Relationship of intracellular free Ca2+ concentration and calcium-activated chloride channels of pulmonary artery smooth muscle cells in rats under hypoxic conditions

Summary To investigate the relationship between intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and calcium-activated chloride (Cl_CB) 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 perfusionin vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs under normal and chronic hypoxic condition. The effect of Cl_CB channels on PASMCs proliferation was assessed by MTT assay. The Cl_CB 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, [Ca²⁺]_i was increased. Under normoxic condition, [Ca²⁺]_i was (123.63±18.98) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (281.75±16.48) nmol/L (P<0.01). Under normoxic condition, [Ca²⁺]_i showed no significant change and no effect on Cl_CB channels was observed (P>0.05 Chronic hypoxia increased [Ca²⁺]_i which opened Cl_CB channels. The NFA and IAA-94 blocked the channels and decreased [Ca²⁺]_i from (281.75±16.48) nmol/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 [Ca²⁺]_i which opened Cl_CB channels and had a positive-feedback in [Ca²⁺]_i. This may play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition, Cl_CB channel may play a part in the regulation of proliferation of PASMCs. YANG Zhao, female, born in 1967, Doctor in Charge     

Rapid Inhibition of the Glutamate-induced Increase of Intracellular Free Calcium by Magnesium in Rat Hippocampal Neurons

Inrecentyears ,largenumbersofrelativestud iesonthemagnesium (Mg2 )andneurologicaldis easesverifiedinmanyneurologicaldiseases ,suchascerebrovasculardiseases,chronicalcoholpoisoning ,Wilsondiseaseetc .,wererelatedwiththeMg2 defi ciency .Magnesiumreagentsc…     

Effects of L-THP on Ca2+ overload of cultured rat cardiomyocytes during hypoxia and reoxygenation

Summary The effects of L-tetrahydropalmatine (L-THP) on the cultured rat cardiomyocytes during hypoxia and reoxygenation and the mechanism of L-THP treating reperfusion-arrythmias were studied. The concentration of intracellular free calcium ([Ca²⁺]_i) of single cultured ventricular myocyte was determined by using EPC-9 light-electricity measurement system. It was found that L-THP (100 μmol/L) could reduce the [Ca²⁺]_i augmentation in single cultured ventricular myocyte during hypoxia and reoxygenation. Verapamil (10 μmol/L) had the similar effect. It was concluded that LTHP could inhibit the Ca²⁺ overload of cultured rat cardiomyocytes during hypoxia and reoxygenation. This study was supported by a grant from the National Research Foundation of the Ministry of Public Health (No. 96-Q-025).     

Effect of SJAMP on human platelet cytoplasmic Ca2+

Summary Using the method of dual-wavelength measurement of platelet [Ca²⁺]_i and Fura-2 as the Ca²⁺ fluorophore probe, we measured the effect of acidic Mucopolysaccharide fromSticopus Japonicus Selenka (SJAMP) on platelet [Ca²⁺]_i. The results showed that the most significant increase in platelets [Ca²⁺]_i was seen when the concentration of SJAMP was 100μg/ml and the elevation of normal platelet [Ca²⁺]_i was 93.96 ± 10.24 nmol/L (n=10). In the presence of extracellular Ca²⁺ (1 mmol/L), the magnitude of platelet [Ca²⁺]_i response to SJAMP was increased and the [Ca²⁺]_i could reach 116.72±10. 66 nmol/L (n = 10). On the other hand, the magnitude of increased platelet [Ca²⁺]_i induced by SJAMP was smaller and the duration of [Ca²⁺]_i reaching the highest level was longer when compared with other platelet aggregation agents. In the mean time, if platelets were first incubated with cyclooxygenase inhibitor, the rise of [Ca²⁺]_i evoked by SJAMP was inhibited. The results indicated that the mechanism of the rise of [Ca²⁺]_i induced by SJAMP might be dependent upon the generation of prostaglandin endoperoxides and(or) TXA₂. This project was supported by grant from the National Nature Science Foundation of China (No. 39370322).     

Effects of cyclosporine A on angiotensin II-induced cardiomyocyte hypertrophy in neonatal rats

Summary The effects of cyclosporine A (CsA) on Angiontensin II (Ang II)-induced protein contents, c-fos protein levels and cytosolic Ca²⁺ level ([Ca²⁺]i) in cultured cardiomyocytes of neonatal rats were observed. Total protein contents were determined by Bradford method. The expression of c-fos protein was detected by Western blot. [Ca²⁺]i labeled with fluorescent probe Fluo-3/AM was measured under a laser scanning confocal microscope. The results revealed that as compared with control, the total protein contents were increased in cardiomyocytes treated with Ang II (10⁻⁷ mol/ L), which could be inhibited by CsA in a dose-dependent manner. It was found that Ang II could increase the c-fos protein expression, which could be inhibited by CsA in a dose-dependent manner. Ang II induced the [Ca²⁺]i elevation in cardiomyocytes. CsA did not influence the resting intracellular Ca²⁺, but inhibited significantly the Ang II-induced [Ca²⁺]i elevation. It was concluded that CsA can suppress the Ang II-induced c-fos protein expression and [Ca²⁺]i elevation in single cardiomyocyte, which might pay a role in the prevention of Ang II-induced cardiomyocyte hypertrophy by CsA. Han Zhaomin, female, born in 1974, Pharmacist     

