Inhibitory Effects of Blockage of Intermediate Conductance Ca^(2+) -Activated K^+ Channels on Proliferation of Hepatocellular Carcinoma Cells

The roles of intermediate conductance Ca2＋-activated K＋ channel （IKCal） in the pathogene- sis of hepatocellular carcinoma （HCC） were investigated. Immunohistochemistry and Western blotting were used to detect the expression of IKCal protein in 50 HCC and 20 para-carcinoma tissue samples. Real-time PCR was used to detect the transcription level of IKCal mRNA in 13 HCC and 11 para-carcinoma tissue samples. The MTT assay was used to measure the function of IKCal in human HCC cell line HepG2 in vitro. TRAM-34, a specific blocker of IKCal, was used to intervene with the function of IKCal. As compared with para-carcinoma tissue, an over-expression of IKCal protein was detected in HCC tissue samples （P〈0.05）. The mRNA expression level of IKCal in HCC tissues was 2.17 times higher than that in para-carcinoma tissues. The proliferation of HepG2 cells was suppressed by TRAM-34 （0.5, 1.0, 2.0 and 4.0 pxnol/L） in vitro （P〈0.05）. Our results suggested that IKCal may play a role in the proliferation of human HCC, and IKCal blockers may represent a potential therapeutic strategy for HCC.

Relationship of Intracellular Free Ca^(2+) Concentration and Calcium-activated Chloride Channels of Pulmonary Artery Smooth Muscle Cells in Rats under Hypoxic Conditions

To investigate the relationship between intracellular free Ca^2＋ concentration （[Ca^2＋ ]i ） and calcium-activated chloride （Clca） channels of pulmonary artery smooth muscle cells （PASMCs） in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca^2＋ indicator Fura-2/AM was used to observe [Ca^2＋ ]i of rat PASMCs under normal and chronic hypoxic condition. The effect of Clca channels on PASMCs proliferation was assessed by MTT assay. The Clca channel blockers niflumic acid （NFA） and indaryloxyacetic acid （IAA-94） exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca^2＋ ]i was increased. Under normoxic condition, [Ca^2＋ If was （123.634-18.98） nmol/ L, and in hypoxic condition, [Ca^2＋]i wag （281. 754-16.48） nmol/L （P〈0. 01）. Under normoxic condition, [Ca^2＋ ]i showed no significant change and no effect on Clca channels was observed （P〉 0. 05）. Chronic hypoxia increased [Ca^2＋ ]i which opened Clca channels. The NFA and IAA-94 blocked the channels and decreased [Ca^2＋ ]i from （281.75± 16.48） nmot/L to （117.66 ±15.36） nmol/L （P〈0.01）. MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency （A value） from 0. 459±0. 058 to 0. 224±0. 025 （P〈0. 01）. Hypoxia increased [Ca^2＋ ]i which opened Cl~ channels and had a positive-feedback in [Ca^2＋ ]i. This may play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition, Clca channel may play a part in the regulation of proliferation of PASMCs.

Measuring Ca^(2+) influxes of TRPC1-dependent Ca^(2+) channels in HL-7702 cells with Non-invasive Micro-test Technique

AIM:To explore the possibility of using the Non-invasive Micro-test Technique(NMT)to investigate the role of Transient Receptor Potential Canonical 1(TRPC1) in regulating Ca 2+ influxes in HL-7702 cells,a normal human liver cell line.METHODS:Net Ca 2+ fluxes were measured with NMT, a technology that can obtain dynamic information of specific/selective ionic/molecular activities on material surfaces,non-invasively.The expression levels of TRPC1 were increased by liposomal transfection,whose effectiveness was evaluated by Western-blotting and single cell reverse transcription-polymerase chain reaction. RESULTS:Ca 2+ influxes could be elicited by adding 1 mmol/L CaCl2 to the test solution of HL-7702 cells.They were enhanced by addition of 20μmol/L noradrenalin and inhibited by 100μmol/L LaCl3(a non-selective Ca 2+ channel blocker);5μmol/L nifedipine did not induce any change.Overexpression of TRPC1 caused increased Ca 2+ influx.Five micromoles per liter nifedipine did not inhibit this elevation,whereas 100μmol/L LaCl3 did.CONCLUSION:In HL-7702 cells,there is a type of TRPC1-dependent Ca 2+ channel,which could be detected via NMT and inhibited by La3+.

