Digoxin-like immunoreactivity in cord blood plasma extracts is not only due to endogenous corticosteroids

Methods for quantitative extraction and enrichment of digoxin-like immunoreactive substances (DLIS) from human cord blood plasma (CBP) using organic solvents combined with a solid state absorbent are presented. Sephadex LH-20 column chromatography is more suitable than sephadex G-25 for DLIS separation and purification from CBP extract. The elution pattern of sephadex LH-20 chromatography of deproteinized and desalted CBP shows two distinct DLIS peaks. The first peak coelutes with the steroids aldosterone, cortisol, progesterone, 17-OH-progesterone, testosterone and estradiol, while the second DLIS peak coelutes with dehydroepiandrosterone sulfate (DHEA-S). The Na+,K(+)-ATPase inhibitory activity peak partially overlaps with the first DLIS peak. Coeluted steroids could account only partially for digoxin-like immunoreactivity in the first DLIS peak; however, they did not contribute to the Na+,K(+)-ATPase inhibitory activity, whereas the second DLIS peak could be ascribed to endogenous DHEA-S.     

异丙酚在第三丁基过氧化氢诱导脐静脉内皮细胞氧化应激中的保护作用

目的:研究异丙酚对第三丁基过氧化氢(t-BHP)诱导的脐静脉内皮细胞(HUVECs)氧化应激的保护作用和机制。方法:体外培养的HUVECs分为对照组、异丙酚组、t-BHP组、异丙酚预处理+t-BHP组,给予相应处理后,Westernblot检测p38MAPK磷酸化水平变化,RT-PCR检测iNOS、eNOS表达。结果:t-BHP处理后,能显著诱导p38MAPK磷酸化,激活iNOS、eNOS表达,而异丙酚预处理后能减轻这些变化。结论:异丙酚通过抑制p38MAPK,减少iNOS、eNOS表达,减轻氧化应激从而起到保护HUVECs的作用。     

Modifications induced by diabetes on the physicochemical and functional properties of erythrocyte plasma membrane

Increasing evidence suggests that in experimental diabetes an impairment in Na+,K+-ATPase activity plays a central role in the pathophysiology of diabetic complications, while only a few data are available with regard to human subjects. We studied the erythrocyte membrane Na+,K+-ATPase activity and membrane fluidity in insulin-dependent and non-insulin-dependent diabetic subjects. A significant decrease in the enzyme activity and in fluorescence polarization values was found in both groups compared with normal subjects. Neither Na+,K+-ATPase activity nor membrane fluidity was found to be related to metabolic control, assessed by means of fasting blood glucose levels and HbA1c. On the contrary, a significant correlation was observed between Na+,K+-ATPase activity and membrane fluidity in both insulin-dependent and non-insulin-dependent diabetic subjects. The present work provides evidence that a reduction in the Na+,K+-ATPase activity is present in the plasma membranes of insulin-dependent and non-insulin-dependent diabetics. Furthermore, it suggests that the change in enzyme activity might be related to modifications in membrane fluidity.     

Diabetes mellitus and subjects' ageing: a study on the ATP content and ATP-related enzyme activities in human erythrocytes

Na⁺/K⁺- and Ca²⁺-ATPase are the major ATP-dependent membrane-bound enzymes that regulate the cation transmembrane gradient which is altered both in red blood cell (RBC) senescence and in RBCs of diabetic patients. In an attempt to clarify the possible connection between diabetes mellitus and ageing, we investigated the relationship between RBC ATP content, Na⁺/K⁺-ATPase, Ca²⁺-ATPase activities and ageing in healthy, insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) subjects. A significant correlation was found (r = ?0.82; P < 0.001) between RBC ATP content and subject's age only in the control group. A significant reduction in Na⁺/K⁺-ATPase activity was observed in the older group (C₂) of control subjects, in comparison with the younger (C₁) one. In both IDDM and NIDDM subjects, the enzymatic activity was significantly decreased when compared with healthy subjects of similar age (P < 0.001). A significant negative correlation was found between age and enzymatic activity in healthy subjects (r = ?0.60; P < 0.001). No difference was observed in the RBC membrane Ca²⁺-ATPase activity between younger (C₁) and older (C₂) healthy subjects. Ca²⁺-ATPase activity was significantly increased both in IDDM patients compared with C₁ (P < 0.001) and in NIDDM patients compared with C₂ (P < 0.001). The present data indicate that ageing causes a reduction in the erythrocyte ATP content in both healthy and diabetic subjects. In diabetic patients Na⁺/K⁺-ATPase activity decreases independently of age.     

