石油生物脱硫菌Agrobacterium tumefaciens UP-3的固定化研究*

对能降解二苯并噻吩(DBT)的根癌土壤杆菌AgrobacteriumtumefaciensUP3菌株进行了固定化研究,以聚乙烯醇(PVA)和海藻酸钠(SA)混合物为包埋法固定化载体,固定化最佳操作条件为4℃交联,PVA和SA混合物总浓度7%,两者最佳浓度比为6,细胞浓度为0.05g/mL。当DBT加入量为2.7mmol/L时,UP-3的静息细胞最高脱硫率为13%,而固定化细胞的脱硫效率超过了60%;固定化细胞的最佳使用条件为降解5d,温度28℃～32℃。     

ARTP诱变对三角帆蚌外套膜细胞生长活性及其生物矿化相关因子的影响

为获取高活力的外套膜细胞, 研究通过常压室温等离子体(Atmospheric and room temperature plasma, ARTP)诱变和流式细胞术等技术分析了不同诱变气量组(10、12和15 SLM组)和处理时间对三角帆蚌(Hyriopsis cumingii)体外培养的外套膜细胞的细胞活性及生物矿化相关功能的影响。结果表明: ARTP诱变360—900s能显著升高各组三角帆蚌外套膜细胞活力, 且在900s时达到最大值(P<0.05); 渗透压稳定剂的添加, 显著提高了诱变过程中12和15 SLM组在360—900s作用时间下的细胞活力(P<0.05), 其中12 SLM组外套膜细胞增殖指数显著上升至最大值(P<0.05); 诱变后细胞体外培养24h时结果显示, 12 SLM组720s的外套膜细胞活力显著达到最高(P<0.05); 15 SLM组(诱变时间为720s, 下同)SOD活力随着诱变气量的增大呈显著下降趋势, 且在15 SLM组显著降至最低水平(P<0.05), 相反, 微核率在15 SLM组达到最大值; 生物矿化分析表明, 外套膜细胞Ca2+的浓度、生物矿化相关的关键酶(碳酸酐酶、碱性磷酸酶)和钙调蛋白基因(Calmodulin, CAM)基因均在12 SLM组达到最大值(P<0.05), 而EFCB1(EF-hand calcium-binding domain-containing protein 1)基因结果显示在10 SLM组达到最大值(P<0.05), 12 SLM次之; 以上分析表明, 氦气诱变在气量为12 SLM, 处理720s时与渗透压稳定剂连用对外套膜细胞活性及其他生物学活性影响最为显著, 暗示氦气诱变可有效作用于外套膜细胞的离体培养, 为三角帆蚌建立细胞系提供生物学基础与新思路。     

PARP-1抑制剂3-AB对肝癌细胞增殖及凋亡的影响*

目的:探讨PARP-1抑制剂3-AB对肝癌细胞系MHCC97-H和SMMC7721及正常肝细胞系L02的增殖与凋亡的影响。方法:细胞增殖试验观察不同浓度3-AB对三种不同细胞系细胞的增殖作用。Annexin V荧光探针标记,流式细胞学检查观察不同浓度3-AB对不同细胞系细胞凋亡的影响。结果:当3-AB浓度分别为5 mM、10 mM与20 mM时,与对照组(0 mM)相比,在培养第6天时开始出现增殖明显减慢,出现统计学差异(p0.05),第九天差异明显(p0.05)。随着浓度增加,其对肿瘤细胞系MHCC97-H和SMMC7721细胞增殖的抑制程度增加,细胞数均逐渐减少;而同样浓度梯度3-AB对人类肝细胞系L02生长则无明显的抑制作用。进一步实验发现,当3-AB浓度为5mM、10 mM与20 mM时,均可诱导肝癌细胞株MHCC97-H和SMMC7721凋亡,与对照组(0 mM)比较均有统计学差异(p0.05),且细胞凋亡率与3-AB的药物浓度相关:浓度越高,凋亡越明显。而同等浓度3-AB对肝脏细胞系L02无明显的促进凋亡作用。结论:3-AB可以抑制肝癌肿瘤细胞的增殖,促进肿瘤细胞的凋亡,对正常肝脏细胞无明显毒害作用,具有治疗肝癌的的潜在应用价值。     

