NPPB和尼弗灭酸对H2O2损伤C6胶质细胞谷氨酸半胱氨酸合成酶及CLIC4表达的影响

目的:观察氯通道阻断剂5-硝基-2-(3-苯丙胺)-苯甲酸(NPPB)、尼弗灭酸(NFA)对H2O2诱导的神经胶质瘤C6细胞损伤的影响。方法:MTT法检测NPPB、NFA、H2O2作用的C6细胞生存率;紫外分光光度法检测乳酸脱氢酶(LDH)释放率以及谷胱甘肽GSH水平;RT-PCR检测谷氨酸半胱氨酸合成酶(GCL)亚单位GCLC、GCLM及线粒体氯通道(CLIC4)mRNA表达;Western blotting检测CLIC4的蛋白水平。结果:与对照组相比,H2O2组C6细胞存活率和GSH含量明显降低;LDH释放率增加;GCLC、GCLM、CLIC4 mRNA表达降低;CLIC4蛋白水平明显增强。NPPB或NFA与H2O2联合作用于C6细胞组,与单独应用H2O2组相比,细胞存活率和GSH含量未见明显变化;LDH释放率降低;GCLC、GCLM mRNA表达未见明显差异;CLIC4蛋白表达下降。结论:氯通道阻断剂NPPB或NFA能够在一定程度上减轻氧化应激引起的C6细胞损伤,可能与调节细胞膜功能和下调CLIC4蛋白表达有关。     

抗P-选择素单抗的荧光素标记及其临床应用

目的:探讨各种血栓形成相关性疾病患者的P-选择素的表达变化及其临床意义。方法:用异硫氰酸荧光素(FITC)标记抗P-选择素单抗SZ-51-IgG和流式细胞术(FCM)分析了42例糖尿病患者、33例高血脂患者、23例脑梗塞患者和20例正常人的血小板膜P-选择素表达,并与间接FCM和酶标记免疫吸附分析法(ELISA)作比较。结果:FCM测得的血小板膜上P-选择素表达阳性率,在糖尿病组为23.92%±15.83%,高血脂组为18.34%±9.46%,脑梗塞组为19.32%±10.38%,均显著高于正常对照3.38%±1.11%(P值均小于0.01)。间接FCM测定血小板膜P-选择素表达在糖尿病和脑梗塞患者中均显著增高,ELISA法测得的血浆中P-选择素在糖尿病、高血脂和脑梗塞患者也显著高于正常对照。结论:FITC标记的SZ-51-IgG可用于FCM,可成为测定血小板活化的一个新的敏感的方法。     

顺铂通过ROS介导ClC-3上调诱导鼻咽癌细胞凋亡

目的:探讨ClC-3氯通道在顺铂诱导的鼻咽癌细胞凋亡中的作用及机制。方法:将不同浓度的顺铂作用于人低分化鼻咽癌细胞(CNE-2Z细胞);MTT法检测不同浓度的顺铂处理24 h和48 h后细胞活力;采用ClC-3-siRNA下调ClC-3的表达,Annexin V-FITC/PI双染法检测细胞凋亡,多功能微孔板检测仪和流式细胞术检测细胞不同状态下的ROS水平。结果:(1)顺铂呈时间和浓度依赖性抑制CNE-2Z细胞生长,氯通道阻断剂DIDS显著抑制顺铂引起的细胞凋亡(P0.01);(2)顺铂促进CNE-2Z细胞ClC-3表达,下调ClC-3蛋白表达后,顺铂诱导的细胞凋亡率下降(P0.05);(3)顺铂促进CNE-2Z细胞ROS产生,用抗氧化剂抑制细胞产生ROS后,顺铂诱导的ClC-3蛋白表达和凋亡被抑制,而下调ClC-3蛋白表达对ROS水平影响不大。结论:顺铂可通过提升CNE-2Z细胞ROS水平,上调氯通道ClC-3蛋白水平,从而诱导细胞凋亡。     

