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.

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.

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.

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.

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.

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.

Effects of Tiaomaiyin and Its Disassembled Prescription on Expression of L-type Calcium Channel β2 Subunit in Rat Model of Tachyarrhythmia

[Objectives] To study the effects of Tiaomaiyin and its disassembled prescription on expression of L-type calcium channel β2 subunit in rat model of tachyarrhythmia. [Methods] Sixty Wistar rats were randomly divided into model group,Tiaomaiyin prescription group( whole prescription group),main efficacy group of removing heat to cool blood( blood cooling group),and auxiliary drug efficacy group of benefiting qi and nourishing heart( qi benefiting group),auxiliary efficacy group of promoting flow of qi and blood circulation( qi flow promoting group),and amiodarone group( western medicine group). Aconitine was given 7 d after the intragastric administration of the corresponding drugs,and the time of occurrence of arrhythmia in each group was observed. The left ventricular myocardium was subjected to reverse transcription-polymerase chain reaction and Western blotting. [Results] The ventricular premature beats( VPB) time in the whole prescription group and western medicine group was significantly longer than that in the model group. Ventricular tachycardia( VT),ventricular fibrillation( VF),and cardiac arrest( CA) were longer in the whole prescription group,blood cooling group,and western medicine group. The mRNA and protein expression of L-type calcium channel β2 subunit in the whole prescription group,blood cooling group and western medicine group were significantly decreased. [Conclusions] Tiaomaiyin whole prescription group and blood cooling group can reduce the occurrence time of tachyarrhythmia and reduce the expression of LTCC β2 in myocardium.

The roles and relations of calpastatin,calmodulin and an undefined cytoplasmic factor in the regulation of cardiac L-type Ca^(2+) channels

Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down".The factors include ATP,calpastatin and H fraction(a high molecular fraction of bovine cardiac cytoplasm).Methods Single Ca2+ channel activities were recorded with patch clamp technique in guinea-pig cardiac myocytes.Run-down was induced by the inside-out patch formation.Calpastatin(CS),calmodulin(CaM)and three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2,CT-1(amino acids number 1509-1791),CT-2(1777-2003)and CT-3(1944-2169)were produced as GST fusion proteins.Results(1)CaM + ATP or CS + ATP restored the channels after run-down;however,the CaM or CS's effects became smaller with the longer run-down time.(2)After run down,CaM-dependent protein kinase(CaMKII)produced Ca2+ channel activity to only 2-10% of the basal activity,however,in the presence of CaMKII,the time-dependent nature of the CaM effect was abolished.(3)In pull-down assay,CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase.(4)CaMKII was detected in the H fraction of bovine cardiac cytoplasm.Conclusions The results show that CS,CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2+ channels,and that there might be cross talks among the four factors(CS,CaM,CaMKII and the undefined cytoplasmic factor).

Antiepileptic Drug-Induced Apoptosis Was Prevented by L-Type Calcium Channel Activator in Cultured Rat Cortical Cells

Experimental data have shown that antiepileptic drugs cause neurodegeneration in developing rats. Valproate (VPA) is the drug of choice in primary generalized epilepsies, and carbamazepine (CBZ) is one of the most prescribed drugs in partial seizures. These drugs block sodium channels, thereby reducing sustained repetitive neuronal firing. The intracellular mechanisms whereby AEDs induce neuronal cell death are unclear. We examined whether AEDs induce apoptotic cell death in cultured cortical cells and whether calcium ions are involved in the AED-induced cell death. VPA and CBZ increased apoptotic cell death and induced morphological changes that were characterized by cell shrinkage and nuclear condensation or fragmentation. Incubation of cortical cultures with VPA or CBZ decreased phospho-Akt levels. CBZ decreased the intracellular calcium levels. On the other hand, FPL64176, an L-type calcium channel activator, increased the intracellular calcium levels and prevented the AED-induced apoptosis. Glycogen synthase kinase-3 inhibitors, such as alsterpaullone and azakenpaullone, prevented the AED-induced apoptosis. These results suggest that intracellular calcium level changes are associated with AEDs and apoptosis and that the activation of glycogen synthase kinase-3 is involved in the death of rat cortical neurons.