合浦珠母贝PU3基因的克隆及功能鉴定

目的:克隆获得合浦珠母贝PU3基因的序列,并研究其在生物矿化中的功能。方法:使用RACE获得PU3基因的全长;利用实时荧光定量PCR的方法检测PU3基因在不同组织中的表达分布;利用实时荧光定量PCR的方法检测贝壳损伤修复过程中PU3基因的表达量的变化;通过RNAi实验,抑制PU3基因的表达,之后用扫描电子显微镜观察合浦珠母贝贝壳表面的变化。结果:合浦珠母贝PU3基因的cDNA全长为2361bp,编码618个氨基酸。氨基酸序列的功能结构域分析表明其含有4个FN3结构域。该基因在外套膜中高表达,且在外套膜边缘区的表达量高于外套膜中心区。在贝壳损伤修复的过程中,该基因的表达水平呈现上升的趋势。利用RNAi技术抑制PU3基因的表达后,贝壳的棱柱层结构发生了变化,缝隙变宽,且出现空洞。结论:PU3基因所表达的蛋白作为正调控因子参与生物矿化的过程,并主要作用于贝壳的棱柱层,抑制其表达会影响棱柱层的框架结构。     

Cu(C6H9N3O2)2Cl2对小麦的生态毒理效应

以冬小麦为实验材料,比较研究了(1)不同浓度配合物对小麦生长的影响;(2)相同浓度的CuC l2、配体C6H9N3O2和配合物Cu(C6H9N3O2)2C l2对冬小麦种子萌发、苗期生长及其保护酶活性的影响。结果表明:与对照相比,(1)不同浓度新配合物对小麦生长具有不同程度的抑制作用,随着浓度的增高抑制作用逐渐增大;(2)CuC l2、配合物Cu(C6H9N3O2)2C l2对小麦种子总淀粉酶活性、蛋白酶活性、萌发率、生长势、根长、株高、总生物量均具有显著的抑制作用,配合物Cu(C6H9N3O2)2C l2的抑制作用小于CuC l2,而配体C6H9N3O2对上述生物学参数具有促进作用;(3)CuC l2、配合物Cu(C6H9N3O2)2C l2处理引起膜脂过氧化,显著的提高了幼苗的M DA浓度,导致SOD、POD、CAT活性降低,CuC l2的抑制作用大于配合物Cu(C6H9N3O2)2C l2,而配体C6H9N3O2处理对SOD、POD、CAT活性的提高有促进作用。上述结果说明C6H9N3O2对CuC l2生理胁迫具有保护作用,结合态的Cu2 (配合物Cu(C6H9N3O2)2C l2)的毒性显著的降低。在此基础上探讨了配合物抑制小麦生长发育的生物学机制。     

氯化钴对原代大鼠肺动脉成纤维细胞的影响

目的:探究氯化钴对于原代大鼠肺动脉成纤维细胞(PAF)的增殖、迁移、表型转化作用的影响。方法:分离培养大鼠肺动脉成纤维细胞,利用氯化钴刺激PAF细胞,并通过MTT、细胞划痕、Transwell、表型转化标志蛋白测定以及PI3K/Akt信号通路蛋白的变化研究氯化钴对PAF的影响。结果:MTT结果显示,与对照组相比,氯化钴可以抑制大鼠肺动脉成纤维细胞的增殖活性(P0.001),并呈浓度依赖性。划痕实验及Transwell实验提示较高氯化钴(200μmol·L~(-1))处理PAF细胞后可以抑制细胞迁移。随浓度增加,氯化钴可以抑制PAF的P110α、p-Akt蛋白表达。结论:氯化钴对于体外培养的原代大鼠肺动脉成纤维细胞的增殖、迁移和表型转化特性有一定的抑制作用,这可能与氯化钴抑制PI3K/Akt信号通路的表达有关。     

