安定在家兔体内的肠胃循环药代动力学分析

6只家兔iv安定5 mg·kg^-1后,浓度—时间数据呈现双峰形。本文提出肠胃循环动力学模型,用于分析实测数据,得到了一般动力学参数:T_1/2(α)=0.21±=0.15 h,T_1/2(β)=2.2+0.6 h,K_e=1.5±0.6 h^-1,K₁₂=2.0±1.0 h^-1,K₂₁=1.0±0.4 h^-1,V₁=3.1±1.6 L·kg^-1,AUC=1.7±0.5μg·h^-1·ml^-1。此外,还求得有关肠胃循环的参数,即:重吸收滞后时间T′=0.25±0.24h,重吸收速率常数K_A=3.5±1.4 h^-1,重吸收率R_A=24±7%。     

柴胡皂甙和甘草甜素抑制Na+,K+-ATP酶活性的构效关系

研究在离体条件下各种单体柴胡皂甙和甘草甜素抑制Na⁺,K⁺-ATP酶活性的构效关系。实验结果表明,各种柴胡皂甙抑制Na⁺,K⁺-ATP酶活性的作用强度依次为:b₁>d>b₂>b₄>a>b₃>e>c。柴胡皂甙化学结构中的C₂₃-OH,C₁₆-OH及C₁₁和C₁₃的共轭双烯可能对其抑制活性起重要作用。甘草甜素(GL),甘草次酸(GA)和生胃酮(18-β-甘草次酸半琥珀酸双钠盐,CX)抑制Na⁺,K⁺-ATP酶活性的作用强度依次为GA≥CX>GL。研究还证明,柴胡皂甙d对Na⁺,K⁺-ATP酶的抑制为非竟争性抑制。     

A K+ single channel and whole-cell clamp study on the effects of levcromakalim in guinea pig portal vein cells

Single channel cell-attached patch and whole-cell clamp experiments on the mode of action of the K⁺ channel opener (KCO), levcromakalim, were performed in guinea pig isolated portal vein cells. At +20 mV (135/23 mM K⁺ in bath/pipette), 10 μM levcromakalim activated K⁺ channels with a chord conductance of 23.2 pS (K_KCO), which were sensitive to the blocker of ATP-dependent K⁺ channels (K_ATP), glibenclamide. Voltage steps from –80 mV to +20 mV activated 4-aminopyridine-sensitive K⁺ channels of 6.5 pS with properties of delayed rectifier K⁺ channels (K_v). In patches which upon a previous voltage step had revealed the existence of K_v, levcromakalim reduced the open-probability of K_v, but it did not concomitantly activate K_KCO. During the course of the experiments, but unrelated to the presence of levcromakalim, large conductance K⁺ channels (BK_Ca) appeared which could be inhibited by iberiotoxin, a selective blocker of BK_Ca, and by the membrane-permeant calcium buffer, BAPTA/AM, but not by glibenclamide. Whole-cell current-voltage (i-V) relations were established in response to voltage ramps from +50 mV to –100 mV; on subtraction of control i-V curves from i-V curves obtained in the presence of 10 μM levcromakalim, the KCO-induced K⁺ current remained which was proportional to voltage. This is not compatible with the upward-bent curvature predicted by the GHK current equation for purely resistive channels at high [K⁺]_i versus low [K⁺]_o. In conclusion, in the guinea pig portal vein cells, no evidence could be established for the hypotheses that KCOs may act via conversion of K_v to K_ATP (Beech and Bolton 1989; Edwards et al. 1993) or by activation of BK_Ca (Balwierczak et al. 1995). In these cells, mild inward rectification of the levcromakalim-induced current was observed which underlines their relationship to K_ATP in other tissues. Received: 1 August 1997 / Accepted: 7 June 1998     

蝙蝠葛碱和粉防己碱对[3H]WEB 2086与体外牛脑前动脉平滑肌细胞结合的影响(英文)

用标记的血小板活化因子拮抗剂[³H]WEB 2086,在培养的牛脑前动脉平滑肌细胞上鉴定了血小板活化因子受体。结果表明在25℃时该细胞上存在两种与配基具有不同亲和力的受体结合位点,其中K_d-1=22.8±5.0 nmol·L^-1,K_d-2=186+20.5 nmol·L^-1;B_max-1=2.1±0.3 pmol/10⁴细胞,B_max-2=12.1±1-5 pmol/10⁶细胞。蝙蝠葛碱和粉防己碱均能抑制[³H]WEB2086与上述细胞的结合。     

