Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats

We have previously shown that diabetes impaired cAMP-mediated endothelium independent vasodilation of rat small coronary arteries. Inhibition of Kv channel activity plays an important role in the decrease of cAMP mediated vasodilation. The present study investigated the effect of streptozotocin (STZ)-induced diabetes on mRNA and protein expressions of Kv1.2 and Kv1.5 channels in vascular smooth muscle cells of rat small coronary artery using RT-PCR, Western blot and immunohistochemistry methods. STZ-induced diabetes obviously impaired mRNA expression of Kv1.2 and Kv1.5 channel. The mRNA levels of Kv1.2 channel were 0.65 +/- 0.08 and 1.02 +/- 0.17 in STZ rats and control rats, respectively (n = 7, P < 0.05). Whereas the levels of Kv1.5 channel were 0.58 +/- 0.05 and 0.94 +/- 0.13 in STZ rats and control rats, respectively (n = 7, P < 0.05). Western blotting analysis showed that protein expression of Kv1.2 channel was decreased significantly but not Kv1.5 channel. Protein expressions of Kv1.2 channel were 0.49 +/- 0.04 and 0.70 +/- 0.06 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 0.61 +/- 0.12 and 0.59 +/- 0.14 in STZ rats and control rats, respectively (n = 5, P > 0.05). Immunohistochemistry identification indicated that immunological reaction of Kv1.2 channel protein was attenuated, but Kv1.5 channel protein was not altered. Positive staining intensity normalized by gray values of Kv1.2 channel were 173 +/- 13 and 131 +/- 11 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 139 +/- 16 and 141 +/- 12 in STZ rats and control rats, respectively (n = 5, P > 0.05). These results suggested that impairment of cAMP-mediated endothelium independent vasodilation of rat small coronary artery by STZ-induced diabetes was resulted from decrease of mRNA and protein expressions of Kv channels, and which eventually leads to a reduced current from Kv channels.     

Elevated glucose impairs cAMP-mediated dilation by reducing Kv channel activity in rat small coronary smooth muscle cells

Hyperglycemia impairs endothelium-dependent vasodilation. In this study, we examined the effect of high glucose (HG) on vascular smooth muscle function. Rat small coronary arteries were freshly isolated or incubated for 24 h with normal glucose (NG; 5.5 mmol/l) or HG (23 mmol/l). In freshly isolated arteries, dilation to isoproterenol (Iso) was reduced by 3 mmol/l 4-aminopyridine (4-AP; 44 +/- 10% vs. 77 +/- 4%; P < 0.05) and further reduced by 4-AP + iberiotoxin (IbTX; 100 nmol/l; 17 +/- 2%). Dilation to forskolin was abolished by 4-AP (-3 +/- 17 vs. 73 +/- 9%). cAMP production was similar in NG and HG vessels. Dilations to Iso and forskolin were significantly reduced in HG arteries (Iso, 41 +/- 5% vs. 70 +/- 6%; forskolin, 40 +/- 4% vs. 75 +/- 4%) compared with NG arteries. A similar reduction was also observed to the dilation to papaverine. Endothelial denudation had no effect on Iso-induced dilation. In HG vessels, the reduced 4-AP-sensitive component of Iso-induced dilation was greater compared with the IbTX-sensitive component. Iso increased whole cell K+ current in NG cells but had little effect in HG cells. Similarly, 4-AP-, but not IbTX-sensitive, K+ currents were reduced in HG cells. These results suggest that HG impairs cAMP-mediated dilation primarily by reducing Kv channel function. We speculate that in addition to the endothelial dysfunction, altered smooth muscle function may also contribute to the reduced coronary vasodilation in diabetes.     

Mitochondria-dependent regulation of Kv currents in rat pulmonary artery smooth muscle cells

Voltage-gated K(+) (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen-sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence, an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes, were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised an enhancement of Kv current in a negative potential range (manifested as a 5- to 14-mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on Kv channel currents (I(Kv)) revealed the presence of a mitochondria-mediated Mg(2+) and ATP-dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species.     

