Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca2+-activated K+ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice / 吉林大学学报(医学版)

Objective:To investigate the relationship between the level of oxidative stress and the expression of intermediate conductance calcium activated potassium channel (KCa3.1) protein in the cardiac fibroblasts (CFs) during hypertension process,and to clarify the role of KCa3.1 in cardiac fibrosis and its mechanism.Methods:The CFs of male C57B6 and AGT-REN double transgenic hypertension (dTH) mice were cultured and the wild C57B6 mouse CFs were used as control group.The CFs of dTH mice were randomly divided into high blood pressure group (dTH) and N-acetyl cysteine group (NAC).The CFs were treated with different concentrations of NAC for 24 h.The cell proliferation was detected by MTT and double dichlorofluorescein (DCFH-DA) probe was used for the detection of cellular reactive oxygen species (ROS) expression;Western blotting was employed to detect the expressions of collagen Ⅰ,collagen Ⅲ,Kca3.1 channel protein and the changes of PI3K signaling pathway protein phosphorylation.Results:The ROS production and protein expression of Kca3.1 channel of the dTH mice on 4,8,12 months were increased compared with 2 months (P＜0.05 or P＜0.01);the results of MTT suggested that the proliferation rates of CFs were 165.9％,138.72％,110.92％ and 109.82％ after administration of 1×10-6,1× 10 5,1× 10 4 and 1 × 10-3 mol · L-1 NAC in the dTH mice,and 1 × 10-4 and 1 × 10 3 mol · L-1 NAC significantly inhibited the proliferation of CFs.Compared with control group,the secretion of collagen Ⅰ and Ⅲ of CFs in the TH mice was decreased in 1 × 10-4 mol · L-1 NAC group (P＜0.01).The results of Western blotting showed that compared with control group,the expression level of Kca3.1 channel protein in CFs of the TH mice in 1 × 10-4 mol · L-1 NAC group was decreased (P＜0.01).Compared with control group,the p-AKT/T-AKt in CFs of the dTH mice was increased (P＜0.01);but in NAC group,the p-AKT/T-AKt was lower than that in dTH group (P＜0.01).Conclusion:NAC can inhibit the expression of KCa3.1 channel protein in CFs of the dTH hypertensive mice,which may be related to increasing the phosphorylation of AKt/PI3K signaling pathway.

Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca2+-activated K+ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice / 吉林大学学报(医学版)

Objective:To investigate the relationship between the level of oxidative stress and the expression of intermediate conductance calcium activated potassium channel (KCa3.1) protein in the cardiac fibroblasts (CFs) during hypertension process,and to clarify the role of KCa3.1 in cardiac fibrosis and its mechanism.Methods:The CFs of male C57B6 and AGT-REN double transgenic hypertension (dTH) mice were cultured and the wild C57B6 mouse CFs were used as control group.The CFs of dTH mice were randomly divided into high blood pressure group (dTH) and N-acetyl cysteine group (NAC).The CFs were treated with different concentrations of NAC for 24 h.The cell proliferation was detected by MTT and double dichlorofluorescein (DCFH-DA) probe was used for the detection of cellular reactive oxygen species (ROS) expression;Western blotting was employed to detect the expressions of collagen Ⅰ,collagen Ⅲ,Kca3.1 channel protein and the changes of PI3K signaling pathway protein phosphorylation.Results:The ROS production and protein expression of Kca3.1 channel of the dTH mice on 4,8,12 months were increased compared with 2 months (P＜0.05 or P＜0.01);the results of MTT suggested that the proliferation rates of CFs were 165.9％,138.72％,110.92％ and 109.82％ after administration of 1×10-6,1× 10 5,1× 10 4 and 1 × 10-3 mol · L-1 NAC in the dTH mice,and 1 × 10-4 and 1 × 10 3 mol · L-1 NAC significantly inhibited the proliferation of CFs.Compared with control group,the secretion of collagen Ⅰ and Ⅲ of CFs in the TH mice was decreased in 1 × 10-4 mol · L-1 NAC group (P＜0.01).The results of Western blotting showed that compared with control group,the expression level of Kca3.1 channel protein in CFs of the TH mice in 1 × 10-4 mol · L-1 NAC group was decreased (P＜0.01).Compared with control group,the p-AKT/T-AKt in CFs of the dTH mice was increased (P＜0.01);but in NAC group,the p-AKT/T-AKt was lower than that in dTH group (P＜0.01).Conclusion:NAC can inhibit the expression of KCa3.1 channel protein in CFs of the dTH hypertensive mice,which may be related to increasing the phosphorylation of AKt/PI3K signaling pathway.

