Secretin dissipates red aridine orange fluoescence from pancreatic duct epithelium

This study was undertaken to elucidate whether duct cells in the pancreas contain acidic cytoplasmic compartments regulated by secretion. Microdissected pancreatic ducts from pigs were examined by acridibe orange (AO) and 2′, 7′-biscarboxyethyl-5(6)-carboxyfluorescein/tetraacetioxymethyl ester (BCECF/AM) epifluorescence microscopy. Estimated cytoplasmic pH using BCECF fluorescence was 7.43pL0.04 and was not changed by altering CO₂ tension in the incubation mdium. The epithelium of acridine orange incbated peripheral interlobular pancreatic ducts exhibited green and fluorescence was sen in resting pancreatic ducts and was greatly accentuated by raising CO₂ in the incubation medium with chloroqyuine or NH₄Cl or the protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or carbonyl cyanide M-chlorophenylhydrazone (CCCP), leaving uniform gren fluoresence. These findings suggest that pancreatic duct cells contain CO₂-dependent acidic compartments which vanishduring seceatin stimulation and which may be cytoplasmic tubulovesicles.     

人类鱼际肌的肌纤维型分布

取 5例 (男 4 ,女 1) ,2 1～ 37岁 ,死亡后 12h内的人体两侧手鱼际肌组织 50块 ,用肌球蛋白ATP酶法 ,系统研究了各肌的肌纤维型分布。结果表明 :人类鱼际肌中I型 (慢缩 )纤维的平均百分率在 59%～ 67%之间 ,平均为 62 %显著高于上肢其它各肌群I型纤维的平均百分率 (P<0 0 5)。鱼际肌的深、浅层间 ,多数肌I型纤维百分率接近。左、右侧别比较 ,除拇收肌横头右侧者显著高于左侧外 ,其余肌均未见显著的I型纤维侧别差异 ,结合肌电图和拇指功能 ,对这些差异的意义进行了分析讨论     

Differentiation of proton-pumping activity in cultured renal inner medullary collecting duct cells

Cultured inner medullary collecting duct (IMCD) cells have been shown to secrete protons (H⁺) by two mechanisms: anN-ethylmaleimide-and dicyclohexyl-carbodiimide-sensitive electrogenic H⁺-ATPase or H⁺ pump, and an amiloride-sensitive, secondary active Na⁺/H⁺ exchanger. These cells also express Cl^–/HCO₃ ^– exchange and carbonic anhydrase activity in common with other renal epithelial cells involved in acid-base transport. Video fluorescence microscopy of individual cells using 2, 7-biscarboxyethyl-5(6)-carboxyfluorescein has demonstrated that adjacent-cultured IMCD cells show substantial functional intercellular heterogeneity. The development of H⁺-pumping activity is associated with high-baseline intracellular pH and peanut agglutinin (PNA) affinity, and loss of mitotic activity and of Na⁺/H⁺ exchange. The H⁺-pumping activity may be further enhanced by removal of fetal calf serum for 6–54 h or by selecting cells with high PNA affinity. IMCD cells in their most differentiated state form domes, which consistently showed the highest rates of H⁺-pumping activity, as well as high affinity for peanut lectin. When IMCD were plated at low density, domes developed relatively late (2–4 weeks), at which time cells located in the center of nests of contiguously growing cells were quiescent and showed H⁺-pumping activity but no Na⁺/H⁺ exchange. On the other hand, dense plating was associated with early development of domes (end of 1st week), at which time adjacent cells showed a high mitotic activity and Na⁺/H⁺ exchange, but no H⁺-pumping activity. We speculate that differentiation of IMCD cells results in the development of cell polarity. This could include either loss of the apical Na⁺/H⁺-exchange activity, or localization of this exchanger only to the basolateral membrane, while the H⁺ pump differentiates at the apical membrane.     