四氢异喹啉类生物碱对大鼠脑内α肾上腺素受体的作用

应用放射受体结合法研究了近30种四氢异喹啉类(TIQs)生物碱对大鼠脑内α肾上腺素受体的作用。其中l-CBN,l-THC和l-STP对α₁受体亲和力最高,K_i值为～2.0×10^-7mol/L。其次是DHS,XLP和l-DCT,K_i值分别为4.7×10^-7,6.5×10^-7和7.6×10^-7mol/L。DHS对α₂受体亲和力最高(K_i=1.25×10^-6mol/L),l-REM次之。对α受体亚型亲和力选择比K_i(alpha-2)/Ki(alpha-1)最高的是l-STP(357) 和XLP(154),它们对α₂受体几无亲和力(K_i>10^-4mnl/L)。提示l-STP和XLP对α₁受体有较高的选择性。l-SPD和l-THP对α₁和α₂受体亲和力相近,均为中等强度。THJ,DRC及l-TTD等6种TIQs对α₁和α₂受体均无亲和力(K_i>10^-4mnl/L)。     

粉防己碱,小檗胺等四氢异喹啉类生物碱对大鼠脑内M-胆碱受体的作用

用放射受体结合方法,研究了36种四氢异喹啉类生物碱及其半合成衍生物对大鼠脑内M-胆碱受体的结合特性。实验发现,粉防己碱对M-胆碱受体的亲和力最高,其K_i值为7.3×10^-8mol/L。小檗胺、四氢黄连碱和半合成原小檗碱衍生物B₁亦具有较高亲和力,K_i值分别为1.9×10^-7,6.8×10^-7和8.1×10^-7mol/L。四氢小檗碱半合成原小檗碱衍生物B₂,B₃,B₄,B₅,B₆和半合成小檗胺衍生物E₁及半合成蝙蝠葛碱衍生物D₁对M-胆碱受体的亲和力为中等强度,K_i值在1～2×10^-6mol/L之间。实验结果提示,某些四氢异喹啉类生物碱的药理作用可能与M-胆碱受体有关。     

大环骨架结构的倍半萜化合物——Ⅵ.马兜铃新内酯的化学结构测定

自绵毛马兜铃(Aristolochia mollissima Hance)根茎中分得九个化合物,其中已报道的七个化合物是尿囊素、马兜铃内酯、绵毛马兜铃内酯、β-谷甾醇、马兜铃酸A、9-乙氧基马兜铃内酯和9-乙氧基马兜铃内酰胺,本文报道结晶K₃的化学结构,经光谱分析(IR,¹H-NMR,¹³C-NMR,2D-NMR和MS),化学反应及X-衍射晶体分析,确证K₃为一个新骨架结构的倍半萜化合物,命名为马兜铃新内酯。     

Interaction of the diuretics torasemide and U-37883A with the KATP channel in rat isolated aorta