Effect of Chengzai Pill on the L-Type Ca^(2+) Channel Currents of Osteoblasts Pretreated with Methylprednisolone

Objective: To observe the effect of the serum containing Chengzai Pill on the L-type voltage-sensitive calcium channels current (L-VSCCsC) of osteoblastic MC3T3-E1 cells pretreated with methylprednisolone (mPSL). Methods: A control group, a model group, a low dose group and a high dose group were set up. The whole cell patch clamp technique was used to record L-VSCCsC of 10 osteoblastic MC3T3-E1 cells in each group and their peak currents were determined. Results: The peak current of the control group was 0.2284±0.0209 nA; the peak current of the model group was 0.1839±0.0179 nA; decreased by 19.5% as compared with the control group (P<0.01); the peak current of the low and high dose groups was 0.2526± 0.0093 nA and 0.2671±0.0120 nA respectively, increased by 37.4% and 45.2% as compared with the model group (P<0.01); the difference between the low and high dose groups was P<0.05. Conclusion: 1. mPSL inhibits L-VSCCsC of osteoblasts; and 2. The serum containing Chengzai Pill increases L-VSCCsC of osteoblasts pretreated with mPSL.

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.

Caribbean maitotoxin elevates [Ca^(2+)]i and activates non-selective cation channels in HIT-T15 cells

AIM:To investigate the cytotoxic mechanism of caribbean maitotoxin(MTX-C) in mammalian cells.METHODS:We used whole-cell patch-clamp techniques and fluorescence calcium imaging to determine the cellular toxic mechanisms of MTX-C in insulin secreting HIT-T15 cells,which is a system where the effects of MTX have been observed.HIT-T15 cells stably express L-type calcium current,making it a suitable model for this study.Using the fluorescence calcium indicator Indo-1 AM,we found that there is a profound increase in HIT-T15 intracellular free calcium 3 min after application of 200 nmol/L MTX-C.RESULTS:About 3 min after perfusion of MTX-C,a gradual increase in free calcium concentration was observed.This elevation was sustained throughout the entire recording period.Application of MTX-C did not elicit the L-type calcium current,but large cationiccurrents appeared after applying MTX-C to the extracellular solution.The current-voltage relationship of the cation current is approximately linear within the voltage range from-60 to 50 mV,but flattened at voltages at-80 and-100 mV.These results indicate that MTX-C induces a non-voltage activated,inward current under normal physiological conditions,which by itself or through a secondary mechanism results in a large amount of cationic influx.The biophysical mechanism of MTX-C is different to its isoform,pacific maitotoxin(MTX-P),when the extracellular calcium is removed.CONCLUSION:We conclude that MTX-C causes the opening of non-selective,non-voltage-activated ion channels,which elevates level of intracellular calcium concentration and leads to cellular toxicities.

2-aminoethoxydiphenyl borate or lanthanum potentiates transient receptor potential-like channels in rat CA1 hippocampal neurons

Expression of transient receptor potential （TRP） channels is widespread with transcripts distributed throughout the brain. All TRP channel subunits are activated following phospholipase C activation and form cation-selective ion channels. Previous studies examining the existence of TRP channels in hippocampal CA1 pyramidal neurons were based on cultured neurons. Therefore, their relevance for living tissue remains unclear. In the present study, patch-clamp recordings were conducted from CA1 pyramidal neurons in hippocampal slices from 7-day-old rats. Whole-cell currents were obtained from CA1 hippocampal neurons with potentiation effects of 2-aminoethoxydiphenyl borate and lanthanum, revealing that recorded experimental currents were characteristic TRP-like channel currents. Identification of rat hippocampal mRNA transcripts of TRPC4, TRPC5, TRPV1, TRPV2, and TRPV3 channels further verified the expression of characteristic TRP-like channels on rat CA1 hippocampal neurons.