酸中毒致大鼠离体冠状动脉收缩的机制

目的:探讨细胞外酸中毒(pH_(ex)6.8)致大鼠离体冠状动脉(RCA)收缩的机制。方法:采用离体微血管张力法,通过观察Na~+-H~+交换体1(NHE-1)选择性抑制剂cariporide(HOE-642)或Na~+-HCO_3~-共同转运体(NBC)抑制剂S0859预孵对pH_(ex)6.8所致RCA收缩的影响,探讨酸碱转运体在酸中毒收缩RCA中的作用;通过观察氯通道抑制剂NPPB和尼氟酸(NFA)预孵及细胞外去除氯离子对pH_(ex)6.8所致RCA收缩的影响,探讨氯离子通道在酸中毒收缩RCA中的作用。结果:pH_(ex)6.8引起RCA静息张力升高,最大张力为(3.90±0.95)mN。30μmol/L HOE-642和100μmol/L S0859均抑制pH_(ex)6.8引起的RCA收缩(P0.01)。NPPB和NFA均呈浓度依赖性地抑制pH_(ex)6.8引起的RCA收缩和KCl(60 mmol/L)引起的收缩。100μmol/L的NPPB和NFA均抑制U46619(1μmol/L)引起的RCA收缩(P0.01)。等摩尔NaBr代替细胞外液中NaCl后,几乎完全抑制pH_(ex)6.8引起的RCA收缩(P0.01),但是对KCl(60 mmol/L)或U46619(1μmol/L)引起的RCA收缩无显著影响。结论:细胞外液酸化所致的RCA收缩与激活NHE-1和NBC及促进氯离子跨细胞膜运动有关。     

Pam3CSK4对血小板活化及其TLR2表达的影响

目的 探讨Toll样受体2(TLR2)在血小板激活及免疫方面的作用.方法 取健康人(n=5)全血6 ml,以密度梯度离心法制备洗涤血小板.用1、5、10μg/ml的TLR2激动剂Pam3CSK4(一种合成的细菌脂蛋白)刺激人洗涤血小板,然后测定血小板聚集率,血小板表面蛋白CD62p和TLR2表达的变化.结果 Pam3CSK4以浓度0、1、5和10μg/ml激活血小板:聚集率增加分别为(12.83±2.43)%、(28.32±5.67)%、(52.56±8.54)%、(76.24±11.23)%,P<0.01;血小板表面CD62p表达量增加分别为(11.20±1.67)%、(18.45±2.66)%、(22.45±2.04)%、(29.53±4.08)%,P<0.01.Pam3CSK4在1μg/ml时TLR2表达为(16.85±6.10)%,与对照组(10.81±3.99)%相比差异无统计学意义(P>0.05),在5 μg/ml、10μg/ml时TLR2表达量分别为(21.15±9.90)%和(22.52±9.26)%,与对照组比较差异有统计学意义(P<0.05).结论 细菌脂蛋白Pam3CSK4通过激活TLR2引起血小板聚集、活化,是血小板参与抑制革兰阳性细菌感染的免疫应答机制之一.     

免疫活化血小板对人脐静脉内皮细胞COX-2及PPAR-α表达与活性的影响及LDL的作用

目的:观察免疫活化血小板对人脐静脉内皮细胞(HUVECs)环氧合酶2(COX-2)及过氧化物酶体增殖物活化受体α(PPAR-α)表达及活性的影响,并探讨低密度脂蛋白(LDL)对其作用。方法:利用腺苷二磷酸(ADP)刺激血小板免疫活化,然后利用免疫活化血小板与HUVECs加或不加LDL共孵育,利用实时定量RT-PCR、Westernblotting分别检测HUVECsCOX-2及PPAR-αmRNA及其蛋白的表达,ELISA法检测PGE2浓度反映COX-2活性、核因子活性检测试剂盒检测PPAR-α活性。结果:血小板活化组COX-2及PPAR-αmRNA表达量较血小板对照组均有较明显升高(1.49±0.27vs0.68±0.21,1.45±0.21vs1.17±0.16,均P0.01);血小板活化组COX-2及PPAR-α蛋白表达量较血小板对照组亦有较明显升高(1.600±0.145vs0.700±0.073,1.630±0.143vs0.960±0.073,均P0.01),血小板活化组孵育液中PGE2浓度较血小板对照组明显升高[(42.46±5.57)ng/Lvs(23.73±2.53)ng/L,P0.01],但血小板活化组PPAR-α活性较血小板对照组无明显变化;LDL本身无明显增加HUVECsCOX-2及PPAR-α的表达及活性,但其能增加免疫活化血小板的这一作用。结论:免疫活化血小板显著促进HUVECsCOX-2表达及活性增加,亦能显著促进HUVECsPPAR-α表达,但并不改变PPAR-α活性;一般浓度的LDL并不对HUVECsCOX-2、PPAR-α的表达及活性产生影响,但能促进免疫活化血小板对内皮细胞的这一作用。     