Effect of colchiceine and ursodeoxycholic acid on hepatocyte and erythrocyte membranes and liver histology in experimentally induced carbon tetrachloride cirrhosis in rats

Colchiceine and ursodeoxycholic acid (UDCA) are drugs currently in use as therapy for different types of liver damage. We evaluated their ability to reverse the damage induced by carbon tetrachloride (CCl₄) in rats. Six groups were analysed: (1) CCl₄ (0.4 g kg⁻¹, i.p., three times a week) for 13 weeks; (2) CCl₄ for 8 weeks followed by colchiceine (60 μg kg⁻¹) + CCl₄ for 5 weeks; (3) CCl₄ for 8 weeks and thereafter UDCA (25 mg kg⁻¹) + CCl₄ for 5 weeks. Groups 4, 5 and 6 were appropriate controls of colchiceine, UDCA and vehicles respectively. Na⁺,K⁺- and Ca²⁺-ATPase activities and the cholesterol–phospholipid (CH/PL) ratio from erythrocyte and hepatocyte membranes were quantified. Membrane enzymatic activities and CH/PL ratios were affected more in group 1 than groups 2 and 3. We concluded that colchiceine and UDCA were effective drugs in this model of liver damage.     

Physicochemical and functional modifications induced by obesity on human erythrocyte membranes

BACKGROUND: Na+,K(+)-ATPase activity was evaluated in relation to membrane composition and molecular organization in erythrocyte membranes from obese patients by the amphyphylic molecule 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). Its possible relationship with fat distribution and hyperinsulinaemia was also investigated. DESIGN: Subjects were 10 obese men (OM), 12 women with subcutaneous obesity (FSO), 10 women with abdominal obesity (FAO) and 41 healthy lean subjects, 26 women (FC) and 15 men (MC). An oral glucose tolerance test was administered to all subjects to evaluate insulin secretion and glucose tolerance. RESULTS: Na+,K(+)-ATPase activity was increased in all obese patients. Values were higher in FSO and FAO than in FC (with FAO greater than FSO) and in OM than in MC. The erythrocyte membrane cholesterol-to-phospholipid ratio was increased in obese patients and was significantly different in FSO patients compared with FC. The erythrocyte membrane protein-to-phospholipid ratio was also increased in all obese subjects, reaching statistical significance only in FSO vs. FC. The liquid crystalline phase, as tested by Laurdan generalized polarization (GP), was decreased in obese patients, indicating the presence of greater molecular environmental order; all patients groups showed lower GP values than control subjects, but only FAO reached statistical significance compared with FC. There was no evident correlation between membrane Na+,K(+)-ATPase activity and insulin levels, nor did membrane composition and properties show any evident relationship with insulin levels. CONCLUSION: Both increased Na+,K(+)-ATPase activity and altered fluidity and lipid composition were observed in the erythrocyte membrane of all obese patients. These findings are in line with previous observations by our group and indicate that the changes in Na+,K(+)-ATPase activity observed in obese patients could be related to changes in plasma membrane organization and composition.     

Effect of colchiceine and ursodeoxycholic acid on hepatocyte and erythrocyte membranes and liver histology in experimentally induced carbon tetrachloride cirrhosis in rats

Colchiceine and ursodeoxycholic acid (UDCA) are drugs currently in use as therapy for different types of liver damage. We evaluated their ability to reverse the damage induced by carbon tetrachloride (CCl₄) in rats. Six groups were analysed: (1) CCl₄ (0.4 g kg^?1, i.p., three times a week) for 13 weeks; (2) CCl₄ for 8 weeks followed by colchiceine (60 μg kg^?1) + CCl₄ for 5 weeks; (3) CCl₄ for 8 weeks and thereafter UDCA (25 mg kg^?1) + CCl₄ for 5 weeks. Groups 4, 5 and 6 were appropriate controls of colchiceine, UDCA and vehicles respectively. Na⁺,K⁺- and Ca²⁺-ATPase activities and the cholesterol–phospholipid (CH/PL) ratio from erythrocyte and hepatocyte membranes were quantified. Membrane enzymatic activities and CH/PL ratios were affected more in group 1 than groups 2 and 3. We concluded that colchiceine and UDCA were effective drugs in this model of liver damage.     

