Endosomes from kidney collecting tubule cells contain the vasopressin-sensitive water channel

The mechanism by which vasopressin rapidly and dramatically increases the water permeability of target epithelial cell membranes is thought to involve a cycle of exo- and endocytosis during which vesicles carrying 'water channels' are successively inserted into, and removed from the apical plasma membrane of epithelial cells. Clusters of intramembranous particles, visible by freeze-fracture electron microscopy and presumed to represent water channels, appear on apical membranes in parallel with increased transepithelial water flow. In the collecting duct, these clusters are located in clathrin-coated pits which are subsequently internalized. There has been no direct evidence, however, that subcellular membranes in vasopressin-sensitive epithelia contain functional water channels. In this report, we have used fluorophores that are sensitive to volume and do not pass through membranes to label and to measure directly the osmotic water permeability of endocytosed vesicles isolated from renal papilla. We present direct evidence that vasopressin induces the appearance of a population of endocytic vesicles whose limiting membranes contain water channels.     

Chloride and potassium channels in cystic fibrosis airway epithelia

Cystic fibrosis, the most common lethal genetic disease in Caucasians, is characterized by a decreased permeability in sweat gland duct and airway epithelia. In sweat duct epithelium, a decreased Cl- permeability accounts for the abnormally increased salt content of sweat. In airway epithelia a decreased Cl- permeability, and possibly increased sodium absorption, may account for the abnormal respiratory tract fluid. The Cl- impermeability has been localized to the apical membrane of cystic fibrosis airway epithelial cells. The finding that hormonally regulated Cl- channels make the apical membrane Cl- permeable in normal airway epithelial cells suggested abnormal Cl- channel function in cystic fibrosis. Here we report that excised, cell-free patches of membrane from cystic fibrosis epithelial cells contain Cl- channels that have the same conductive properties as Cl- channels from normal cells. However, Cl- channels from cystic fibrosis cells did not open when they were attached to the cell. These findings suggest defective regulation of Cl- channels in cystic fibrosis epithelia; to begin to address this issue, we performed two studies. First, we found that isoprenaline, which stimulates Cl- secretion, increases cellular levels of cyclic AMP in a similar manner in cystic fibrosis and non-cystic fibrosis epithelial cells. Second, we show that adrenergic agonists open calcium-activated potassium channels, indirectly suggesting that calcium-dependent stimulus-response coupling is intact in cystic fibrosis. These data suggest defective regulation of Cl- channels at a site distal to cAMP accumulation.     

Rapid gating and anion permeability of an intracellular aquaporin

Aquaporin (AQP) water-channel proteins are freely permeated by water but not by ions or charged solutes. Although mammalian aquaporins were believed to be located in plasma membranes, rat AQP6 is restricted to intracellular vesicles in renal epithelia. Here we show that AQP6 is functionally distinct from other known aquaporins. When expressed in Xenopus laevis oocytes, AQP6 exhibits low basal water permeability; however, when treated with the known water channel inhibitor, Hg2+, the water permeability of AQP6 oocytes rapidly rises up to tenfold and is accompanied by ion conductance. AQP6 colocalizes with H+-ATPase in intracellular vesicles of acid-secreting alpha-intercalated cells in renal collecting duct. At pH less than 5.5, anion conductance is rapidly and reversibly activated in AQP6 oocytes. Site-directed mutation of lysine to glutamate at position 72 in the cytoplasmic mouth of the pore changes the cation/anion selectivity, but leaves low pH activation intact. Our results demonstrate unusual biophysical properties of an aquaporin, and indicate that anion-channel function may now be explored in a protein with known structure.     

An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.     

Molecular identification of the gene responsible for congenital nephrogenic diabetes insipidus.

Antidiuretic hormone (arginine vasopressin) binds to and activates V2 receptors in renal collecting tubule cells. Subsequent stimulation of the Gs/adenylyl cyclase system promotes insertion of water pores into the luminal membrane and thereby reabsorption of fluid. In congenital nephrogenic diabetes insipidus (CNDI), an X-linked recessive disorder, the kidney fails to respond to arginine vasopressin. Here we report that an affected male of a family with CNDI has a deletion in the open reading frame of the V2 receptor gene, causing a frame shift and premature termination of translation in the third intracellular loop of the receptor protein. A normal receptor gene was found in the patient's brother. Both the normal and the mutant allele were detected in his mother. A different mutation, causing a codon change in the third transmembrane domain of the V2 receptor, was found in the open reading frame of an affected male but not in the unaffected brother belonging to another family suffering from CNDI.     

