A cross-sectional study of basal platelet intracellular free calcium concentration in normotensive and hypertensive primigravid pregnancies

1. The intracellular free calcium concentration ([Ca2+]i) in washed human platelets was measured using the fluorescent indicator, fura-2, in a cross-sectional study of 36 normotensive, primigravid volunteers, 12 in each trimester of pregnancy and a further 12 at 6 weeks post partum. The results were compared with those obtained from 30 normal female volunteers not using oral contraception. 2. The mean basal [Ca2+]i in the platelets of the pregnant women in the first two trimesters (115.6 +/- 6.7 and 120.1 +/- 5.7 nmol/l, respectively) was not shown to differ significantly from that of normal non-pregnant volunteers (112.3 +/- 2.9 nmol/l). However, during the third trimester a significant increase in [Ca2+]i was noted (134.0 +/- 4.9 nmol/l; P less than 0.05), with a return to normal values in the post-partum period (108.2 +/- 6.1 nmol/l). 3. [Ca2+]i was also measured in the platelets of a group of 12 primigravid pregnant women in the third trimester whose pregnancies were complicated by gestational hypertension (pregnancy-induced hypertension and pre-eclampsia). A significant rise in basal [Ca2+]i was noted in the platelets of primigravidae whose pregnancies were complicated by pre-eclampsia (163.6 +/- 8.8 nmol/l) as compared with normotensive, third-trimester primigravidae (P less than 0.02). However, no correlation could be demonstrated between [Ca2+]i and systemic blood pressure.     

Correlation of blood pressure in normotensive and hypertensive individuals with platelet but not lymphocyte intracellular free calcium concentrations

1. Fifty-two normotensive and essential hypertensive subjects were studied. Intracellular free calcium concentration [( Ca2+]i) was measured in lymphocytes (37 subjects) and platelets (18 subjects) by means of the fluorescent indicators, quin 2 and fura-2. In 31 subjects, plasma ionized calcium concentration was also measured. 2. There was a positive correlation between platelet [Ca2+]i and systolic blood pressure (r = 0.485, P less than 0.05), diastolic blood pressure (r = 0.542, P less than 0.02) and mean blood pressure (r = 0.534, P less than 0.02). 3. No statistically significant relationship was observed between plasma ionized calcium and blood pressure. 4. No relationship was found between lymphocyte [Ca2+]i and blood pressure, or between lymphocyte [Ca2+]i and plasma ionized calcium. 5. There was no relationship between [Ca2+]i of lymphocytes and platelets measured simultaneously from the same subject. 6. These findings reconcile previous conflicting reports and show a relationship between platelet but not lymphocyte [Ca2+]i and blood pressure in man.     

Cytosolic free calcium and intracellular calcium stores in neutrophils from hypertensive subjects

Alterations in intracellular calcium have been implicated in the pathogenesis of essential hypertension. To see whether this is a generalized phenomenon we assessed cytosolic free calcium and intracellular calcium stores in neutrophils from normo- and hypertensive subjects, by trapping the fluorescent calcium indicator quin2 in intact cells. Ten patients with untreated essential hypertension were compared with 10 age- and sex-matched normotensive subjects. The levels of cytosolic free calcium and intracellular calcium stores releasable by the calcium ionophore ionomycin did not differ. No significant relationship was found between blood pressure and the calcium parameters in all 20 subjects studied. The results indicate that essential hypertension is not associated with a membrane defect in calcium handling of all human cell systems, leading to generalized increases in resting values of cytosolic free calcium. Neutrophils do not appear to be a good model for intracellular calcium handling in vascular smooth muscle.     

Platelet cytosolic free calcium concentration, total plasma calcium concentration and blood pressure in human twins: a genetic analysis.