干姜吴茱萸水煎液对大鼠结肠平滑肌细胞L型Ca^(2+)通道的影响

目的:探讨干姜吴茱萸水煎液对大鼠结肠平滑肌收缩及平滑肌细胞L型Ca^(2+)通道的影响机制。方法:采用0、0.01、0.1、1、10 g/L干姜吴茱萸(1∶1)水煎液依次作用于正常结肠平滑肌,10 g/L水煎液及L型Ca^(2+)通道激动剂BAY-K-8644依次作用于氯化乙酰胆碱(Ach)诱导后的正常平滑肌,检测平滑肌收缩张力、频率和振幅;全细胞膜片钳记录L型钙电流;Western blot检测各组细胞中L-型电压依赖钙离子通道α1C亚基(α1c)和钙调蛋白(CaM)蛋白表达情况。结果:1、10 g/L水煎液组能显著降低离体结肠平滑肌的收缩张力(P<0.05),对频率及振幅并无显著影响(P>0.05);与0 g/L水煎液组相比,Ach促进平滑肌收缩张力、频率和振幅显著增加(P<0.05);与Ach组相比,10 g/L水煎液显著降低平滑肌的收缩张力、频率和振幅(P<0.05);与10 g/L水煎液组相比,BAY-K-8644显著增加平滑肌的收缩张力、频率和振幅(P<0.05);与0 g/L水煎液组相比,Ach可显著增加ICa-L峰值电流密度(P<0.05);与Ach组相比,10 g/L水煎液显著降低ICa-L峰值电流密度(P<0.05);与10 g/L水煎液组相比,BAY-K-8644可显著增加ICa-L峰值电流密度(P<0.05);与对照组相比,Ach组结肠平滑肌细胞Ca^(2+)相对荧光强度峰值、α1c、CaM表达显著增加(P<0.05);Ach组相比,10 g/L组Ca^(2+)相对荧光强度峰值、α1c和CaM表达水平显著降低(P<0.05);与10 g/L组相比,BAYK-8644组Ca^(2+)相对荧光强度峰值、α1c、CaM表达显著增加(P<0.05)。结论:干姜吴茱萸水煎液可抑制大鼠结肠平滑肌收缩,并阻滞L型Ca^(2+)通道。

Effects of isoflurane and ethanol on large conductance Ca^(2+)-activated K^+ channels

Objective: To study the effect of isoflurane and ethanol on large conductance Ca 2+-activated K + channels(BK channels). Methods: The cRNA of mslo1 encoding BK channels was injected into Xenopus oocytes. Oocytes were incubated in ND96 (96 mmol/L NaCl, 2.0 mmol/L KCl, 1.8 mmol/L CaCl 2, 1.0 mmol/L MgCl 2, and 5.0 mmol/L HEPES, pH 7.4) at 4 ℃. Patch clamp recording (outside-out) were performed after 2-3 d. Isoflurane was administrated by the vaporizer driven by air, ethanol was applied by a closed, manual-controlled administration system. Different test potentials from 0 to 10 mV were given to observe changes of currents. Results: 0.7 mmol/L and 1.2 mmol/L of isoflurane could inhibit BK currents obviously at different command potentials, but 50 mmol/L, 100 mmol/L, or 200 mmol/L of ethanol had no any effect on BK currents. Conclusion: Clinical concentration of isoflurane can distinctly inhibit isolating BK currents.