H2O2 activates redox- and 4-aminopyridine-sensitive Kv channels in coronary vascular smooth muscle

Previously, we demonstrated that coronary vasodilation in response to hydrogen peroxide (H(2)O(2)) is attenuated by 4-aminopyridine (4-AP), an inhibitor of voltage-gated K(+) (K(V)) channels. Using whole cell patch-clamp techniques, we tested the hypothesis that H(2)O(2) increases K(+) current in coronary artery smooth muscle cells. H(2)O(2) increased K(+) current in a concentration-dependent manner (increases of 14 +/- 3 and 43 +/- 4% at 0 mV with 1 and 10 mM H(2)O(2), respectively). H(2)O(2) increased a conductance that was half-activated at -18 +/- 1 mV and half-inactivated at -36 +/- 2 mV. H(2)O(2) increased current amplitude; however, the voltages of half activation and inactivation were not altered. Dithiothreitol, a thiol reductant, reversed the effect of H(2)O(2) on K(+) current and significantly shifted the voltage of half-activation to -10 +/- 1 mV. N-ethylmaleimide, a thiol-alkylating agent, blocked the effect of H(2)O(2) to increase K(+) current. Neither tetraethylammonium (1 mM) nor iberiotoxin (100 nM), antagonists of Ca(2+)-activated K(+) channels, blocked the effect of H(2)O(2) to increase K(+) current. In contrast, 3 mM 4-AP completely blocked the effect of H(2)O(2) to increase K(+) current. These findings lead us to conclude that H(2)O(2) increases the activity of 4-AP-sensitive K(V) channels. Furthermore, our data support the idea that 4-AP-sensitive K(V) channels are redox sensitive and contribute to H(2)O(2)-induced coronary vasodilation.     

平滑肌细胞上的钙库操纵性通道

钙库操纵性通道(SOC)是目前研究较热门的一种离子通道,其开放与关闭受内质网中Ca2 贮量调控。SOC参与机体许多重要生理功能的调节,尤其在平滑肌紧张性变化的调节中起重要作用。果蝇瞬时受体电位(transient receptor potential,TRP)蛋白在光信号传递中发挥重要作用,在哺乳动物中,发现TRP蛋白的同系物TRPC1蛋白是SOC的组成成分。研究并深入了解SOC的特性对于开发一类新的钙通道拮抗剂具有重要的理论意义。     

Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

大鼠结肠平滑肌细胞的钙库操纵性通道

目的:探讨大鼠结肠平滑肌细胞是否存在钙库操纵性通道(SOC)。方法:荧光探针Fura-2/AM标记细胞内游离Ca2+后,用荧光分光光度计检测毒胡萝卜素(thapsigargin)和咖啡因(caffeine)耗竭胞内钙库后激活的SOC通道对酶解分离的大鼠结肠平滑肌细胞[Ca2+]i的影响。结果:在无Ca2+缓冲液中,thapsigargin(1μmol/L)以及caf-feine(10 mmol/L)分别使[Ca2+]i由静息时(68.32±3.43)nmol/L升高至(240.85±12.65)nmol/L(、481.25±34.77)nmol/L,继之,向细胞外液中引入两种浓度的Ca2+(1.5 mmol/L和3.0 mmol/L),导致[Ca2+]i进一步升高,分别为(457.55±19.80)nmol/L、(1005.93±54.62)nmol/L;(643.88±34.65)nmol/L、(920.16±43.25)nmol/L。且上述升高效应对维拉帕米(verapamil,5μmol/L)以及KCl引起的细胞膜去极化不敏感,但可被La3+(1 mmol/L)抑制。结论:在酶解分离的大鼠结肠平滑肌细胞上,存在胞内钙库耗竭激活的SOC通道,为支持在电兴奋性细胞上存在库容性Ca2+内流提供了实验和理论依据。     

Voltage-dependent anion-selective channels in cultured smooth muscle cells of the rat aorta

1. Properties of the voltage-dependent anion-selective channel in cultured smooth muscle cells of the rat aorta were studied using the patch-clamp technique. 2. The channel had a single channel conductance of 346 +/- 4 pS (n = 43, mean +/- SEM) with symmetrical 142 mM-Cl- solution in inside-out patch configurations. 3. The channel was activated spontaneously at a potential range -20 approximately +20 mV and inactivated more rapidly with increases to more positive or negative potentials. 4. The channel was selective for anions and the permeability ratio for monovalent anion was Br-:Cl-:HCOO-:CH3COO-:propionate-:aspartate- = 1.1:1:0.7:0.4: less than 0.02: less than 0.02. 5. The openings of the channels were observed more frequently in inside-out membrane patches than in cell-attached ones, and were independent of intracellular free Ca concentrations. 6. The density of this channel was estimated to be 1.3/micron2. 7. Physiological roles of the channel were discussed.     