In this study we have investigated the interaction of the loop diuretics torasemide and furosemide and of the eukalemic diuretic U-37883A (4-morpholinocarboximidine-N–1-adamantyl-N’-cyclohexylhydrochloride) with the ATP-sensitive K⁺ channel (K_ATP channel) in rat aortic rings. Torasemide contains a sulphonylurea group which might enable the compound to interfere with K_ATP channels; this group is lacking in furosemide. U-37883A blocks several types of K_ATP channels. The interaction with the vascular K_ATP channel was probed in binding studies, ⁸⁶Rb⁺ efflux experiments and vasorelaxation assays. Torasemide inhibited the binding of the K_ATP channel inhibitor [³H]glibenclamide and of the opener [³H]P1075 with IC₅₀ values of 19 and 45 μM, respectively; furosemide and U-37883A were inactive or interfered with binding in a nonspecific way. In ⁸⁶Rb⁺ efflux experiments, the loop diuretics, at μM concentrations, inhibited basal tracer efflux to 50% whereas U-37883A had no effect. P1075-stimulated ⁸⁶Rb⁺ efflux, a qualitative measure of K_ATP channel opening, was inhibited by U-37883A and torasemide with IC₅₀ values of 0.06 and 130 μM, respectively; furosemide induced only a small (23%) inhibition. In experiments measuring isometric force, torasemide and furosemide partially relaxed endothelium-denuded aortic rings precontracted with noradrenaline or KCl with EC₅₀ values between 6 and 10 μM. The vasorelaxant effect of P1075 was inhibited in a noncompetitive manner by torasemide (300 μM) but unaffected by furosemide. U-37883A increased noradrenaline-induced force and inhibited the vasorelaxant effect of P1075 in an apparently competitive manner with an inhibition constant of 0.4 μM. The data show that torasemide interferes specifically with the binding of the K_ATP channel modulators [³H]glibenclamide and [³H]P1075 and with the K_ATP channel opening and vasorelaxant effects of P1075 whereas furosemide is inactive. This suggests that the interaction of torasemide with the vascular K_ATP channel is due to the sulphonylurea group present in torasemide. U-37883A, which does not inhibit P1075 binding, is one of the most potent blockers of P1075-induced ⁸⁶Rb⁺ efflux yet described but is relatively weak as an inhibitor of P1075-mediated vasorelaxation. The opposite vascular actions of torasemide and U-37883A are expected to contribute to the renal effects of these drugs. Received: 28 January 1998 / Accepted: 20 April 1998     

牛蒡子苷代谢动力学与分布研究

目的研究牛蒡子苷代谢动力学与分布。方法单剂量随机灌胃,高效液相色谱法测定大鼠体内牛蒡子苷浓度及其在脏器中的分布,代谢动力学分析采用3P87软件。结果口服牛蒡子苷300 mg/kg在大鼠体内呈二室模型分布,其主要动力学参数为A=(37.374 5±8.964 7)μg·mL^-1;B =(6.210 6±1.489 3)μg·mL^-1;α=(0.004 3±0.000 9)min^-1;β=(0.000 4±0.000 2)min^-1;K_α==(0.420 2±0.167 5)min^-1;t_1/2α=(115.192 6±14.382 4)min ;t_1/2β=(1 485.578 1±161.173 3)min;K₁₀=(0.001 0±0.000 4)min^-1;K₂₁=(0.001 4±0.000 6)minn^-1;K₁₂=(0.002 3±0.001 3)min-1;C_max=(41.786 3±7.521 7)μg·mL^-1;T_max=(9.891 9±4.341 4)min;AUC =(22 503.272 7±4 120.182 8)μg·min·mL^-1。牛蒡子苷在心、肝、肾、脑等脏器也有分布,以肝脏中最高。结论高效液相色谱法测定牛蒡子苷在动物体内代谢变化,快速、受杂质干扰小,且稳定性和重现性较好,适合牛蒡子苷代谢产物含量测定。牛蒡子苷在体内吸收很快,消除也快。     

慢性孵育β-淀粉样肽(25-35)对培养大鼠海马神经元外向钾电流的影响

目的　研究慢性孵育β-淀粉样肽_(25-35) (β-AP_25-35)对海马神经元上瞬时外向钾电流(I_A)和延迟整流钾电流(I_K)的影响。方法　在培养的大鼠海马神经元上用膜片钳全细胞记录钾通道电流。结果　β-AP_25-35 3μmol·L^-1 孵育细胞24h ,I_K 电流幅度增加(44.3±5.4)% ,电流密度由(30.4±6.4)pA·PF^-1 增加至(43.8±4.7)pA·PF^-1 ;β-AP_25-3510μmol·L^-1 孵育12h ,I_K 电流幅度增加(69.8±4.1) % ,电流密度增加至(51.6±7.9)pA·PF^-1,呈浓度依赖性;β-AP_25-35引起的I_K 增加对TEA 5mmol·L^-1 敏感;β-AP_25-35上调I_K 的作用主要发生在β-AP_25-355用药后48h内。β-AP_25-35对I_A无显著性影响。结论　β-AP_25-35选择性地增加海马神经元上I_K,这一作用可能与β-AP的神经毒性有关     

维生素K3中枢镇痛效应的探讨

经由大鼠、小鼠甩尾及兔甩头法测痛,证实k₃具有剂量依赖的镇痛作用。兔侧脑室微量注射k₃亦有显著镇痛效应。k₃的镇痛作用可被阿片拮抗剂纳络酮所拮抗。实验观察到k₃与吗啡的镇痛效应间存在交叉耐受现象。一定浓度的k₃可抑制电场刺激所致豚鼠回肠纵肌标本的收缩,这一效应亦可被纳络酮部分逆转。小鼠经k₃预处理后对k₃的镇痛产生耐受;连续k₃大剂量预处理后纳络酮激发不产生跳跃。     

KV7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries

BACKGROUND AND PURPOSE

Hypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca²⁺ concentration ([Ca²⁺]_i) by opening of K channels and release of H₂S.

