Potassium Channels: A Potential Therapeutic Target for Parkinson's Disease / 神经科学通报·英文版

The pathogenesis of the second major neurodegenerative disorder, Parkinson's disease (PD), is closely associated with the dysfunction of potassium (K) channels. Therefore, PD is also considered to be an ion channel disease or neuronal channelopathy. Mounting evidence has shown that K channels play crucial roles in the regulations of neurotransmitter release, neuronal excitability, and cell volume. Inhibition of K channels enhances the spontaneous firing frequency of nigral dopamine (DA) neurons, induces a transition from tonic firing to burst discharge, and promotes the release of DA in the striatum. Recently, three K channels have been identified to protect DA neurons and to improve the motor and non-motor symptoms in PD animal models: small conductance (SK) channels, A-type K channels, and K7/KCNQ channels. In this review, we summarize the physiological and pharmacological effects of the three K channels. We also describe in detail the laboratory investigations regarding K channels as a potential therapeutic target for PD.

Nobiletin attenuates neurotoxic mitochondrial calcium overload through K⁺ influx and ΔΨ(m) across mitochondrial inner membrane

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (ΔΨ(m)). Therefore, pharmacological manipulation of ΔΨ(m) can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ΔΨ(m) against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity (100 µM, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 µM)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca²⁺ (5 µM). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ΔΨ(m) were completely abolished in K⁺-free medium on pure isolated mitochondria. Taken together, results demonstrate that K⁺ influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K⁺ influx is probably mediated, at least in part, by activation of mitochondrial K⁺ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

Possible mechanisms involved in the vasorelaxant effect produced by clobenzorex in aortic segments of rats

Clobenzorex is a metabolic precursor of amphetamine indicated for the treatment of obesity. Amphetamines have been involved with cardiovascular side effects such as hypertension and pulmonary arterial hypertension. The aim of the present study was to investigate whether the direct application of 10-9-10-5 M clobenzorex on isolated phenylephrine-precontracted rat aortic rings produces vascular effects, and if so, what mechanisms may be involved. Clobenzorex produced an immediate concentration-dependent vasorelaxant effect at the higher concentrations (10-7.5-10-5 M). The present outcome was not modified by 10-6 M atropine (an antagonist of muscarinic acetylcholine receptors), 3.1×10-7 M glibenclamide (an ATP-sensitive K+ channel blocker), 10-3 M 4-aminopyridine (4-AP; a voltage-activated K+ channel blocker), 10-5 M indomethacin (a prostaglandin synthesis inhibitor), 10-5 M clotrimazole (a cytochrome P450 inhibitor) or 10-5 M cycloheximide (a general protein synthesis inhibitor). Contrarily, the clobenzorex-induced vasorelaxation was significantly attenuated (P<0.05) by 10-5 M L-NAME (a direct inhibitor of nitric oxide synthase), 10-7 M ODQ (an inhibitor of nitric oxide-sensitive guanylyl cyclase), 10-6 M KT 5823 (an inhibitor of protein kinase G), 10-2 M TEA (a Ca2+-activated K+ channel blocker and non-specific voltage-activated K+ channel blocker) and 10-7 M apamin plus 10-7 M charybdotoxin (blockers of small- and large-conductance Ca2+-activated K+ channels, respectively), and was blocked by 8×10-2 M potassium (a high concentration) and removal of the vascular endothelium. These results suggest that the direct vasorelaxant effect by clobenzorex on phenylephrine-precontracted rat aortic rings involved stimulation of the NO/cGMP/PKG/Ca2+-activated K+ channel 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.

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.