Effect of calmodulin and voltage-dependent Ca<Superscript>2+</Superscript> channel on the proliferation of heptoma cells induced by epidermal growth factor

Summary The effect of thyrosine kinase, calmodulin and voltage-dependent Ca²⁺ 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 H-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 Ca²⁺ 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 Ca²⁺-calmodulin-dependent pathway may play a critical role on the proliferation of heptoma cells induced by EGF and voltage-dependent Ca²⁺ channel is independent of the effect of EGF. WU Binwen, male, born in 1968, M. D., Ph.D.     

Mechanism of anti-β-adrenoceptor antibody mediated myocardial damage in dilated cardiomyopathy

Summary Antibodies against β₁-adrenoceptor can be detected in serum of patients with dilated cardiomyopathy (DCM), which have β-agonist-like activity, and induce a positive chronotropic effect on cardiac myocytes by its persistence at full strength. Effects of the antibodies against β-adrenoceptor from sera of patients with DCM on myocardial cytotoxicity and cytoplasmic free Ca²⁺-concentration ([Ca²⁺]_i) were observed in the cultured single layer SD rat ventricular cells by using the cytotoxicity assay and fluorescent Ca²⁺- indicator fura-2/AM. The positive sera of the anti-β-adrenoceptor antibodies from patients with DCM markedly enhanced myocardial [Ca²⁺]_i. Betaloc, a β_i-receptor blocker, might inhibit the increase of the antibody-mediated myocardial [Ca²⁺]_i, and the sera from healthy donors had no effect on myocardial [Ca²⁺]_i. Our results suggest that the anti-β-adrenoceptor antibody might increase myocardial [Ca²⁺]_i and result in myocardial damage. The antibodies might activate receptor-gating [Ca²⁺]-channel, thereby causing myocardial [Ca²⁺]_i rise and calcium overload. Early use of betaloc is recommended in the treatment of dilated cardiomyopathy. This project was supported by the National Natural Science Fundation of China (No. 39370317).     

Effects of Total Flavonoids ofHippophae Rhamnoides L. on intracellular free calcium in cultured vascular smooth muscle cells of spontaneously hypertensive rats and Wistar-Kyoto rats

Objective: To explore the effects of total flavonoids ofHippophae rhamnoides L. (TFH), quercetin (Que) and isorhamnetin (Isor) on the intracellular free calcium ([Ca²⁺ ]_i) in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY).Methods: Fluo 3-acetoxymethylester(Fluo-3/AM) was used to observe the effects of TFH (100mg/L) and its essential monomers, namely Que (l0^-4 mol/L) and Isor (l0^-4 mol/L) on changes of [Ca²⁺]_i in cultured SHR and WKY VSMC (abbr. to Ca-SHR & Ca-WKY) following exposure to high K⁺, norepinephrine (NE) and angiotensin II (Ang II), and to compare with the effects of verapamil (Ver).Results: (1) TFH, Que and Isor had inhibitory effects on resting Ca-SHR (P<0.05), but had no significant effects on Ca-WKY (P>0.05). (2) High K⁺ could increase Ca-SHR more significantly than Ca-WKY (P<0.05); TFH, Que and Isor could inhibit the elevation of [Ca²⁺]_i induced by high K⁺-depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY (P<0.05). (3) NE and Ang II could increase Ca-SHR more significantly than Ca-WKY (P<0.05), TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or Ang II. (4) In the absence of extracellular Ca²⁺, TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter(P<0. 05).Conclusion: TFH, Que and lsor might decrease the levels of [Ca²⁺]_i in VSMCs by blocking both voltage-dependent calcium channels (VDC) and receptor-operated calcium channels (ROC) in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension. Supported by One-hundred-people Plan of Hygiene System in Shanghai (No. 990122)     

K<Superscript>+</Superscript> channels and their effects on membrane potential in rat bronchial smooth muscle cells