NPPB和尼弗灭酸对H2O2损伤C6胶质细胞谷氨酸半胱氨酸合成酶及CLIC4表达的影响

目的：观察氯通道阻断剂5-硝基-2-（3-苯丙胺）-苯甲酸(NPPB)、尼弗灭酸(NFA)对H₂O₂诱导的神经胶质瘤C6细胞损伤的影响。方法：MTT法检测NPPB、NFA、H₂O₂作用的C6细胞生存率；紫外分光光度法检测乳酸脱氢酶(LDH)释放率以及谷胱甘肽GSH水平；RT-PCR检测谷氨酸半胱氨酸合成酶（GCL）亚单位GCLC、GCLM及线粒体氯通道（CLIC4） mRNA表达；Western blotting检测CLIC4的蛋白水平。结果：与对照组相比，H₂O₂组C6细胞存活率和GSH含量明显降低；LDH释放率增加；GCLC、GCLM、CLIC4 mRNA表达降低；CLIC4蛋白水平明显增强。NPPB或NFA与H₂O₂联合作用于C6细胞组，与单独应用H₂O₂组相比，细胞存活率和GSH含量未见明显变化；LDH释放率降低；GCLC、GCLM mRNA表达未见明显差异；CLIC4蛋白表达下降。结论：氯通道阻断剂NPPB或NFA能够在一定程度上减轻氧化应激引起的C6细胞损伤，可能与调节细胞膜功能和下调CLIC4蛋白表达有关。     

沉默ClC-3氯通道基因对HeLa细胞周期分布的影响

目的:探讨沉默HeLa细胞的ClC-3氯通道基因后细胞周期分布的变化及其作用机制。方法:依照siRNA设计原则构建沉默ClC-3基因的ClC-3 siRNA并转染HeLa细胞;实验分为空白对照组(control组)、转染试剂对照组(Lipo组)、阴性对照组(negative siRNA组)和ClC-3 siRNA组。采用real-time PCR检测ClC-3 siRNA的沉默效率;流式细胞术检测细胞周期分布情况;Western blot检测ClC-3蛋白及相关细胞周期蛋白(cyclin)D1、细胞周期蛋白依赖激酶(cyclin-dependent kinase,CDK)4、CDK6、P21和P27等表达。结果:CIC-3 siRNA成功沉默HeLa细胞的ClC-3基因。和其它组相比,ClC-3 siRNA组的细胞周期被阻抑在G_0/G_1期。CIC-3 siRNA组的cyclin D1、CDK4和CDK6蛋白表达水平明显下降,P21和P27蛋白表达水平明显上升。结论:沉默HeLa细胞ClC-3氯通道基因可影响cyclin D1、CDK4、CDK6、P21和27蛋白的表达水平胆抑HeLa细胞周期停滞在G_0/G_1期。     

Cl-/HCO-3交换器对星状孢子素诱导的小鼠心肌细胞凋亡的调节

目的:观察Cl-/HCO-3交换器是否参与了星状孢子素（STS）诱导的心肌细胞凋亡过程及其作用。方法：在建立的STS诱导原代培养的乳鼠心肌细胞凋亡模型上,通过使用Cl-通道阻断剂（NPPB）、Cl-通道阻断剂并Cl-/ HCO-3交换器阻断剂（DIDS）或更换含或无HCO-3成分的培养基,观察Cl-/HCO-3交换器对STS诱导的心肌细胞凋亡的影响。结果：(1) 在含HCO-3成分培养基中,DIDS和NPPB组在细胞存活率、caspase-3激活水平分别为59.7％和47.2％、175.0％和212.0％;两组比较具有显著差异（P<0.01,n=20）。在无HCO-3成分培养基中,DIDS与NPPB组在细胞存活率、caspase-3活性分别是62.1%与61.8%、176.5%与181.6%,两组比较无显著差异（P>0.05,n=20）。(2)STS阳性组中在含有与无HCO-3成分培养基中细胞存活率、caspase-3活性分别是29.8%与41.6%、553.4%与424.7%,两组相比有显著差异（P<0.01,n=20）,但两组均可观察到DNA凋亡片段。结论：Cl-/HCO-3交换器通过氯交换参与了STS诱导的心肌细胞凋亡过程,并非是细胞凋亡过程中氯离子交换的主体。     

