Antidiarrheal potential of Distemonanthus benthamianus Baillon. extracts via inhibiting voltage-dependent calcium channels and cholinergic receptors

Objective:To evaluate spasmolytic mechanisms of aqueous and methanolic extracts from Distemonanthus benthamianus trunk-bark.Methods:Spasmolytic activities of extracts were evaluated in vitro on spontaneous and potassium chloride-induced jejunum contractions,or against cholinergic[acetylcholine(0.3μmol/L)]stimulations.High performance liquid chromatography analysis of both extracts was performed in reference to standard compounds.Results:Extracts developed concentration-dependent inhibitory activities.The methanolic extract,which revealed better activity,produced spasmolytic and myorelaxant effects at concentrations of 0.01-0.30 mg/mL with EC(50)of 0.06 and 0.09 mg/mL(95%CI:0.03-0.3 mg/mL),respectively.Its anticholinergic effect was obtained at the same concentrations with EC(50)of 0.11 mg/mL(95%CI:0.03-0.3 mg/mL).Chromatograms showed the presence of gallic acid in both extracts,rutin being only detected in the aqueous extract.Conclusions:Distemonanthus benthamianus extracts exhibit verapamil and atropine-like activities,thus highlighting calcium channels and muscarinic receptors blocking potentials,which may be conveyed by some phenolic compounds.These results confirm the antidiarrheal activity of Distemonanthus benthamianus extracts.

Types of voltage-dependent calcium channels involved in high potassium depolarization-induced amylase secretion in the exocrine pancreatic tumour cell line AR4-2J

In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted large amount of amylase. High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation. High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel antagonists with an order of potency as follows: nifedipine > ω-agatoxin IVA > ω-conotoxin GVIA. In contrast, the L-type calcium channel antagonist nifedipine almost completely inhibited potassium-induced amylase secretion, whereas the N-type channel antagonist ω-conotoxin GVIA was without effect. The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect, but this inhibition was not significant at the level of amylase secretion. In conclusion, the AR4-2J cell line possesses different voltage-dependent calcium channels (L, P,N) with the L-type predominantly involved in depolarization induced amylase secretion.

Pharmacological Investigation of Voltage-dependent Ca^(2+) Channels in Human Ejaculatory Sperm in vitro

The types of the voltage-dependent calcium channels （VDCCs） in human ejaculatory sperm and the effects of calcium channel blocker （CCB） on human sperm motility parameters in vitro were investigated. The human sperm motility parameters in vitro in response to the pharmacological agents nifedipine （NIF, inhibitor of L-type VDCC） and ω-conotoxin （GVIA, inhibitor of N-type VDCC） were compared and analyzed statistically. The results showed that NIF （1, 5, 10 μmol/L） could not only significantly affect human sperm＇s shape but also spermatozoa motility after incubated at least 10 rain in vitro （P〈0.001）. GVIA （0.1, 0.5 and 1 μmol/L） could just only significantly affect human sperm＇s progressive motility （a %＋b %） after incubated for 20 min in vitro （P〈0.01）, but they both could not significantly affect spermic abnormality rate. It is suggested that L-type VDCC, non L-type VDCCs and isoform of L-type VDCC exist in the cell membrane of human sperm solely or together, and they participate in the spermic physiological processes especially the spermic motility.

Effects of Rhynchophyline on L-type Calcium Channels in Isolated Rat Cortical Neurons During Acute Hypoxia

目的：研究钩藤碱对急性分离的Ｗｉｓｔａｒ大鼠大脑皮层神经元低氧状态下Ｌ型钙通道的作用。方法：细胞贴附膜片钳技术，从－４０ｍＶ去极化至０ｍＶ激活Ｌ型钙通道。结果：低氧可激活皮层神经元Ｌ型钙通道开放，使平均开放时间延长，平均关闭时间缩短，开放概率增加。钩藤碱（１５和３０μｍｏｌ·Ｌ－１）可浓度依赖性抑制低氧状态下此钙通道的激活，缩短其平均开放时间（从８８７ｍｓ缩至３０３ｍｓ和２２３ｍｓ，Ｐ＜０００１），延长其平均关闭时间（从９２３ｍｓ延至３８８４和５４４３ｍｓ，Ｐ＜０００１）并降低其开放概率（从０１４２降至００３１和００２５，Ｐ＜０００１）此作用与维拉帕米（１５μｍｏｌ·Ｌ－１）相似，但稍弱。结论：钩藤碱对低氧大鼠大脑皮层神经元Ｌ型钙通道有阻滞作用，从而降低细胞内钙超载。

