1-磷酸鞘氨醇对心肌细胞内游离钙离子浓度的影响

目的研究1-磷酸鞘氨醇（sphingosine-1-phosphate,SIP）对体外培养的乳鼠心肌细胞内游离钙离子浓度的作用。方法应用钙离子荧光指示剂Fluo-3AM负载原代培养的大鼠心肌细胞,通过激光共聚焦扫描显微镜上机检测,记录其荧光强度变化。结果@SIP能降低[Ca2＋]i平均荧光强度值,对照组由（743．15±34．78）nmol／L降至（446．89±55．36）nmol／L（P〈O．01,n=7）;而S1P（1．1μmol／L）＋苏拉明（Suramin）（200μmol／L）组[ca2＋]i平均荧光强度值由（778．39±42．15）nmol／L降至（759．41±38．17）nmol／L,差异无统计学意义（P〉O．05,n=7）。结论S1P可降低乳鼠心肌细胞内游离钙离子浓度,这种作用可能通过其特异性的G蛋白耦联S1P受体介导产生。     

黄芪总黄酮对心肌细胞内游离钙离子浓度的影响

目的研究黄芪总黄酮（Total flavonoids of Ast ragalus,TFA）对体外培养的乳鼠心肌细胞内游离钙离子浓度的作用。方法实验应用钙离子荧光指示剂Fluo-3AM负载原代培养的乳鼠心肌细胞,通过激光共聚焦扫描显微镜上机检测,记录其荧光强度变化,同时观察黄芪总黄酮对乳鼠心肌细胞及化学处理因素H2O2损伤大鼠心肌细胞的荧光强度的作用。结果①H2O2损伤的乳鼠心肌细胞游离钙离子[Ca^2＋]i平均荧光强度值为（1346．89±65．36）nmol／L,与对照组平均荧光强度值（743．15±34．78）nmol／L相比,差异有统计学意义（P〈0．01,n=7）;同时H2O2（0．3mmol／L）使予给TFA（20mg／kg）组乳鼠心肌细胞的[Ca^2＋]i平均荧光强度值由（668．29±41．15）nmol／L下降到（649．31±39．17）nmol／L,差异无统计学意义（P〉0．05,n=7）。结论H2O2可明显升高乳鼠心肌细胞游离钙离子浓度,黄芪总黄酮可对抗H2O2的这种作用。     

中性粒细胞与白细胞介素8在哮喘发病中的作用

目的探讨中性粒细胞与白细胞介素8（IL-8）在支气管哮喘(简称哮喘)发病中的作用。方法对轻度哮喘患者行气道激发试验,对哮喘患者中性粒细胞超氧阴离子（O(*)/(2)）产生、血浆及诱导痰IL-8、丙二醛(MDA)水平进行测定分析。结果中、重度哮喘患者每106个周围血中性粒细胞O(*)/(2)产生水平为［（20.9±5.1）nmol/L］,与轻度哮喘患者［(15.2±4.2)nmol/L］比较,差异有显著性（P<0.01）,轻度哮喘组与正常人［(11.3±2.4)nmol/L］比较,差异有显著性(P<0.05);周围血中性粒细胞O(*)/(2)产生与气道反应性指标一秒钟用力呼气容积下降20%时所需的累积量(PD20FEV1)呈显著负相关(r=-0.693,P<0.05);急性发作期哮喘患者血浆及诱导痰IL-8水平［(585±75)ng/L、(791±103)ng/L］、MDA水平［(6.3±1.6)mmol/L、(21.8±6.3)mmol/L］,与缓解期哮喘患者血浆及诱导痰IL-8水平［(227±54)ng/L］、［(322±95)ng/L］、MDA水平［(5.4±1.0)mmol/L］、［(15.1±5.6)mmol/L］比较,差异有显著性（P<0.01）;缓解期哮喘患者与正常人血浆及诱导痰IL-8水平［(188±46)ng/L］、［(224±51)ng/L］及MDA水平［(4.1±0.4)mmol/L］、［(9.5±4.2)mmol/L］比较,差异有显著性（P<0.05）,诱导痰MDA水平与PD20FEV1呈显著负相关(r=-0.708,P<0.01);诱导痰IL-8水平与诱导痰中性粒细胞百分数呈显著正相关（r=0.838,P<0.01）。结论中性粒细胞产生的氧自由基可能参与哮喘气道炎症及气道高反应的形成。     