心房颤动患者血浆血小板P-选择素浓度变化的研究

目的探讨心房颤动引起血小板活化机理及其程度.方法用放射免疫学方法对76例不同类型的心房颤动(21例阵发孤立性房颤LPAF,28例孤立持续性房颤LSAF,27例风心病并心房颤动VAF)患者于清晨、空腹、清醒时抽取外周静脉血(LPAF分别于AF发作及终止1周后各采血一次)测定外周血血浆血小板a颗粒膜蛋白(血小板P-选择素)的浓度,相互并与PSVT病人及健康对照组进行比较.结果(1):LSAF、VAF与LPAF组AF发作时血小板P-选择素浓度无显著性差别,较LPAF组AF终止后一周,PSVT组及正常对照组明显升高;LPAF组AF终止一周后与PSVT组、正常对照组血小板P-选择素浓度无显著差别(表1).(2)LPAF组AF持续时间与血浆血小板P-选择素浓度呈正相关(表2).(3)VAF组血小板P-选择素与射血分数(EF值)呈负相关(表3).结论房颤发作引起血小板活化并释放活性物质参与形成血栓前状态,房颤持续时间与活性物质释放程度呈正比.     

Prolonged platelet activation in individuals with elevated blood pressure in response to a moderate exercise challenge

We examined the magnitude of 20-min moderate exercise-induced platelet activation in 50 volunteers with normal ( n =31) or elevated blood pressure (EBP; n =19). Blood was drawn before, immediately after, and 25 min after exercise. Antibody-staining for platelet activation markers, P-selectin, and fibrinogen receptors was done with and without adenosine diphosphate (ADP) stimulation in whole blood for flow cytometric analyses. Exercise led to increases in percent aggregated platelets and percent platelets expressing P-selectin or PAC-1 binding ( p s≤.001). This increase in percent platelets expressing P-selectin continued even after a 25-min rest only in the EBP group ( p ≤.01) accompanied by an increase in percent of aggregated platelets ( p ≤.05). Although ADP stimulation led to increased platelet activation at rest, it was attenuated following exercise, even among EBP individuals. A moderate exercise challenge induced prolonged platelet activation in individuals with EBP but attenuation in activation to further stimulation by an agonist. Findings suggest that a recovery period after physical stress appears critical in individuals with high BP regarding platelet activation and aggregation, which can lead to an acute coronary syndrome in vulnerable individuals.     

Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.     

High levels of LDL-C combined with low levels of HDL-C further increase platelet activation in hypercholesterolemic patients

High levels of low-density lipoprotein cholesterol (LDL-C) enhance platelet activation, whereas high levels of high-density lipoprotein cholesterol (HDL-C) exert a cardioprotective effect. However, the effects on platelet activation of high levels of LDL-C combined with low levels of HDL-C (HLC) have not yet been reported. We aimed to evaluate the platelet activation marker of HLC patients and investigate the antiplatelet effect of atorvastatin on this population. Forty-eight patients with high levels of LDL-C were enrolled. Among these, 23 had HLC and the other 25 had high levels of LDL-C combined with normal levels of HDL-C (HNC). A total of 35 normocholesterolemic (NOMC) volunteers were included as controls. Whole blood flow cytometry and platelet aggregation measurements were performed on all participants to detect the following platelet activation markers: CD62p (P-selectin), PAC-1 (GPIIb/IIIa), and maximal platelet aggregation (MPAG). A daily dose of 20 mg atorvastatin was administered to patients with high levels of LDL-C, and the above assessments were obtained at baseline and after 1 and 2 months of treatment. The expression of platelets CD62p and PAC-1 was increased in HNC patients compared to NOMC volunteers (P<0.01 and P<0.05). Furthermore, the surface expression of platelets CD62p and PAC-1 was greater among HLC patients than among HNC patients (P<0.01 and P<0.05). Although the expression of CD62p and PAC-1 decreased significantly after atorvastatin treatment, it remained higher in the HLC group than in the HNC group (P<0.05 and P=0.116). The reduction of HDL-C further increased platelet activation in patients with high levels of LDL-C. Platelet activation remained higher among HLC patients regardless of atorvastatin treatment.     