Gene Expression of L-type Calcium Channel α1c Subunit in the Atrial Tissue of Patients with Atrial Fibrillation

Objective To investigated themessenger ribonucleic acid (mRNA) expression of L -type calcium channelaic subunit in the atrial tissue of patients with atrial fibrillation due to rheumatic heart disease (RHD) . Methods A total of 50 patients with mitral valvular disease due to RHD were included. Atrial tissue was obtained during cardiac surgery in all patients from the right atrial appendage and the right atrial free wall, and in 35 patients of from the left atrial appendage, respectively. The mRNA amount of L -type calcium channel a1c subunit was measured by reverse transcription - polymerase chain reaction and normalized to the mRNA levels of glyceraldehyde3 -phosphate dehydrogenase (GAPDH) . Results The mRNA of L - type calcium channelaic subunit was significantly decreased in patients with persistent AF for more than 3 months compared to patients with sinus rhythm ( P all <o.o1). There were no difference of the mRNA expression of L - type calcium channelaic sub-unit among the three atrial tissue sampling sites ( P all <o. o5) .Age, gender, left ventricular ejection fraction, mean pulmonary arterial pressure, left atrial diameter, and right atrial diameter had no significant effects on the mRNA expression of L - type calcium channelaic subunit. Conclusions The mRNA expression of L - type calcium channelau subunit was down - regulated only in patients with long - term persistent AF. Such abnormalities may be involved in the initiatioin and/or perpetuation of AF.

The Effect of Extrogenous Phosphocreatine on L-type Calcium Current in Ischemic Guinea Pig Ventricular Myocytes

Objectives Heart failure （HF） is one of the most common outcome for all kinds of heart diseases, the effects of energetic therapy on HF remains controversial, especially to ischemic HF. The aim of this study was to explore the effect of exogenous phosphocreatine with different concentration on L-type calcium（I Cc-L） current in ischemic ventricular myocytes of guinea pig and to investigate its underlying electrophysiological mechanism for the treatment of ischemic HF. Methods Single ventricular myocytes were isolated enzymatically from left ventricle of guinea pig. Peak I Ca-L current were recorded using patch clamp techniques in the whole-cell configuration when myocytes had been superfused with normal Tyrode solution, simple ischemic solution, ischemic solution containing phosphocreatine with different concentration for 10 minutes respectively. Results Peak I Ca-L current density of myocytes superfused with simple simulated ischemic solution was remarkably inhibited by 80.6 ± 5.2% compared with myocytes superfused with normal Tyrode solution（P〈0.05）. Ischemic solution containing phosphocreatine of 5, 10, 20, 30mmol/L inhibited Peak I Ca-L current density by （53.8±6.7）%, （41.8 ± 8.2）%, （38.1±7.4）%, （36.6±9.7）% respectively. There was no statistical significance among phosphocreation of 10, 20, 30 mmol / L. Conclusions Extrogenous phosphocreatine could reverse the inhibition of I Ca-L current under ischemic condition, which could be the ionic basis for the treatment of ischemic heart failure. 0-10 mmol/L phosphocreatine exerted significant dose-effect relationship which no longer existed as concentration more than 10 mmol/L. It is supposed that phosphocreatine increased I Ca-L current by many pathways rather than simple substrate for ATP synthesis.

Effect of Shenmai Injection on L-type Calcium Current of Diaphragmatic Muscle in Rats

In this study, whole cell patch clamp recording technique was employed to investigate the effect of Shenmai Injection (SMI) on L-type calcium current ofdiaphragmatic muscle in rats. The result showed that when the diaphragmatic muscle cell was held at —80 mV and depolarized to +60 mV, 10 μl/ml, 50 μl/ml and 100 μl/ml SMI enhanced the inner peak L-type calcium current from -(6.8±0.7) pA/pF (n=7) to -(7.3±0.8) pA/pF (P>0.05, n=7), -(8.6±1.0) pA/pF (P<0.05, n=7) and -(9.4±1.2) pA/pF (P<0.05, n=7), respectively. The rates of L-type calcium current were increased by (7.34±2.37) %, (25.72±5.94)% , and (38.16±7.33)% , respectively. However, it had no significant effect on maximal activation potential and reversal potential. Our results suggested that SMI could activate the calcium channel of the diaphragmatic fibers of the rats, increase the influx of Ca 2+, and enhance the contractility of diaphragmatic muscles.

