Influence of isolation media on synaptosomal properties: Intracellular pH,pCa, and Ca2+ uptake

Preparations of synaptosomes isolated in sucrose or in Na⁺-rich media were compared with respect to internal pH (pH₁), internal Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and⁴⁵Ca²⁺ uptake due to K⁺ depolarization and Na⁺/Ca²⁺ exchange. We found that synaptosomes isolated in sucrose media have a pH_i of 6.77±0.04 and a [Ca²⁺]_i of about 260 nM, whereas synaptosomes isolated in Na⁺-rich ionic media have a pH_i of 6.96±0.07 and a [Ca²⁺]_i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca²⁺ only by voltage sensitive calcium channels (VSCC'S) when K⁺-depolarized, while the Na⁺-rich synaptosomes take up⁴⁵Ca²⁺ both by VSCC'S and by Na⁺/Ca²⁺ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca²⁺ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca²⁺ uptake by VSCC'S, but not by Na⁺/Ca²⁺ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca²⁺ flux through channels and through Na⁺/Ca²⁺ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca²⁺ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF 2,7-Biscarboxyethyl-5(6)-carboxyfluorescein - BCECF/AM acetoxymethyl ester of BCECF - [Ca²⁺]_i Internal free calcium ion concentration - CBZ-DMB 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil - DMB 2, 4-dimethylbenzamil - DMSO dimethyl sulfoxide - Indo-1/AM acetoxymethyl ester of Indo-1 - MES 2-|N-Morpholino|ethanesulfonic acid - NMG N-methyl-D-glucamine - pH_i internal pH - TPP⁺ tetraphenylphosphonium - _p plasma membrane potential     

消炎痛对大鼠肝线粒体微粒体^45Ca摄取及膜流动性的影响

我们曾报道消炎痛预处理在大鼠能引起明显的肝保护作用,为了进一步探讨消炎痛肝保护作用的机制,本工作观察了它对大鼠肝线粒体、微粒体钙调节作用及膜流动性的影响。结果表明,经消炎痛整体预处理的大鼠肝线粒体和微粒体的钙摄取及膜流动性均明显增加,但是,将消炎痛直接加入由正常大鼠分离的线粒体或微粒体中,则反而使膜的流动性降低。这些变化可能与消炎痛的肝保护作用有关。     

EMA,an epithelial membrane-associated antigen during early development and morphogenesis ofXenopus laevis

Summary We raised monoclonal antibodies against a membrane fraction ofXenopus neurulae in order to detect tissue-specific cell-surface markers. Here we describe a monoclonal antibody that recognizes an epithelial membrane-associated antigen (EMA) in immunohistological stainings. The tissue-specific and membrane-associated antigen detected in immunohistological stainings could serve as useful marker in epithelium differentiation and membrane organization of the early embryo. In tadpoles and adults EMA was found in specific epithelial tissues derived from different germ layers such as kidney, skin, gut, pancreas, epiphysis and choroid plexus. In the cleaving embryo this antibody stained newly formed membranes between blastomeres from the two-cell stage onwards. Cytoplasmic staining in large oocytes and early embryos was also observed. The possibility that the cytoplasmic signal represents a maternal store of membrane material is discussed.     

Further purification and partial characterization of the rat lung cytoplasmic factors modulating adenylate cyclase activity in plasma membrane

Summary The adult rat lung cytoplasm contains some factors which markedly stimulate adenylate cyclase activity in plasma membranes (Nijjar, M. S. Biochim. Biophys. Acta 584:43–50, 1979). Adenylate cyclase activator (ACA) was purified from rat lungs by DEAE-cellulose chromatography, preparative isoelectric focusing and by repeated high-performance liquid chromatography on a Sepharogel TSK 2000SW column. The final preparation showed about 200 fold purification in ACA activity over the original lung supernatant, and appeared to be homogeneous on the basis of its migration into a single band on SDS-polyacrylamide gel electrophoresis, and co-elution of ACA activity with protein from a gel exclusion column. ACA is an acidic (pl 4.8 ± 0.1), heat labile, monomeric protein of 40000 ± 2000 dalton molecular weight, and does not resemble calmodulin.     