Role of Ca2+-activated K+channels in alkalinization and β-glycerophosphate induced vascular smooth muscle cells calcification / 中华肾脏病杂志

Objective To observe the role of intermediate conductance calcium?activated potassium channels (KCa3.1) in alkalinization and β?glycerophosphate induced vascular calcification. Methods Vascular smooth muscle cells (VSMCs) and aortic rings were obtained from rat thoracic aorta, and then randomly divided into control group (pH was provided into 7.4, 8.0), high phosphorus groups (pH was provided into 7.4, 7.7 and 8.0, VSMCs in three groups were treated with 10 mmol/L β?glycerophosphate; HCl and NaHCO3 were used to adjust the pH) and TRAM?34 group (20 nmol/L was added into pH8.0 high phosphorus dulbecco's modified eagle's medium). Calcium deposition and alkaline phosphatase (ALP) activity were measured by Alizarin red staining, calcium content and enzyme linked immunosorbent assay after cells were simulated for 12 days. Intracellular free Ca2 + was measured by ELISA. The expression of KCa3.1, runt?related transcription factor 2 (Runx2) were detected by RT?PCR and Western blotting 4 days after cells were stimulated. Calcium deposition was measured by von Kossa staining and calcium content after aortic rings were cultured for 12 days. The expressions of KCa3.1 and Runx2 were detected by immunohistochemistry after aortic rings were cultured for 4 days. Results Compared with control group, calcification in VSMCs and aortic rings were significantly increased in high phosphorus group (P<0.05) while decreased in TRAM?34 group (P<0.05). Compared with control group, the expressions of KCa3.1, Runx2 and the activity of ALP in high phosphorus groups were increased (P<0.05) while decreased in TRAM?34 group (P<0.05). Besides, expressions of Runx2 and KCa3.1 were augmented as the pH was higher (P<0.05). The expression of Runx2 in aortic rings was the same situation. Besides, the Ca2+ influx was blocked by TRAM?34 (P<0.05). Conclusions Alkalinization contributes to β?glycerophosphate induced VSMCs calcification through increase of Ca2 + influx, up?regulation of KCa3.1 and promotion of osteogenic/chondrogenic differentiation.

Tacrolimus inhibits vasoconstriction by increasing Ca(2+) sparks in rat aorta / 华中科技大学学报（医学）（英德文版）

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.

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.

Expression of Ca2+ -activated K+ Channels and Their Role in Proliferation of Rat Cardiac Fibroblasts

Cardiac fibroblasts constitute one of the largest cell populations in the heart, and contribute to structural, biochemical, mechanical and electrical properties of the myocardium. Nonetheless, their cardiac functions, especially electrophysiological properties, have often been disregarded in studies. Ca2+-activated K+(KCa) channels can control Ca2+influx as well as a number of Ca2+-dependent physiological processes. We, therefore, attempted to identify and characterize KCa channels in rat Cardiac fibroblasts. First, we showed that the cells cultured from the rat ventricle were cardiac fibroblasts by immunostaining for discoidin domain receptor 2 (DDR-2), a specific fibroblast marker. Secondly, we detected the expression of various KCa channels by reverse transcription polymerase chain reaction (RT-PCR), and found all three family members of KCa channels, including large conductance KCa (BK-alpha 1- and -beta 1~4 subunits), intermediate conductance KCa (IK), and small conductance KCa (SK1~4 subunits) channels. Thirdly, we recorded BK, IK, and SK channels by whole cell mode patch clamp technique using their specific blockers. Finally, we performed cell proliferation assay to evaluate the effects of the channels on cell proliferation, and found that the inhibition of IK channel increased the cell proliferation. These results showed the existence of BK, IK, and SK channels in rat ventricular fibroblasts and involvement of IK channel in cell proliferation.

Role of KCa Channels in SNAP-Induced Relaxation of Aorta from Renal Hypertensive Rats / 대한신장학회지

PURPOSE: S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, is thought to relax vascular smooth muscle by stimulation of soluble guanylate cyclase, accumulation of its product cyclic GMP (cGMP) level. Evidence has emerged that NO-induced vasodilatation is also mediated by stimulating Ca2+-activated K+ (KCa) channels directly or indirectly through cGMP. The aim of the present study was to investigate possible involvement or alteration of KCa channels in the mechanism of vasodilation induced by SNAP in two-kidney, one-clip (2K1C) hypertensive rats. METHODS: 2K1C hypertension was made by clipping the left renal artery and age-matched control rats received a sham treatment. Using rings prepared from thoracic aortae, we studied changes in isometric tension of the rings in response to SNAP to evaluate effects of a soluble guanylate cyclase inhibitor methylene blue (MB), and a specific blocker of KCa channel iberiotoxin (ITX). RESULTS: Aortic rings from 2K1C hypertensive and sham-clipped control rats precontracted with phenylephrine showed similar relaxation to SNAP. MB markedly suppressed the SNAP-induced relaxation in both groups, leaving about 30% of MB-resistant relaxation. ITX nearly completely eliminated the MB-resistant relaxation in control rats, but it did not affect 2K1C rats. CONCLUSION: These results suggest that SNAP-induced vasorelaxation is mediated through cGMP- dependent and cGMP-independent KCa channel involving mechanisms, the latter may be altered in 2K1C renal hypertension.