L-type Calcium Channels are Involved in Iron-induced Neurotoxicity in Primary Cultured Ventral Mesencephalon Neurons of Rats

In the present study,we investigated the mechanisms underlying the mediation of iron transport by Ltype Ca^2+ channels(LTCCs)in primary cultured ventral mesencephalon(VM)neurons from rats.We found that cotreatment with 100 lmol/L FeSO4 and MPP^+(1-methyl-4-phenylpyridinium)significantly increased the production of intracellular reactive oxygen species,decreased the mitochondrial transmembrane potential and increased the caspase-3 activation compared to MPP^+ treatment alone.Co-treatment with 500 lmol/L CaCl2 further aggravated the FeSO4-induced neurotoxicity in MPP^+-treated VM neurons.Co-treatment with 10 lmol/L isradipine,an LTCC blocker,alleviated the neurotoxicity induced by co-application of FeSO4 and FeSO4/CaCl2.Further studies indicated that MPP^+treatment accelerated the iron influx into VM neurons.In addition,FeSO4 treatment significantly increased the intracellular Ca^2+ concentration.These effects were blocked by isradipine.These results suggest that elevated extracellular Ca^2+ aggravates ironinduced neurotoxicity.LTCCs mediate iron transport in dopaminergic neurons and this,in turn,results in elevated intracellular Ca^2+ and further aggravates iron-induced neurotoxicity.

Ferulic acid enhances insulin secretion by potentiating L-type Ca^(2+)channel activation

Objective To investigate the effect of ferulic acid,a natural compound,on pancreatic beta cell viability,Ca^(2+)channels,and insulin secretion.Methods We studied the effects of ferulic acid on rat insulinoma cell line viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay.The whole-cell patch-clamp technique and enzyme-linked immunosorbent assay were also used to examine the action of ferulic acid on Ca^(2+)channels and insulin secretion,respectively.Results Ferulic acid did not affect cell viability during exposures up to 72 h.The electrophysiological study demonstrated that ferulic acid rapidly and concentration-dependently increased L-type Ca^(2+)channel current,shifting its activation curve in the hyperpolarizing direction with a decreased slope factor,while the voltage dependence of inactivation was not affected.On the other hand,ferulic acid have no effect on T-type Ca^(2+)channels.Furthermore,ferulic acid significantly increased insulin secretion,an effect inhibited by nifedipine and Ca^(2+)-free extracellular fluid,confirming that ferulic acid-induced insulin secretion in these cells was mediated by augmenting Ca^(2+)influx through L-type Ca^(2+)channel.Our data also suggest that this may be a direct,nongenomic action.Conclusion This is the first electrophysiological demonstration that acute ferulic acid treatment could increase L-type Ca^(2+)channel current in pancreaticβcells by enhancing its voltage dependence of activation,leading to insulin secretion.

Blocking Effect of Salvianolic Acid A on Calcium Channels in Isolated Rat Ventricular Myocytes

Objective： To study the effect of salvianolic acid A （SAA） on L-type calcium current （I-CaL） in isolated ventdcular myocytes of Sprague-Dawley rats. Methods： SPA powder was dissolved in normal Tyrode＇s solution to reach the concentrations of 1, 10, 100, and 1000 μmol/L. The traditional whole-cell patch-clamp recording technique was employed to evaluate the effects of SAA on I-CaL in single ventricular myocytes which were prepared by Langendorff perfusion apparatus from Sprague-Dawley rats. Results： SPA （1, 10, 100, and 1000 μmol/L） inhibited I-CaL peak value by 16.23%± 1.3% （n=6, P〈0.05）, 22.9% ± 3.6% （n=6, P〈0.05）, 53.4% ± 3.0% （n=8, P〈0.01）, and 62.26% ± 2.9% （n=8, P〈0.01）, respectively. SAA reversibly inhibited I-CaL in a dose-dependent manner and with a half-blocking concentration （IC50） of 38.3 μmol/L. SAA at 100 μmol/L elevated the I-V curve obviously, and shifted the half-active voltage （V0.5） from （-15.78± 0.86） mV to （-11.24 ± 0.77） mV （n=6, P〈0.05） and the slope （K） from 5.33 ±0.74 to 4.35±0.74 （n=6, P〉0.05）. However, it did not alter the shapes of I-V curve, steady-state inactivation curve, or recovery from inactivation curve. Conclusions： SAA inhibited I-CaL in a dose-dependent manner. It shifted the steady-state activation curve to a more positive voltage, which indicated that the drug affected the activated state of calcium channels, and suggested that the Ca2. antagonistic effect of SPA be beneficial in the treatment of myocardial ischemia reperfusion injury.