黄芪抑制去甲肾上腺素诱导大鼠心肌细胞内游离钙含量升高的作用

目的 :研究黄芪对去甲肾上腺素 (NE)诱导大鼠心肌细胞内游离钙 [Ca2 + ]i 含量升高的作用。方法 :应用AR CM MIC阳离子检测系统 ,采用Ca2 + 指示剂Fura 2 /AM检测培养新生大鼠单个心肌细胞内游离钙的浓度 ,并观察Am对NE诱导大鼠心肌细胞内 [Ca2 + ]i 升高的影响。结果 :当细胞外Ca2 + 浓度为 1 .3mmol/L时 ,大鼠心肌细胞 [Ca2 + ]i 为 ( 97.1 1 +8.2 1 )nmol/L ,给予 1 0 -5mol/L的NE ,可使大鼠心肌细胞 [Ca2 + ]i 增至 ( 2 91 .73± 1 5 .0 3 )nmol/L,而经黄芪处理的大鼠心肌细胞的 [Ca2 + ]i则由 ( 95 .98±8.1 4)nmol/L增至 ( 1 1 4.2 8± 9.2 9)nmol/L。结论 :NE能诱导大鼠心肌细胞内游离钙含量明显升高 ,黄芪对心肌细胞静息 [Ca2 + ]i无明显影响 ,但能抑制NE诱导大鼠心肌细胞 [Ca2 + ]i 升高     

高原红细胞增多症患者中性粒细胞[Ca2＋]i及NADPH氧化酶活性变化

目的 观察高原红细胞增多症（HAPC）患者外周血中性粒细胞[Ca2＋]i及NADPH氧化酶活性变化.方法 HAPC患者30例（HAPC组）,世代居住的健康者29例（健康组）,分离两组外周血中性粒细胞,两组细胞各添加fM-LP（终浓度为0、0.001、0.01、0.1、1、10 μmol/L）及PMA（终浓度为0、10、20、30、40、50 μmol/L）后,采用钙离子荧光探针（Fura-2/AM）法检测两组中性粒细胞内[Ca2＋]i;ELISA法检测中性粒细胞NADPH氧化酶活性.结果 fMLP终浓度为0.1、1、10 μmol/L时,健康组中性粒细胞[Ca2＋]i分别为（223.6 ±34.6）、（260.7 ±30.0）、（316.6 ±53.8） nmol/L;HAPC组分别为（203.2±25.6）、（235.5 ±35.9）、（278.1 ±47.1） nmol/L,两组比较,P＜0.05或＜0.01.fMLP终浓度为0.1、1、10 μmol/L时,健康组中性粒细胞NADPH氧化酶活性分别为（81.3±16.9）、（97.6±23.7）、（119.5 ±33.8）pmol/（10-6个细胞·30 min）;HAPC组分别为（72.0±16.9）、（83.6±15.3）、（102.9±26.3） pmol/（10-6个细胞·30min）,两组比较,P＜0.05或＜0.01.PMA终浓度为30、40、50 μmol/L时,健康组中性粒细胞[Ca2＋]i分别为（224.9±33.1）、（326.7 ±45.3）、（646.9 ±78.1） nmol/L;HAPC组分别为（205.5±26.3）、（293.9 ±52.6）、（567.9±103.1）nmol/L,两组比较,P ＜0.05或＜0.01.PMA终浓度为30、40、50 μmol/L时,健康组中性粒细胞NADPH氧化酶活性分别为（258.2±25.1）、（327.3 ±38.5）、（383.1±36.7）pmol/（10-6个细胞·30 min）;HAPC组分别为（213.8 ±36.3）、（291.3 ±50.5）、（337.2±36.0） pmol/（10-6个细胞·30 min）,两组比较,P＜0.01.结论 高原缺氧降低人外周血中性粒细胞NADPH活性及[Ca2＋]i浓度升高幅度.     