Summary In order to investigate the K⁺ channels and their effects on resting membrane potential (Em) and excitability in rat bronchial smooth muscle cells (BSMCs), the components of outward K⁺ channel currents and the effects of K⁺ channels on Em and tension in rat bronchial smooth muscle were observed by using standard whole-cell recording of patch clamp and isometric tension recording techniques. The results showed that under resting conditions, total outward K⁺ channel currents in freshly isolated BSMCs were unaffected by ATP-sensitive K⁺ channel blocker. There were two types of K⁺ currents: voltage-dependent delayed rectifier K⁺ channel (K_v) and large conductance calcium-activated K⁺ channel (BK_Ca) currents. 1 mmol/L 4-aminopyridine (4-AP, an, inhibitor of K_V) caused a significant depolarization (from −8.7±5.9 mV to −25.4±3.1 mV,n=18,P<0.001). In contrast, 1 mmol/L tetraethylammonium (TEA, an inhibitor of BK_Ca) had no significant effect on Em (from −37.6±4.8 mV to −36.8±4.1 mV,n=12,P>0.05). 4-AP caused a concentration-dependent contraction in resting bronchial strips. TEA had no effect on resting tension, but application of 5 mmol/L TEA resulted in a left shift with bigger pD₂ (the negative logarithm of the drug concentration causing 50% of maximal effect) (from 6.27±0.38 to 6.89±0.54,n=10,P<0.05) in the concentration-effect curve of endothine-1, and a right shift with smaller pD₂ (from 8.10±0.23 to 7.69±0.08,n=10,P<0.05) in the concentration-effect curve of isoprenaline. It was suggested that in rat BSMCs there may be two types of K⁺ channels, K_v and BK_Ca, which serve distinct roles. K_v participates in the control of resting Em and tension. BK_Ca is involved in the regulation of relaxation or contraction associated with excitation. LIU Xiansheng, male, born in 1969, M. D., Ph. D. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30270583).     

2-甲基-3-羟基蒽醌诱导人乳腺癌MCF-7细胞凋亡的机制

为探讨2-甲基-3-羟基蒽醌抗肿瘤作用及其机制,本研究采用锥虫蓝法检测细胞活力,流式细胞仪检测细胞周期变化、细胞凋亡率、线粒体膜电位及细胞内游离钙的变化,Western blot方法检测凋亡相关蛋白caspase-4、caspase-7、caspase-9、Bcl-2、Bax、JNK、细胞色素C的表达。结果发现：2-甲基-3-羟基蒽醌时间依赖性地抑制乳腺癌细胞的生长,升高细胞内游离钙含量,降低线粒体膜电位并诱导其凋亡;药物上调Bax并下调Bcl-2蛋白的表达;诱导caspase-4、caspase-7、caspase-9、calpain的活化及细胞色素C的释放。结果提示2-甲基-3-羟基蒽醌可能通过Ca²⁺/calpain/caspase-4途径诱导人乳腺癌MCF-7细胞凋亡。     

Prediction of Trend between Water Environment Pollution of D Lake and Death Rate of Malignancy in Population

Waterisoneofthemostnecessarybasicsforhu manlifeandproductionactivities .Alotofattentionhasbeenpaidto predictionbetweenwaterenviron mentpollutionandpopulationhealth[1- 3] .Thee cosystemwherehumanexistsisverycomplexandthemechanismsbywhichwatercontaminationaffectpopulationhealtharealsoverycomplicated .Re searcheshaveshownthatbladdercancer ,stomachcancer ,coloncancer ,rectumcancer ,esophaguscan cerandlivercancerareamongthecommoncancerswhichhaveshownstatisticalsignificancebetweenpopulationandwatero…     

A study of endothelium-dependent proliferation of pulmonary smooth muscle cells in vitro

Summary The direct effect of hypoxia and the effect of hypoxic endothelial cells conditioned medium on cultured pulmonary arterial smooth muscle cells in vitro were studied with phase contrast microscopy,³H-thymidine labelled technique and flow cytometric measurements. The results showed that direct hypoxia inhibited proliferation of pulmonary arterial smooth muscle cells, retained pulmonary arterial smooth muscle cells in the Go/G₁ phase and decreased³H-thymidine incorporation into pulmonary arterial smooth muscle cells and that hypoxic endothelial cells conditioned medium stimulated proliferation of pulmonary arterial smooth muscle cells, promoted pulmonary arterial smooth muscle cells from G₀/G₁ phase to S phase and increased³H-thymidine incorporation into pulmonary arterial smooth muscle cells. It was reasonable to believe that hypoxia might enable pulmonary arterial endothelial cells to secrete some growth factors which could stimulate proliferation of pulmonary arterial smooth muscle cells, thereby playing an important role in structural remodeling of the pulmonary arteries and in the development of hypoxic pulmonary hypertension.     

