Characterization of synaptic vesicles and related neuronal features in nerve growth factor and ras oncogene differentiated PC12 cells

PC12 cells can differentiate into neuron-like cells after treatment with either nerve growth factor (NGF) or transduction with a retrovirus which expresses the K-ras oncogene. The concomitant treatment of NGF plus ras differentiates PC12 cells further than either agent alone with respect to neurite outgrowth, acetylcholinesterase levels, and most strikingly, the number of synaptic vesicle (SV) clusters. These SV clusters in PC12 cell neurites closely resemble those in the presynaptic terminals of neurons. Such SV clusters have not been described in cell lines previously. The SV clusters from all three differentiated groups (NGF, ras, and NGF plus ras) were similar in size, shape, and configuration, except that the ones in the doubly treated group occur in higher frequency and have more vesicles. The synaptic nature of these vesicle clusters was demonstrated by their regulated depletion after potassium stimulation. Furthermore, these vesicle clusters stained positively for two SV-associated proteins, synapsin I and synaptophysin, by EM immunocytochemistry (ICC). Such SV clusters in a cell line are very useful for characterizing the regulated release of SVs and the distribution of SV-related antigens in intact cells. Analysis by SDS-gel electrophoresis and immunoblotting indicated that synapsin I levels are higher in all three differentiated groups compared to untreated cells; whereas synaptophysin levels are lower in cells exposed to NGF alone or with NGF and ras double treatment. Possible convergence and/or divergence on the mechanisms of NGF and ras differentiation in PC12 cells are discussed. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

离子通道镶嵌点过程模型在K+通道研究中的应用

目的：用作者提出的离子通道镶嵌点过程模型拟合海马锥体细胞钾离子单通道记录数据分析１７β雌二醇的效应。方法：用协方差密度度量通道记忆,从３个方面做拟合优度检验。结果：该模型与观察资料拟合效果良好;１７β雌二醇能改变海马锥体细胞钾离子单通道的记忆;１７β雌二醇对关闭的影响比对开放的影响大,从而表现为对通道具有抑制作用：浓度为１０ｎｍｏｌ／Ｌ组的１７β雌二醇对通道记忆的影响比对浓度为０１ｎｍｏｌ／Ｌ组的大。结论：Ｍａｒｋｏｖ模型的常规分析只能得到１７β雌二醇对海马锥体细胞钾离子单通道具有抑制作用,而用本模型分析能得到更多通道的信息     

Tissue potassium,selenium, and iron levels associated with gastric cancer progression

The contents of 10 minor and trace elements in histologically confirmed gastric adenocarcinomas and their corresponding normal gastric mucosal tissues obtained from 39 patients at the time of gastric resection were simultaneously determined by instrumental neutron activation analysis. Specimens were irradiated by reactor neutrons and subsequently subject to direct analysis using a high-resolution HPGe -spectrometer. Univariate analysis revealed that gastric cancer tissues had significantly higher concentrations of Fe, K, Mg, Na, Rb, Se, and Zn than normal gastric mucosal tissues. However, multivariate analysis found that Fe, K, and Se were independent elements that associated with gastric cancer. Upon further evaluation of their clinical significance, we found a high tissue K level was related to lymphatic duct metastasis. High Se tissue levels were linked to intestinal type adenocarcinoma. A positive correlation was found between high Fe levels and vascular involvement. These findings suggest that Fe and K are associated with gastric cancer progression. Se is involved in carcinogenesis of stomach in high-risk areas. The mechanisms that underlie the corresponding pathohistological features deserve further study.     