人脐静脉内皮细胞分离培养及鉴定技术

目的探讨人脐静脉内皮细胞(HUVEC)体外分离原代培养的方法,并总结对培养的细胞鉴定方法。方法通过胰酶灌注法从人脐带获取内皮细胞进行分离培养,并采用免疫组化法及光镜和透射电镜观察超微结构鉴定所获得的细胞系。结果分离的HUVEC在体外7-10天左右可长成单层,光镜下胞体为单层铺路石状排列。第VⅢ因子相关抗原的检测为阳性。透射电镜下观察培养的内皮细胞胞浆内可见Weibel-Palade小体。结论用胰酶灌注法是获得脐静脉内皮细胞的有效方法。本方法为血管内皮细胞的研究提供了实验模型。     

富血小板血浆对血管内皮细胞增殖及血管形成的影响

目的:探讨富血小板血浆(platelet-rich plasma,PRP)对人脐静脉血管内皮细胞(human umbilicalvein vessel endothelial cells,HUVECs)增殖及体外形成血管的影响.方法:体外培养HUVECs 细胞系,二次离心法制备自体富血小板血浆,将细胞分为实验组与对照组,实验组以含5%、10%、20%自体富血小板血浆的条件培养液干预,对照组不进行干预.采用四甲基偶氮唑蓝(XTT)法测定HUVECs 的增殖情况,流式细胞仪检测细胞周期,体外成管实验检测细胞形成血管的能力.结果:富血小板血浆实验组较对照组细胞形态饱满、密度增大;XTT 比色法显示各浓度实验组细胞增殖与对照组比较明显增高(P ＜ 0.01);流式细胞仪检测实验组S 期细胞明显增多(P ＜ 0.01);体外成管实验显示实验组细胞能形成完整的管状结构,小管面积明显大于对照组(P ＜ 0.01),对照组小管形成结构不完整、面积小于实验组(P ＜ 0.05).结论:PRP 能在体外明显促进HUVECs 的增殖及形成毛细血管腔,在组织再生或修复过程中可能发挥促新生血管形成的作用.     

10-Hydroxy-2-decenoic acid, a major fatty acid from royal jelly, inhibits VEGF-induced angiogenesis in human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF) is reported to be a potent pro-angiogenic factor that plays a pivotal role in both physiological and pathological angiogenesis. Royal jelly (RJ) is a honeybee product containing various proteins, sugars, lipids, vitamins and free amino acids. 10-Hydroxy-2-decenoic acid (10HDA), a major fatty acid component of RJ, is known to have various pharmacological effects; its antitumor activity being especially noteworthy. However, the mechanism underlying this effect is unclear. We examined the effect of 10HDA on VEGF-induced proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). Our findings showed that, 10HDA at 20 microM or more significantly inhibited such proliferation, migration and tube formation. Similarly, 10 microM GM6001, a matrix metalloprotease inhibitor, prevented VEGF-induced migration and tube formation. These findings indicate that 10HDA exerts an inhibitory effect on VEGF-induced angiogenesis, partly by inhibiting both cell proliferation and migration. Further experiments will be needed to clarify the detailed mechanism.     

血管抑素对人脐静脉内皮细胞及体外微血管模型的作用

目的:观察血管抑素对人脐静脉内皮细胞及体外微血管模型的抑制作用。方法:实验于2005-01/2006-12在中国医科大学基础医学院实验病理研究室(省部级)完成。①采用改进的Jaffe式培养法体外培养人脐静脉内皮细胞。②MTT法检测不同质量浓度(1,2,4,8,16,32mg/L)、不同时间(24,48,72h)血管抑素对人脐静脉内皮细胞增殖的影响。③流式细胞仪检测不同质量浓度(2,4,8,16,32mg/L)的血管抑素对人脐静脉内皮细胞生长周期的影响。④建立肿瘤微血管体外生成模型,培养4,8,12h倒置显微镜观察血管网形成情况;血管网未形成之前加16mg/L血管抑素观察对血管形成的影响;肿瘤微血管体外生成模型经24h培养形成血管网后加16mg/L血管抑素观察对新生血管的影响。结果:①血管抑素对人脐静脉内皮细胞增殖的影响:8,16,32mg/L血管抑素能明显抑制人脐静脉内皮细胞的生长(P<0.01),血管抑素作用于人脐静脉内皮细胞24,48,72h后细胞明显受抑制(P<0.01),这种作用具有剂量-时间依赖性。②血管抑素对人脐静脉内皮细胞生长周期的影响:血管抑素作用于人脐静脉内皮细胞后能诱导细胞凋亡。③血管抑素对体外血管模型的影响:光镜下血管抑素可抑制新生血管的形成,且能破坏新生的血管网。结论:血管抑素可能抑制人脐静脉内皮细胞增殖,从而破坏新生血管形成,抑制肿瘤的生长和转移。     