Carbon-dioxide-induced exocytotic insertion of H+ pumps in turtle-bladder luminal membrane: role of cell pH and calcium

The contents of endocytic vesicles and other intracellular organelles (such as Golgi and microsomes) are acidified by an electrogenic proton-translocating ATPase that is remarkably similar to that found in urinary epithelia. We recently found that the number of H+ ATPases in the apical plasma membrane of these epithelia is regulated by exocytotic insertion of endocytic vesicles whose membranes contain this H+ pump. Carbon dioxide, a major stimulus for urinary acidification, causes rapid fusion of these vesicles with the luminal membrane, thereby inserting these pumps there and increasing the rate of net transepithelial H+ secretion; CO2 also inhibits endocytic retrieval of the pumps from the luminal membrane. Such reciprocal regulation of endocytosis and exocytosis by a physiological modulator makes this system particularly attractive for studying the cellular events regulating membrane fusion. Here we present evidence that CO2 induces exocytosis by a cascade of events, the first step of which is cytoplasmic acidification. Cell acidification then increases calcium activity, which causes the fusion event.     

The appearance of single-ion channels in unmodified lipid bilayer membranes at the phase transition temperature

Ion-specific channels in artificial membranes have been formed by the addition of gramicidin A, alamethicin, polyene antibiotics and some proteins to the solution surrounding the bilayer lipid membrane. Until now there have been no reports of single-ion channels in unmodified lipid membranes. We have now studied the electrical conductance of planar lipid bilayers membranes made of synthetic distearoylphosphorylcholine (DSPC). Current fluctuations of amplitude approximately 1pA and duration approximately 1 s have been discovered at phase transition temperature, which shows that the appearance of ionic channels may be the result of lipid domain interactions. This would explain the dramatic increase in ion permeability observed in liposomes during phase transition. We suggest that these channels could conduct the transmembrane ionic current in biological membranes without the involvement of peptides and proteins.     

Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium

Chloride (Cl-) secretion by the airway epithelium regulates, in part, the quantity and composition of the respiratory tract fluid, thereby facilitating mucociliary clearance. The rate of Cl- secretion is controlled by apical membrane Cl- channels. Apical Cl- channels are opened and Cl- secretion is stimulated by a variety of hormones and neurotransmitters that increase intracellular levels of cyclic AMP (cAMP). In cystic fibrosis (CF), a common lethal genetic disease of Caucasians, airway, sweat-gland duct, secretory-coil and possibly other epithelia are anion impermeable. This abnormality may explain several of the clinical manifestations of the disease. The Cl- impermeability in CF-airway epithelia has been localized to the apical cell membrane, where regulation of Cl- channels is abnormal: hormonal secretagogues stimulate cAMP accumulation appropriately but Cl- channels fail to open. Here we report that the purified catalytic subunit of cAMP-dependent protein kinase plus ATP opens Cl- channels in excised, cell-free patches of membrane from normal cells, but fails to open Cl- channels in CF cells. These results indicate that in normal cells, the cAMP-dependent protein kinase phosphorylates the Cl- channel or an associated regulatory protein, causing the channel to open. The failure of CF Cl- channels to open suggests a defect either in the channel or in such an associated regulatory protein.     

黄缘盒龟肝脏与肾脏的超微结构

在透射电子显微镜下观察黄缘盒龟的肝脏和肾脏的超微结构.结果显示肝实质内结缔组织少,肝小叶分界不清楚;肝细胞具两种形态,即L细胞和D细胞.肾小球由毛细血管构成;肾小囊外壁由扁平细胞构成,内壁可见足细胞附于其上;颈段由单层纤毛立方上皮细胞构成,细胞核较大,细胞游离面有很多纤毛伸向管腔,细胞核周围排列密集的线粒体;近曲小管由单层柱状上皮细胞构成,细胞核圆形,位于细胞中部,胞质内含有丰富的线粒体;中间段由单层纤毛立方上皮构成;远曲小管由单层柱状上皮细胞构成,细胞核位于细胞中部,其周围分布有许多线粒体,细胞底部可见丰富的质膜内褶;收集管由单层柱状上皮细胞组成,胞质内可见较丰富的膜迷路.黄缘盒龟肝脏的超微结构与其他爬行动物相似.肾脏结构与其生态习性有关.     

