cell viability significantly, with a statistical significance (P < 0.01); all the KCa specific blockers (Paxilline, Tram-34, Apamin) could reduce LDH leakage and IL-1β release, with a statistical significance (P < 0.05); meanwhile, BK and IK blockers (Paxilline,Tram-34) were able to reduce TNF-α release,with a statistical significance (P < 0.05).Reduction of IL-1β and TNF-α by Tram-34 was dose-dependent, but not so in the other two blockers.</p><p><b>CONCLUSION</b>Blocking calcium-activated potassium channels (KCa) could reduce cell membrane damage as well as IL-1β and TNF-α release induced by crystal quartz in the rat alveolar macrophages cell line in vitro, which might serve as a signal in the early regulation of inflammatory responses by quartz.</p>

The large-conductance calcium-activated potassium channel holds the key to the conundrum of familial hypokalemic periodic paralysis / 소아과

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.

Differential effect of calcium-activated potassium and chloride channels on rat basilar artery vasomotion / 华中科技大学学报（医学）（英德文版）

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. We investigated the cellular mechanisms underlying vasomotion in the cerebral basilar artery (BA) of Wistar rats. Pressure myograph video microscopy was used to study the changes in cerebral artery vessel diameter. The main results of this study were as follows: (1) The diameters of BA and middle cerebral artery (MCA) were 314.5±15.7 μm (n=15) and 233.3±10.1 μm (n=12) at 10 mmHg working pressure (P<0.05), respectively. Pressure-induced vasomotion occurred in BA (22/28, 78.6%), but not in MCA (4/31, 12.9%) from 0 to 70 mmHg working pressure. As is typical for vasomotion, the contractile phase of the response was more rapid than the relaxation phase; (2) The frequency of vasomotion response and the diameter were gradually increased in BA from 0 to 70 mmHg working pressure. The amplitude of the rhythmic contractions was relatively constant once stable conditions were achieved. The frequency of contractions was variable and the highest value was 16.7±4.7 (n=13) per 10 min at 60 mmHg working pressure; (3) The pressure-induced vasomotion of the isolated BA was attenuated by nifedipine, NFA, 18β-GA, TEA or in Ca(2+)-free medium. Nifedipine, NFA, 18β-GA or Ca(2+)-free medium not only dampened vasomotion, but also kept BA in relaxation state. In contrasts, TEA kept BA in contraction state. These results suggest that the pressure-induced vasomotion of the isolated BA results from an interaction between Ca(2+)-activated Cl(-) channels (CaCCs) currents and K(Ca) currents. We hypothesize that vasomotion of BA depends on the depolarizing of the vascular smooth muscle cells (VSMCs) to activate CaCCs. Depolarization in turn activates voltage-dependent Ca(2+) channels, synchronizing contractions of adjacent cells through influx of extracellular calcium and the flow of calcium through gap junctions. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates K(Ca) channels and hyperpolarizes VSMCs, which provides a negative feedback loop for regenerating the contractile cycle.

Mechanisms of phytoestrogen biochanin A-induced vasorelaxation in renovascular hypertensive rats

BACKGROUND: The plant-derived estrogen biochanin A is known to cause vasodilation, but its mechanism of action in hypertension remains unclear. This study was undertaken to investigate the effects and mechanisms of biochanin A on the thoracic aorta in two-kidney, one clip (2K1C) renovascular hypertensive rats. METHODS: Hypertension was induced by clipping the left renal artery, and control age-matched rats were sham treated. Thoracic aortae were mounted in tissue baths to measure isometric tension. RESULTS: Biochanin A caused concentration-dependent relaxation in aortic rings from 2K1C hypertensive and sham-treated rats, which was greater in 2K1C rats than in sham rats. Biochanin A-induced relaxation was significantly attenuated by removing the endothelium in aortic rings from 2K1C rats, but not in sham rats. Nomega-Nitro-L-arginine methylester, a nitric oxide synthase inhibitor, or indomethacin, a cyclooxygenase inhibitor, did not affect the biochanin A-induced relaxation in aortic rings from 2K1C and sham rats. By contrast, treatment with glibenclamide, a selective inhibitor of adenosine triphosphate-sensitive K+ channels, ortetraethy-lammonium, an inhibitor of Ca2+-activated K+ channels, significantly reduced biochanin A-induced relaxation in aortic rings from both groups. However, 4-aminopyridine, a selective inhibitor of voltage-dependent K+ channels, inhibited the relaxation induced by biochanin A in 2K1C rats, whereas no significant differences were observed in sham rats. CONCLUSION: These results suggest that the enhanced relaxation caused by biochanin A in aortic rings from hypertensive rats is endothelium dependent. Vascular smooth muscle K+ channels may be involved in biochanin A-induced relaxation in aortae from hypertensive and normotensive rats. In addition, an endothelium-derived activation of voltage-dependent K+ channels contributes, at least in part, to the relaxant effect of biochanin A in renovascular hypertension.