1. We used path analysis and maximum-likelihood model fitting to evaluate the relative contributions of genetic and environmental factors to the relationships observed between level of blood pressure and both total plasma calcium concentration and platelet cytosolic free calcium concentration in 109 twin pairs. 2. Total plasma calcium concentration was positively associated with systolic (r = 0.26, P less than 0.001) but not diastolic blood pressure, a relationship which remained significant after adjustment for albumin, age and body mass index. A relationship between platelet cytosolic free calcium concentration and both systolic and diastolic blood pressure (r = 0.17 and r = 0.13, respectively, P less than or equal to 0.05) was no longer significant after adjustment for age and body mass index. 3. Additive genetic influences, unique environmental effects and age contributed to 60%, 30% and 10% of the variance in systolic blood pressure, respectively. Additive genetic effects explained 78% of the variance in plasma total calcium concentration and at least 48% of the variance in platelet cytosolic free calcium concentration in females and 37% in males. 4. Bivariate factor models provided evidence of genetic, but not environmental, co-variation of total plasma calcium concentration and systolic blood pressure, suggesting that a common genetic factor (or factors) contributes to their univariate relationship. In contrast, there was evidence of environmental, but not genetic, covariation of platelet cytosolic free calcium concentration and systolic blood pressure, suggesting that some of the individual experiences specific to each twin may be causing these two traits to vary together. 5. The possible confounding effects of adiposity and environmental factors should be considered in future studies investigating the role of intracellular calcium levels in the pathogenesis of hypertension.     

大蒜素对血管紧张素Ⅱ诱发人心房肌细胞钙电流和游离钙离子浓度的影响

背景近年来发现血管紧张素Ⅱ可诱发心房电重构,而大蒜素具有相应的拮抗作用,可阻止或减轻心房电重构的发生.目的观察大蒜素对血管紧张素Ⅱ诱发人心房肌细胞膜钙通道电流和细胞内游离钙离子浓度的影响.设计以急性分离的人的心房肌细胞为实验对象,随机对照设计.单位一所军医大学医院心内科. 方法实验于2003-06/2004-06在第四军医大学西京医院心脏内科实验室完成.选择在此期间接受心脏体外循环手术的先天性心脏病患者10例;男6例,女4例,平均年龄(15±6)岁.术中取患者右心耳标本送至实验室后,急性分离单个人心房肌细胞,实验分4组对照组(不加任何干预措施);血管紧张素Ⅱ组加入终浓度为0.1μmol/L的血管紧张素Ⅱ;大蒜素组加入终浓度为50 μmol/L的大蒜素;血管紧张素Ⅱ+大蒜素组大蒜素50 μmol/L与血管紧张素Ⅱ0.1 μmol/L同时加入.采用全细胞膜片钳方法记录L型钙电流;以钙荧光探针荧光指示剂负载,应用激光共聚焦显微镜技术,分别于加入干预药物后即刻与15 min检测游离钙离子浓度变化.主要观察指标各组人心房肌细胞L型钙电流峰值电流密度及钙离子游离钙离子的荧光强度变化率.结果①血管紧张素Ⅱ组人心房肌细胞膜L型钙电流峰值电流密度明显高于对照组[(-12.77±1.61),(-5.78±0.81)pA/pF,P＜0.05).②大蒜素组人心房肌细胞膜L型钙电流峰值电流密度与对照组比较,无明显差异[(-5.69±0.83)pA/pF,P＞0.05].③血管紧张素Ⅱ+大蒜素组人心房肌细胞膜L型钙电流峰值电流密度明显低于血管紧张素Ⅱ组[(-8.75±0.97)pA/pF,P＜0.05).④血管紧张素Ⅱ组人心房肌细胞内游离钙离子荧光强度和游离钙离子荧光强度变化率明显高于对照组和大蒜素组[(2610.1±112.6,(299.2±27.3)%;653.9±42.5,0;639.5±44.7,(-2.2±0.6)%,P＜0.05].血管紧张素Ⅱ+大蒜素组人心房肌细胞游离钙离子荧光强度和游离钙离子荧光强度变化率明显低于血管紧张素Ⅱ组[(1284.9±85.2,(96.5±8.4)%,P＜0.05].结论大蒜素可拮抗血管紧张素Ⅱ致人心房肌细胞膜L型钙电流峰值电流密度的增加,拮抗人心房肌细胞内钙超载,产生减轻心房肌细胞电重构的作用.     