EXPERIMENTAL APPROACH

Porcine coronary arteries without endothelium were mounted for measurement of isometric tension and [Ca²⁺]_i, and the expression of voltage-gated K channels K_V7 channels (encoded by KCNQ genes) and large-conductance calcium-activated K channels (K_Ca1.1) was examined. Voltage clamp assessed the role of K_V7 channels in hypoxia.

KEY RESULTS

Gradual reduction of oxygen concentration from 95 to 1% dilated the precontracted coronary arteries and this was associated with reduced [Ca²⁺]_i in PGF_2α (10 μM)-contracted arteries whereas no fall in [Ca²⁺]_i was observed in 30 mM K-contracted arteries. Blockers of ATP-sensitive voltage-gated potassium channels and K_Ca1.1 inhibited hypoxia-induced dilatation in PGF_2α-contracted arteries; this inhibition was more marked in the presence of the K_v7 channel blockers, XE991 and linopirdine, while a K_V7.1 blocker, failed to change hypoxic vasodilatation. XE991 also inhibited H₂S- and adenosine-induced vasodilatation. PCR revealed the expression of K_V7.1, K_V7.4, K_V7.5 and K_Ca1.1 channels, and K_Ca1.1, K_V7.4 and K_V7.5 were also identified by immunoblotting. Voltage clamp studies showed the XE991-sensitive current was more marked in hypoxic conditions.

CONCLUSION

The K_V7.4 and K_V7.5 channels, which we identified in the coronary arteries, appear to have a major role in hypoxia-induced vasodilatation. The voltage clamp results further support the involvement of K_V7 channels in this vasodilatation. Activation of these K_V7 channels may be induced by H₂S and adenosine.     

Modulation of K2P2.1 and K2P10.1 K+ channel sensitivity to carvedilol by alternative mRNA translation initiation

Background and Purpose

The β-receptor antagonist carvedilol blocks a range of ion channels. K_2P2.1 (TREK1) and K_2P10.1 (TREK2) channels are expressed in the heart and regulated by alternative translation initiation (ATI) of their mRNA, producing functionally distinct channel variants. The first objective was to investigate acute effects of carvedilol on human K_2P2.1 and K_2P10.1 channels. Second, we sought to study ATI-dependent modulation of K_2P K⁺ current sensitivity to carvedilol.

Experimental Approach

Using standard electrophysiological techniques, we recorded currents from wild-type and mutant K_2P2.1 and K_2P10.1 channels in Xenopus oocytes and HEK 293 cells.

Key Results

Carvedilol concentration-dependently inhibited K_2P2.1 channels (IC_50,oocytes = 20.3 μM; IC_50,HEK = 1.6 μM) and this inhibition was frequency-independent. When K_2P2.1 isoforms generated by ATI were studied separately in oocytes, the IC₅₀ value for carvedilol inhibition of full-length channels (16.5 μM) was almost 5-fold less than that for the truncated channel variant (IC₅₀ = 79.0 μM). Similarly, the related K_2P10.1 channels were blocked by carvedilol (IC_50,oocytes = 24.0 μM; IC_50,HEK = 7.6 μM) and subject to ATI-dependent modulation of drug sensitivity.

Conclusions and Implications

Carvedilol targets K_2P2.1 and K_2P10.1 K⁺ channels. This previously unrecognized mechanism supports a general role of cardiac K_2P channels as antiarrhythmic drug targets. Furthermore, the work reveals that the sensitivity of the cardiac ion channels K_2P2.1 and K_2P10.1 to block was modulated by alternative mRNA translation initiation.     