Hydrogen sulfide-induced enhancement of gastric fundus smooth muscle tone is mediated by voltagedependent potassium and calcium channels in mice

AIM:To investigate the effect of hydrogen sulfide(H2S)on smooth muscle motility in the gastric fundus.METHODS:The expression of cystathionineβ-synthase(CBS)and cystathionineγ-lyase(CSE)in cultured smooth muscle cells from the gastric fundus was examined by the immunocytochemistry technique.The tension of the gastric fundus smooth muscle was recorded by an isometric force transducer under the condition of isometric contraction with each end of the smooth muscle strip tied with a silk thread.Intracellular recording was used to identify whether hydrogen sulfide affects the resting membrane potential of the gastric fundus in vitro.Cells were freshly separated from the gastric fundus of mice using a variety of enzyme digestion methods and whole-cell patch-clamp technique was used to find the effects of hydrogen sulfide on voltage-dependent potassium channel and calcium channel.Calcium imaging with fura-3AM loading was used to investigate the mechanism by which hydrogen sulfide regulates gastric fundus motility in cultured smooth muscle cells.RESULTS:We found that both CBS and CSE were expressed in the cul tured smooth muscle cel ls from the gastric fundus and that H2S increased the smooth muscle tension of the gastric fundus in mice at low concentrations.In addition,nicardipine and aminooxyacetic acid(AOAA),a CBS inhibitor,reduced the tension,whereas Nω-nitro-L-arginine methyl ester,a nonspecific nitric oxide synthase,increased the tension.The AOAA-induced relaxation was significantly recovered by H2S,and the Na HS-induced increase in tonic contraction was blocked by 5 mmol/L4-aminopyridine and 1μmol/L nicardipine.Na HS significantly depolarized the membrane potential and inhibited the voltage-dependent potassium currents.Moreover,Na HS increased L-type Ca2+currents and caused an elevation in intracellular calcium([Ca2+]i).CONCLUSION:These findings suggest that H2S may be an excitatory modulator in the gastric fundus in mice.The excitatory effect is mediated by voltagedependent potassium and L-type calcium channels.

Polydatin attenuated food allergy via store-operated calcium channels in mast cell

AIM: To investigate the effect of polydatin (PD), a resveratrol glucoside, on mast cell degranulation and antiallergic activity. METHODS: After the rats were orally sensitized with ovalbumin (OVA) for 48 d and underwent PD treatment for 4 d, all the rats were stimulated by 100 mg/mL OVA for24 h and then sacrificed for the following experiments. The small intestines from all the groups were prepared for morphology examination by hematoxylin and eosin staining. We also used a smooth muscle organ bath to evaluate the motility of the small intestines. The OVA-specific immunoglobulin E (IgE) production and interleu-kin-4 (IL-4) levels in serum or supernatant of intestinal mucosa homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA). Using toluidine blue stain, the activation and degranulation of isolated rat peritoneal mast cells (RPMCs) were analyzed. Release of histamine from RPMCs was measured by ELISA, and regulation of PD on intracellular Ca 2+ mobilization was investigated by probing intracellular Ca 2+ with fluo-4 fluo-rescent dye, with the signal recorded and analyzed. RESULTS: We found that intragastric treatment with PD significantly reduced loss of mucosal barrier integrity in the small intestine. However, OVA-sensitization caused significant hyperactivity in the small intestine of allergic rats, which was attenuated by PD administration by 42% (1.26 ± 0.13 g vs OVA 2.18 ± 0.21 g, P < 0.01). PD therapy also inhibited IgE production (3.95 ± 0.53 ng/mL vs OVA 4.53 ± 0.52 ng/mL, P < 0.05) by suppressing the secretion of Th2-type cytokine, IL-4, by 34% (38.58 ± 4.41 pg/mLvs OVA 58.15 ± 6.24 pg/mL, P < 0.01). The ratio of degranulated mast cells, as indicated by vehicles (at least five) around the cells, dramatically increased in the OVA group by 5.5 fold (63.50% ± 15.51% vs phosphate-buffered saline 11.15% ± 8.26%, P < 0.001) and fell by 65% after PD treatment (21.95% ± 4.37% vs OVA 63.50% ± 15.51%, P < 0.001). PD mediated attenuation of mast cell degranulation was further confirmed by decreased histamine levels in both serum (5.98 ± 0.17 vs OVA 6.67 ± 0.12, P < 0.05) and intestinal mucosa homogenates (5.83 ± 0.91 vs OVA 7.35 ± 0.97, P < 0.05). Furthermore, we demonstrated that administration with PD significantly decreased mast cell degranulation due to reduced Ca 2+ influx through store-operated calcium channels (SOCs) (2.35 ± 0.39vs OVA 3.51 ± 0.38,P < 0.01).CONCLUSION: Taken together, our data indicate that PD stabilizes mast cells by suppressing intracellular Ca 2+ mobilization, mainly through inhibiting Ca 2+ entry via SOCs, thus exerting a protective role against OVA-sensitized food allergy.