哇巴因与地高辛对大鼠肝脏钠泵A亚单位基因表达的影响

目的　观察哇巴因与地高辛对大鼠肝脏钠泵基因表达的影响 .方法　分别给大鼠注射哇巴因 (2 0 μg· kg- 1·d- 1 )、地高辛 (32 μg·kg- 1·d- 1 )和生理盐水 (1m L· kg- 1·d- 1 ) ,观察给药后 6wk大鼠血压的动态变化 ;并分别应用分子生物学 RT- PCR及免疫组织化学技术 ,探讨各组大鼠肝脏钠泵 α1 ,α2 及 α3 亚单位 m RNA及蛋白水平基因表达的改变 .结果　给予哇巴因 2 wk后大鼠血压开始升高 ,至 6 wk时明显高于生理盐水组 (17.7± 1.2 ) vs(15 .4± 1.1) k Pa,P<0 .0 1) ,而实验过程中地高辛组血压与对照组比较差异不明显 .无论是在 m RNA水平还是在蛋白水平 ,哇巴因对大鼠肝脏钠泵α1 亚单位表达无影响 ,而地高辛使α1 亚单位表达增强 ;两组大鼠 α2 亚单位表达均无改变 ;哇巴因使 α3 亚单位蛋白水平表达减弱 ,而 m RNA水平增强 ,地高辛组大鼠肝脏钠泵 α3 亚单位无论在 m RNA水平及蛋白水平表达均增强 .结论　哇巴因在高血压发病中可能起着重要作用 ;哇巴因与地高辛可导致不同的钠泵基因表达改变 ,为进一步揭示内源性哇巴因生理与病理作用以及洋地黄类药物药理与毒理作用的分子机制提供了理论及实验依据     

酚妥拉明对兔去甲肾上腺素性心肌损伤的保护作用

目的：探讨酚妥拉明对去甲肾上腺毒（ＮＥ）性心肌损伤时保护作用的机理。方法：采用静滴超生理量去甲肾上腺素致心肌损伤,观察兔外周血红细胞内ａ＾２＋、Ｍｇ＾２＋、心肌细胞膜Ｃａ＾２＋－Ｍｇ＾２＋ＡＴＰａｓｅ,Ｎａ－Ｋ－ＡＴＰａｓｅ的变化及酚妥拉明的保护作用。结果：酚妥拉明组各次指标均与ＮＥ组有显著性差异。结论：Ｓ寻拉明可防止或减轻ＮＥ心肌损伤时Ｃａ＾２＋超载、Ｍｇ＾２＋丢失及能量耗竭。     

持续顺行灌注温血停搏液对心肌的保护作用

研究持续灌注温血停搏液对心肌的保护作用,探讨理想的心肌保护策略,方法６９只猫随机分为对照组、损伤组和保护组。观察测各组不同时间点心功能、心肌细胞膜ＡＴＰａｓｅ活性心肌细胞膜蛋白颗粒的形态变化。结果组Ⅰ各时间点各项指标无明显变化。升主动脉阻断期间,组Ⅱ－ⅢａＫ－ＡＴＰａｓｅ活性均显著下降（ＡＣＣ１５ｍｉｎ ｕｓ ＡＣＣ６０ｍｉｎ,Ｐ〈０．０１）,组ⅡＣａＭｇ－ＡＴＰａｓｅ活性单位面积心肌细胞膜蛋白颗     

Interaction of cyclosporin derivatives with the ATPase activity of human P-glycoprotein