低温缺血-再灌注对离体大鼠心房肌Kir2.1和CaMKⅡ表达的影响

目的探讨再灌注房性心律失常心房肌复极时程延长的分子机制。方法采用随机数字表法将16只由雄性SD大鼠制备的Langendorff离体心脏灌注模型分为对照组(C组,n=8)和低温缺血-再灌注组(IR组,n=8)。根据再灌注后是否发生房性心律失常,将IR组进一步细分为再灌注非房性心律失常亚组(N-RAA组)和再灌注房性心律失常亚组(R-AA组)。C组使用37℃K-H液平衡灌注120 min。IR组使用37℃K-H液平衡灌注30 min后停止,注射4℃Thomas液(20 mL/kg)使心脏停跳60 min,停跳30 min时使用半量4℃Thomas液(10 mL/kg)对离体心脏进行复灌[停跳期间用低温Thomas液(4℃)对心脏进行保护],后再次灌注37℃K-H液30 min。记录平衡灌注30 min(T_(0))、平衡灌注105 min/再灌注15 min(T_(1))和平衡灌注120 min/再灌注30 min(T_(2))时右心房单相动作电位(MAP),测量单相动作电位复极50%和90%的时程(MAPD_(50)和MAPD_(90))。电生理指标监测完后用Western blot检测右心房组织内向整流钾通道2.1(Kir2.1)和Ca^(2+)/CaM依赖激酶Ⅱ(CaMKⅡ)的表达。结果与T_(0)时点比较,R-AA组T_(1)、T_(2)时MAPD_(50)、MAPD_(90)明显延长(P<0.05);与C组比较,R-NAA组和R-AA组T_(1)、T_(2)时MAPD_(90)明显延长(P<0.05);与R-NAA组比较,R-AA组T_(1)、T_(2)时MAPD_(50)、MAPD_(90)明显延长(P<0.05)。Western blot结果显示,R-NAA组和R-AA组Kir2.1表达明显少于C组(P<0.05),且R-AA组明显少于R-NAA组(P<0.05);R-NAA组和R-AA组CaMKⅡ表达较C组明显增加(P<0.05),且R-AA组CaMKⅡ表达较R-NAA组明显增加(P<0.05)。结论低温缺血-再灌注房性心律失常大鼠心房肌复极时程延长可能与Kir2.1表达下调和CaMKⅡ表达增加有关。

Diabetes-induced changes in cardiac voltage-gated ion channels

Diabetes mellitus affects the heart through various mechanisms such as microvascular defects,metabolic abnormalities,autonomic dysfunction and incompatible immune response.Furthermore,it can also cause functional and structural changes in the myocardium by a disease known as diabetic cardiomyopathy(DCM)in the absence of coronary artery disease.As DCM progresses it causes electrical remodeling of the heart,left ventricular dysfunction and heart failure.Electrophysiological changes in the diabetic heart contribute significantly to the incidence of arrhythmias and sudden cardiac death in diabetes mellitus patients.In recent studies,significant changes in repolarizing K+currents,Na+currents and L-type Ca^(2+)currents along with impaired Ca^(2+ )homeostasis and defective contractile function have been identified in the diabetic heart.In addition,insulin levels and other trophic factors change significantly to maintain the ionic channel expression in diabetic patients.There are many diagnostic tools and management options for DCM,but it is difficult to detect its development and to effectively prevent its progress.In this review,diabetes-associated alterations in voltage-sensitive cardiac ion channels are comprehensively assessed to understand their potential role in the pathophysiology and pathogenesis of DCM.

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.

MicroRNA-155 mediates endogenous angiotensin II type 1 receptor regulation:implications for innovative type 2 diabetes mellitus management