Basolateral KCl co-transport in a NaCl-absorbing epithelium

In NaCl-absorbing epithelia such as proximal renal tubule, small intestine and gallbladder, Na+-dependent Cl- entry across the luminal membrane is an electroneutral transport process that has been attributed to Na-Cl symport, Na-K-Cl symport, or a double (Na-H, Cl-HCO3) antiport. At the basolateral (antiluminal) membrane, Na+ extrusion can be attributed to the Na+-K+ pump, and Cl- transport could be explained in principle by electrodiffusion since the intracellular Cl- activity is higher than predicted for equilibrium distribution. However, in Necturus gallbladder, as in other epithelia, the electrodiffusional Cl- permeability of the membrane (PCl) is too low to account for the transepithelial Cl- transport rate. Because K+ is at a higher chemical potential in the cell than in the extracellular fluid, and because serosal Cl- substitutions have only small effects on membrane potential, the hypothesis of carrier-mediated electroneutral KCl co-transport was proposed. The experiments reported here were designed to test this hypothesis in Necturus gallbladder epithelium. Intracellular Cl- and K+ activities (aCli, aKi) were measured with ion-sensitive intracellular microelectrodes before, during and after ionic substitutions of the serosal (basolateral) bathing medium. The results demonstrate a Na+-independent basolateral membrane KCl symport.     

Segregation of receptor and ligand regulates activation of epithelial growth factor receptor

Interactions between ligands and receptors are central to communication between cells and tissues. Human airway epithelia constitutively produce both a ligand, the growth factor heregulin, and its receptors--erbB2, erbB3 and erbB4 (refs 1-3). Although heregulin binding initiates cellular proliferation and differentiation, airway epithelia have a low rate of cell division. This raises the question of how ligand-receptor interactions are controlled in epithelia. Here we show that in differentiated human airway epithelia, heregulin-alpha is present exclusively in the apical membrane and the overlying airway surface liquid, physically separated from erbB2-4, which segregate to the basolateral membrane. This physical arrangement creates a ligand-receptor pair poised for activation whenever epithelial integrity is disrupted. Indeed, immediately following a mechanical injury, heregulin-alpha activates erbB2 in cells at the edge of the wound, and this process hastens restoration of epithelial integrity. Likewise, when epithelial cells are not separated into apical and basolateral membranes ('polarized'), or when tight junctions between adjacent cells are opened, heregulin-alpha activates its receptor. This mechanism of ligand-receptor segregation on either side of epithelial tight junctions may be vital for rapid restoration of integrity following injury, and hence critical for survival. This model also suggests a mechanism for abnormal receptor activation in diseases with increased epithelial permeability.     

毛果冷杉湿心材的闭塞纹孔及细菌对木材结构的降解

扫描电镜观察到毛果冷杉湿心材部位管胞具缘纹孔纹孔膜上存在着水痕状、片状或块状的细菌,而在正常材部位没有发现。细菌的活动不但使湿心材部位的部分射线薄壁细胞发生降解,而且纹孔膜塞缘的微纤丝束也偶尔被降解而产生断裂。生材时,湿心材管胞间的具缘纹孔纹孔膜严重结壳并闭塞,早、晚材管胞具缘纹孔的闭塞率分别为77.7%和72.1%,而正常材大多数是非闭塞的,早、晚材的闭塞程度分别只有6.8%和13.4%。湿心材中存在的大量闭塞纹孔,是导致木材干燥困难的主要原因。     

Molecular cloning of the receptor for human antidiuretic hormone.

Antidiuresis, the recovery of water from the lumen of the renal collecting tubule, is regulated by the hypothalamic release of antidiuretic hormone (ADH), which binds to specific receptors on renal collecting tubule cells, stimulates adenylyl cyclase and promotes the cyclic AMP-mediated incorporation of water pores into the luminal surface of these cells. We report here the isolation of the human ADH receptor gene using a genomic expression cloning approach. The gene was used to clone the complementary DNA from a human renal library. The deduced amino-acid sequence of the receptor yields a hydropathy profile characteristic of receptors with seven putative transmembrane regions. This and the comparison with other cloned receptors indicates that the ADH receptor is a member of the superfamily of G-protein-coupled receptors.     

成膜温度对磺化聚醚醚酮质子交换膜性能的影响

以浓硫酸为磺化试剂制备相同磺化度的磺化聚醚醚酮(SPEEK),采用等体积的水/醇混合溶剂通过溶液成膜的方法制备SPEEK质子交换膜,采用交流阻抗法、扩散池法评价膜的质子传导能力和阻醇性能,探讨溶液成膜温度对质子交换膜性能的影响规律。结果表明,采用水/醇混合物为溶剂的质子交换膜,随着成膜温度的提高,膜的吸水率、溶胀率、质子电导率和甲醇渗透率均降低。     

聚乙二醇-丝素共混膜物理性能的研究

不同相对分子质量的聚乙二醇(PEG)以不同比例混入丝素可形成共混丝素膜，并具有良好的拉伸性、透水、透；气、透光性和一定的生物降解与溶解性。实验结果表明：虽PEG和丝素的可混合性稍差，但在一定的透水、透汽性和较好的柔韧性前提下，可达到创面膜的基本要求。     