探讨胃动素、神经降压素和一氧化氮合酶与Oddi括约肌运动功能障碍的关系

目的 探讨胃动素（MTL）、神经降压素（NT）及一氧化氮合酶（NOS）对胆囊切除后Oddi括约肌（SO）运动功能的影响。 方法 在动物实验中，豚鼠造模组（胆囊切除）和对照组（开关术）术后12周行SO压力测定（SOM），计算受试者工作特征（ROC）曲线下面积值（AUC）及界限值，从造模组中筛选出符合条件的豚鼠，作为SO运动功能障碍（SOD）模型组。采用放射免疫法检测SOD模型组和对照组血浆MTL、NT的含量及采用积分光密度值方法检测SO组织中MTL、NT及NOS蛋白表达量。在临床研究中，比较SOD组（SO压力＞40 mmHg）和对照组患者血浆MTL、NT含量。 结果 根据ROC曲线计算AUC为0.75，豚鼠SO压力≥29.8 mmHg（界限值）为SOD模型组。SOD模型组（n=10）血浆MTL［（193.16±29.2） pg／mL］和NT含量［（104.57±19.52） pg／mL］均分别高于对照组（n=11） 血浆MTL［（154.24±27.69）pg／mL］和血浆NT含量［（79.65±11.24）pg／mL］（P＜0.01）； SO中MTL（3 556.71±455.80）和NT蛋白表达量（6 321.74±203.54）高于对照组MTL（3 075.92±350.06）和NT蛋白表达量（5 843.57±344.00），而NOS蛋白表达量（2 954.21±173.54）低于对照组（3 314.91±246.67）（P＜0.05）。临床研究SOD组（n=15）血浆MTL［（350.98±24.31）pg／mL］和NT含量［（102.39±19.56）pg／mL］分别高于对照组（n=15）MTL［（319.56±23.54）pg／mL］和NT含量［（80.45±12.35）pg／mL］（P＜0.05）。 结论 血浆MTL和NT含量以及SO中MTL、NT和NOS蛋白表达量与SOD发生有关。检测血浆MTL和NT可能对胆囊切除术后SOD有辅助诊断作用。     

5种黄酮醇类化合物对大鼠心肌细胞内钙离子的影响

目的研究山萘酚（Kaempferol，KA）、杨梅素（Myricetin，MY）、二氢杨梅素（Dihydromyricetin，DMY）、槲皮素（Quercetin，Qu）、二氢槲皮素（Dihydroquercetin，DQU）5种黄酮醇类化合物对大鼠心肌细胞内游离钙离子（[Ca^2＋]i）的影响。方法采用Fluo-3／AM同时加入PluronicF-127作为细胞内游离钙离子的荧光探针，负载H9C2心肌细胞系，应用激光扫描共聚焦显微技术，测定5种黄酮醇类化合物（50μmol／L）在静息状态和加入60mmol／L氯化钾（KCl）时心肌细胞胞浆内游离钙离子的荧光强度（FI）。结果静息状态下，KA和Qu能够明显降低心肌细胞内[Ca^2＋]i（P〈0．05），MY能够短暂而明显升高[Ca^2＋]i（P〈0．05），而DMY和DQU对心肌细胞内[Ca2＋li无明显影响（P〉0．05）；KA、MY、DMY、Qu和DQU对KCl介导的高钙有抑制作用（P〈0．05）。结论5种黄酮醇类化合物对电压依赖性钙通道（VDC）有明显抑制作用，这可能是产生药理活性机制之一．有利于进一步研究其对心肌的保护作用和探讨其构效关系。     