Expression of platelet P-selectin and detection of soluble P-selectin,NPY and RANTES in patients with inflammatory bowel disease

OBJECTIVE AND DESIGN: P-selectin, a membrane glycoprotein which is expressed on activated platelets and endothelial cells, plays a crucial role in the inflammatory response. The main action is adhesion of leukocytes, facilitation of diapedesis and induction of cytokine production from monocytes (MCP-1 and IL-8), mediated via RANTES released from activated platelets. An abnormal platelet activity has been reported in patients with ulcerative colitis (UC) and Crohn's disease (CD), jointly referred to as inflammatory bowel disease (IBD), which could have an aggravating influence on the inflammatory response. In addition, an up-regulation of platelet IL-8 receptors among patients with IBD has been reported. To reveal a presumptuous platelet dysfunction we analysed the expression of platelet surface P-selectin at resting state and after stimulation with thrombin, collagen, epinephrine and interleukin 8 (IL-8), and plasma levels of soluble P-selectin, neuropeptide Y (NPY) and RANTES in patients with IBD. SUBJECTS: Blood from twelve healthy subjects (control group) and twenty-one patients with IBD who had not taken any anti-platelet drugs or steroids were analysed. METHODS: Patients were sub-grouped according to disease entity, disease activity and 5ASA medication. Surface P-selectin expression on isolated human platelets and plasma P-selectin, NPY and RANTES were analysed with ELISA. All values are presented as mean +/- standard error of the mean (SEM). Mann-Whitney U test and Wilcoxon matched rank test were used for statistical analyses. RESULTS: Patients with IBD in remission (n = 9) had higher basal P-selectin expression, 0.38+/-0.04, compared to the control group (n = 12), 0.22+/-0.03,p < 0.01. UC patients (n = 16) showed down-regulation of P-selectin expression after stimulation with IL-8, 0.26+/-0.03 to 0.22+/-0.02, p < 0.05. No significant differences could be observed concerning soluble P-selectin and NPY in plasma. Patients with 5ASA (n = 12) had lower levels of plasma RANTES, 2.39+/-0.06 microg/l, compared to the control group (n = 12), 3.29+/-0.19 microg/l, p < 0.01, and patients without 5ASA (n = 9), 2.90+/-0.17 microg/l, p < 0.05. CONCLUSIONS: Patients with IBD in remission have higher basal platelet surface P-selectin expression. An exaggerated platelet activity with increased expression of platelet P-selectin and release of inflammatory mediators such as RANTES, which is chemotactic and induce chemokine production, could have a reinforcing and aggravating influence on the inflammatory response and increase the susceptibility to IBD. In addition IL-8 has a down-regulating effect on platelet surface P-selectin expression and 5ASA medication seems to lower plasma RANTES. If 5ASA is responsible for lowering the concentration of RANTES this could be one of the beneficial outcomes of 5ASA medication.     

葡萄籽原花青素体外抗血小板聚集的机制研究

目的: 研究葡萄籽原花青素(PC)体外抗血小板聚集的可能机制。方法: 应用血小板聚集仪研究PC、二亚苯基碘鎓(DPI,非特异性NADPH氧化抑制剂)和夹竹桃麻素(apocynin,特异性NADPH氧化酶抑制剂)对胶原诱导的健康志愿者血小板最大聚集率的影响。用化学发光仪检测PC对血小板NADPH氧化酶活性、一氧化氮(NO)含量和超氧阴离子(O₂^-·)水平的影响。用流式细胞术观察PC对血小板活化标志物PAC-1和CD62P表达率的影响。结果: PC(100 μmol/L)、apocynin(10 μmol/L)和DPI(100 μmol/L )均可显著抑制胶原蛋白诱导的血小板最大聚集率(P<0.01)。胶原蛋白激活的血小板NO含量显著降低,O₂^-·含量显著增加,100 μmol/L PC可使二者明显恢复(P<0.05)。添加了100 μmol/L PC的样本中,血小板NADPH氧化酶活性受到明显的抑制(P<0.01),血小板PAC-1 和CD62P表达率也显著降低(P<0.05)。结论: 葡萄籽原花青素可能通过抑制NADPH氧化酶的活性,进而影响血小板NO和O₂^-·含量,在一定程度上阻断血小板活化过程,最终达到抗血小板聚集的作用。     