1. P-glycoprotein, a 170–180 kDa membrane glycoprotein that mediates multidrug resistance, hydrolyses ATP to efflux a broad spectrum of hydrophobic agents. In this study, we analysed the effects of three MDR reversing agents, verapamil, cyclosporin A and [3′-keto-Bmt]-[Val^*]-cyclosporin (PSC 833), on the adenosine triphosphatase (ATPase) activity of human P-glycoprotein.
2. P-glycoprotein was immunoprecipitated with a monoclonal antibody (MRK-16) and the P-glycoprotein-MRK-16-Protein A-Sepharose complexes obtained were subjected to a coupled enzyme ATPase assay.
3. While verapamil activated the ATPase, the cyclosporin derivatives inhibited both the substrate-stimulated and the basal P-glycoprotein ATPase. No significant difference was observed between PSC 833 and cyclosporin A on the inhibition of basal P-glycoprotein ATPase activity. PSC 833 was more potent than cyclosporin A for the substrate-stimulated activity.
4. Kinetic analysis indicated a competitive inhibition of verapamil-stimulated ATPase by PSC 833.
5. The binding of 8-azido-[α-³²P]-ATP to P-glycoprotein was not altered by the cyclosporin derivatives, verapamil, vinblastine and doxorubicin, suggesting that the modulation by these agents of P-glycoprotein ATPase cannot be attributed to an effect on ATP binding to P-glycoprotein.
6. The interaction of the cyclosporin derivatives with ATPase of P-glycoprotein might present an alternative and/or additional mechanism of action for the modulation of P-glycoprotein function.

     

Real-time visual detection of Pi released by flagellar dynein ATPase

 Using a novel fluorescent probe for P_i, a method for the direct visualization of P_i release from reactivated flagellar dynein ATPase has been developed. The probe undergoes a fluorescence increase when it binds P_i. The technique involves simultaneous imaging of demembranated sperm tails by epi-fluorescence and dark-field microscopy, and the use of the caged ATP technique for axoneme reactivation. To limit diffusion and thus maintain the released P_i within the observed field of view, the assay is carried out within a minute droplet under oil (volume 5–15 pl). The video output of a recursively filtered ICCD camera is used to visualize the fluorescence signal, which is subsequently digitized and automatically analysed on a PC. A major advantage of this technique is that it enables simultaneous analysis of the ATP-utilization rate and the motility of the reactivated axonemes. Received: 24 November 1998 / Received after revision: 21 January 1999 / Accepted: 22 January 1999     

Distribution and metabolic fate of adenosine nucleotides in the membrane of storage vesicles from bovine adrenal medulla

Summary The reactions of adenosine ¹⁴C- and ³²P-labelled ATP with isolated membranes from catecholamine storage vesicles of the bovine adrenal medulla were studied. In presence of Mg²⁺ about twice as much of ³²P-radioactivity combined with the membrane as ¹⁴C-adenosine compounds at 31°C and also at 0°C, while in the absence of Mg²⁺ the amounts of ¹⁴C and ³²P incorporated were similar for both substances. Autoradiography of the SDS-polyacrylamide gel after electrophoresis of the ³²P-ATP-treated membrane protein showed two distinct zones corresponding to protein bands. Sonication released twice as much ³²P-ATP as ¹⁴C-ATP from the space within the membrane particles indicating that at least half of the ATP present in this space did not contain its original terminal phosphate group. About 40–45% of the ³²P-radioactivity was incorporated in the membrane lipids, whereas only small amounts of ¹⁴C-radioactivity were extracted with the lipids. About 1/3 of the incorporated ¹⁴C-radioactivity was not extractable with acids. The same amount remained in the ³²P-ATP treated preparation acid-stably bound after extraction of the lipids and thus must be firmly bound ATP. When the reaction of the membrane preparation with labelled ATP was performed at 0°C the fractions of the acid-stably bound ³²P- and ¹⁴C-radioactivity increased. About 1 nmole/mg of protein (10–15%) of the bound ³²P-radioactivity was exchangeable against unlabelled ATP, while only a very small fraction (<0.5 nmole/mg protein) of the ¹⁴C-radioactivity was exchanged against unlabelled ATP. Preincubation of the membrane particles with ATP-Mg²⁺ at 0°C induced 30% inhibition of the ATPase activity and abolition of the net uptake of catecholamines. Different K _m values obtained from initial velocity studies of ATPase activity and the overall-incorporation of ³²P-radioactivity indicated that a direct correlation between these processes did not exist. Different strong inhibitory effects exerted by ADP on the ATPase activity and net uptake of catecholamine at the one hand and the overall ³²P- and ¹⁴C-incorporation at the other hand supported that view. It is concluded that only small fractions of the observed ³²P-and ¹⁴C-incorporation can be involved in the ATP-hydrolyzing reaction.