高渗枸橼酸腺嘌呤液灌注对猪胰腺泡细胞[Ca~(2 )]i的影响

目的探讨高渗枸橼酸腺嘌呤液（ＨＣＡ）灌注对猪胰腺泡细胞［Ｃａ２＋］ｉ的影响．方法以Ｆｕｒａ＿２为细胞内钙离子的荧光探剂，采用精密的ＡＲ＿ＣＭ＿ＭＩＣ阳离子测定系统，检测ＨＣＡ灌注前后猪胰（ｎ＝８）腺泡细胞［Ｃａ２＋］ｉ的变化．结果ＨＣＡ灌注后猪胰腺泡细胞［Ｃａ２＋］ｉ／１０－７ｍｏｌ／Ｌ为１８５１±０１９１，ＨＣＡ灌注前猪胰腺泡细胞［Ｃａ２＋］ｉ为１３３７±００８９（Ｐ＜００１）．结论ＨＣＡ灌注使猪胰腺泡细胞［Ｃａ２＋］ｉ升高     

钾依赖钠钙交换蛋白6对人胚胎肾细胞钙离子浓度的影响

目的观察钾依赖钠钙交换蛋白6（NCKX6）对人胚胎肾细胞钙离子浓度的影响。方法RT—PCR克隆NCKX6基因全长,将NCKX6基因构建到真核表达载体pEGFP-C3,转染HEK293细胞,荧光分光光度仪观察细胞内钙离子浓度变化。结果重组真核表达载体pEGFP-C3-NCKX6构建成功,荧光分光光度仪观察到NCKX6过表达后HEK293细胞内游离Ca2＋浓度明显高于正常对照组,并有显著统计学差异。当细胞外145mmol／L的Na＋完全由145mmol／L的Li＋置换后,NCKX6过表达的HEK293细胞内Ca2＋浓度出现明显升高。结论NCKX6的双向转运模式主要以逆向转运模式即将细胞外Ca2＋转运入细胞内为主。     

Thapsigargin-insensitive calcium pools in vascular smooth muscle cells.

Since sarcoplasmic Ca2+-ATPase may play an important role for the regulation of cytosolic free calcium concentration ([Ca2+]i) and may be altered in primary hypertension, the effects of thapsigargin and bradykinin on intracellular calcium pools in cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats of the Münster strain (SHR) and normotensive Wistar-Kyoto (WKY) rats were investigated. VSMC were cultured on glass cover slips and [Ca2+]i was measured using the fluorescent dye fura2. To exclude transplasmamembrane calcium influx all experiments were performed in a calcium free medium. Thapsigargin, a selective inhibitor of the sarcoplasmic Ca2+-ATPase, and bradykinin, that is known to induce inositol trisphosphate release, dose dependently caused an increase of [Ca2+]i by emptying intracellular Ca2+ stores. The peak increase of [Ca2+]i after addition of saturation doses of thapsigargin (1 micromol/L) was not significantly different in the two strains (SHR: 69 +/- 11 nmol/L, n=24; WKY: 58 +/- 12 nmol/L, n=20; mean +/- SEM). When 10 micromol/L bradykinin was added after depletion of the thapsigargin-sensitive pools, still a release of [Ca2+]i could be observed. The bradykinin-induced [Ca2+]i increase was similar in the absence and presence of thapsigargin in VSMC from SHR (62 +/- 12 nmol/L, n=20; vs 52 +/- 18 nmol/L, n=22). In contrast, in the VSMC from WKY a significant reduction of the bradykinin induced [Ca2+]i-increase could be observed after the depletion of the thapsigargin sensitive calcium pools (70 +/- 8 nmol/L, n=21, vs. 33 +/- 7, n=20; p<0.002). It is concluded that bradykinin releases calcium from a pool that is not refilled by the common, thapsigargin-sensitive Ca2+-ATPase. In contrast to VSMC from normotensive WKY, in VSMC from spontaneously hypertensive rats thapsigargin and bradykinin sensitive pools may be regulated separately.     

Effect of dietary sodium intake on intracellular calcium in lymphocytes of salt-sensitive hypertensive patients.