Type 2 diabetes mellitus(T2DM)is a lifelong condition and a threat to human health.Thorough understanding of its pathogenesis is acutely needed in order to devise innovative,preventative,and potentially curative pharmacological interventions.MicroRNAs(miRNA),are small,non-coding,one-stranded RNA molecules,that can target and silence around 60%of all human genes through translational repression.MiR-155 is an ancient,evolutionarily well-conserved miRNA,with distinct expression profiles and multifunctionality,and a target repertoire of over 241 genes involved in numerous physiological and pathological processes including hematopoietic lineage differentiation,immunity,inflammation,viral infections,cancer,cardiovascular conditions,and particularly diabetes mellitus.MiR-155 Levels are progressively reduced in aging,obesity,sarcopenia,and T2DM.Thus,the loss of coordinated repression of multiple miR-155 targets acting as negative regulators,such as C/EBPβ,HDAC4,and SOCS1 impacts insulin signaling,deteriorating glucose homeostasis,and causing insulin resistance(IR).Moreover,deranged regulation of the renin angiotensin aldosterone system(RAAS)through loss of Angiotensin II Type 1 receptor downregulation,and negated repression of ETS-1,results in unopposed detrimental Angiotensin II effects,further promoting IR.Finally,loss of BACH1 and SOCS1 repression abolishes cytoprotective,anti-oxidant,anti-apoptotic,and anti-inflam matory cellular pathways,and promotesβ-cell loss.In contrast to RAAS inhibitor treatments that further decrease already reduced miR-155 Levels,strategies to increase an ailing miR-155 production in T2DM,e.g.,the use of metformin,mineralocorticoid receptor blockers(spironolactone,eplerenone,finerenone),and verapamil,alone or in various combinations,represent current treatment options.In the future,direct tissue delivery of miRNA analogs is likely.

Functional remodeling of Ca^(2+)-activated Cl^-channel in pacing induced canine failing heart

To determine whether Ca2+ activated Cl-current (ICl（Ca）) contributes to the functional remodeling of the failing heart. Methods Whole cell patch-clamp recording technique was employed to record the ICl（Ca） in cardiac myocytes enzymatically isolated from rapidly pacing induced canine failing hearts at room temperature and compared that of the normal hearts （Nor）.Results The current density of DIDS （200M） sensitive ICl（Ca） induced by intracellular Ca2+ release trigged by L-type Ca2+ current (ICa,L) was significantly decreased in heart failare （HF） cells compared to Nor cells.At membrane voltage of 20mV,the ICl（Ca） density was 3.02±0. 54 pA/pF in Nor （n=6） vs.1.31±0.25 pA/pF in HF （n=8） cells,（P【0.01）,while the averaged ICa,L density did not show difference between two groups.The time constant of current decay of ICl（Ca） was similar in both types of cells.On the other hand,in intra cellular Ca2+ clamped mode,where the [Ca2+]i was maintained at 100nmol/L,ICl（Ca） density be increased significantly in HF cells when the membrane voltage at +30mV or higher.Conclusions Our results suggest that ICl（Ca） density was decreased in pacing induced failing heart but the channel function be enhanced.Impaired Ca2+ handing in HF cells rather than reduced ICl（Ca） channel function itself may have caused this abnormality.The ICl（Ca） density reduction might contribute to the prolongation of action potential in failing heart.The ICl（Ca） channel function up-regulation is likely to cause cardiac arrhythmia by inducing a delayed after depolarization,when Ca2+ overload occurred in diastolic failing heart cells.

Inhibitory actions of mibefradil on steroidogenesis in mouse Leydig cells： involvement of Ca^2＋ entry via the T-type Ca^2＋ channel

Intracellular cAMP and Ca^2＋ are involved in the regulation of steroidogenic activity in Leydig cells, which coordinate responses to luteinizing hormone （LH） and human ehorionic gonadotropin （hCG）. However, the identification of Ca^2＋ entry implicated in Leydig cell steroidogenesis is not well defined. The objective of this study was to identify the type of Ca^2＋ channel that affects Leydig cell steroidogenesis. In vitro steroidogenesis in the freshly dissociated Leydig cells of mice was induced by hCG incubation. The effects of mibefradil （a putative T-type Ca^2＋ channel blocker） on steroidogenesis were assessed using reverse transcription （RT）-polymerase chain reaction analysis for the steroidogenic acute regulatory protein （STAR） mRNA expression and testosterone production using radioimmunoassay. In the presence of 1.0 mmol L-1 extracellular Ca^2＋, hCG at 1 to 100 IU noticeably elevated both StAR mRNA level and testosterone secretion （P 〈 0.05）, and the stimulatory effects of hCG were markedly diminished by mibefradil in a dose-dependent manner （P 〈 0.05）. Moreover; the hCG-induced increase in testosterone production was completely removed when external Ca^2＋ was omitted, implying that Ca entry is needed for hCG-induced steroidogenesis. Furthermore, a patch-clamp study revealed the presence of mibefradil-sensitive Ca^24- currents seen at a concentration range that nearly paralleled those inhibiting steroidogenesis. Collectively, Our data provide evidence that hCG-stimulated steroidogenesis is mediated at least in part by Ca^2＋ entry carried out by the T-type Ca^2＋ channel in the Leydig cells of mice.