Mechanism of quercetin improving rat coronary artery myogenic re-sponse under high glucose / 中国病理生理杂志

AIM:To investigate the mechanism of quercetin improving rat coronary artery myogenic response under high glucose ( HG) by measuring muscle tension of coronary arterial ring and recording voltage -gated K +channel ( Kv) current of coronary artery smooth muscle cells by whole cell patch clamp .METHODS:The coronary rings from the normal SD rats were acutely isolated , and then divided into 6 groups:(1) control group;(2) HG group;(3) HG+low dose (3 μmol/L) of quercetin group;(4) HG+moderate dose (10 μmol/L) of quercetin group; (5) HG+high dose (30 μmol/L) of quercetin group;(6) HG+C6303 (PKC inhibitor) +high dose of quercetin group.Determinations of coronary artery response to vasoconstrictor (60 mmol/L KCl or 0.1 mmol/L U46619) or vasodilator (Ach at 10 -9 ~10 -5 mol/L) were performed, and the percentage of coronary ring tension was calculated using the contraction as 100%caused by 60 mmol/L KCl.The rat coronary artery smooth muscle cells were acutely isolated for recording the Kv current using whole cell patch clamp .RESULTS:Compared with control group , the contraction amplitudes to 60 mmol/L KCl or 0.1 mmol/L U46619 were significantly increased under HG incubation .Quercetin intervention concentration-dependently re-duced the coronary artery contraction amplitude .Incubation of PKC specific inhibitor C 6303 attenuated the effect of querce-tin.Compared with control group , the diastolic amplitude to Ach decreased significantly in HG group , and quercetin inter-vention concentration-dependently increased the coronary artery diastolic amplitude .Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin .Compared with control group , HG incubation inhibited Kv current of coronary ar-tery vascular smooth muscle cells significantly , and quercetin intervention attenuated the inhibitory effect of HG on Kv cur-rent intensity .Incubation of PKC specific inhibitor C 6303 attenuated the effect of quercetin .CONCLUSION: Quercetin has a protective effect on myogenic response of coronary artery under HG and the effects is related to the increase in Kv cur -rent and the activation of PKC in vascular smooth muscle cells .

Research progress on regulation effect of Panax notoginseng saponins on mitochondria / 中国中药杂志

Mitochondria is the key energy source of cells and plays an important role in energy synthesis and release, and maintenance of cellular functions. As the most important active ingredients in Chinese medicine pseudo-ginseng, Panax notoginseng saponins(PNS) have pharmacological effects on protecting against thrombosis, dilating blood vessels, lowering the blood pressure, anti-inflammation, and antioxidant, etc. Domestic and foreign studies have shown that PNS participates in regulating mitochondrial energy metabolism, oxidative stress, biosynthesis, apoptosis, mitophagy and the status of membrane channels. Therefore, the mitochondria is one of the important targets of PNS. In this paper, the regulation effects of P. notoginseng saponins on mitochondria were reviewed.

Mechanism of quercetin improving rat coronary artery myogenic re-sponse under high glucose / 中国病理生理杂志

AIM:To investigate the mechanism of quercetin improving rat coronary artery myogenic response under high glucose ( HG) by measuring muscle tension of coronary arterial ring and recording voltage -gated K +channel ( Kv) current of coronary artery smooth muscle cells by whole cell patch clamp .METHODS:The coronary rings from the normal SD rats were acutely isolated , and then divided into 6 groups:(1) control group;(2) HG group;(3) HG+low dose (3 μmol/L) of quercetin group;(4) HG+moderate dose (10 μmol/L) of quercetin group; (5) HG+high dose (30 μmol/L) of quercetin group;(6) HG+C6303 (PKC inhibitor) +high dose of quercetin group.Determinations of coronary artery response to vasoconstrictor (60 mmol/L KCl or 0.1 mmol/L U46619) or vasodilator (Ach at 10 -9 ~10 -5 mol/L) were performed, and the percentage of coronary ring tension was calculated using the contraction as 100%caused by 60 mmol/L KCl.The rat coronary artery smooth muscle cells were acutely isolated for recording the Kv current using whole cell patch clamp .RESULTS:Compared with control group , the contraction amplitudes to 60 mmol/L KCl or 0.1 mmol/L U46619 were significantly increased under HG incubation .Quercetin intervention concentration-dependently re-duced the coronary artery contraction amplitude .Incubation of PKC specific inhibitor C 6303 attenuated the effect of querce-tin.Compared with control group , the diastolic amplitude to Ach decreased significantly in HG group , and quercetin inter-vention concentration-dependently increased the coronary artery diastolic amplitude .Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin .Compared with control group , HG incubation inhibited Kv current of coronary ar-tery vascular smooth muscle cells significantly , and quercetin intervention attenuated the inhibitory effect of HG on Kv cur-rent intensity .Incubation of PKC specific inhibitor C 6303 attenuated the effect of quercetin .CONCLUSION: Quercetin has a protective effect on myogenic response of coronary artery under HG and the effects is related to the increase in Kv cur -rent and the activation of PKC in vascular smooth muscle cells .