Fetal development: voice processing in normotensive and hypertensive pregnancies

Recent observation of maternal voice recognition provides evidence of rudimentary memory and learning in healthy term fetuses. However, such higher order auditory processing has not been examined in the presence of maternal hypertension, which is associated with reduced and/or impaired uteroplacental blood flow. In this study, voice processing was examined in 40 fetuses (gestational ages of 33 to 41 weeks) of hypertensive and normotensive women. Fetuses received 2 min of no sound, 2 min of a tape-recorded story read by their mothers or by a female stranger, and 2 min of no sound while fetal heart rate was recorded. Results demonstrated that fetuses in the normotensive group had heart rate accelerations during the playing of their mother's voice, whereas the response occurred in the hypertensive group following maternal voice offset. Across all fetuses, a greater fetal heart rate change was observed when the amniotic fluid index was above compared to below the median (i.e., 150 mm), indicating that amniotic fluid volume may be an independent moderator of fetal auditory sensitivity. It was concluded that differential fetal responding to the mother's voice in pregnancies complicated by maternal hypertension may reflect functional elevation of sensorineural threshold or a delay in auditory system maturation, signifying functional differences during fetal life or subtle differences in the development of the central nervous system.     

Erythropoietin stimulates a rise in intracellular free calcium concentration in single early human erythroid precursors.

Erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate the differentiation and proliferation of erythroid cells. To determine the cellular mechanism of action of these growth factors, we measured changes in intracellular free calcium concentration [( Cac]) in single human erythroid precursors in response to recombinant erythropoietin and GM-CSF. [Cac] in immature erythroblasts derived from cultured human cord blood erythroid progenitors was measured with fluorescence microscopy digital video imaging. When stimulated with erythropoietin, [Cac] in the majority of erythroblasts increased within 3 min, peaked at 5 min, and returned toward baseline at 10 min. The percentage of cells that responded to erythropoietin stimulation increased in a dose-dependent manner. Additional stimulation with GM-CSF in cells previously exposed to erythropoietin resulted in a second [Cac] increase. Immature erythroblasts treated with GM-CSF followed by erythropoietin responded similarly to each factor with a rise in [Cac]. The source of transient calcium is intracellular since erythroblasts were incubated in medium devoid of extracellular calcium. Our observations suggest that changes in [Cac] may be an intracellular signal that mediates the proliferative/differentiating effect of hematopoietic growth factors.     

Maternal heart rate variability and fetal behavior in hypertensive and normotensive pregnancies

The relation between maternal heart rate variability (HRV) and fetal behavior was examined in hypertensive and normotensive pregnant women. A total of 40 mother-fetal pairs (n = 20 normotensive mothers; n = 20 hypertensive mothers) at 33-41 weeks' gestation were observed using a standardized procedure lasting approximately 50 min. It included the following measurements: maternal beat-by-beat arterial blood pressure and HRV; spontaneous fetal heart rate (HR), body and breathing movements; and an estimate of amniotic fluid volume. The women in the hypertensive group had higher average body mass index (BMI) (33.7 vs. 28.8 kg/m2) than the normotensive group. In the normotensive group, there was no association between maternal HRV and fetal gestational age, HR, body or breathing movements. In the hypertensive group, maternal HRV measures of low-frequency, high-frequency, and total power were associated with fetal gestational age; also, there was an association between maternal autonomic modulation of HR and fetal spontaneous HR. These findings suggest that the maternal autonomic system influences fetal cardiac function in pregnancies complicated by hypertension.     

Effect of substance P and its precursor α-protachykinin on intracellular free calcium concentration in human polymorphonuclear leukocytes

The increase in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation. The undecapeptide substance P can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, phagocytosis, and respiratory burst. In this study we investigated the ability of low-concentration (that can occur in vivo) substance P (10⁻⁷ M) and its precursor α-protachykinin (3×10⁻⁷ M) to increase the intracellular free calcium concentration in human polymorphonuclear leukocytes. Cells isolated from ten healthy donors were incubated with substance P or α-protachykinin in 1 mM calcium medium for 5 min and the intracellular free calcium concentration was monitored using the fluorescent calcium indicator Fura-2am. Polymorphonuclear leukocytes from 40% of donors responded to both agonists. The substance P- and α-protachykinin-induced increase in intracellular free calcium concentration was 59±13 nM and 58±12 nM and the extracellular calcium influx contributed to 87±8% and 54±8% of the calcium response, respectively. α-Protachykinin released almost all the calcium from intracellular stores, while substance P mobilized only 24±5% of this calcium pool. Finally, cells that responded to a single challenge with substance P and α-protachykinin were able to increase their intracellular free calcium concentration in response to each of three consecutive stimulations with these agonists. This may be an additional mechanism by which substance P and its precursor modify the function of human polymorphonuclear leukocytes.     