Effects of calcium-activated chloride channels on proliferation of pulmonary artery smooth muscle cells in rats under chronic hypoxic condition

Objective：To investigate the effects of calcium-activated chloride （ClCa） channels on proliferation of pulmonary artery smooth muscle cells（PASMCs） in rats under chronic hypoxic condition. Methods：The cultured PASMCs were placed under normoxic and chronic hypoxic conditions：The cells were observed by light and electron microscope; The cell cycles were observed by flow-cytometry; Immunocytochemistry staining was used to detect the expressions of PCNA, c-fos and c-jun of PASMCs; Cytoplasmic free Ca^2＋ concentration （[Ca^2＋]i） in PASMCs was investigated by fluorescent quantitation using fluorospectrophotometer. Results：The PASMCs were contractile phenotype under normoxic conditions. Observation by transmission electron microscope： In kytoplasm of contractile phenotype cells, myofilament bundles were abundant and the content of cell organs such as Golgi＇s bodies were rare. The PASMCs were synthetic phenotype under chronic hypoxic condition. There were increased free ribosomes, dilated rough endoplasmic reticulums, highly developed Golgi complexes, decreased or disappeared thick filaments and dense body in kytoplasm of synthetic phenotype cells. After NFA and IAA-94, the situations were reversed The number of S ＋G2M PASMCs were significantly increased in chronic hypoxic condition; The NFA and IAA-94 were shown to significantly decrease them from （28.6±1.0）% to （16.0±1.6）% and the number of G0G1 PASMCs significantly increased from （71.4± 1.9）% to （83.9 ± 1.6）% （P〈 0.01）. In chronic hypoxic conditions, the expression of proliferating cell nucleus antigen was significantly increased; The NFA and IAA-94 were shown to significantly decrease it from （81 ± 6）% to （27 ± 7）%（P 〈 0.01）. The expression of c-fos and c-jun were significantly increased in＇chronic hypoxic conditions; The NFA and IAA-94 were shown to significantly decrease them from 0.15 ±0.02, 0.32 ± 0.05 to 0.05 ± 0.01, 0.12 ± 0.05, respectively （P〈 0.01）; Under chronic hypoxic conditions, [Ca^2＋]i was increased; The NFA and IAA-94 decreased it from （281.8±16,5）nmol/L to （117.7 ± 15.4）nmol/L（P 〈 0.01）. Conclusion：Hypoxia initiated the change of PASMCs from contractile to synthetic phenotype and increased proliferation of PASMCs. NFA and IAA-94 depressed cell proliferation by blocking ClCa channels in hypoxic condition. These may play an important role in proliferation of PASMCs under chronic hypoxic conditions.

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.