毛竹材导管分子的纹孔特征

【目的】探究毛竹材导管细胞壁上纹孔结构的主要特征,为深入认识竹类植物生理输导作用和竹材横向渗透机制提供理论依据。【方法】通过观测毛竹(Phyllostachys edulis(Carr.)J.Houz.)材导管及导管树脂铸型的扫描电镜图像,研究纹孔的几何形态、分布规律和纹孔膜结构等特征。【结果】依据纹孔缘与纹孔口的相对位置和大小关系,将毛竹导管具缘纹孔分为两侧均没有纹孔缘(PⅠ)、仅在单侧有纹孔缘(PⅡ)和两侧都有纹孔缘(PⅢ)3种类型。纹孔内口和外口(短轴方向)尺寸分别为0.9～2.7 μm和1.1～3.8 μm; 纹孔膜有微纤丝暴露与被覆盖两种状态,多数具渗透性; 后生木质部导管上的纹孔呈选择性分布,总是密集于靠近维管束几何中心的一侧管壁; 毛竹维管束内分散有小导管,且相互以具缘纹孔对连接; 部分导管内含有侵填体。【结论】综合运用常规解剖方法和树脂铸型技术,不仅可以较系统地研究导管主要特征,还发现了毛竹材中特殊的导管结构和纹孔分布规律。     

Structure and Properties of Polytetrafluorethylene and Polyurethane Layered Membrane for Protective Clothing

IntroductionProtective clothing articles used for wear in wetconditions ; in outdoor activities ; in handling hazardouschemicals , in preventing contamination, in avoidinginfection, should in each instance protect the wearer bypreventing leakage of water or other fluids andmicroorganisms into the article while keeping the wearercomfortable by allowing perspiration to evaporate from thewearer to the outside of the article .In additionto the above ,the amount of stretch and its recovery and the …     

改性聚酰亚胺膜的水蒸气渗透性和空气除湿性能

考察了改性聚酰亚胺(P I)膜的亲水性、水蒸气渗透性、制膜混合溶液的表观粘度。结果表明:随着季胺盐含量的增加,膜的亲水性增强,水分子的渗透系数增加,同时水分子的渗透受膜的厚度和相对湿度的影响,制膜混合溶液的表观粘度增大,出现了盐增粘现象。测试了一系列共混改性中空纤维膜组件的除湿性能,增大压力和增加吹扫气流量,对组件除湿十分有利,尽管在低吹扫气量的情况下,组件的除湿性能并不是一直随着膜的亲水性增强而提高,但是适当增加亲水性盐的含量,膜的除湿性能会得到有效改善。     

水分胁迫对枣树组培苗渗透调节物质的影响

水分胁迫条件下分别测定了继代苗抗性不同的品种叶片中脯氨酸、可溶性总糖、丙二醛含量、叶片质膜相对透性。结果表明:随着干旱胁迫程度的加剧,不同品种间叶片脯氨酸、可溶性总糖、丙二醛含量、叶片质膜相对透性存在较大差异。表现出较抗旱的品种叶片中脯氨酸、可溶性糖含量变化表现出较高的趋势;而丙二醛含量和质膜相对透性变化表现出较低的趋势。     

Fc receptor phosphorylation during receptor-mediated control of B-cell activation

It is well known that Fc receptors for IgG (FcRII) on macrophages mediate the endocytosis of antibody-antigen complexes and signal the release of inflammatory and cytotoxic agents. FcRII are also expressed at high levels on B cells where they are less involved in endocytosis than in modulating B-cell activation by membrane immunoglobulins. Although crosslinking of membrane immunoglobulins can result in B-cell differentiation and proliferation through stimulation of phospholipase C, mobilization of intracellular Ca2+, and activation of protein kinase C, crosslinking FcR with membrane immunoglobulins confers a dominant inhibitory signal that prevents or aborts activation. This form of regulation may have a role in the induction of tolerance by IgG and in controlling the B-cell repertoire by anti-idiotypes. The different functions of FcR on B cells and macrophages may reflect the fact that these cell types express closely related but distinct FcR isoforms. We have recently found that the main lymphocyte FcR isoform, FcRII-B1, is unable to mediate endocytosis by way of coated pits and coated vesicles owing to an in-frame insertion of 47 amino acids in its cytoplasmic tail. Here we show that this insert, absent from the FcRII-B2 macrophage isoform, also contains serine phosphorylation sites that may have a role in the ability of FcR to regulate B-cell activation through membrane immunoglobulins.