Molecular topography of the Ca2+ — ATPase of sarcoplasmic reticulum

Summary The Ca²⁺ binding site region of the Ca²⁺ — ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas [1, 2], in the absence of Ca²⁺ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca²⁺ occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16 - > 12- > 10- > 7- > 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I^– indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca²⁺ binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- > 7- > 10- > 12- > 16-NS. The trp residue(s) which changes its fluorescence upon Ca²⁺ binding is shown to be near the membrane surface.     

Time and site of assembly of the peritrophic membrane of the mosquito Aedes aegypti

Summary We determined the time and site of secretion of the precursors of the peritrophic membrane (PM) in Aedes aegypti and when the structure is assembled. The fine structure of the developing membrane of blood-feed females was described, and the pattern of secretion of injected tritiated glucosamine analyzed autoradiographically. Immediately following blood feeding, ingested red cells rapidly become compressed, such that the surrounding plasma is extruded to the margin of the midgut contents. Thereby, ingested fluids form a narrow margin separating the blood mass from the midgut epithelium. By electron microscopy, the PM first becomes evident at about 4 to 8 h after blood is ingested, and the membrane attains mature texture by 12 h. The compacted mass of ingested erythrocytes seems to serve as a template for the forming structure. In contrast, tritiated glucosamine, injected into freshly engorged mosquitoes, begins to concentrate on the midgut microvilli by 2 h after feeding. By 8 h the label assumes the layered appearance that characterizes the fine structure of the mature membrane. In contrast to the prevailing concept that the PM of mosquitoes first assumes texture anteriorly immediately after blood is ingested, we find that this potential barrier to pathogen development forms no earlier than 4 h after feeding and that it is formed from precursors secreted along the entire length of the epithelium overlying the food mass.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

Chromosomes and their relationship to nuclear components during the cell cycle in Chinese hamster cells

Summary Chromosomes and their relationship to nuclear components during various phases of the cell cycle were studied with different fixation, embedding, and enzyme techniques. The results showed that interphase chromosomes may have oriented in such a way that a given locus became associated with the nuclear membrane. Some chromosomes also appeared to interact with the nucleolus. The nuclear matrix materials, however, were distributed between the chromosomes and formed a delineating boundary for the chromosomes. These matrix materials, furthermore, formed channel-like structures within the nucleus and towards the cytoplasm through their interaction with nuclear pore complexes. During mitosis, chromosomes were encapsulated with material that appeared to be derived from the matrix, disintegrated residues and fragments of the nuclear envelope, the lamina, and nucleolar material. These chromosome-associated materials seen in mitosis appeared to serve as foci for formation of new nuclear components in subsequent interphase.     

Lipid detection by malachite green-aldehyde in the dental basement membrane in the rat incisor

Summary Developing rat incisors were treated with malachite green-aldehyde fixative solution (MGA), which retains and stains lipids. We observed positive staining occurring as dots in the basement membrane. Most of these dots (2–3.5 nm in diameter) were grouped in the lamina densa but some were also present in the lamina lucida and the lamina fibroreticularis. These data provide evidence for the existence of lipids in the dental basement membrane and suggest that they are distributed together with the various groups of proteins so far detected.     

Epidermal growth factor (EGF)-induced morphological changes in the basement membrane of chick embryonic skin

Summary The effect of epidermal growth factor (EGF) on the basement membrane structure of chick embryonic skin cultured in a chemically defined medium (BGJb) containing 20 mM hydrocortisone, and EGF at 10, 50, or 100 ng/ml supplemented with 5% delipidized fetal calf serum, was examined by electron microscopy. During development of the epidermis in vitro, EGF (100 ng/ml) caused striking changes to occur in the basement membrane structure and in the keratinization process. The basement membrane frequently became discontinuous with many gaps apparent in section, and occasionally became folded following detachment from the basal surface of the epidermis and protruded into the underlying dermis. In the basal and intermediate cells of EGF-treated epidermis, tonofilament bundles were decreased in number, while desmosomes and hemidesmosomes revealed no significant changes in morphology.