Chronic Ca²⁺ influx through voltage-dependent Ca²⁺ channels enhance delayed rectifier K⁺ currents via activating Src family tyrosine kinase in rat hippocampal neurons

Excessive influx and the subsequent rapid cytosolic elevation of Ca²⁺ in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic Ca²⁺ level in normal as well as pathological conditions. Delayed rectifier K⁺ channels (I(DR) channels) play a role to suppress membrane excitability by inducing K⁺ outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under Ca²⁺-mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of IDR channels to hyperexcitable conditions induced by high Ca²⁺ pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high Ca²⁺-treatment significantly increased the amplitude of IDR without changes of gating kinetics. Nimodipine but not APV blocked Ca²⁺-induced IDR enhancement, confirming that the change of I(DR) might be targeted by Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated I(DR) enhancement was not affected by either Ca²⁺-induced Ca²⁺ release (CICR) or small conductance Ca²⁺-activated K⁺ channels (SK channels). Furthermore, PP2 but not H89 completely abolished I(DR) enhancement under high Ca²⁺ condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for Ca²⁺-mediated I(DR) enhancement. Thus, SFKs may be sensitive to excessive Ca²⁺ influx through VDCCs and enhance I(DR) to activate a neuroprotective mechanism against Ca²⁺-mediated hyperexcitability in 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.

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.

Opening of ATP-sensitive K+channels protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting TLR4/NF-κB pathway / 中国病理生理杂志

AIM:To investigate whether the opening of ATP-sensitive K+(KATP) channels protects H9c2 car-diac cells against high glucose ( HG)-induced injury and inflammation by inhibiting the Toll-like receptor 4 ( TLR4 )/nu-clear factor-κB ( NF-κB) pathway.METHODS:The protein levels of TLR4 and NF-κB p65 were determined by Western blot.The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by ELISA.The cell viabil-ity was measured by CCK-8 assay.Mitochondrial membrane potential (MMP) was examined by rhodamine 123 (Rh 123) staining followed by photofluorography.The intracellular levels of reactive oxygen species ( ROS) were detected by 2′, 7′- dichlorfluorescein-diacetate (DCFH-DA) staining followed by photofluorography.The number of apoptotic cells was ob-served by Hoechst 33258 nuclear staining followed by photofluorography.RESULTS: After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the protein levels of TLR4 and phosphorylated NF-κB p65 ( p-NF-κB p65) were significantly increased.Pretreatment of the cells with 100 μmol/L diazoxide ( DZ, a KATP channel opener) for 30 min before exposure to HG considerably blocked the up-regulation of the TLR4 and p-NF-κB protein levels induced by HG.Moreover, co-treatment of the cells with 30 μmol/L TAK-242 (an inhibitor of TLR4) obviously inhibited the HG-in-duced up-regulation of the p-NF-κB p65 protein level.On the other hand, pretreatment of the cells with 100 μmol/L DZ had a clear myocardial protection effect, which attenuated the HG-induced cytotoxicity, inflammatory response, mitochon-drial damage, oxidative stress and apoptosis, evidenced by an increase in the cell viability, and decreases in the levels of IL-1βand TNF-α, MMP loss, ROS generation and the number of apoptotic cells.Similarly, co-treatment of H9c2 cardiac cells with 30μmol/L TAK-242 or 100μmol/L PDTC ( an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries and inflammation induced by HG.CONCLUSION: The opening of KATP channels protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the TLR4/NF-κB pathway.