久强脑立清对人脐静脉内皮细胞一氧化氮和内皮素分泌的影响

目的观察久强脑力清 (JNQ)含药血清对人脐静脉内皮细胞 (HUVECs)分泌一氧化氮 (NO)和内皮素 (ET 1)的影响 ,探讨其对HUVECs血管活性分子分泌的调节作用。方法取原始JNQ含药血清体积分数的 80 %、40 %、2 0 %、10 %和 5 %作用于原代培养的HUVECs 2 4h后 ,分别检测上清中的NO和EI 1的含量。结果不同浓度的JNQ含药血清作用组的NO、ET 1含量与正常血清对照组比较增加 (P <0 0 5 ) ;NO/ET 1比值随着含药血清浓度的增加 ,比正常血清组增高 (P <0 0 5 )。结论JNQ含药血清通过提高HUVECs分泌NO和ET 1,具有保护内皮细胞功能和维持NO/ET 1平衡的作用     

利鲁唑对体外培养人脐静脉内皮细胞及小鼠视网膜新生血管形成的影响

背景:研究已证实利鲁唑具有较强的抑制蛋白激酶C-βⅡ单体介导血管内皮细胞生长因子促内皮细胞增殖效应的作用,因此推测其可用于抑制视网膜新生血管的形成.目的:观察利鲁唑对培养的人脐静脉内皮细胞增殖及小鼠视网膜新生血管形成的抑制及其作用途径.设计、时间及地点:单一样本观察及随机对照动物实验,于2007-08/12在重庆医科大学动物实验室及基础研究实验室完成.材料:清洁级出生第3天的健康C57BL/6小鼠30只60眼,雌雄不限,以改良法建立高氧诱导血管增生性视网膜病变小鼠模型.利鲁唑原料物质为北京万全医药科技有限公司产品,批号:060630,含量≥98.5%.方法:常规传代培养人脐静脉内皮细胞,随机分为3组.高氧模犁组及利鲁唑治疗组为造模小鼠,利鲁唑治疗组小鼠腹腔注射利鲁唑10 mg/(kg·d),正常对照组小鼠腹腔注射等量等渗的生理盐水.主要观察指标:以四甲基偶氮唑盐比色法检测利鲁唑对血管内皮生长因子诱导的人脐静脉内皮细胞增殖的影响.以组织切片苏木精-伊红染色观察并计数突破视网膜内界膜的血管内皮细胞核数目.以SABC法免疫组织化学染色观察视网膜组织中蛋白激酶C-βⅡ及血管内皮生长因子的表达.结果:利鲁唑在0.1～10 μ mol/L浓度范围内.可抑制培养的人脐静脉内皮细胞增殖,且呈剂量效应依赖关系.高氧模型组视网膜组织切片上见较多突破视网膜内界膜的血管内皮细胞核,治疗组突破视网膜内界膜的内皮细胞核数目明显减少(P<0.01),免疫组织化学染色检测高氧模型组小鼠视网膜蛋白激酶C-βⅡ及血管内皮生长因子表达比正常对照组明显增强,治疗组比模型组明显减弱.结论:利鲁哗腹腔注射能抑制氧致视网膜新生血管的形成,对视网膜蛋白激酶C-βⅡ及血管内皮生长因子的表达有抑制作用,利鲁唑对体外培养人脐静脉内皮细胞增殖的抑制作用与其抑制蚩白激酶C-βⅡ的活性进而抑制血管生长因子的表达有关.     

胰蛋白酶消化法体外培养和鉴定人脐静脉血管内皮细胞

背景:体外建立稳定可靠的人脐静脉血管内皮细胞模型,目前培养方法缺乏统一的标准。目的:探索脐静脉内皮细胞的体外培养的方法。方法:采用2.5g/L胰蛋白酶和0.02%EDTA消化、分离、体外原代培养及消化传代脐静脉内皮细胞,简化完全培养液组分(不添加血管内皮细胞生长因子、肝素等辅助因子),当原代培养细胞80%以上汇合,根据细胞特有的形态学特征和Ⅷ因子进行内皮细胞的鉴定。结果与结论:种植在培养瓶中的内皮细胞2h贴壁生长,24h换液后内皮细胞80%融合,细胞状态好,内皮细胞呈单层铺路石样外观,经过镜下观察和Ⅷ因子相关抗原鉴定证明是脐静脉内皮细胞。证实用胰蛋白酶灌注脐静脉是一种简单、实用的获得人脐静脉血管内皮细胞的方法,可靠性大,成功率高,可以构建体外研究血管内皮细胞的模型。     