High dietary Na+ raises mean arterial pressure (MAP) by more than 10% in salt-sensitive (SS) patients with essential hypertension. To test whether the rise in MAP in these patients is caused by a Na(+)-linked increase in [Ca2+]i in vascular smooth muscle cells, we measured [Ca2+]i in the lymphocytes of 14 patients with essential hypertension kept on a Na+ intake of 20 mEq/day for 9 days, and 200-mEq/day for 14 days. Nifedipine gastrointestinal transport system (GITS) (30 mg/day) was given during the last 4 days of each diet. We isolated lymphocytes on Ficoll-Hypaque gradient and measured [Ca2+]i levels using Fura-2 fluorescent dye. During low Na+ intake, there was no difference in MAP (102 +/- 3.5 v 93 +/- 3.8 mm Hg) and in lymphocytes [Ca2+]i (80 +/- 3.0 v 87 +/- 5.4 nmol/L) between the seven salt-sensitive and the seven salt-resistant patients. During high Na+ intake, MAP (92 +/- 2.8 mm Hg) and [Ca2+]i (85 +/- 6.8 nmol/L) did not change in salt-resistant patients. On the contrary, MAP (115 +/- 3.4 mm Hg) and [Ca2+]i (130 +/- 11.1 nmol/L) increased significantly (P less than .01) in the salt-sensitive patients. Nifedipine did not significantly alter MAP and [Ca2+]i in both groups of patients during low Na+ and in salt-resistant patients during high Na+ intake. On the contrary, during high Na+ intake, nifedipine decreased significantly (P less than .01) both MAP (104 +/- 2.4 mm Hg) and [Ca2+]i (89 +/- 5.7 nmol/L) in salt-sensitive patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+/Ca2+ exchanger: target for oxidative stress in salt-sensitive hypertension

The Na+/Ca2+ exchanger regulates intracellular calcium ([Ca2+]i), and attenuation of Na+/Ca2+ exchange by oxidative stress might lead to dysregulation of [Ca2+]i. We have shown that the Na+/Ca2+ exchanger differs functionally and at the amino acid level between salt-sensitive and salt-resistant rats. Therefore, the purpose of these studies was to determine how oxidative stress affects the activities of the 2 Na+/Ca2+ exchangers that we cloned from mesangial cells of salt-resistant (RNCX) and salt-sensitive (SNCX) Dahl/Rapp rats. The effects of oxidative stress on exchanger activity were examined in cells expressing RNCX or SNCX by assessing 45Ca2+ uptake (reverse mode) and [Ca2+]i elevation (forward mode) in the presence and absence of H2O2 and peroxynitrite. Our results showed that 45Ca2+ uptake in SNCX cells was attenuated at 500 and 750 micromol/L H2O2 (63+/-12% and 25+/-7%, respectively; n=16) and at 50 and 100 micromol/L peroxynitrite (47+/-9% and 22+/-9%, respectively; n=16). In RNCX cells, 45Ca2+ uptake was attenuated at only 750 and 100 micromol/L H2O2 and peroxynitrite (61+/-9% and 63+/-6%, respectively; n=16). In addition, the elevation in [Ca2+]i was greater in SNCX cells than in RNCX cells in response to 750 micromol/L H2O2 (58+/-5.5 vs 17+/-4.1 nmol/L; n=13) and 100 micromol/L peroxynitrite (33+/-5 vs 11+/-6 nmol/L; n=19). The enhanced impairment of SNCX activity by oxidative stress might contribute to the dysregulation of [Ca2+]i that is found in this model of salt-sensitive hypertension.     

Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+ and cell apoptosis in Caco-2 cells

AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry. RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase Ⅺ and dispase Ⅰ. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 μmol/L) increased the FI of Caco-2 cells from 27.50±13.18 (control, n = 150) to 35.71±13.99 (n = 150, P<0.01), 72.19±35.40 (n = 150, P<0.01) and 211.34±29.03 (n = 150,P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 μmol/L). The FI value of Caco-2 cells treated by FAC was 185.85±33.77 (n = 150, P<0.01), 122.73±58.47 (n = 150, P<0.01), and 53.29±19.82 (n= 150,P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187(0.1,1.0 and 10 μmol/L) increased the FI of Caco-2 cells from 40.45±13.95 (control, n = 150) to 45.19±21.95 (n = 150, P<0.01), 89.87±43.29 (n = 150, P<0.01) and 104.64±51.07 (n = 150,P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i. CONCLUSION: Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.     

Intracellular pattern of cytosolic Ca2+ changes during adhesion and multiple phagocytosis in human neutrophils. Dynamics of intracellular Ca2+ stores

The subcellular pattern of cytosolic free Ca2+ ([Ca2+]i) changes in human polymorphonuclear neutrophils (PMNs) was studied using imaging of fura-2 fluorescence (time resolution 12.5 ratios/s) to determine whether PMNs could obtain directional information from the [Ca2+]i signal. [Ca2+]i changes were observed during initial adherence, the subsequent chemotactic movement, and the phagocytosis of opsonized yeast particles. Initial adherence was followed by a rapid increase in [Ca2+]i (from 90 +/- 10 to 290 +/- 40 nmol/L in 6.5 +/- 2.5 seconds; +/- SEM, n = 10), apparently homogeneously distributed over the entire cytoplasm, which preceded the spreading of the PMNs. [Ca2+]i increases after the contact of the PMNs with yeast particles were of lower mean amplitude; [Ca2+]i increased simultaneously throughout the cytosol. In the absence of extracellular Ca2+, multiple phagocytotic events could proceed normally without a mandatory [Ca2+]i transient. In PMNs polarized on phagocytosis, gradients in [Ca2+]i could be observed. [Ca2+]i was more elevated in the periphagosomal area than in the remaining parts. Taken together, these data show that [Ca2+]i waves do not provide the neutrophil with directional information during chemotaxis and phagocytosis. Sustained small inhomogeneity of [Ca2+]i levels are consistent with a proposed redistribution of releasable Ca2+ stores on phagocytosis.     

Effect of inhibition of Na, K-ATPase on cytosolic free sodium and calcium in platelets of spontaneously hypertensive rats.

Cytosolic free sodium concentrations ([Na+]i) in intact platelets of 18 spontaneously hypertensive rats (SHR) and of 18 age-matched normotensive Wistar-Kyoto rats (WKY) were measured using the sodium-sensitive fluorescent dye sodium-binding-benzofuran-isophthalate. In resting platelets [Na+]i tended to be higher in SHR compared to WKY (20.5 +/- 3.5 mmol/L v 15.1 +/- 1.9 mmol/L, mean +/- SEM), but the differences were not statistically significant. Stimulation of the Na-H-exchange by 1.0 U/mL thrombin increased [Na+]i in SHR by 22.9 +/- 4.3 mmol/L and in WKY by 35.0 +/- 5.6 mmol/L in a similar way. After inhibition of Na, K-ATPase by 1 mmol/L ouabain there was a significant rise of [Na+]i both in platelets of SHR to 38.0 +/- 5.1 mmol/L (P < .01 compared to resting platelets) and in platelets of WKY to 26.5 +/- 4.3 mmol/L (P < .01). However, no significant difference could be observed between these two groups. Using the calcium-sensitive dye fura-2, resting cytosolic free calcium concentrations ([Ca2+]i) were found to be significantly higher in platelets of SHR compared to WKY (171.9 +/- 21.5 nmol/L v 93.14 +/- 19.7 nmol/L, P < .05). After the addition of ouabain [Ca2+]i was significantly higher in SHR compared to WKY (245.5 +/- 32.6 nmol/L v 159.6 +/- 22.5 nmol/L, P < .05). The results do not support the hypothesis that altered sodium-calcium exchange causes elevated cytosolic free calcium in SHR.     