茶树OSCA基因家族的鉴定及表达分析

钙通透性阳离子通道蛋白(OSCA)在植物调节高渗胁迫中发挥着重要作用。为了解OSCA基因家族在茶树响应干旱胁迫中的作用,本研究基于茶树全基因组数据对OSCA基因家族进行鉴定,分析CsOSCAs基因和蛋白结构以及启动子顺式作用元件,并对CsOSCAs在茶树不同组织、不同抗旱性品种间和干旱胁迫下的表达模式进行分析。结果表明:茶树基因组包含12个OSCA基因家族成员,分别命名为CsOSCA1~CsOSCA12;CsOSCAs基因编码的氨基酸序列长度为667~831 bp,蛋白分子量在76630.55~93563.99 kDa之间,等电点在6.15~9.33之间,含有9~12个跨膜结构域,均含有特征保守结构域DUF221,根据系统进化关系可以分为4个亚族;10个CsOSCAs基因定位于茶树7条染色体上,2个CsOSCAs基因定位于未锚定染色体的contig上;CsOSCAs基因编码的蛋白二级结构含有32%~38%的跨膜结构和60%~68%的α-螺旋;CsOSCAs基因具有组织表达特异性,CsOSCA2、CsOSCA3、CsOSCA11、CsOSCA12基因在干旱敏感的品种CN98中的表达量显著高于其他2个耐旱品种,9个基因响应PEG胁迫,其中CsOSCA2、CsOSCA3、CSOSCA5、CsOSCA8、CsOSCA10和CsOSCA12受干旱胁迫强烈诱导;进一步分析启动子顺式作用元件显示,6个基因含有干旱诱导响应元件,11个基因含有脱落酸响应元件。由此推测茶树OSCA基因家族在茶树响应干旱胁迫中发挥着重要的作用,此研究为茶树OSCA基因功能分析与茶树抗逆性研究提供参考。

Correlation of large conductance Ca2+ activated K+ channelα andβ subunit expression in uterine smooth muscle with the postpartum hemorrhage induced by uterine inertia

Objective:To study the correlation of large conductance Ca2+ activated K+ channel (BKCa)α andβ subunit expression in uterine smooth muscle with the postpartum hemorrhage induced by uterine inertia.Methods: The puerperae who underwent cesarean section and had postpartum hemorrhage induced by uterine inertia in Panzhihua Women and Children Health Hospital between March 2015 and May 2017 were selected as the hemorrhage group of the study, and the puerperae who underwent cesarean section and were without postpartum hemorrhage in Panzhihua Women and Children Health Hospital during the same period were selected as the control group. Proper amount of uterine muscle tissue was collected during the cesarean section to measure the expression of BKCaα andβ subunits and the levels of contraction-related proteins in uterine muscle as well as the contraction characteristic parameters of the uterine muscle.Results: The mRNA expression and protein expression of BKCaα andβ subunits in uterine muscle tissue of hemorrhage group were significantly higher than those of control group;the contraction amplitude, contraction frequency and contraction activity of uterine muscle tissue as well as the OTR, COX2, CX43 and HSP27 levels in uterine muscle tissue of hemorrhage group were significantly lower than those of control group;the BKCaα andβ subunit expression in uterine muscle tissue of hemorrhage group were negatively correlated with the contraction amplitude, contraction frequency and contraction activity as well as the OTR, COX2, CX43 and HSP27 levels.Conclusion: The high expression of BKCa in uterine smooth muscle can reduce the uterine muscle contractility and decrease the levels of contraction-related proteins, and it is closely related to the occurrence of postpartum hemorrhage induced by uterine inertia.

三七皂甙单体Rb_1对心肌细胞Ca^（2＋）内流作用的研究

应用Ｆｕｒａ－２荧光技术及放射配基结合实验探讨三七皂甙单体Ｒｂ１对心肌细胞胞外Ｃａ２＋内流的作用。０．０４ｍｍｏｌ·Ｌ－１Ｒｂ１可以抑制高钾引起的大鼠心肌细胞胞浆Ｃａ２＋浓度的升高，且呈浓度依赖性；可以完全阻断高钾基础上的异丙肾上腺素的作用。其作用与钙通道拮抗剂维拉帕米类似。Ｒｂ１对大鼠心肌细胞膜上β受体亲和力和受体数均无明显影响。结果提示：Ｒｂ１对心肌细胞电压依赖性钙通道开放引起的胞内钙升高有抑制作用；对β受体相关联的钙通道开放引起的胞浆Ｃａ２＋升高也有抑制作用。而不影响β受体本身。