Calcium signaling and T-type calcium channels in cancer cell cycling

Regulation of intracellular calcium is an important signaling mechanism for cell proliferation in both normal and cancerous cells. In normal epithelial cells, free calcium concentration is essential for cells to enter and accomplish the S phase and the M phase of the cell cycle. In contrast, cancerous cells can pass these phases of the cell cycle with much lower cytoplasmic free calcium concentrations, indicating an alternative mechanism has developed for fulfilling the intracellular calcium requirement for an increased rate of DNA synthesis and mitosis of fast replicating cancerous cells. The detailed mechanism underlying the altered calcium loading pathway remains unclear; however, there is a growing body of evidence that suggests the T-type Ca2+ channel is abnormally expressed in cancerous cells and that blockade of these channels may reduce cell proliferation in addition to inducing apoptosis. Recent studies also show that the expression of T-type Ca2+ channels in breast cancer cells is proliferation state dependent, i.e. the channels are expressed at higher levels during the fast-replication period, and once the cells are in a non-proliferation state, expression of this channel isminimal. Therefore, selectively blocking calcium entry into cancerous cells may be a valuable approach for preventing tumor growth. Since T-type Ca2+ channels are not expressed in epithelial cells, selective T-type Ca2+ channel blockers may be useful in the treatment of certain types of cancers.

Voltage-dependent K^+-channel Responses during Activation and Damage in Alveolar Macrophages Induced by Quartz Particles

The roles of voltage-dependent K^＋ channels during activation and damage in alveolar macrophages （AMs） exposed to different silica particles were examined. Rat AMs were collected by means of bronchoalveolar lavage, and were adjusted to 5× 10^5/mL. After AMs were exposed to different concentrations （0, 25, 50, 100, 200 μg/mL） of quartz particles and 100 μg/mL amorphous silica particles for 24 h, the voltage-depended K^＋ current in AMs was measured by using patch clamp technique. Meanwhile the leakage of lactate dehydrogenase （LDH） and the viability of AMs were detected respectively. Patch clamp studies demonstrated that AMs possessed outward delayed and inward rectifying K^＋ current. Exposure to quartz particles increased the outward delayed K^＋ current but it had no effect on inward rectifier K^＋ current in AMs. Neither of the two K^＋ channels in AMs was affected by amorphous silica particles. Cytotoxicity test showed that both silica particles could damage AM membrane and result in significant leakage of LDH （P〈0.05）. MTT studies, however, showed that only quartz particles reduced viability of AMs （P〈0.05）. It is concluded that quartz parti- cles can activate the outward delayed K^＋ channel in AMs, which may act as an activating signal in AMs to initiate an inflammatory response during damage and necrosis in AMs induced by exposure to quartz particle. K^＋ channels do not contribute to the membrane damage of AMs.