Modulation of P-selectin expression on isolated human platelets y an NO donor assessed by a novel ELISA application

Adhesion molecules such as P-selectin are potential markers for evaluating platelet activation and studying the role of cell-cell interactions in numerous biological processes related to hemostasis and inflammation. The expression of P-selectin and related molecules has previously been quantified with different techniques. As an alternative to the most common method, flow cytometry, we have developed a useful ELISA method to simultaneously analyse 96 samples for platelet expression of P-selectin. Samples may be stored for at least 7 days at 4°C prior to analysis. The method is simple, reproducible, flexible and requires only standard equipment. Washed platelets (WP) from healthy male volunteers, at a concentration of 1 × 10⁷/microtiter plate well, were stimulated with various known platelet activators and fixed with 0.1% formaldehyde for 10 min. The fixed WP were centrifuged to form a confluent layer in the wells and then incubated with optimal dilutions of primary antibodies (1/2000) directed against P-selectin, CD41, CD9 and secondary antibodies conjugated with alkaline phosphatase. Our results show that P-selectin expression on WP increases significantly upon stimulation with thrombin (0.1–1.0 U/ml), ADP (10 μM) and epinephrine (100 μM). The induction of P-selectin expression by thrombin is fast and has different kinetics depending on the concentration of the agonist. Prior incubation with the nitric oxide donor SNAP (10 μM) inhibits the up-regulation of P-selectin induced by sub-maximal concentrations of thrombin (p < 0.05). This ELISA is suitable for studying the expression and regulation of P-selectin and other surface molecules on human platelets in various pathological states.     

Effects of methacholine and uridine 5'-triphosphate on tracheal mucus rheology in mice

We compared the action of methacholine (MCh) and uridine 5'-triphosphate (UTP) with and without pretreatment with the chloride channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) on the transepithelial potential difference (PD), the mucus collection rate (MCR), and tracheal mucus rheology using anesthetized C57BL/6 mice. The cystic fibrosis transmembrane conductance regulator (CFTR) blocker 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) was also used as a pretreatment for MCh. After collecting baseline mucus for 1.5 h, mucus secretion was stimulated by instilling 5 microl of 10(-2) M MCh or UTP around the upper trachea. There was a significant increase in PD after MCh or UTP stimulation (-21.3+/-2.0 mV MCh versus -14.1+/-1.6 mV control; -25.4+/-2.5 mV UTP versus -19.2+/-1.9 mV control). When UTP administration was preceded by DIDS, PD shifted from -15.2+/-2.9 to -12.0+/-2.2 mV. When MCh was preceded by DIDS or by NPPB, there was no change in PD. There was a significant decrease in mucus rigidity index, logG*, with MCh (2.54+/-0.09 versus 2.99+/-0.14 for control), similar to that previously reported in other species. With UTP, 14 of 16 mice responded in terms of PD becoming more negative, and of these, there was a significant difference in logG* after UTP administration (2.29 +/-0.10 versus 2.57+/-0.10 for control), whereas there was no change in logG* with DIDS administration before UTP. When DIDS administration preceded MCh, there was a diminished but still significant decrease in logG* from control, whereas there was no change in logG* when NPPB was preadministered. The control mucus collection rate was 0.19+/-0.09 mg/h, whereas after MCh stimulation, it increased to 2.83+/-0.78 mg/h. No significant difference was measured in the MCR after either UTP or DIDS+UTP stimulation. DIDS+MCh and NPPB+MCh both resulted in significant increases in MCR, but of a much smaller magnitude than that for MCh alone. We conclude that hypersecretion owing to UTP in C57BL/6 mice is less vigorous than with MCh, reflecting the limited population of Ca(2+)-dependent Cl(-) channels stimulated by UTP P(2) receptors. The action of MCh on tracheal mucus secretion in mice appears to involve both CFTR- and non-CFTR-dependent chloride channels.