Effect of cytochalasin B on intracellular free calcium concentration in human polymorphnuclear leukocytes after repeated stimulation withn-formyl-methionyl-leucyl-phenylalanine

Summary Cytochalasin B can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, lysosomal enzyme release, and reactive oxygen species generation. In this study we investigated the effect of cytochalasin B on the increase in intracellular free calcium concentration after three consecutive additions of 10⁻⁷ MN-formyl-methionyl-leucyl-phenylalanine. The interval between stimulations was 5 min. Intracellular free calcium concentration was monitored using the fluorencent calcium indicator FURA-2AM. Cytochalasin B (3.3 μg/ml) added 60 s before the cell stimulation enhanced all three polymorphonuclear leukocyte calcium responses by increasing theN-formyl-methionyl-leucylphenylalanine-induced calcium influx from the extracellular space. Cytochalasin B increased the peak intracellular free calcium concentration and elevated the plateau phase level, but had no influence on its shape. In addition, pretreatment with cytochalasin B of polymorphonuclear leukocytes suspended in low calcium medium restored their capacity to respond to a third stimulation withN-formyl-methiolnyl-leucyl-phenylalanini. Finally, in resting cells cytochalasin B caused a moderate increase in intracellular free calcium concentration which was independent of extracellular calcium.     

Corticosteroid binding globulin in normotensive and hypertensive human pregnancy

Corticosteroid binding globulin (CBG) concentrations in maternal plasma have been measured throughout pregnancy in a series of 100 singleton pregnancies in 89 normotensive women. Plasma CBG concentrations were monitored also in 10 women with essential or renovascular hypertension. Plasma albumin, cortisol and oestriol were measured concurrently. Plasma CBG increased two and a half to three times during pregnancy. In those women who developed hypertension in pregnancy (mean arterial pressure greater than 107 mmHg), the plasma CBG concentrations were significantly lower than in those who remained normotensive. In women who developed hypertension, the CBG either failed to increase at the same rate as in normal pregnancies or the level fell before the appearance of hypertension. The earlier the onset of hypertension, the greater the decline in CBG. In all subjects, the CBG concentration at 34-36 weeks gestation was directly related to the birthweight of the infant. Plasma cortisol levels were depressed in hypertension relative to that in the normotensive women. Whilst plasma albumin levels decreased at least 30% in most women during pregnancy, the fall tended to be less in hypertensive women, but there was marked overlap between patient groups. Plasma oestriol concentrations were depressed only in the very severely affected cases. It is suggested the CBG concentration is a further reflection of the metabolic abnormalities associated with hypertension in pregnancy, and that it can be used as a marker to identify and monitor those patients at risk.     

盐敏感性高血压大鼠细胞内游离钙变化及螺内脂对其影响

目的观察盐敏感性高血压动物模型之细胞内游离钙的变化;螺内脂对该模型细胞内游离钙的影响;并对它们的作用机理作初步探讨。方法新生雄性Wistar大鼠在第1天和第2天分别皮下注射辣椒辣素以建立感觉神经损伤性盐敏感性高血压大鼠模型,以螺内脂治疗四个月前后及与同龄对照鼠比较,测量大鼠血压及进行细胞内游离钙检测。结果(1)注射辣椒辣素和高盐饮食可使大鼠血压高达(166.4±3.8)mmHg,明显高于其他各组血压(P<0.01)。(2)该组大鼠细胞内钙达(175.8±23.6)nmol/L,明显高于其他各组(P<0.01)。(3)经螺内脂治疗后,该模型鼠尾收缩压降低,由(166.4±3.8)mmHg降至(116.4±8.3)mmHg,细胞内钙亦由(175.8±23.6)nmol/L降至(73.5±9.8)nmol/L,P<0.01,生化指标有改善。结论(1)辣椒辣素处理的新生大鼠在高盐饮食时可迅速建立起一种盐敏感性高血压模型。(2)该模型存在细胞内钙超载现象。(3)螺内脂能有效阻止盐敏感性高血压的产生及减轻细胞内钙超载。     

Measurement of intracellular calcium concentration in intact monolayers of human endothelial cells