The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa： differences from the zona pellucida

acrosome 反应(AR ) ，为精子的一个绝对要求和卵熔化，通过电压依赖者 Ca2+ 隧道和操作店的隧道要求 Ca2+ 的流入进精子。Maitotoxin (MTx ) ，一个动员 Ca2+ 代理人，被显示了是老鼠精子 AR 的有势力 inducer 与类似于带 pellucida (ZP ) 的药理学，可能为两 inducers 建议一条普通小径。使用的 recombinant 人 ZP3 (rhZP3 ) ，老鼠 ZP 和二 MTx 隧道 blockers (U73122 和 U73343 ) ，我们在人和老鼠精子调查了并且比较导致 MTx 、导致 ZP 的艺术。此处，我们报导 MTx 导致了 AR 并且提高了细胞内部的 Ca2+([Ca2+] 我) 在人的精子，哪个被 U73122 和 U73343 堵住。这二混合物也在老鼠精子禁止了导致 MTx 的 AR。在有我们的以前的建议的争论， AR 由 rhZP3 被触发或老鼠 ZP 没被 U73343 堵住，显示在人和老鼠精子，由生理的 ligands 或由 MTx 的 AR 正式就职通过不同小径发生了。U73122，然而并非 U73343 (不活跃的类似物) ，能堵住 phospholipase C (PLC ) 。另一个 PLC 禁止者， edelfosine，也堵住了导致 rhZP3 、导致 ZP 的艺术。这些调查结果在人和老鼠带证实了一条 PLC 依赖的发信号小径的参予导致蛋白质的 AR。尤其是， edelfosine 也禁止了导致 MTx 的老鼠精子 AR 然而并非人的，建议导致毒素的 AR 在老鼠是 PLC 依赖的并且在人 PLC 独立。

Calcium channels and iron uptake into the heart

Iron overload can lead to iron deposits in many tissues,particularly in the heart.It has also been shown to be associated with elevated oxidative stress in tissues.Elevated cardiac iron deposits can lead to iron overload cardiomyopathy,a condition which provokes mortality due to heart failure in iron-overloaded patients.Currently,the mechanism of iron uptake into cardiomyocytes is still not clearly understood.Growing evidence suggests L-type Ca2+channels(LTCCs)as a possible pathway for ferrous iron(Fe2+)uptake into cardiomyocytes under iron overload conditions.Nevertheless,controversy still exists since some findings on pharmacological interventions and those using different cell types do not support LTCC’s role as a portal for iron uptake in cardiac cells.Recently,T-type Ca2+channels (TTCC)have been shown to play an important role in the diseased heart.Although TTCC and iron uptake in cardiomyocytes has not been investigated greatly,a recent finding indicated that TTCC could be an important portal in thalassemic hearts.In this review,comprehensive findings collected from previous studies as well as a discussion of the controversy regarding iron uptake mechanisms into cardiomyocytes via calcium channels are presented with the hope that understanding the cellular iron uptake mechanism in cardiomyocytes will lead to improved treatment and prevention strategies,particularly in iron-overloaded patients.

Differential Effect of Calcium-Activated Potassium and Chloride Channels on Rat Basilar Artery Vasomotion

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. We investigated the cellular mechanisms underlying vasomotion in the cerebral basilar artery （BA） of Wistar rats. Pressure myograph video microscopy was used to study the changes in cerebral artery vessel diameter. The main results of this study were as follows： （1） The diameters of BA and middle cerebral artery （MCA） were 314.5±15.7 μm （n=15） and 233.3±10.1 μm （n=12） at 10 mmHg working pressure （P〈0.05）, respectively. Pressure-induced vasomotion occurred in BA （22/28, 78.6%）, but not in MCA （4/31, 12.9%） from 0 to 70 mmHg working pressure. As is typical for vasomotion, the contractile phase of the response was more rapid than the relaxation phase; （2） The frequency of vasomotion response and the diameter were gradually increased in BA from 0 to 70 mmHg working pressure. The amplitude of the rhythmic con- tractions was relatively constant once stable conditions were achieved. The frequency of contractions was variable and the highest value was 16.7±4.7 （n=13） per 10 min at 60 mmHg working pressure; （3） The pressure-induced vasomotion of the isolated BA was attenuated by nifedipine, NFA, 181]-GA, TEA or in Ca2＋-free medium. Nifedipine, NFA, 18^-GA or Ca2＋-free medium not only dampened vasomotion, but also kept BA in relaxation state. In contrasts, TEA kept BA in contraction state. These results sug- gest that the pressure-induced vasomotion of the isolated BA results from an interaction between Ca2＋-activated C1- channels （CaCCs） currents and Kca currents. We hypothesize that vasomotion of BA depends on the depolarizing of the vascular smooth muscle cells （VSMCs） to activate CaCCs. Depolarization in turn activates voltage-dependent Ca2＋ channels, synchronizing contractions of adjacent cells through influx of extracellular calcium and the flow of calcium through gap junctions. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates Kca channels and hyperpo- larizes VSMCs, which provides a negative feedback loop for regenerating the contractile cycle.