Role of Kv7 Channels in Vascular Dysfunction associated with Metabolic Syndrome

Vasoconstriction is regulated by various ion channels expressed in the plasma membrane of vascular smooth muscle cells. In particular, potassium (K+) channel activity determines resting membrane potential and regulates intracellular calcium (Ca2+) signaling. A number of studies have suggested that dysregulation of K+ channel activity is associated with increased myogenic tone or diminished vasorelaxation. Among the various families of K+ channels, voltage-dependent K+ channels (Kv channels) encoded by the KCNQ gene family (Kv7 channels or M channels) are widely expressed in various blood vessels isolated from mouse, rat, and human. Recent studies have demonstrated that a subunit of the Kv7 channel, Kv7.4, is down-regulated in the aorta and mesenteric and renal arteries of the Spontaneously Hypertensive Rat (SHR) model. Previous studies have also suggested that Kv7 channels play an important role in the regulation of vasorelaxation/vasoconstriction in response to activators/blockers. In addition, previous studies have indicated that hypertension, diabetes mellitus, and cerebrovascular disease result in development of vascular dysfunction associated with Kv7 abnormalities in various animal models. This review focuses on the potential role of the Kv7 channel in vascular dysfunction.

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.

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.

Vasorelaxant effect of novel Rho-kinase inhibitors on isolated thoracic aorta rings and underlying mechanisms / 中国药理学通报

Aim To evaluate the vasorelaxant effect of two new chemical entities, J35242 and J35243, on iso-lated rat thoracic aorta rings as Rho-kinase inhibitors, and further to explore the underlying mechanisms of these two compounds. Methods Isolated rat thoracic aorta rings pre-contracted by KCl or norepinephrine ( NE) were used to evaluate the vasodilatory effect of J35242 and J35243 . Through the interventions of sev-eral tool drugs, the mechanisms of compounds concern-ing endothelium, K+ channels and Ca2+ were studied. Results J35242 and J35243 showed potent relaxant effect on both KCl and NE pre-contracted vessels, and exhibited partial endothelium dependency. L-NAME and Methylene Blue( MB) could influence the relaxant effect of these compounds. Meanwhile, the compounds could inhibit intracellular Ca2+ release and extracellu-lar Ca2+ influx, which indicated that the compounds might block the calcium channels to relax the vessels. In addition, the two compounds probably did not dilate the aorta rings through opening potassium channels. Conclusions J35242 and J35243 have vasorelaxant effects on vessels in vitro and the potency of J35242 is stronger than that of J35243 . The underlying mecha-nisms might be endothelium-dependent. Also the com-pounds might block Ca2+ channels, lowering intracel-lular Ca2+ concentration to relax the vessels.

Vasorelaxant effect of Rho kinase inhibitor DL0805-0 on isolated rat aortic rings and its underlying mechanisms / 中国药理学通报