Intracellular calcium ([Ca++]i) plays an important role in signal transduction and cell activation. The measurement of [Ca++]i in intact monolayers of human umbilical vein endothelial cells with the fluorescent calcium-sensitive probe fura 2 has been evaluated. Monolayers provide a more physiologic cell preparation than suspensions and allow a greater variety of experimental manipulation. Basal [Ca++]i was 117 +/- 5 nmol/L, with a range from 40 to 280 nmol/L that was not affected by cell age (days of primary culture) or degree of confluence. Thrombin in concentrations of 0.005 to 5 NIH units/ml produced a dose-dependent increase in [Ca++]i up to a maximum of 1500 +/- 147 nmol/L; this increase was shown to depend in part on the concentration of extracellular calcium. The presence of antithrombin III at physiologic concentrations abolished responses to 0.5 NIH units/ml thrombin but had no effect on 5 NIH units/ml. The potential of this technique was demonstrated further by our ability to examine [Ca++]i responses in endothelial cells following infection with herpes simplex virus type 1, a virus implicated in vascular injury. After 18 hours' infection, the response to both thrombin and histamine was dramatically reduced despite a normal resting [Ca++]i. It is concluded that this method may be useful for detecting early and subtle changes in endothelial cell function under a variety of physiologic and pathologic conditions.     

External bioenergy increases intracellular free calcium concentration and reduces cellular response to heat stress.

BACKGROUND: External bioenergy (energy emitted from the body) can influence a variety of biological activities. It has been shown to enhance immunity, promote normal cell proliferation, increase tumor cell death, and accelerate bone fracture recovery. In this study, we investigated whether external bioenergy could alter intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. METHODS: A practitioner emitted bioenergy toward tubes of cultured Jurkat cells for one 15-minute period. [Ca2+]i was measured spectrofluorometrically using the fluorescent probe indo-1. The heat shock protein 72 kd was detected using 35S-methionine prepulse and Western blot analysis. RESULTS: The resting [Ca2+]i in Jurkat T cells was 90+/-3 nM in the presence of external calcium. The removal of external calcium decreased the resting [Ca2+]i to 54+/-2 nM, indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. In the presence of external Ca2+, treatment of Jurkat T cells with external bioenergy for 15 minutes increased [Ca2+]i by 22+/-3%. [Ca2+]i remained elevated in these cells for 2 hours. Surprisingly, we also observed that [Ca2+]i increased by 11+/-1% if cells were simply placed in the area where external bioenergy had been used. This lingering effect of external bioenergy dissipated within 24 hours. Treatment with external bioenergy reduced cellular responses to heat stress and did not induce the production of heat shock protein 72 kd, which is known to provide cytoprotection. CONCLUSIONS: These results suggest that externally applied bioenergy can upregulate [Ca2+]i and downregulate the cellular response to stress. The association between the external bioenergy and increases in [Ca2+]i may be a good index for detecting presence of bioenergy.     

Erythropoietin induces rapid increases in intracellular free calcium in human bone marrow cells

Elevation of intracellular free calcium (Ca++) is an early activation event that occurs as a result of ligand binding in several human cell systems. In this report, erythropoietin, the major hormone governing erythroid differentiation, was found to elicit an increase in Ca++ in human bone marrow mononuclear cells. Two chelators of intracellular calcium, quin 2 and the more specific and sensitive analogue, fura-2, were used to characterize the response evoked by both recombinant and native hormone. Erythropoietin caused a rapid, dose-dependent rise (within seconds) in Ca++ in bone marrow mononuclear cells, which could be prevented by preincubation of hormone with a rabbit erythropoietin antiserum. The erythropoietin response did not occur in purified populations of T- or B-lymphocytes. These studies suggested that increased Ca++ on erythropoietin binding may be an early transmembrane signal in hormone action.     