Effects of Pharmacological Modulators of Ca2+ -activated K+ Channels on Proliferation of Human Dermal Fibroblast

Employing electrophysiological and cell proliferation assay techniques, we studied the effects of Ca2+ -activated K+ channel modulators on the proliferation of human dermal fibroblasts, which is important in wound healing. Macroscopic voltage-dependent outward K+ currents were found at about -40 mV stepped from a holding potential of -70 mV. The amplitude of K+ current was increased by NS1619, a specific large-conductance Ca2+ -activated K+ (BK) channel activator, but decreased by iberiotoxin (IBTX), a specific BK channel inhibitor. To investigate the presence of an intermediate-conductance Ca2+ -activated K+ (IK) channels, we pretreated the fibroblasts with low dose of TEA to block BK currents, and added 1-EBIO (an IK activator). 1-EBIO recovered the currents inhibited by TEA. When various Ca2+ -activated K+ channel modulators were added into culture media for 1~3 days, NS1619 or 1-EBIO inhibited the cell proliferation. On the other hand, IBTX, clotrimazole or apamin, a small conductance Ca2+ -activated K+ channel (SK) inhibitor, increased it. These results suggest that BK, IK, and SK channels might be involved in the proliferation of human dermal fibroblasts, which is inversely related to the channel activation.

The alteration of Ca2+-activated K+ channels in coronary arterial smooth muscle cells isolated from isoproterenol-induced cardiac hypertrophy in rabbit

It has been proposed that Ca2+-activated K+ (KCa) channels play an essential role in vascular tone. The alterations of the properties of coronary KCa channels have not been studied as a possible mechanism for impaired coronary reserve in cardiac hypertrophy. The present studies were carried out to determine the properties of coronary KCa channels in normal and hypertrophied hearts. These channels were measured from rabbit coronary smooth muscle cells using a patch clamp technique. The main findings of the present study are as follows: (1) the unitary current amplitudes and the slope conductance of coronary KCa channels were decreased without changes of the channel kinetics in isoproterenol-induced cardiac hypertrophy; (2) the sensitivity of coronary KCa channels to the changes of intracellular concentration of Ca2+ was reduced in isoproterenol-induced cardiac hypertrophy. From above results, we suggest for the first time that the alteration of KCa channels are involved in impaired coronary reserve in isoproterenol-induced cardiac hypertrophy.

Fatty acids directly increase the activity of Ca(2+)-activated K+ channels in rabbit coronary smooth muscle cells

The large conductance Ca2+ activated K+ channel (BK channel) has been considered to play an important role in the excitability and contractility of vascular smooth muscle cells. Activation of the BK channel causes the hyperpolarization and relaxation of vascular smooth muscle cells. It has been reported that fatty acids can affect the BK channel activity and its concentration is increased significantly during myocardial ischemia. These reports suggest that fatty acids may contribute to the ischemic coronary vasodilation by increasing the BK channel activity. However, the underlying mechanism of fatty acid-induced activation of the BK channel is still uncertain. In the present study, we measured the effect of fatty acids on the BK channel activity in rabbit coronary smooth muscle cells by using patch clamp method and also examined its underlying mechanism. Arachidonic acid (AA) dissolved in DMSO activated the BK channel in a dose-dependent manner (from 0.5 to 10 microM), and DMSO (0.1%) alone had no effect on the activity of the BK channel. Arachidonic acid activated BK channels in both cell-attached and inside-out patches, but the onset and recovery of this effect were slower in the cell-attached patch configuration. The BK channel activity was also increased by other fatty acids, including myristic acid, linoleic acid, palmitoleic acid and palmitic acid. Long chain fatty acids were more effective than short chain fatty acids (myristic acid), and there was no statistical difference between the effect of saturated (palmitic acid) and unsaturated fatty acids (palmitoleic acid) on the BK channel activity. The concentration of Ca2+ and Mg2+ in the bathing solution had no appreciable effects on the AA-induced increase of BK channel activity. From the above results, it may be concluded that fatty acids directly increase the BK channel activity and may contribute to the ischemic coronary vasodilatation in rabbit coronary smooth muscle cells.