Warfarin and metabolism of vitamin K1

To further examine the hypothesis that warfarin inhibits prothrombin synthesis by interfering with the cyclic interconversion of vitamin K₁ and phylloquinone epoxide, the metabolism of tracer doses of labeled vitamin K₁ was studied in anticoagulant-treated rats. A tracer dose of [³H]K₁ initially turned over with a half-life of 2.2 hr in the liver, but after 5 hr the degradation rate decreased considerably. Warfarin caused hepatic [³H]K₁ levels to drop to 50 per cent of controls 5 min after administration of the vitamin, and over 10 hr the concentration of [³H]K₁ was 26–36 per cent of control levels. This decrease in vitamin K₁ was not large enough to account for the inhibition of prothrombin synthesis by warfarin. Thirty-five min after warfarin administration, there was more [³H]phylloquinone epoxide in the liver than labeled vitamin and over 10 hr the [³H]epoxide: [³H]K₁ ratio varied from 1.6 to 3.0. In addition to increasing the relative amount of [³H]epoxide, warfarin and phenylindanedione also increased hepatic metabolites more polar than vitamin K₁ or epoxide. However, warfarin did not increase plasma or urinary radioactivity. The urine was the principal excretory route for metabolites of vitamin K₁. 2-Diethylaminoethyl-2,2, diphenylvalerate hydrochloride (SKF 525-A) inhibited the turnover of [³H]K₁ but did not decrease the [³H]polar metabolites in the liver in control or warfarin-treated rats. The drug also did not inhibit the epoxidation of the vitamin to phylloquinone epoxide. The metabolism of vitamin K₁ was not altered by vitamin K deficiency or chronic administration of the vitamin. After a small dose of warfarin (35 μg/100 g body wt), plasma prothrombin decreased for 14 hr and then slowly rose but did not reach 90 per cent of normal until 3 days after administration of anticoagulant. The epoxide: K₁ ratio in the liver, measured by injecting a tracer dose of [³H]K₁, was 0.90 at 6 hr when prothrombin synthesis was completely blocked. The ratio dropped to 0.52 just before prothrombin started to rise at 14 hr and remained around 0.5 during the slow climb of plasma prothrombin toward the normal level. These low ratios were unexpected in animals in which prothrombin synthesis was partially or completely blocked. Hepatic epoxide: K₁ ratios of 11.4 and 1.3 were observed in warfarin-treated rats in which prothrombin synthesis was stimulated by injections of 0.1 mg [³H]K₁ plus 0.4 mg [³H]epoxide and 0.1 mg [³H]K₁ respectively. Therefore, there appears to be a lack of correlation of epoxide: K₁ ratios and prothrombin synthesis.     

Characteristics and molecular basis of celecoxib modulation on K(v)7 potassium channels

BACKGROUND AND PURPOSE

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used for the treatment of pain and inflammation. Emerging and accumulating evidence suggests that celecoxib can affect cellular targets other than COX, such as ion channels. In this study, we characterized the effects of celecoxib on K_v7 K⁺ channels and compared its effects with the well-established K_v7 channel opener retigabine.

EXPERIMENTAL APPROACH

A perforated whole-cell patch technique was used to record K_v7currents expressed in HEK 293 cells and M-type currents from rat superior cervical ganglion neurons.

KEY RESULTS

Celecoxib enhanced K_v7.2–7.4, K_v7.2/7.3 and K_v7.3/7.5 currents but inhibited K_v7.1 and K_v7.1/KCNE1 currents and these effects were concentration dependent. The IC₅₀ value for inhibition of K_v7.1 channels was approximately 4 µM and the EC₅₀ values for activation of K_v7.2–7.4, K_v7.2/K_v7.3 and K_v7.3/K_v7.5 channels were approximately 2–5 µM. The effects of celecoxib were manifested by increasing current amplitudes, shifting the voltage-dependent activation curve in a more negative direction and slowing the deactivation of K_v7 currents. 2,5-Dimethyl-celecoxib, a celecoxib analogue devoid of COX inhibition activity, has similar but greater effects on K_v7currents. K_v7.2(A235T) and K_v7.2(W236L) mutant channels, which have greatly attenuated responses to retigabine, showed a reversed response to celecoxib, from activation to inhibition.

CONCLUSIONS AND IMPLICATIONS

These results suggest that K_v7 channels are targets of celecoxib action and provide new mechanistic evidence for understanding the effects of celecoxib. They also provide a new approach to developing K_v7 modulators and for studying the structure–function relationship of K_v7 channels.     

Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling

BACKGROUND AND PURPOSE

Controlling vascular tone involves K⁺ efflux through endothelial cell small- and intermediate-conductance calcium-activated potassium channels (K_Ca2.3 and K_Ca3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling.

EXPERIMENTAL APPROACH

Transgenic mice expressing enhanced green fluorescent protein (eGFP) in astrocytes were used to assess K_Ca2.3 and K_Ca3.1 expression by immunohistochemistry and RT-PCR. K_Ca currents in eGFP-positive astrocytes were determined in situ using whole-cell patch clamp electrophysiology. The contribution of K_Ca3.1 to neurovascular coupling was investigated in pharmacological experiments using electrical field stimulation (EFS) to evoke parenchymal arteriole dilatation in FVB/NJ mouse brain slices and whisker stimulation to evoke changes in cerebral blood flow in vivo, measured by laser Doppler flowmetry.

KEY RESULTS

K_Ca3.1 immunoreactivity was restricted to astrocyte processes and endfeet and RT-PCR confirmed astrocytic K_Ca2.3 and K_Ca3.1 mRNA expression. With 200 nM [Ca²⁺]_i, the K_Ca2.1-2.3/K_Ca3.1 opener NS309 increased whole-cell currents. CyPPA, a K_Ca2.2/K_Ca2.3 opener, was without effect. With 1 µM [Ca²⁺]_i, the K_Ca3.1 inhibitor TRAM-34 reduced currents whereas apamin (K_Ca2.1-2.3 blocker) had no effect. CyPPA also inhibited currents evoked by NS309 in HEK293 cells expressing K_Ca3.1. EFS-evoked Fluo-4 fluorescence confirmed astrocyte endfoot recruitment into neurovascular coupling. TRAM-34 inhibited EFS-evoked arteriolar dilatation by 50% whereas charybdotoxin, a blocker of K_Ca3.1 and the large-conductance K_Ca channel, K_Ca1.1, inhibited dilatation by 82%. TRAM-34 reduced the cortical hyperaemic response to whisker stimulation by 40%.

CONCLUSION AND IMPLICATIONS

Astrocytes express functional K_Ca3.1 channels, and these contribute to neurovascular coupling.

LINKED ARTICLES

This article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-3     

Differential effects of Kv11.1 activators on Kv11.1a, Kv11.1b and Kv11.1a/Kv11.1b channels

BACKGROUND AND PURPOSE

K_v11.1 channels are involved in regulating cellular excitability in various tissues including brain, heart and smooth muscle. In these tissues, at least two isoforms, K_v11.1a and K_v11.1b, with different kinetics, are expressed. K_v11.1 activators are potential therapeutic agents, but their effects have only been tested on the K_v11.1a isoform. In this study, the effects of two different K_v11.1 activators, NS1643 and RPR260243, were characterized on K_v11.1a and K_v11.1b channels.

EXPERIMENTAL APPROACH

K_v11.1a and K_v11.1b channels were expressed in Xenopus laevis oocytes, and currents were measured using two-electrode voltage clamp. I/V curves and channel kinetics were measured before and after application of 30 µM NS1643 or 10 µM RPR260243.

KEY RESULTS

NS1643 increased steady-state currents through Kv11.1b several fold more than through K_v11.1a channels, without affecting EC₅₀ values. NS1643 increased activation rates and decreased rates of inactivation, recovery from inactivation and deactivation for both channels. Except for activation, where effect of NS1643 was comparable, relative changes were greater for Kv11.1b than for K_v11.1a. RPR260243 increased steady-state currents only through Kv11.1a channels, but slowed the process of deactivation for both channels primarily by decreasing time constant of slow deactivation. This effect was greater on K_v11.1b than on K_v11.1a. Effects of both compounds on heteromeric K_v11.1a/K_v11.1b channels were similar to those on K_v11.1a.

CONCLUSIONS AND IMPLICATIONS

Both NS1643 and RPR260243 displayed differential effects on K_v11.1a and K_v11.1b channels, the effects being relatively more pronounced on K_v11.1b channels. This affirms the importance of testing the effect of K_v11.1 activators on different channel isoforms.     