Luteinizing hormone (LH) drives diverse intracellular calcium second messenger signals in isolated porcine ovarian thecal cells: preferential recruitment of intracellular Ca2+ oscillatory cells by higher concentrations of LH

The present study examines Ca2+ second messenger signaling driven by LH in isolated porcine thecal cells. To this end, we implemented semiquantitative fluorescent (fura-2) videomicroscopic imaging of single thecal cells in vitro. Stimulation of 388 cells with LH (5 microg/ml) elicited an intracellular Ca2+ ([Ca2+]i) signal in 85+/-5.3% of individual thecal cells (n = 11 experiments). Among 337 LH-responsive cells, we identified four predominant temporal modes of [Ca2+]i signaling: 1) [Ca2+]i oscillations with periodicities of 0.5 to 4.5 min(-1) (63+/-4.5%), 2) a [Ca2+]i spike followed by a sustained plateau (17+/-2.6%), 3) a [Ca2+]i spike only (5.8+/-2.6%); and 4) a [Ca2+]i plateau only (3.8+/-1.5%). The prevalence, but not the amplitude or frequency, of LH-induced [Ca2+]i oscillations in thecal cells was dependent on the agonist concentration. Reduced availability of extracellular Ca2+ induced by treatment with EGTA or cobaltous chloride did not block the initiation, but reversibly abolished ongoing [Ca2+]i oscillations (72% of cells) or increased the mean [Ca2+]i interspike periodicity from 1.09+/-0.16 to 0.59+/-0.07 min(-1) (P < 0.05). Putative phospholipase C inhibition with U-73122 (10 microM) also abolished or frequency-damped LH-driven [Ca2+]i oscillations in 95+/-4.7% of cells. [Ca2+]i oscillations in thecal cells were not abrogated by overnight pretreatment with pertussis toxin. We conclude that 1) thecal cells (unlike earlier findings in granulosa cells) manifest a diverse array of [Ca2+]i signaling responses to LH at the single cell level; 2) LH can dose dependently recruit an increasing number of individually [Ca2+]i oscillating thecal cells; 3) extracellular Ca2+ is required for LH to sustain (but not initiate) frequent and high amplitude [Ca2+] oscillations in thecal cells; and 4) these signaling actions of LH are mediated via phospholipase C, but not a pertussis-toxin sensitive mechanism. Accordingly, the present data extend the apparent complexity of LH-induced [Ca2+]i second messenger signaling and identify at the single cell level LH's dose-responsive drive of [Ca2+]i oscillations in gonadal cells.     

Effects of nitric oxide on P2Y receptor resensitization in spontaneously hypertensive rat mesangial cells

BACKGROUND: Cellular responses to agonists of G protein-coupled receptors are usually rapidly attenuated - a process known as 'receptor desensitization'. The mechanisms that attenuate signalling are important both physiologically and therapeutically. OBJECTIVE: To evaluate the effects of nitric oxide on the P2Y receptor resensitization in cultured glomerular mesangial cells in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. METHODS: The cytosolic calcium concentration ([Ca2+]i ) in cultured mesangial cells was determined with a fluorescence digital imaging system, using the intracellular fluorescent indicator, Fura 2-AM. RESULTS: The first ATP-stimulated [Ca2+]i measured was significantly greater in SHRs (1330.25 +/- 360.31 nmol/l) than in WKY rats (974.28 +/- 397.72 nmol/l; 0.05). Spermine- -[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]-butyl-1,3-propanediamine (spermine NONOate) and L-arginine significantly increased the fourth ATP-stimulated [Ca2+]i in WKY rats ( P<0.01, 0.05, respectively). In SHRs, only spermine-NONOate was able to restore the fourth ATP-challenged [Ca2+]i value significantly. Nomega-nitro-L-arginine methyl ester (L-NAME) greatly reduced the second, third and fourth ATP-stimulated [Ca2+]i in WKY rats (P< 0.01), but not in SHRs. When the cells from WKY rats were superfused with L-NAME, L-arginine or spermine-NONOate for a period of 5 min before and during one single ATP challenge, the responses observed were not significantly different from those in controls. CONCLUSIONS: L-Arginine and spermine-NONOate are able to increase P2Y receptor resensitization in rat mesangial cells, an effect that is less potent in SHRs than in WKY rats. The presence of >l-NAME enhanced receptor desensitization in WKY rats, but not in SHRs.     