Effects of oxymatrine on calcium channels and GABA release in mice under neuropathic pain condition

OBJECTIVE To investigate effects of oxymatrine,an alkaloid from Sophora flavescens Ait.,on high-voltage dependent calcium channel and inhibitory neurotransmitter GABA under neuropathic pain condition.METHODS The partial sciatic nerve ligation(PSNL)was executed on C57/BL6 mice to produce neuropathic pain.Oxymatrine(150 mg·kg-1)was administrated intraperitoneally to PSNL mice.Mechanical hindpaw withdral threshold(MWT)was measured under Von-Frey filament stimulation with up-and-down method.In brain tissue,GABA concentration was measured with ELISA.Change of GABAAreceptor protein expression,N-type calcium channel(Cav2.2)and L-type calcium channel(Cav1.3)protein expressions were detected with Western-blot;intracellular calcium concentration was measured in cultured cortical neurons with Fluo-3/AM fluorescent probe.RESULTS Compared to saline,oxymatrine significantly increased ED50 of MWT on PSNL mice(P<0.05).GABA concentration and GABAAreceptor protein level in brain tissue were decreased in PSNL mice,while administration of oxymatrine increased both GABA concentration and GABAA receptor expression.Intracellular calcium concentration was increased in cultured cortical neurons by oxymatrine treatment,but this phenomenon was not seen under calcium-free condition.Protein expression of Cav2.2,but not Cav1.3,was found to be decreased in the brains of PSNL mice and to be restored to a normal level with oxymatrine administration.CONCLUSION Oxymatrine has analgesic effect on PSNL-induced neuropathic pain in mice.This phenominon relates to the increase of GABA release,GABAAreceptor expression,and also the restoration of expression level of Cav2.2 but not Cav1.3 in brain tissues,which suggesting that Ca2+ flow through Cav2.2 calcium channel may be the key point underlying oxymatrine analgesia.

The Effect of Levobunolol Hydrochloride on the Calcium andPotassium Channels in Isolated Ventricular Myocytes of Guinea Pig

The effects of levobunolol hydrochlorid (Bun) on the type L calciumchannel currents (Ica) and delayed rectifier potassium channel currents (Ik) in isolated ventricular myocytes of guinea pig were studied by using patch clamp wholecell recording techniques. The results were showed that: 1) Bun caused a dosedependent decrease in Ica and a dose-dependent increase in Ik of the ventricular myocytes.The threshold concentrations of Bun for Ica and Ik were 10-8 mol/L and10-7 mol/L respectively. The maximum effective concentration of Bun for both Ica and Ik was 3 × 10-5 mol/L, and half-maximal concentration was 3 × 10-6 mol/L;2 ) Ik was blocked by 2× 10-6mol/L tetraethylammonium (TEA). A concentration of 3 × 10-6 mol/L Bun showed a decreasing effect on the Ica as revealed by the current-voltage relationship curve, i. e., Bun caused an elevation of the curve; 3)When Ica was blocked by 2 × 10-6 mol/L Isoptin (Verapamil), at a concentrationof 3 × 106- mol/L Bun showed an increasing effect on Ik and the effect could be blocked by TEA. The above-mentioned results indicated that Bun had an inhibito-ry effect on Ica and a fascilitatory effect on Ik The results suggested that themolecular mechanisms of antihypertensive, heart rate slowing and β-receptorblocking effects of Bun might be due to decrease of Ica and increase of Ik.