丹参酮II-A对万古霉素所致肾小管上皮细胞KIM-1及TGF-β1表达的影响

目的:研究丹参酮II-A(TSII-A)对万古霉素(vancomycin,VAN)诱导的人肾近曲小管上皮细胞(HK-2)的存活、氧化应激水平和肾损伤分子1(KIM-1)及转化生长因子-β(TGF-β1)表达的影响。方法:将体外培养的HK-2细胞株接种于6孔培养板,分为空白组、模型组、阳性药物组和TSII-A高、中、低剂量组。加入终浓度为100μg·L-1万古霉素建立VAN损伤HK-2细胞模型,空白组、模型组加入等体积的生理盐水,阳性药物组加入终浓度为2.5 mg·L~(-1)的氨磷汀溶液,TSII-A各剂量组分别予不同浓度的TSII-A处理48 h。进一步通过MTT法测定细胞存活率;裂解细胞取上清液,紫外分光光度法监测细胞内谷胱甘肽(GSH-PX)含量,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性、硫代巴比妥酸(TBA)法测定丙二醛(MDA)含量、硝酸还原酶法测定一氧化氮(NO)活性;ELISA法测定细胞上清液中KIM-1、TGF-β1的浓度;RT-PCR监测KIM-1、TGF-β1m RNA表达。结果:与空白组比较,模型组的细胞存活率、细胞悬液中的SOD酶活性和GSH-PX含量明显减低,细胞悬液中MDA含量及NO水平明显升高,上清液中KIM-1的浓度明显升高,KIM-1m RNA的相对表达量明显上调(P0.05);TGF-β1的浓度及其m RNA的表达差异无统计学意义(P0.05);与模型组比较,阳性组和TSII-A高、中、低剂量组的细胞存活率、细胞悬液中的SOD酶活性和GSH-PX含量明显升高,细胞悬液中MDA含量及NO水平明显降低,上清液中KIM-1的浓度明显降低,KIM-1m RNA的相对表达量明显下调(P0.05),且作用均有浓度依赖性。TGFβ1的浓度及其m RNA的表达差异无统计学意义(P0.05)。结论:本研究结果显示TSII-A以剂量依赖性方式减轻VAN所致HK-2细胞损伤,可能机与其减轻氧化应激有关。     

运用微电极技术测定三角帆蚌外套膜Ca2+流量的研究

Ca既是珍珠和贝壳中的主要组成成分(CaCO3占95%以上),又是普遍存在的细胞内第二信使,在生物体内承担着非常重要生理功能。生物矿化的研究表明外套膜细胞在珍珠和贝壳生长中起着非常重要的作用[1,2],并且     

Yb3+-儿茶素配合物的抑菌活性及其机理探究

本文对Yb~(3+)-儿茶素配合物(Yb~(3+)-C)的合成、抑菌活性及抑菌机理展开了系统研究.结果表明,合成配合物Yb~(3+)-C的最适摩尔比为1∶4.Yb~(3+)-C的最小抑菌浓度(MIC)均显著低于Yb~(3+)和C,对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、绿脓杆菌(Pseudomonas aeruginosa)及沙门氏菌(Salmonella)均有较强的抑菌作用,而对金黄色葡萄球菌抑制作用更强,其MIC值低至0.0963 mmol/L.进一步研究表明,Yb~(3+)-C仅需要2 h就能杀灭细菌,可有效缩短杀菌时间.此外,扫描电镜(SEM)、透射电镜(TEM)观察结果以及Yb~(3+)在细胞膜和细胞内的吸收和分布表明,Yb~(3+)-C对细胞具有更强的亲和力与穿透力,使Yb~(3+)更容易在细胞质中累积至有效浓度,从而造成细胞结构的破坏,并引起细胞凋亡.     