Effect of the Ito activator NS5806 on cloned Kv4 channels depends on the accessory protein KChIP2

BACKGROUND AND PURPOSE

The compound NS5806 increases the transient outward current (I_to) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I_to is thought to be mediated by K_V4.3 and various ancillary proteins, yet, the exact subunit composition of I_to channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative I_to channel subunits and other potassium channels.

EXPERIMENTAL APPROACH

Cloned K_V4 channels were co-expressed with KChIP2, DPP6, DPP10, KCNE2, KCNE3 and K_V1.4 in Xenopus laevis oocytes or CHO-K1 cells.

KEY RESULTS

NS5806 increased K_V4.3/KChIP2 peak current amplitudes with an EC₅₀ of 5.3 ± 1.5µM and significantly slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated K_V4.3 currents and the response to NS5806, but current decay was slowed only in complexes containing KChIP2. The effect of NS5806 on K_V4.2 was similar to that on K_V4.3, and current decay was only slowed in presence of KChIP2. However, for K_V4.1, the slowing of current decay by NS5806 was independent of KChIP2. K_V1.4 was strongly inhibited by 10 µM NS5806 and K_V1.5 was inhibited to a smaller extent. Effects of NS5806 on kinetics of currents generated by K_V4.3/KChIP2/DPP6 with K_V1.4 in oocytes could reproduce those on cardiac I_to in canine ventricular myocytes. K_V7.1, K_V11.1 and K_ir2 currents were unaffected by NS5806.

CONCLUSION AND IMPLICATIONS

NS5806 modulated K_V4 channel gating depending on the presence of KChIP2, suggesting that NS5806 can potentially be used to address the molecular composition as well as the physiological role of cardiac I_to.     

Effect of tyrphostin AG879 on Kv4.2 and Kv4.3 potassium channels

Background and Purpose

A-type potassium channels (I_A) are important proteins for modulating neuronal membrane excitability. The expression and activity of K_v4.2 channels are critical for neurological functions and pharmacological inhibitors of K_v4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a K_v4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned K_v4.2/K_v channel-interacting protein 2 (KChIP2) channels.

Experimental Approach

To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with K_v4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing K_v4.2/KChIP2 channels using a whole-cell patch-clamp technique.

Key Results

Tyrphostin AG879 selectively and dose-dependently inhibited K_v4.2 and K_v4.3 channels. In K_v4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of K_v4.2 channels without apparent effect on the V_1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons.

Conclusion and Implications

AG879 was identified as a selective and potent inhibitor the K_v4.2 channel. AG879 inhibited K_v4.2 channels by preferentially interacting with the open state and further accelerating their inactivation.     

Activation of endothelial and epithelial K(Ca) 2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

BACKGROUND AND PURPOSE

Small (K_Ca2) and intermediate (K_Ca3.1) conductance calcium-activated potassium channels (K_Ca) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we investigated the expression of K_Ca2.3 and K_Ca3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries.

EXPERIMENTAL APPROACH

Channel expression and functional studies were conducted in human isolated small pulmonary arteries and bronchioles. K_Ca2 and K_Ca3.1 currents were examined in human small airways epithelial (HSAEpi) cells by whole-cell patch clamp techniques.

RESULTS

While K_Ca2.3 expression was similar, K_Ca3.1 protein was more highly expressed in pulmonary arteries than bronchioles. Immunoreactive K_Ca2.3 and K_Ca3.1 proteins were found in both endothelium and epithelium. K_Ca currents were present in HSAEpi cells and sensitive to the K_Ca2.3 blocker UCL1684 and the K_Ca3.1 blocker TRAM-34. In pulmonary arteries contracted by U46619 and in bronchioles contracted by histamine, the K_Ca2.3/ K_Ca3.1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the K_Ca2 channel blocker apamin, while the K_Ca3.1 channel blocker, charybdotoxin failed to reduce relaxation to NS309 (0.01–1 µM).

CONCLUSIONS AND IMPLICATIONS

K_Ca2.3 and K_Ca3.1 channels are expressed in the endothelium of human pulmonary arteries and epithelium of bronchioles. K_Ca2.3 channels contributed to endo- and epithelium-dependent relaxations suggesting that these channels are potential targets for treatment of pulmonary hypertension and chronic obstructive pulmonary disease.