Aetiology of bipolar disorder: contribution of the L-type voltage-gated calcium channels

LTCCS ARE IMPLICATED IN THE PATHOLOGY OF BIPOLAR DISORDER Bipolar disorder (BPD) is a common mental illness with significant morbidity and mortality.1 Although evidence have suggested changes in oxidative stress, dopamine and inflammation in BPD, it is hard to define the aetiological mechanism of BPD clearly. Recently, some but not all candidate gene association studies, family-based association studies, linkage studies, genome-wide association studies (GWASs) and meta-analyses showed that mutation of L-type voltage-gated calcium chan? nels (LTCCs) gene CACNAlCis implicated in the mechanism of BPD.'-8 These findings support the possibility that BPD might have calcium channelopathy.

Argemone mexicana extract alleviates gastrointestinal disorders by stimulating muscarinic receptors and blocking voltage-gated L-type calcium channels

Objective:To investigate the pharmacological potential of Argemone mexicana in treating constipation and emesis by using in vitro and in vivo models.Methods:The spasmogenic and spasmolytic effects were evaluated on isolated rabbit jejunum fragments loaded in a tissue organ bath.The response was recorded with an isotonic transducer attached with Power Lab Data Acquisition System.The laxative and antiemetic activities were assessed in BALB-c mice and poultry chicks challenged with carbamylcholine and copper sulphate stimulated emesis,respectively.Results:The total phenolic and total flavonoids contents of the extract were(267.75±5.77)mg GAE/g and(73.86±6.01)mg QE/g,respectively.Argemone mexicana extract exerted spasmogenic effect on isolated rabbit jejunum segments with an EC_(50)value of 0.016 mg/m L,which was blocked by atropine(0.3μM).Argemone mexicana extract exerted spasmolytic effect in atropine treated jejunum fragments with an EC_(50)value of 2.185 mg/mL.Furthermore,Argemone mexicana extract relaxed potassium(80 mM)-induced contractions(EC_(50):9.07 mg/mL),similar to a standard drug verapamil.The calcium channel blocker activity was confirmed by a rightward shift of concentration-response curve of calcium in the presence of Argemone mexicana extract(1-5 mg/mL)and verapamil(0.1-1μM).In addition,the extract increased the distance travelled by a charcoal in the gastrointestinal tract and exhibited antiemetic effect on copper sulphate induced emesis in chicks.Conclusions:Argemone mexicana shows cholinergic agonist and calcium channel blocker activities,as well as antiemetic effect.It may be used as a potential agent for treating gastrointestinal disorders.

Voltage-dependent potassium channel Kv4.2 promotes neurogenesis and alleviates isch⁃emic stroke impairments