Fe3O4-PEG-CD56/Avastin@Ce6纳米探针的合成及细胞MR成像初步研究

目的:探讨Fe_3O_4-PEG-CD56/Avastin@Ce6靶向探针与NK92细胞的结合能力并进行细胞体外MRI成像。方法:制备Fe_3O_4-PEG-CD56/Avastin@Ce6纳米探针,对合成的材料进行表征。应用凋亡试剂盒测定不同浓度的材料对NK92的细胞毒性,通过流式细胞术分析纳米材料与NK92细胞的结合能力和应用MRI对细胞进行体外成像并分析其T2信号强度的改变。结果:合成的纳米探针具有较好的生物相容性,且对NK92细胞的影响较小,不同浓度下细胞凋亡水平基本一致,与NK92细胞结合的材料随浓度的增加而逐渐增加。MRI检查提示不同浓度探针孵育的NK92细胞T2加权像(T2WI)的信号均降低。结论:Fe_3O_4-PEG-CD56/Avastin@Ce6探针对NK92细胞具有靶向性,3.0 T MR扫描仪可对其进行体外监测。     

Loss and Ca2+-Dependent Retention of Scinderin in Digitonin-Permeabilized Chromaffin Cells: Correlation with Ca2+-Evoked Catecholamine Release

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.     

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 microM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) could not replace GTP but prevented the action of GTP. The effects of GTP and GTP gamma S were reversible. Neither GTP nor GTP gamma S induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 microM free Ca2+, a half-maximal Ca2+ no Ca2+ release was observed with 0.1 microM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 microM) were required to evoke Ca2+ release. At 8 microM free Ca2+, even 0.25 microM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 microM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.     

Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport.

Ca2+ treatment renders the outer membrane of Escherichia coli reversibly permeable for macromolecules. We investigated whether Ca2+-induced uptake of exogenous protein into the periplasm occurs by mechanisms similar to Ca2+-induced uptake of DNA into the cytoplasm during transformation. Protein import through the outer membrane was monitored by measuring reconstitution of maltose transport after the addition of shock fluid containing maltose-binding protein. DNA import through the outer and inner membrane was measured by determining the efficiency of transformation with plasmid DNA. Both processes were stimulated by increasing Ca2+ concentrations up to 400 mM. Plasmolysis was essential for a high efficiency; reconstitution and transformation could be stimulated 5- and 40-fold, respectively, by a high concentration of sucrose (400 mM) in cells incubated with a suboptimal Ca2+ concentration (50 mM). The same divalent cations that promote import of DNA (Ca2+, Ba2+, Sr2+, Mg2+, and Ni2+) also induced import of protein. Ca2+ alone was found to be inefficient in promoting reconstitution; successive treatment with phosphate and Ca2+ ions was essential. Transformation also was observed in the absence of phosphate, but could be stimulated by pretreatment with phosphate. The optimal phosphate concentrations were 100 mM and 1 to 10 mM for reconstitution and transformation, respectively. Heat shock, in which the cells are rapidly transferred from 0 to 42 degrees C, affected the two processes differently. Incubation of cells at 0 degrees C in Ca2+ alone allows rapid entry of protein, but not of DNA. Transformation was observed only when exogenous DNA was still present during the heat shock. Shock fluid containing maltose-binding protein inhibited transformation (with 6 microgram of DNA per ml, half-maximal inhibition occurred at around 300 microgram of shock fluid per ml). DNA inhibited reconstitution (with 5 microgram of shock fluid per ml, half-maximal inhibition occurred at around 3 mg of DNA per ml).     

The role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes.

To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.     