Effects of adenovirus-mediated sorcin overexpression on excitation-contraction coupling in isolated rabbit cardiomyocytes

To evaluate the effect of sorcin on cardiac excitation-contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad-sorcin). A beta-galactosidase adenovirus (Ad-LacZ) was used as a control. Fractional shortening in response to 1-Hz field stimulation (at 37 degrees C) was significantly reduced in Ad-sorcin-transfected myocytes compared with control myocytes (2.10+/-0.05% [n=311] versus 2.42+/-0.06% [n=312], respectively; P<0.001). Action potential duration (at 20 degrees C) was significantly less in the Ad-sorcin group (458+/-22 ms, n=11) compared with the control group (520+/-19 ms, n=10; P<0.05). In voltage-clamped, fura 2-loaded myocytes (20 degrees C), a reduced peak-systolic and end-diastolic [Ca2+]i was observed after Ad-sorcin transfection. L-type Ca2+ current amplitude and time course were unaffected. Caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na+-Ca2+ exchanger (NCX) current revealed a significantly lower SR Ca2+ content and faster Ca2+-extrusion kinetics in Ad-sorcin-transfected cells. Higher NCX activity after Ad-sorcin transfection was confirmed by measuring the NCX current-voltage relationship. beta-Escin-permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca2+] using a confocal microscope. Under these conditions, caffeine-mediated SR Ca2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin-overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca2+-transient amplitude predominantly by lowering SR Ca2+ content via increased NCX activity. The effect of sorcin overexpression on Ca2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation-contraction coupling.     

Differential effects of luteinizing hormone on intracellular free Ca2+ in small and large bovine luteal cells

The effect of LH on the intracellular free Ca2+ concentration ([Ca2+]i) was investigated in highly purified small and large bovine luteal cell populations. Luteal cells were obtained from midcycle corpora lutea dispersed with collagenase and separated by flow cytometry into large and small cells. Resting levels of Ca2+ were higher (P less than 0.05) in the large than small cells [314 +/- 25 nM (n = 5) vs. 186 +/- 13 nM (mean +/- SE; n = 13) for large and small cells, respectively]. LH rapidly increased [Ca2+]i in both small and large cells loaded with the fluorescent Ca2+ probe fura-2. In the small cells, [Ca2+]i was immediately increased 2- to 6-fold (from 176 +/- 8 to 468 +/- 8 nM; n = 5) after adding LH. The LH induced [Ca2+]i rise occurred in two phases: an initial peak due to intracellular Ca2+ mobilization and a secondary rise due to Ca2+ influx from extracellular sources. Preincubation of the small cells with EGTA reduced the initial phase and abolished the secondary rise in [Ca2+]. Both forskolin and 8-bromo-cAMP increased [Ca2+]i in the small cells. In contrast, only a single phase of [Ca2+]i rise was observed in LH-treated large cells, and the response was 1.5- to 2-fold greater than the resting Ca2+ levels [314 +/- 25 vs. 435 +/- 60 nM (n = 4), for resting vs. LH-treated values, respectively]. The addition of both LH and prostaglandin F2 alpha (PGF2 alpha) to the large cells resulted in increases in [Ca2+]i that were greater than those induced by each hormone separately (2.0-fold for LH and 2.7-fold for PGF2 alpha vs. 7- to 9-fold in the presence of both hormones). These findings demonstrate that LH induces rapid increases in intracellular [Ca2+]i that differ in magnitude and profile between the small and large bovine luteal cells. Furthermore, LH and PGF2 alpha interacted to promote increases in [Ca2+]i in the large cells, that were higher than the sum of [Ca2+]i induced by each hormone separately.