OBJECTIVE Stroke has become the top ten leading cause of death in China.Isch⁃emic stroke accounts for 85%of stroke cases,and insufficiency of cerebral blood supply caused by atherosclerosis is one of the important causes of ischemic stroke.Therefore,it is of posi⁃tive significance to study the molecular mecha⁃nism of stroke injury caused by hypoperfusion in the search for drug targets.Voltage-dependent potassium channels are a family of potassium channels widely expressed in the central ner⁃vous system.However,their roles in neurogene⁃sis after stroke insults have not been clearly illus⁃trated.The purpose of this experiment is to explore the expression changes of different sub⁃families of voltage-dependent potassium chan⁃nels after the occurrence of ischemic stroke and their influence on neuroregeneration,to study the molecular mechanism of stroke injury caused by hypoperfusion,and to find potential targets for drug therapy of ischemic stroke.METHODS C57BL/6 mice aged 7-8 weeks and C17.2 cells were used in vivo and in vitro in the experiment.The mice in the experimental group were suf⁃fered from bilateral common carotid artery occlu⁃sion(BCCO)for 1 h and reperfusion for 7 d.In the control group,bilateral carotid artery was dis⁃sected without occlusion.Behavioral assay of suspension test were performed to assess the motor deficits of the mice.In this assay,the time of the first drop(latency),the number of drops within one minute(frequency),and the final scores were recorded as the results of athletic ability.A lower score indicated more severe motor damage of the mice.TTC staining was used to observe the cerebral infarction areas caused by ligation of bilateral common carotid arteries.After seven days,mice were sacrificed and brain tissue protein samples were collected for real-time quantitative PCR(RT-PCR)and Western blotting test to detect the changes of potassium channel subfamily expression levels in different brain regions.Neuronal injuries in all brain regions were detected using Nissl staining methods 7 d following model establishment.To detect the effects and the underlying mechanism of the related potassium channel on neurogene⁃sis,recombinant plasmids of the potassium chan⁃nels were transfected in cultured C17.2 neural stem cells.Afterwards,oxygen glucose depriva⁃tion experiments were performed.RESULTS Behavioral tests showed that BCCO can cause impaired motor performance.TTC staining showed that cerebral infarction existed in the stri⁃atum region,and the motor function decline caused by the injury in this region was consistent with the behavioral experiment results which veri⁃fied the effectiveness of our surgical operation.Nissl staining revealed a large amount of neuronal cell necrosis in the cortex and striatum regions,and dense neuronal cells in the lateral ventricular limbic region,suggesting that neurogenesis may have occurred in this region.The results of real-time quantitative RT-PCR showed that among the detected potassium channels distributed in the measured nervous system,the expression of voltage-dependent potassium channel Kv4.2 decreased significantly in all brain regions after stroke,suggesting that it may be involved in the pathological process of stroke.Immunohisto⁃chemical staining showed that there was neuro⁃genesis in the subgranular zone(SGZ)and sub⁃ventricular zone(SVZ)of the mice,and Kv4.2 expression was significantly changed in the regions,suggesting that it may be involved in the regulation of neuro regeneration after stroke.The transfected Kv4.2 plasmid enhanced the dif⁃ferentiation of the C17.2 neural stem cells to neu⁃rons and astrocytes under normoxia and the oxy⁃gen-glucose deprivation,suggesting that Kv4.2 may induce the differentiation of neural stem cells after stroke.Kv4.2 could induce the neural stem cells to differentiate into neurons in vitro and in vivo,and Western blotting assay showed that Kv4.2 could up-regulate the expression level of ERK1/2,p-ERK1/2,p-STAT3,NGF,p-TtkA,and BDNF.Moreover,the calcium ions and CAMKⅡwas also increased by Kv4.2 in vitro.CONCLUSION BCCO insults can induce the expressions of the potassium channels in the brains,among which the expression of Kv4.2 is down-regulated in the cerebral cortex,hippocam⁃pus and striatum.In vitro experiments confirmed that Kv4.2 can induce the differentiation of C17.2 neural stem cells into neurons and astrocytes under the condition of normoxia and oxygen-glucose deprivation.We concluded that Kv4.2 possibly promoted neurogenesis through ERK1/2/STAT3,NGF/TrkA,and Ca2+/CAMKⅡsignal pathways after stroke.Regulating the physiologi⁃cal functions of Kv4.2 channel might contribute to the rehabilitation of neuronal damage after stroke.

Effect of Calmodulin and Voltage-dependent Ca^(2+) Channel on the Proliferation of Heptoma Cells Induced by Epidermal Growth Factor

The effect of thyrosine kinase, calmodulin and voltage-dependent Ca 2+ channel on the proliferation of hepatoma cells induced by EGF was studied. Hepatoma cell line SMMC7721 was cultured in RPMI1640 serum-free medium. DNA synthesis rate of hepatoma cells was measured by 3H-TdR incorporation. 10 -9 mol/L EGF could significantly stimulate the proliferation of hepatoma cells (P<0.05), and this effect might be significantly inhibited by tyrosine kinase inhibitor (P<0.001). Calmodulin inhibitor W-7 had no effect on the basic phase of cultured hepatoma cells (P> 0.05), but it had very significantly inhibitory effect on the proliferation of hepatoma cells induced by EGF (P<0.001). Voltage-dependent Ca 2+ channel inhibitor Varapamil had no inhibition on the proliferation of hepatoma cells induced by EGF (P>0.05). It had no effect on the basic phase of cultured hepatoma cells (P>0.05). It is suggested that tyrosine kinase and Ca 2+-calmodulin-dependent pathway may play a critical role on the proliferation of heptoma cells induced by EGF, and voltage-dependent Ca 2+ channel is independent of the effect of EGF.