The role of neutrophil activation in pathogenesis of preeclampsia

Pre- eclampsia,a common pregnancy complica-tion,isone of the leading causesof maternal mortali-ty and perinatal mortality and modality.The patho-genesis of this disease remains unclear.Endothelialdamage,which appears to be common to the patho-physiologicalfeatures of pre- eclampsia,has been doc-umented[1] .However,neutrophil activation may playan importantrole in vascular endothelium damage[2 ] .In this study,the CD1 1 b expression was examinedby using flow cytometry,and the levelsof plasma e…     

低氧通过Ca~(2+)-NFAT信号通路促进肺动脉平滑肌细胞增生

目的通过观察低氧对肺动脉平滑肌细胞增生的影响及作用机制,探讨低氧在肺动脉高压发病过程中的作用,为肺动脉高压的进一步干预治疗提供可靠的理论依据。方法培养人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cell,HPASMCs)并分为以下几组:正常对照组、低氧组(24h、48h、72h)、低氧并处理组(低氧48h并在细胞外液中加入EDTA2mmol/L或VIVIT4μmol/L)。分别应用细胞计数法检测细胞的增生情况、荧光钙成像法测定细胞内游离钙离子浓度([Ca2+]i)、激光共聚焦显微镜观察活化T细胞核因子c3(nuclear factor of activated Tcells,NFATc3)在细胞核内外分布的变化。结果低氧呈时间依赖性促进HPASMCs增生;低氧处理的细胞静息[Ca2+]i和库容性钙内流(capacitative calcium entry,CCE)显著升高,NFATc3的核转位增多。EDTA(Ca2+螯合剂)或VIVIT(NFAT特异性抑制剂)能够明显抑制低氧诱导的细胞增生现象。结论低氧能促进HPASMCs的增生,其机制可能是通过增加静息[Ca2+]i和库容性钙内流,使HPASMCs内的Ca2+异常升高并导致NFATc3转位至核内,进而促进与增生有关的基因的表达。     

Tonic and phasic activity in smooth muscle

We have studied the electrical and mechanical behaviour of two very different smooth muscle preparations, mesenteric lymphatic ducts and proximal urethra. These tissues generate different patterns of spontaneous contraction adapted to fulfil their contrasting functions. While lymphatics undergo regular phasic contractions and relaxations, suited to their role in propelling lymph, the urethra remains in a state of contracture to maintain urinary continence. The challenge is to understand how both of these achieve their respective roles. Interestingly, electrical activity of lymphatics resembles that in the heart in having a one to one relationship between the action potential and phasic contraction. Patch clamp studies have shown that lymphatic cells express 3 ionic currents that are not present in urethral cells, but are shared with cardiac muscle. These are, i) fast Na⁺ current, ii) T-type Ca²⁺ current and iii) a hyperpolarisation-activated cation current, I_f. The fast Na⁺ current is ideally suited to the propagation of the action potential over large distances, as required by a vessel capable of generating a rapid well co-ordinated contraction along its length. The T-current and I_f, on the other hand, appear to be involved in electrical pacemaking as they are in the heart. The urethra does not usually undergo regular phasic contractions and it lacks these currents. Instead, urethral tone may depend on an interaction between L-type Ca²⁺ current and a large Ca²⁺-activated Cl⁻ current. Activation of Cl⁻ channels (perhaps by spontaneous release of Ca²⁺ from intracellular stores) would depolarise the membrane potential to within the ‘window current’ range for L-type Ca²⁺ channels and result in Ca²⁺ influx and contraction. This process may be maintained for a time by positive feedback whereby the influx of Ca²⁺ continues to activate the Cl⁻ channels. Conway Review Lecture     

Comparison on the effects of three sex hormones on the fetal rat calvarial osteoblasts

Despite the overwhelming evidence of estrogenin bone preservation in postmenopausal osteoporosis, the mechanism of action of sex hormones onbone metabolism remains uncertain. In order toexplore the effect of 178--estradiol (E, ), progesterone (P ), and testosterone (T ) on rat osteoblasts proliferation and differentiation and difference of actions of these hormones, rat calvarialosteoblasts were used as culture model, since thisculture system, when cultured in a suitable environment, could mim…     

Involvement of M3 cholinergic receptor signal transduction pathway in regulation of the expression of chemokine MOB-1, MCP-1 genes in pancreatic acinar cells

Summary Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-κB and the expression of chemokine MOB-1, MCP-1 genes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTCin vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-κB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10⁻³ mol/L, 10⁻⁴ mol/L carbachol) could induced a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10⁻³ mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-κB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10⁻³ mol/L carbachol)in vitro for 30 min. Either M3 cholinergic receptor antagonist (10⁻⁵) mol/L atropine) or NF-κB inhibitor 10⁻² mol/L PDTC) could obviously inhibit the activation of NF-κB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-1 genes in pancreatic acinar cellsin vitro through the activation of NF-κB. ZHENG Hai, male, born in 1972, M. D., Ph. D.