三磷酸肌醇对猪冠状动脉平滑肌细胞大电导钙激活钾通道的作用

应用膜片钳单通道电流记录技术,研究三磷酸肌醇(trisphosphateinositol,IP3)对猪冠状动脉平滑肌细胞大电导钙激活钾通道(large-conductanceCa2+-activatedpotassiumchannels,BKchannels)的作用。结果显示:在内面向外式(inside-out)膜片下,IP3(10~50μmol/L)可以浓度依赖性地增加通道的开放概率,而对电流幅值无明显影响,开放概率的增加是通过明显缩短平均关闭时间实现的(n=11,P<0.01);洗去药物后通道活性可以恢复到对照水平;IP3对通道的激活作用不随时间而衰减;IP3的降解产物对通道没有明显的激活作用。结果表明:在inside-out膜片下,IP3能够激活猪冠状动脉平滑肌细胞BK通道。     

Connexin45 gap junction channels in rat cerebral vascular smooth muscle cells

Cells in blood vessel walls express connexin (Cx)43, Cx40, and Cx37. We recently characterized gap junction channels in rat basilar artery smooth muscle cells and found features attributable not only to these three connexins but also to an unidentified connexin, including strong voltage dependence and single channel conductance of 30-40 pS. Here, we report data consistent with identification of Cx45. Immunofluorescence using anti-human Cx45 and anti-mouse Cx45 antibodies revealed labeling between alpha-actin-positive cells, and RT-PCR of mRNA from arteries after endothelial destruction yielded amplicons exhibiting 90-98% identity with mouse Cx45 and human Cx45. Dual-perforated patch clamping was performed after exposure to oligopeptides that interfere with docking of Cx43, Cx40, or Cx45. Cell pairs pretreated with blocking peptides for Cx43 and Cx40 exhibited strongly voltage-dependent transjunctional conductances [voltage at which voltage-dependent conductance declines by one-half (V1/2) = +/-18.9 mV] and small single channel conductances (31 pS), consistent with the presence of Cx45, whereas cell pairs pretreated with blocking peptide for Cx45 exhibit weaker voltage-dependent conductances (V1/2 = +/-37.9 mV), consistent with block of Cx45. Our data suggest that Cx45 is transcribed, expressed, and forms functional gap junction channels in rat cerebral arterial smooth muscle.     

尼古丁调节大鼠冠状动脉平滑肌细胞大电导钙激活钾通道

目的:研究尼古丁对Wistar大鼠冠状动脉平滑肌大电导钙激活钾通道(BKca)活性的抑制作用及其细胞信号转导机制。方法:8周雄性Wistar大鼠随机分为两组:生理盐水组和尼古丁组;分别予以生理盐水和尼古丁2mg/(kg.d)注射21 d,蛋白酶法分离冠状动脉血管平滑肌细胞,将两组平滑肌细胞分别以对氯苯硫基环腺苷酸(CPT-cAMP,100μmol/L)和佛司可林(forskolin,10μmol/L)干预,单通道膜片钳记录干预前后平滑肌细胞单通道电流的平均开放时间(To)、平均关闭时间(Tc)、平均开放概率(Po)。结果:CPT-cAMP和Forskolin均能显著延长生理盐水组大鼠BKca的平均开放时间,缩短平均关闭时间,增加通道开放概率(P均<0.01)。对尼古丁组BKca的To、Tc、Po均无明显影响。结论:尼古丁促使冠状动脉血管收缩的生理机制是通过抑制cAMP/PKA途径诱导的大电导钙激活钾通道活性增加实现的。     

Muscarinic effects on ion channels in smooth muscle cells

Acetylcholine is the most important excitatory neurotransmitter providing depolarization of the membrane and contraction of different smooth muscle cells due to activation of the muscarinic receptors. In our review, we analyze and summarize the published data on the effects of activation of acetylcholine muscarinic receptors on ion channels expressed in smooth muscle cells of different organs and the results of our own studies of this topic. Special attention is paid to the mechanisms of depolarizing effects of acetylcholine mediated by activation of non-selective cationic channels. Intracellular mechanisms underlying modulating influences on calcium, potassium, and chloride channels are also analyzed. Physiological roles of activation and regulation of different ion channels and possible interactions within this complicated system are discussed.     