Angiotensin II Effects on the Cytosolic Free Ca2+ Concentration in N1E-115 Neuroblastoma Cells: Kinetic Properties of the Ca2+ Transient Measured in Single Fura-2-Loaded Cells

The effect of angiotensin II on the cytosolic free Ca2+ concentration was measured in single mouse neuroblastoma N1E-115 cells loaded with fura-2. Angiotensin II induced a transient concentration-dependent increase in Ca2+ and also increased the production of inositol polyphosphates. The Ca2+ increase did not require extracellular Ca2+ and was unaffected by pretreatment with pertussis toxin. These data suggest that angiotensin II increased Ca2+ by an inositol trisphosphate-mediated release of intracellular Ca2+ following activation of phospholipase C via a pertussis toxin-insensitive guanine nucleotide binding protein. Similar results were obtained with bradykinin. The angiotensin II- or bradykinin-induced increase in Ca2+ occurred after a concentration-dependent latent period. Low concentrations of agonist elicited a small increase in Ca2+ following a variable lag that sometimes exceeded 1 min, whereas at maximally effective angiotensin II concentrations a larger, more rapid increase in Ca2+ occurred without a measurable delay. In some cells, oscillatory increases in Ca2+ were induced by angiotensin II and bradykinin. Possible mechanisms to explain the concentration dependency of the latent period and the oscillatory nature of the increases of Ca2+ are discussed. These results indicate that the mouse neuroblastoma N1E-115 cell represents a useful model for studying the signal response transduction mechanisms regulating the effects of angiotensin II in neuronal cells.     

Recording of intracellular Ca2+ from smooth muscle cells by sub-micron tip, double-barrelled CA2+-selective microelectrodes

Novel, double-barrelled Ca2+-selective microelectrodes with tip diameters of approximately 0.1 micron were constructed by using Simon's neutral Ca2+ ligand (ETH 1001). Concentric micropipettes were utilized for the first time for Ca2+-selective microelectrodes in which the Ca2+ ligand was incorporated into a protruding inner pipette, surrounded by an outer reference electrode. In addition, they were made from high resistance aluminosilicate glass tubing (Corning Code 1724). These Ca2+-selective electrodes had linear responses from pCa 3 to pCa 7 in the presence of constant [K+]. They provided on-line observation of changes in intracellular [Ca2+] and in the resting membrane potential in single smooth muscle cells isolated from toad stomach. The mean concentration of intracellular Ca2+ in resting cells was 163.6 +/- 20 nM (+/- SEM, n = 16). Doubling the intracellular Ca2+ level by exposure of cells to elevated [K+] was sufficient to cause shortening.     

Effects of Substance P on Carbachol-Stimulated 45Ca2+ Uptake into Cultured Adrenal Chromaffin Cells

Substance P is known to modulate acetylcholine-induced catecholamine release from adrenal chromaffin cells. To investigate the mechanisms involved in this modulation, the present study examined the effects of substance P on net 45Ca2+ fluxes in cultures of bovine adrenal chromaffin cells. Two effects of substance P were observed: (1) Substance P inhibited carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux and (2) substance P protected against desensitization of carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux. Thus substance P modulates two other cholinergic responses, 45Ca2+ uptake and 45Ca2+ efflux, in a manner similar to its modulation of catecholamine release. The results also indicate that substance P's inhibition of net carbachol-induced 45Ca2+ uptake is due to inhibition of 45Ca2+ uptake rather than enhancement of 45Ca2+ efflux. Substance P almost completely inhibited carbachol-induced 45Ca2+ uptake in both Na+-containing and Na+-free media, suggesting that substance P can inhibit the uptake of 45Ca2+ induced by carbachol regardless of whether 45Ca2+ is taken up through voltage-sensitive or acetylcholine receptor-linked channels. However, substance P produced only a small inhibition of K+-induced 45Ca2+ uptake, indicating that substance P does not interact directly with voltage-sensitive Ca2+ channels. In addition, substance P's inhibition of carbachol-induced 45Ca2+ uptake was noncompetitive with respect to Ca2+, were unable to overcome substance P's inhibition of [3H]-norepinephrine ( [3H]NE) release. It is concluded that substance P does not interact directly with Ca2+ channels in bovine adrenal chromaffin cells.     

Calcium localization in the shell-forming tissue of the freshwater snail,Biomphalaria glabrata: a comparative study of various methods for localizing calcium

Summary The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snailBiomphalaria glabrata (Planorbidae, Basommatophora).In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue.The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.