The cardiotonic bipyridine AWD 122-60 inhibits adenosine triphosphate-sensitive potassium channels of mouse skeletal muscle

Summary The inhibition of ATP-sensitive potassium channels in mouse skeletal muscle by the cardiotonic bipyridine AWD 122-60 was investigated with the patch-clamp technique. In excised patches of the inside-out configuration, internally applied AWD 122-60 (10^–6–10^–3 mol/1) reversibly reduced the open-probability of single ATP-sensitive potassium channels. The agent shortened the periods of channel activity but did not affect the channel conductance. At positive membrane potentials channel inhibition by AWD 122-60 was more pronounced than at negative potentials, the drug concentrations producing 50% channel inhibition were 11 mol/l at + 40 mV and 29 mol/l at –40 mV. The Hill coefficients of the concentration-response curves were in the range between 0.5 and 0.6 for both potentials. milrinone (10^–4 mol/I), had no effects on ATP-sensitive potassium channels in skeletal muscle. potassium channels in heart muscle by AWD 122-60 are discussed. Send offprint requests to B. Neumcke at the above address     

Citrate and recurrent idiopathic calcium urolithiasis

Summary In idiopathic recurrent calcium urolithiasis (RCU) in men (n=37) the metabolic effects of oral tripotassium citrate (PC) were investigated in a longitudinal field study. The patients were either normo- (n=22) or hypocitraturic (n=15). Laboratory examinations were performed before, and after 3, 6, and more than 12 months of medication. Acceptance of PC was poor, mainly because of the salty taste of the tablet preparation chosen, and a number of participants dropped out of the study. In the remaining participants, compliance was acceptable when evaluated on the basis of urinary potassium and undesired side effects did not occur. In the short term (up to 3 months), PC evoked compensated metabolic alkalosis (pH and citrate in urine increased; blood gases remained normal), a drop in urinary calcium, together with increasing oxaluria, hydroxyapatite supersaturation, and calcium phosphate crystalluria. In the long term (>12 months) PC urinary pH and citrate dissociated, in that pH returned to pretreatment baseline values, whereas citrate stayed at high levels. In normocitraturics but not in hypocitraturics, urinary urea and sodium in creased with PC. Hypocitraturics appeared to be less sensitive to the effects of PC, as reflected by the relatively small rise in urinary pH and citrate, and they maintained higher mean levels of indicators of bone metabolism (osteocalcin, alkaline phosphatase, hydroxyproline) despite continuous administration of PC. It was concluded that although the PC tablet preparation was effective it may not be an ideal anti-stone drug treatment in the long term and that, especially in hypocitraturiecs, the intrinsic metabolic defect of RCU may not be sufficiently well controlled.     

Flurazepam interaction with sodium and potassium channels in squid giant axon

External application of 0.05-1.0 mM flurazepam was found to partially block both sodium and potassium currents in voltage-clamped squid giant axons. At the same concentration the fractional block of the potassium current was found to be 3 times greater than that of the sodium current. In the presence of the drug the potassium current appeared to "inactivate', as flurazepam block became more profound during the course of the depolarization. The decay of the potassium current can be explained by a model in which flurazepam enters and blocks the potassium channels only after they have opened. Once bound in the potassium channel, removal of flurazepam from its binding site develops slowly (tau = 48 ms). Thus repetitive stimulation of the nerve produced a cumulative block. When applied inside the axon flurazepam was found to be 1.5 (n = 4) times more potent blocker of potassium channels than following external application. This result suggests that when applied externally, a neutral form of the drug diffuses across the membrane and blocks occurs from the inner end of the channel.     

钾通道阻滞剂对大鼠支气管平滑肌细胞增殖的影响

目的探讨电压依赖性延迟整流钾通道(K_V)、Ca²⁺激活钾通道(K_Ca)和ATP敏感性钾通道(K_ATP)对大鼠支气管平滑肌细胞(BSMC)增殖的影响。 方法应用免疫细胞化学、MTT微量比色分析法及流式细胞术,观察K_V,K_Ca和K_ATP对培养中大鼠BSMC增殖的影响。结果K_V阻断剂4-氨基吡啶(4-AP)显著促进大鼠BSMC增殖细胞核抗原的表达,提高BSMC吸光度值,使S+G₂M期细胞数显著增多,并显著提高基础状态下BSMC内Ca²⁺浓度,而K_Ca阻断剂四乙铵(TEA)和K_ATP阻断剂格列本脲(Glib)均无此效应。 结论大鼠BSMC K_V活性的抑制,可提高细胞内Ca²⁺浓度,促进细胞的增殖,而K_Ca和K_ATP对BSMC的增殖均无明显影响。     