Effects of intracellular calcium alteration on SK currents in atrial cardiomyocytes from patients with atrial fibrillation / 中国应用生理学杂志

<p><b>OBJECTIVE</b>SK channels are existed in hearts of mouse, rat, and human. Biochemical evidence indicates that SK2 channels are expressed more in atrial than in ventricular tissue. SK channels are highly sensitive to the calcium concentration of the pipette solution. In the present study, performed whole-cell patch clamp was used to detect the calcium sensitivity of small conductance Ca(2+)-activated K+ channels (SK) currents between sinus ryhthm (SR) and auricular fibrillation (AF).</p><p><b>METHODS</b>The patients who accepted cardiopulmonary bypass were divided into two groups: 21 patients with SR and 8 patients with AF. The enzymatic dissociation method was improved according to the previous research by our lab. The performed whole cell patch-clamp technique was used to record SK2 currents in both SR and AF groups at room temperature.</p><p><b>RESULTS</b>The SK2 current density was (-2.92 +/- 0.35) pA/pF in SR group (n = 6) vs (-6.83 +/- 0.19) pA/pF in AF group at -130 mV (n = 3, P < 0.05). In SR group, the SK2 current densities in calcium concentration of the pipette solution are (-1.43 +/- 0.33) pA/pF (n = 7), (-2.92 +/- 0.35) pA/pF (n = 6), (-10.11 +/- 2.15) pA/pF (n = 8, P < 0.05); In AF group, the SK2 current densities are (-2.17 +/- 0.40) pA/pF (n = 4), (-6.83 +/- 0.19) pA/pF (n = 3), (-14.47 +/- 2.89 pA/pF) (n = 4, P < 0.05).</p><p><b>CONCLUSION</b>The SK2 currents recorded in this experiment are voltage-independent, inwardly rectifying and apamin-sensitive. When the calcium concentration of the pipette solution is 5 x 10(-7) mol/L, SK2 current density in AF group are significantly larger than those in SR group. It suggests that SK currents involve the cardiomyocytes electric remodeling in AF. In AF group, the SK2 currents are more sensitive to free calcium ion. It shows that the increased sensitivity of SK2 currents to the calcium contribute to the occurrence and maintenance of AF.</p>

Establishing vascular smooth muscle from coronary arteries

Vascular smooth muscle cells [VSMCs] have the ability to transform into non-contractile, proliferating cells which can either be reversible or irreversible [such as that occur in some diseases] and can be accompanied by a change in the function of VSMCs. Isolation and culturing of VSMCs is important to address the mechanisms of such transformation. In the present study, VSMCs were isolated from porcine coronary arteries. RT-PCR, Western blot and immunofluorescence techniques were employed to confirm the identity of the cells. The large- and small-conductance calcium activated potassium channels [BKCa and SK3, respectively] were clearly present in the cells. Data from the present study demonstrate the method of developing VSMCs model from blood vessels which can be used to investigate mechanisms that are involved in the change of cells function in different diseases and identification of pharmacology of several drugs that are of significant importance in the treatment of vascular dysfunction

Comparison of membrane current of vascular smooth muscle cells in brain artery of spontaneously hypertensive rats and Wistar rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the difference in membrane current of vascular smooth muscle cells (VSMCs) in brain artery (BA) of spontaneously hypertensive rats (SHR) and Wistar rats.</p><p><b>METHODS</b>We compared the properties of spontaneous transient outward K+ currents (STOCs), the density and composition of current of VSMCs in BA of SHR and Wistar rats by whole-cell patch clamp technique.</p><p><b>RESULTS</b>(1) When the command voltage was 0, + 20, + 40 and + 60 mV respectively, the current densities of VSMCs in BA of SHR and Wistar rats were significant different (P < 0.01). (2) The whole-cell current of VSMCs was partly inhibited by 1 mmol/L4-AP (voltage-gated K+ channel blocker) or 1 mmol/L TEA (big conductance Ca(2+)-activated K+ channel blocker) respectively. (3) The frequency and amplitude of STOCs in SHR were faster and bigger than those in Wistar rats. 1 mmol/L TEA almostly inhibited the STOCs, but not by 4-AP.</p><p><b>CONCLUSION</b>These results suggest that the current densities of VSMCs in BA of SHR and Wistar rats are significant different, the outward current of VSMCs in BA of SHR and Wistar rats are composed by Kv and BK(Ca). SHR express more STOCs mediated by BK(Ca), than Wistar rats.</p>

Application of recording SK2 current in human atrial myocytes by perforated patch clamp techniques with the mix of beta-escin and amphotericin B / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To establish a perforated patch-clamp technology with amphotericin B and beta-escin and to research the regulation of small conductance calcium-activated potassium channel SK2 currents by calcium ions.</p><p><b>METHODS</b>Single human atrial myocytes were enzymatically isolated from the right atrial appendage. Amphotericin B and / or beta-escin were used by perforated electrode liquid. The regulation of SK2 current by calcium ions in human atrial myocytes was performed with the perforated patch-clamp technique. The intracellular calcium changes were measured by the intracellular calcium test system.</p><p><b>RESULTS</b>Mixed perforated electrode liquid compared with 150 microg/ml amphotericin B or 6.88 microg/ml beta-escin alone, it was easy to seal cells and activate SK2 current by the former method. Moreover, the ration of F340/380 was consistent with the change of intracellular free calcium ion concentration increase after the formation of perforation. The ration of F340/380 was measured by intracellular calcium test system.</p><p><b>CONCLUSION</b>The appropriate concentration of amphotericin B mixed with beta-escin can form a stable whole-cell patch recording technology that is appropriate for the research of SK2 current regulation by intracellular calcium.</p>