Aim To investigate the in vitro vasorelax-ant effect of DL0805-0, a Rho kinase inhibitor, on iso-lated rat thoracic aorta and explore its underlying mechanism. Methods Tension was measured to eval-uate the vasorelaxant effect of DL0805-0 on rat endo-thelium-intact and endothelium-denuded thoracic aorta rings. Rho kinase inhibitor fasudil, nitric oxide syn-thase inhibitor Nω-nitro-L-arginine methyl ester ( L-NAME), guanylate cyclase inhibitor methylene blue, cyclooxygenase inhibitor indomethacin, calcium-activa-ted potassium channel blocker tetraethyl ammonium ( TEA ) , ATP-sensitive potassium channel blocker glibenclamide and voltage-dependent potassium chan-nel blocker 4-aminopyridine ( 4-AP ) were used to il-lustrate the mechanisms of vasorelaxant effect of DL0805-0 . Results DL0805-0 exerted vasorelaxation in a dose-dependent manner in KCl (60 mmol·L-1 ) or NE ( 0. 1 μmol · L-1 ) -induced contraction. DL0805-0-induced vasorelaxation was significantly re-duced by L-NAME. However, methylene blue and in-domethacin did not significantly affect vasorelaxation of DL0805-0. In endothelium-denuded rings, TEA re-markably attenuated the vasorelaxant effect of DL0805-0 , while glibenclamide and 4-AP did not affect vasore laxation of DL0805-0 significantly. DL0805-0 also re-duced NE-induced transient contraction and inhibited contraction induced by increasing extracellular calci-um. Conclusion These results suggest that DL0805-0 induces vasorelaxation through an endothelium-depend-ent pathway. The opening of calcium-activated K+channels and blocking of Ca2+ channels in vascular smooth muscle cells may be one of the mechanisms of DL0805-0-induced vasorelaxation.

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.

Effect of glibenclamide on antinociceptive effects of antidepressants of different classes

OBJECTIVES: The purpose of this work was to determine whether the intraperitoneal administration of glibenclamide as a K ATP channel blocker could have an effect on the antinociceptive effects of antidepressants with different mechanisms of action. METHODS: Three antidepressant drugs, amitriptyline as a dual-action, nonselective inhibitor of noradrenaline and a serotonin reuptake inhibitor, fluvoxamine as a selective serotonin reuptake inhibitor and maprotiline as a selective noradrenaline reuptake inhibitor, were selected, and the effect of glibenclamide on their antinociceptive activities was assessed in male Swiss mice (25-30 g) using a formalin test. DISCUSSION: None of the drugs affected acute nociceptive responses during the first phase. Amitriptyline (5, 10 mg/ kg), maprotiline (10, 20 mg/kg) and fluvoxamine (20 and 30 mg/kg) effectively inhibited pain induction caused by the second phase of the formalin test. Glibenclamide (5 mg/kg) alone did not alter licking behaviors based on a comparison with the control group. However, the pretreatment of animals with glibenclamide (10 and 15 mg/kg) partially reversed the antinociceptive effects of fluvoxamine but not those of maprotiline. In addition, the highest dose of glibenclamide (15 mg/kg) partially prevented the analgesic effect of amitriptyline. CONCLUSION: Therefore, it seems that adenosine triphosphate-dependent potassium channels have a major role in the analgesic activity of amitriptyline and fluvoxamine.

Increased Expression of ATP-sensitive K+ Channels Improves the Right Ventricular Tolerance to Hypoxia in Rabbit Hearts

ATP-sensitive K+ channels (KATP) are major component of preventing ischemia-reperfusion injury. However, there is little information regarding to the expressional difference of K(ATP) and its function between left and right ventricles. In this study, we measured the lactate dehydrogenase release of rabbit heart slices in vitro and determined the difference of the K(ATP) expression at the both ventricles by measuring the level of K(ATP)-forming Kir6.2 (OcKir6.2) mRNA using in situ hybridization. The hearts were preconditioned with 15 min hypoxia and reoxygenated for 15 min before a hypoxic period of 60 min, followed by reoxygenation for 180 min. With hypoxic preconditioning (100% N2) with 15 min, left ventricles (LV) showed higher release of LDH comparing with right ventricles (RV). Adding KATP blocker glibenclamide (10 microM) prior to a hypoxic period of 60 min, hypoxic preconditioning effect of RV was more abolished than LV. With in situ hybridization, the optical density of OcKir6.2 was higher in RV. Therefore, we suggest that different K(ATP) expression between LV and RV is responsible for the different response to hypoxia and hypoxic preconditioning of rabbit hearts.