Delayed Administration of the K+ Channel Activator Cromakalim Attenuates Cerebral Vasospasm after Experimental Subarachnoid Hemorrhage

Summary ? Background. Delayed cerebral vasospasm remains an unpredictable and inadequately treated complication of aneurysmal subarachnoid hemorrhage (SAH). Recent evidence indicates that the potassium channel activator cromakalim is capable of limiting cerebral vasospasm in rabbits when administered immediately after experimental SAH (i.e. before spastic constriction has been initiated). However, the ultimate clinical value of cromakalim for treating vasospasm will depend in part on its effectiveness when administered after SAH-induced constriction has already been initiated. The present study examined the effects of cromakalim on vasospasm when treatment was initiated after SAH-induced constriction was underway.  Methods. New Zealand white rabbits were subjected to experimental SAH by injecting autologous blood into the cisterna magna. Cromakalim (0.03, 0.1 or 0.3 mg/kg) or vehicle was injected intravenously at 8 hour intervals beginning 24 hours post-SAH. Animals were killed by perfusion fixation 48 hours after SAH. Basilar arteries were removed and sectioned, and cross-sectional area was measured.  Findings. The average cross sectional areas of basilar arteries were reduced by 64% and 68% in the SAH-only and SAH+vehicle groups, respectively. Treatment with cromakalim dose-dependently attenuated SAH-induced constriction. The groups treated with 0.03, 0.1, and 0.3 mg/kg cromakalim exhibited average decreases in cross-sectional area of 57%, 42%, and 19%, respectively.  Interpretation. These findings indicate that cromakalim dose-dependently attenuates cerebral vasospasm when administered 24 hours after experimental SAH in the rabbit. The results suggest K_ATP channel activators, such as cromakalim, could be of benefit for reversing cerebral vasospasm after aneurysmal SAH.     

Suppression of surgical hyperaldosteronism by potassium canrenoate during gynecologic surgery under sevoflurane anesthesia

BACKGROUND: Surgical hyperaldosteronism leads to sodium and water retention during surgery and often causes postoperative edema. This study investigated the effect of potassium canrenoate (PC) on pituitary adrenocortical function in lower abdominal surgery under sevoflurane anesthesia. METHODS: Twenty patients were randomized to receive 400 mg of PC (the PC group, n=10) or saline (the control group, n=10) intravenously. The following parameters were determined: plasma aldosterone, adrenocorticotropic hormone (ACTH), plasma renin activity (PRA), serum sodium and potassium, urinary sodium and potassium, and urine output. RESULTS: The aldosterone and ACTH levels showed significant increases in the control group during surgery. Plasma ACTH also increased significantly in the PC group, but plasma aldosterone levels were unchanged during surgery. The urine Na/K ratio of the PC group was significantly higher than that of the control group. CONCLUSION: The present study suggested that PC suppresses the increase of plasma aldosterone caused by surgical stress. That may prevent sodium retention and potassium excretion during surgery.     

Cytochrome P450 epoxygenases as EDHF synthase(s)

The metabolism of arachidonic acid by cytochrome P450 (CYP) epoxygenases generates epoxyeicosatrienoic acids (EETs) which affect numerous cellular process including Ca(2+) signaling and the activity of Ca(2+)-dependent K(+) channels. The expression of the CYP epoxygenase(s) that generate EETs in endothelial cells is not constitutive but is determined by a number of physical (fluid shear stress and cyclic stretch) and pharmacological stimuli which also affect responses attributed to an endothelium-derived hyperpolarizing factor (EDHF). This review summarizes the role played by EETs, and the enzymes that generate and metabolize them, in EDHF-mediated responses.