ADMA对人脐静脉内皮细胞粘附功能影响的体外研究

目的观察非对称性二甲基精氨酸(ADMA)对体外培养的人脐静脉内皮细胞(HUVECs)粘附功能的影响。方法原代获取HUVECs,分别加入ADMA5μmol/L和10μmol/L培养48小时。测定细胞的数目、粘附和产生一氧化氮的能力。流式细胞仪检测细胞表面粘附分子的表达。结果与对照组相比,不同浓度的ADMA可显著抑制HU-VECs产生NO和粘附的能力、增强细胞表面ICAM-1和VCAM-1的表达。结论ADMA可抑制体外培养的HUVECs粘附功能。     

人脐静脉内皮胞细体外培养及鉴定

目的探索人脐静脉内皮细胞体外培养及鉴定的方法。方法采用0.1%Ⅰ型胶原酶分离脐静脉内皮细胞,加入含10%胎牛血清及人AB血清的M1640培养基,在37℃,5%CO2孵箱中培养,以免疫组化方法对内皮细胞进行鉴定。结果原代培养细胞在接种4 h后开始贴壁生长,5～7 d后融合成单层,倒置相差显微镜下观察细胞呈鹅卵石状排列,有接触抑制现象,免疫组化显示细胞胞浆中人Ⅷ因子相关抗原阳性。结论用胶原酶灌注消化脐静脉是获得内皮细胞的一种可靠的方法,有助于体外研究血管内皮细胞模型的构建。     

Study of pro-angiogenic activity of astilbin on human umbilical vein endothelial cells in vitro and zebrafish in vivo

Astilbin is a dihydroflavonol natural product isolated from a variety of food and medicinal herbs (e.g. Smilax glabra Roxb.), and its mechanism of action in vascular pharmacology remains unclear. The aim of this study was to investigate the pro-angiogenic effects of astilbin and its putative mechanism of action. Briefly, our in vitro studies showed a dose-dependent ability of astilbin to increase the ability of HUVECs to proliferate and migrate, and undergo cell invasion and tube formation. Moreover, astilbin significantly increased the expression levels of several major proteins involved in the angiogenesis pathway, e.g. PI3K, Akt, p38 and ERK1/2. Our in vivo studies demonstrated the ability of astilbin to significantly restore the blood vessel loss induced by VRI in a VRI-induced vascular insufficiency zebrafish model. In conclusion, in this study we first demonstrate that astilbin exhibits pro-angiogenic activity in HUVECs and VRI-induced vascular insufficient zebrafish, possibly through the activation of the PI3K/Akt and MAPK/ERK dependent signaling pathways. These findings suggest that astilbin could be further developed as a potential agent in the prevention or treatment of insufficient angiogenesis related diseases in the future.

Pro-angiogenic activity of astilbin on endothelial cells in vitro and zebrafish in vivo.     

WNK4 regulates airway Na+ transport: study of familial hyperkalaemia and hypertension

Background WNK [With No K (lysine)] kinases are essential for regulation of blood pressure and potassium homeostasis. WNK4 expression was recently found not only in the distal nephron but also in chloride‐transporting epithelia. To establish a physiological role for this distribution we studied patients with familial hyperkalaemia and hypertension (FHH), [pseudohypoaldosteronism type II (PHAII)], which is caused by mutations in WNK4. Design Measurement of nasal potential difference (NPD) and sweat electrolytes were performed in controls, in six subjects with FHH and ten subjects with cystic fibrosis (CF). Results Basal NPD was higher in FHH compared with controls (n = 20): 22·8 ± 5·7 vs. 16·2 ± 5·3 mV, respectively (P = 0·014). Maximal response to amiloride was also higher in FHH compared with controls: 14·8 ± 3·5 vs. 10·0 ± 4·8 mV, respectively (P = 0·03). In CF these values were 42·9 ± 9·3 and 29·9 ± 7·4 mV, respectively. The kinetics of the amiloride effect were faster in FHH, and as first reported here also in CF, compared with controls. At 30 s, amiloride‐inhibitable residual PD in FHH was 50 ± 30 vs. 81 ± 9% in controls (P = 0·0003) and 56 ± 7% in CF. The response to chloride‐free and isoproterenol solutions, which determines chloride transport activity, was similar in FHH compared with controls [16·0 ± 8·6 vs. 10·4 ± 5·9 mV (P = 0·08)]. Sweat conductivity in FHH was 49·7 ± 7·3 vs. 38·2 ± 8·1 mmol (NaCl eq) L^?1 in 16 controls (P = 0·007) and 94·0 ± 19·3 in CF. Conclusions Mutant WNK4 increases Na⁺ transport in airways, and therefore it is regulated by wild‐type WNK4. This may be caused by a regulation of ENaC or a K⁺ channel.     