枸杞糖肽对缺氧乳鼠心肌细胞内游离钙浓度的影响

目的:研究低氧心肌细胞内游离钙(犤Ca2+犦i)浓度的变化,枸杞糖肽减轻缺氧诱导的心肌细胞钙超载以及对缺氧心肌的保护作用。方法:实验选用出生两三天的SD乳大鼠80只进行心肌细胞培养,建立心肌缺血模型,实验分3组:对照组;缺氧组:细胞缺氧6h;枸杞糖肽组:先加入终浓度为50mg/L的枸杞多糖,再行缺氧6h。以Fluo/AM荧光指示剂负载,通过激光扫描共聚焦显微系统和Flou-3/AM荧光指示剂标记技术,观察枸杞糖肽对缺氧心肌细胞游离钙含量的影响。结果:心肌细胞经缺氧处理6h后,细胞之间相互连接减少,大部分细胞肿胀,折光性减弱,部分细胞甚至呈圆形,少部分细胞胞膜破裂,提示心肌细胞损伤。枸杞糖肽处理组心肌细胞经缺氧后,可见部分细胞肿胀,内有颗粒,折光性减弱,但较缺氧模型组有一定的改善作用。心肌细胞Fluo-3/AM负载后在激光共聚焦显微镜下观察,细胞的分布和外形与倒置显微镜下所见相符,呈不规则棱形、多角形、星形细胞,轮廓清晰,成群的细胞出现自发性,同步性、有节律的搏动。经Flu3-AM标记后,细胞出现规律性闪烁。对照组心肌细胞犤Ca2+犦i荧光强度和吸光度(A值)较低,缺氧6h荧光强度明显增加(P<0.05);枸杞糖肽组心肌细胞犤Ca2+犦i荧光密度(62.86±28.71)低于缺氧组(156.76±55.39)(P<0.01)。结论:低氧导致心肌细胞钙超     

Platelet cytosolic free calcium concentration and parathyroid hormone: changing relationships with haemodialysis in end-stage renal disease.

1. Twelve patients receiving haemodialysis for end-stage renal failure were studied at a single dialysis session. Platelet cytosolic calcium concentration, plasma ionized calcium concentration and serum parathyroid hormone concentration were measured before dialysis, mid-dialysis and 30 min after dialysis. 2. Plasma ionized calcium concentration increased towards dialysate calcium concentrations, falling insignificantly after cessation of dialysis. Serum parathyroid hormone concentration fell by 39% during dialysis, with incomplete recovery afterwards. There was no overall change in platelet cytosolic calcium concentration. 3. Patients were divided into two subgroups: low parathyroid hormone (serum parathyroid hormone concentration less than 10 pmol/l) and high parathyroid hormone (serum parathyroid hormone concentration greater than 10 pmol/l). Before dialysis, values of platelet cytosolic calcium concentration or plasma ionized calcium concentration were not statistically different between the subgroups, but the platelet cytosolic calcium concentration was higher in the high-parathyroid hormone subgroup during and after dialysis. 4. Before haemodialysis there was a linear correlation between plasma ionized calcium concentration and platelet cytosolic calcium concentration, which disappeared during dialysis. In contrast, there was no relationship between serum parathyroid hormone concentration and platelet cytosolic calcium concentration before dialysis, but after dialysis a hyperbolic relationship was evident. 5. These results suggest that uraemic toxins may interfere with cytosolic calcium homoeostasis, allowing passive diffusion of extracellular calcium to influence the resting concentration, and that this effect is reversible by haemodialysis.     

推拿(扌衮)法样刺激对人脐静脉内皮细胞内钙离子浓度的影响

背景:迄今对推拿(扌衮)按法作用机制的研究多着眼于软组织、骨关节、神经、血管等宏观层面,还需要从微观细胞学水平对手法力学刺激钙离子通道引发的效应机制进行研究.目的:观察推拿(扌衮)法样刺激对人脐静脉内皮细胞内钙离子浓度的影响.方法:将正常培养和缺氧培养的人脐静脉内皮细胞均分为对照组、推拿组、维拉帕米组.对照组细胞不做任何处理,推拿组细胞用Fluo-3/AM标记后给予手法样压力刺激,压力大小为0.4 kg,频率120次/min,时间1min:维拉帕米组细胞用FIuo-3/AM标记后.先加入终浓度为10-5mol/L的维拉帕米,然后给与同样的(扌衮)法样刺激.各组细胞在处理前后分别采用共聚焦激光扫描显微镜观察并测定细胞荧光强度进行比较.结果与结论:手法样刺激和缺氧环境均可引起人脐静脉内皮细胞内游离钙离子浓度升高,但这两种促进人脐静脉内皮细胞内游离钙离子浓度升高的作用均能够被钙离子拮抗剂维拉帕米所抑制.由此推测,推拿(扌衮)法的作用机制是通过机械刺激激活钙离子通道,引发细胞钙离子内流,促使细胞内游离钙离子浓度升高,进而启动并产生后续的生物学治疗效应.