Oxytocin actions on voltage-dependent ionic channels in pregnant rat uterine smooth muscle cells.

The effects of oxytocin, a uterotonic polypeptide hormone, on the voltage-dependent slow calcium, fast sodium, and potassium channel currents were studied using whole-cell voltage clamp of freshly isolated cells from late pregnant (18-21 day) rat myometrium. The calcium current was rapidly inhibited by oxytocin (about 25% inhibition at 20 nM) in a dose-dependent manner, and this inhibitory effect was completely reversible by washout. However, inhibition was not observed when barium was used as the charge carrier. Sodium current and potassium current were not modified by oxytocin, thus sodium and potassium currents may not play important roles in oxytocin-induced augmentation of uterine contraction. It is concluded that oxytocin stimulates uterine contraction by mechanisms other than augmentation of the voltage-dependent calcium current, e.g., by release of Ca from sarcoplasmic reticulum (by inositol triphosphate) or by activation of a receptor-operated Ca channel. The inhibition of the slow calcium current may be induced by the elevation of [Ca]i.     

Effects of streptozotocin-induced diabetes on acetylcholine metabolism in rat brain

The main objective of this study was to determine whether uncontrolled hyperglycemia, as a consequence of diabetes, altered the metabolism of acetylcholine (ACh) in rat brain. To accomplish this, rats received injections of streptozotocin (STZ, 60 mg/kg, i.v.) or vehicle, and were maintained for up to 7 weeks after the injections. Various indices of ACh metabolism were determined in striatum and hippocampus, two brain regions densely innervated by cholinergic neurons. STZ induced diabetes in 96% of the rats injected, as evidenced by glucose spillage into the urine within 48 hours. Serum glucose levels increased to 326% of control values by 1 week and remained at this level for the duration of the study. The steady-state concentrations of ACh and choline, determined in brain tissue from animals killed by head-focused microwave irradiation, did not differ between the control and STZ-injected groups. However, the synthesis and release of neurotransmitter by striatal slices, measured in vitro, decreased in a time-dependent manner. Although the basal release of ACh was unaltered at 1 week, neurotransmitter release decreased significantly by 21% at 5 weeks and by 26% at 7 weeks. The release of ACh evoked by incubation with 35 mM KCl was inhibited significantly by 20% at all time points studied. ACh synthesis by slices incubated under basal conditions decreased by 13% and 27% at 5- and 7-weeks, respectively, the latter significantly less than controls. Synthesis by striatal slices incubated with 35 mM KCl was inhibited by 17% at 7 weeks. Although the synthesis and release of ACh by hippocampal slices from diabetic animals tended to be less than controls, these alterations were not statistically significant. Investigations into the mechanism(s) mediating the deficit in ACh synthesis exhibited by striatal slices indicated that it did not involve alterations in precursor choline availability, nor could it be attributed to alterations in the activities of the synthetic or hydrolytic enzymes choline acetyltransferase or acetylcholinesterase; rather, the decreased turnover of ACh may be secondary to other STZ-induced, hyperglycemia-mediated neurochemical alterations.     

cGMP对原代培养猪冠状动脉平滑肌细胞钙激活钾通道的作用

３′,５′－环－磷酸鸟苷（ｃＧＭＰ）具有激活血管平滑肌细胞膜上钙激活钾通道（ＫＣａ通道）的作用,从而引起血管平滑肌细胞的舒张。但ｃＧＭＰ激活ＫＣａ物机制存在争论。本工作应用膜片箝技术以原代培养猪冠状动脉平滑肌细胞为对象研究了ｃＧＭＰ影响ＫＣａ通道的机制。实验结果显示：（１）在ｃｅｌｌ－ａｔｔａｃｈｅｄ膜片方式下,当溶液内游离Ｃａ＾２＋浓度为１０＾－７ｍｏｌ／Ｌ,膜电位为＋７０ｍＶ时,不同浓度的ｃＧ