明胶对人脐静脉内皮细胞株细胞相容性影响的研究

目的探讨明胶对人脐静脉内皮细胞株(HUVECs)细胞生长的影响。方法建立后干扰(模型1)及预干扰(模型2)的细胞模型,采用MTT法和倒置光镜动态观察细胞生长状态及观察不同分子量明胶(40000道尔顿、60000道尔顿、70000道尔顿)、不同质量分数(5%、10%、15%)对HUVECs细胞相容性的影响。实验分为10组:对照组,明胶Aa、Ab、Ac组(明胶分子量为40000道尔顿,明胶溶液浓度分别为5%、10%、15%),明胶Ba、Bb、Bc组(明胶分子量为60000道尔顿,明胶溶液浓度分别为5%、10%、15%),明胶Ca、Cb、Cc组(明胶分子量为70000道尔顿,明胶溶液浓度分别为5%、10%、15%)。结果 (1)倒置光镜动态观察显示:模型1:对照组HUVECs呈正常生长状态,3个时间段(24、48、72h)细胞形态及密度无明显变化,而与对照组比较,明胶各实验组细胞大量死亡,仅少数细胞贴壁存活;模型2:对照组HUVECs呈正常生长状态,与对照组比较,3个时间段(24、48、72h)内明胶各实验组细胞生长状态好,无明显差异,并于72h时出现细胞密度明显增加的现象。(2)MTT检测:模型1:与对照组比较,明胶各实验组3个时间段(24、48、72h)细胞增殖有明显降低,至72h,HUVECs增殖无明显增加;模型2:与对照组比较,随着观察时间增加,3个时间段(24、48、72h)明胶各实验组细胞增殖逐渐增加,其中48h明胶Aa组、72h明胶Cc组两组与对照组HUVECs细胞增殖比较差异有统计学意义(P0.05)。结论在本研究范围内,采用预干扰模型(模型2)对较高分子量明胶进行细胞实验较适合;较高分子量明胶可能对微血管在一定时间内的修复与重建有促进作用。     

Inhibitory effects of tacrine and physostigmine on catecholamine secretion and membrane currents in guinea-pig adrenal chromaffin cells

Summary— The effects of tacrine and physostigmine on catecholamine secretion induced by veratridine and high K⁺, and on voltage-dependent Na⁺ and Ca²⁺ currents, were investigated in guinea-pig adrenal chromaffin cells. In perfused adrenal glands, tacrine (100 μM) caused an inhibition of veratridine-induced catecholamine secretion, but physostigmine (100 μM) did not. In dispersed cells, both tacrine (1 μM-1 mM) and physostigmine (1 μM-1 mM) decreased catecholamine secretion induced by veratridine in a dose-dependent manner. The inhibitory effect of tacrine was much greater than that of physostigmine. Tacrine alone at a high concentration (such as 1 mM) caused a substantial increase in catecholamine secretion by itself and completely abolished the veratridine-induced secretory response in dispersed cells. High-concentration physostigmine showed a similar effect, but to a much lesser extent. The high K⁺ (46.2 mM)-evoked catecholamine secretion from dispersed cells was not affected by tacrine (1–100 μM) or physostigmine (1 μM-1 mM). In fura-2 loaded cells, tacrine (100 μM) almost abolished [Ca²⁺]i rise induced by veratridine, but only slightly reduced that evoked by high K⁺. In voltage-clamped cells, tacrine (300 μM) depressed the voltage-dependent Na⁺ and Ca²⁺ currents by about 93% and 69%, and physostigmine (300 μM) depressed them by about 30% and 17%, respectively. These results suggest that tacrine decreases the veratridine-induced catecholamine secretion primarily by inhibiting the voltage-dependent Na⁺ channels rather than the Ca²⁺ channels. Physostigmine acts in a manner similar to tacrine, but its potency is much lower than that of tacrine.