Antineoplastic Effect of Calcium Channel Blocker-Verapamil and 5-Fluorouracil Intraperitoneal Chemotherapy on Hepatocarcinoma-Bearing Rats

Objective To study the antineoplastic effect of the calcium channel blocker verapamil and 5-fluorouracil intraperitoneal chemotherapy on hepatocarcinoma-bearing rats,and examine the action between calcium channel blockers and cytotoxic drugs. Methods We adopted the method of subcapsular implantation of carcinoma tissues of walker-256 in the left liver lobe as a model of liver carcinoma-bearing rats.All experimental animals were divided into four groups.On the sixth day post implantation,in group A (control group) 6ml of saline was injected intraperitoneally once a day for 3 days.In group B(single chemotherapy group) 6ml of 5-Fu 75 mg/kg was injected intraperitoneally once a day for 3 days.In group C(combination of treatment group)both 5-Fu(75mg/kg) and verapamil (25mg/kg) were administered simultaneously as in A and B.In group D(simple verapamil group)only 6ml of verapamil(25mg/kg)was administered as above. Results Compared with groups A, B and D,The volume of cancer and the contents of liver cancer DNA and protein were significantly reduced.The rates of inhibiting cancer(89.9% in group C and 35.4% in group B)were significantly increased in groupC. Group C had significantly long survival time compared to groups A, B and D(P<0.05).By light microscopy, a number of focal necroses were found in cancer tissue in group C.Conclusion Calcium channel blockers can enhance the antineoplastic effect of 5-Fu intraperitonea chemotherapy to liver cancer;The use of verapamil can not increase the toxicity of 5-Fu.

Ligustrazine inhibits high voltage-gated Ca^(2+) and TTX-resistant Na^+ channels of primary sensory neuron and thermal nociception in the rat:a study on peripheral mechanism

Objective Ligustrazine, also named as tetramethylpyrazine, is a compound purified from Ligusticum chuanxiong hort and has ever been testified to be a calcium antagonist. The present investigation was to determine the antinociceptive effect of ligustrazine and, if any, the peripheral ionic mechanism involved. Methods Paw withdrawal Latency （PWL） to noxious heating was measured in vivo and whole-cell patch recording was performed on small dorsal root ganglion （DRG） neurons. Results Intraplantar injection of ligustrazine （0.5 mg in 25 μl） significantly prolonged the withdrawal latency of ipsilateral hindpaw to noxious heating in the rat. Ligustrazine not only reversibly inhibited high-voltage gated calcium current of dorsal root ganglion （DRG） neuron in dose-dependent manner with IC50 of 1.89 mmol/L, but also decreased tetrodotoxin （TTX） -resistant sodium current in relatively selective and dose-dependent manner with IC50 of 2.49 mmol/L. Conclusion The results suggested that ligustrazine could elevate the threshold of thermal nociception through inhibiting the high-voltage gated calcium current and TTX-resistant sodium current of DRG neuron .in the rat.

Calcium channel blocker monotherapy versus combination with reninangiotensin system inhibitors on the development of new-onset diabetes mellitus in hypertensive Korean patients

Background In real practice, two or more antihypertensive drugs are needed to achieve target blood pressure. We investigated the comparative beneficial actions of combination therapy of renin-angiotensin system inhibitors (RASI), with calcium channel blockers (CCB) over CCB monotherapy on the development of new-onset diabetes mellitus (NODM) in Korean patients during four-year follow-up periods. Methods A total of 3208 consecutive hypertensive patients without a history of diabetes mellitus who had been prescribed CCB were retrospectively enrolled from January 2004 to December 2012. These patients were divided into the two groups according to the additional use of RASI (the RASI group, n = 1221 and the no RASI group, n = 1987). Primary endpoint was NODM, defined as a fasting blood glucose ≥ 126 mg/dL or hemoglobin A1c ≥ 6.5%. Secondary endpoint was major adverse cardiac events (MACE) defined as total death, myocardial infarction (MI) and percutaneous coronary intervention (PCI). Results After propensity score-matched (PSM) analysis, two propensity- matched groups (939 pairs, n = 1878, C-statistic = 0.743) were generated. The incidences of NODM (HR = 1.009, 95% CI: 0.700–1.452, P = 0.962), MACE (HR = 0.877, 95% CI: 0.544–1.413, P = 0.589), total death, MI, PCI were similar between the two groups after PSM during four years. Conclusions The use of RASI in addition to CCB showed comparable incidences of NODM and MACE compared to CCB monotherapy in non-diabetic hypertensive Korean patients during four-year follow-up period. However, large-scaled randomized controlled clinical trials will be required for a more definitive conclusion.