Role of prostaglandin E2 in regulation of low and high water osmotic permeability in frog urinary bladder.

The water osmotic permeability of frog urinary bladder was found to be increased from 0.08 +/- 0.01 to 1.28 +/- 0.20 microl/min cm2 when serosal bathing medium was changed 4 times for a fresh Ringer solution. High epithelium permeability is accompanied by an increased content of cyclic AMP in the bladder tissue (by 42%, P < 0.01), higher activity of both basal and forskolin-stimulated membrane adenylate cyclase (AC) (by 109% and 74%, respectively, P < 0.05) and by appearance of aggregates of intramembranous particles in the apical membrane. The water flow was inhibited by 10(-9)-10(-5) M prostaglandin E2 (PGE2); the inhibitory effect was eliminated in the presence of 10(-4) M N-ethylmaleimide. The increase of water permeability due to changes of the bathing medium was accompanied by a decrease of serosal PGE2 concentration from 14.8 +/- 1.0 in the 1st solution to 0.6 +/- 0.1 nM in the 5th. 10(-6) M PGE2 in vitro inhibited the activity of membrane AC from highly permeable bladders by 33.4% (P < 0.02). Pretreatment of the membranes with 10 microg/ml pertussis toxin (PT) completely reversed this effect (+149%, P < 0.01). A significant activation of AC was also observed under 10(-10) M PGE2 (by 196%). These data demonstrate that the water permeability could be markedly increased independently of ADH, suggesting that the trigger role in activation of water transport is played by a decreased level of PGE2 which could stimulate AC.

[Analysis of the mechanism of the fixation of membrane structures using osmium tetroxide. II. An electron microscopic and x-ray structural study of myelin frozen and lyophilized at low temperature]. / Analiz mekhanizma fiksatsii membrannykh struktur chetyrekhokis'iu osmiia. II. Elektronnomikroskopicheskoe i rentgenostrukturnoe issledovanie zamorozhennogo i liofilizirovannogo pri nizkoi temperature mielina.

Comparative electron microscope and X-ray studies were made on the frog sciatic nerve myelin after freeze-drying technique. The specimens were fixed with OsO4 before and after freeze-drying. In the latter case, osmium was used as a hydrophobic solution (OsO4 in CCl4), or in the high vacuum during osmium sublimation. The results obtained in this study do not fit in the accepted mechanism operating during osmium fixation of membranes. Another mechanism is proposed by the authors, and the problem of osmium localization within the space of the myelin repeated unit is discussed.

[Method of purifying glutaraldehyde for microscopic research on biological objects]. / Metod ochistki glutarovogo al'degida dlia mikroskopicheskikh issledovanii biologicheskikh ob''ektov.

A simple and reliable method of purification and determination of glutaraldehyde concentration for histochemical fixation is proposed. Purification of glutaraldehyde is provided by vacuum distillation with a rotational-filmy evaporator, and its concentration is determined using refractometer.

[Cholesterol localization in the membranes of granular cells in the bladder epithelium of the frog during stimulated water transport]. / Lokalizatsiia kholesterina v membranakh granuliarnykh kletok épiteliia mochevogo puzyria liagushki pri stimulirovannom transporte vody.

The polyene antibiotic filipin has been used to characterize the cholesterol distribution in the membranes of resting and ADH-stimulated frog urinary bladder in freeze-fracture replicas. In general, the intracellular membranes takes up filipin only insignificantly. An exception is the cholesterol rich granule membrane. Both density and polarity of filipin-induced deformations were evaluated, and the asymmetry in membrane cholesterol was analysed. Upon ADH-stimulation of water flow both density and polarity of filipin-induced deformations altered differently in apical and basolateral regions of the plasma membrane. This difference is presumably due to the stretching of the basolateral membrane as a result of swelling, on the one hand, and to incorporation of aggregate containing membranes into the apical membrane, on the other one. The results obtained may suggest that the appearance of ADH-induced intramembranous particle aggregates in the apical membrane be accompanied with a relative cholesterol decrease in this apical membrane.

[Ultrastructure and elemental composition of frog bladder granular epithelial cells in normal state and upon stimulation of water transport]. / Ul'trastruktura i élementnyi sostav granuliarnykh kletok épiteliia mochevogo puzyria liagushki v norme i pri stimuliatsii transporta vody.

Changes in the frog urinary bladder granular cell ultrastructure were analysed in parallel with those in element composition of these cells after induction of water transport across the urinary bladder wall. Two ultrastructural (ultrathin section and freeze-fracture) methods were used in addition to two methods of object preparation for electron microprobe analysis--freeze-drying and freeze-substitution. It has been shown that arginin-vasotocin stimulation of osmotic water flow across the urinary bladder wall causes certain morphological changes in the granular cells: decrease in electron density of the cytoplasm, depolymerization of the apical submembrane layer of actin microfilaments, increase in the number of sites of specific granules and apical membrane fusion, emergency of intramembrane particle aggregates in the apical membrane P-face. The quantitative electron microprobe analysis made it possible to reveal a statistically significant increase in sodium and calcium concentration and fall in that of potassium and chlorine in granular cells after water transport stimulation. A concentration gradient of sodium and potassium ions was seen to appear along the apical-basal axis in the cytoplasm of granular cells. Possible association between the obvious morphological transformations in granular cells and changes in their elemental composition has been discussed, in addition to some regulatory significance of calcium concentration increase in granular cells after arginin-vasotocin-induced osmotic water transport.

[Change in distribution of actin and intermediate filaments at early stages of action of epidermal growth factor on A431 cells]. / Izmenenie razpredeleniia aktinovykh i promezhutochnykh filamentov na rannikh srokakh deistviia épidermal'nogo faktora rosta na kletki A431.

By electron microscopy using immunocytochemical reactions on actin and EGF-R with mild glycerinization it became possible to reveal a thick layer of actin filaments under the apical membrane within the first minutes of the impact of EGF on A431 cells. Keratin fibrils being located near the nucleus are transported apart from the membrane. It is supposed that this process is connected with retardation of capping on principles of competition.

[Electron microscopic study of the chemical structural asymmetry of myelin sheaths]. / Elektronno-mikroskopicheskoe issledovanie strukturno-khimicheskoi asimmetrii membran mielina.

Osmium washing of ultrathin sections of the frog sciatic nerve myelin fibres performed with 4% acetic acid solution preceded their treatment with the Dragendorf solution and ammonium paramolibdate. This enabled us to localize amine and phosphate groups of myelin membranes. The data obtained suggest that the majority of phospholipids of these membranes are located on the cytoplasmic surfaces, whereas glycolipids and cholesterol--on the extracellular ones. Examination of these surfaces by freeze--fracturing revealed different hydratation degrees in these surfaces. These results show that myelin membranes are most convenient models for the study of structural and chemical asymmetry of biological membranes.

[Tubular structures in Mycoplasma gallisepticum and the localization of a tubulin-like protein]. / Tubuliarnye struktury Mycoplasma gallisepticum i lokalizatsiia tubulinopodobnogo belka.

In all the strains of M. gallisepticum investigated, a protein with apparent molecular weight 40 kDa was revealed by immunoblotting with polyclonal anti-calf brain tubulin antibodies and monoclonal anti-chicken alpha-tubulin antibodies. In other 8 investigated Mycoplasma species no positive reactions with the same antibodies were found. The M. Gallisepticum cells were examined under electron microscope on fine serial sections and on some sections going at different angles to the long cell axis. Undermembrane system of tubules was revealed and the intracellular pattern of the tubular structures were reconstructed. The immunoelectron microscopic data suggest that tubulin-like protein may be included into the structures.

[The ultrastructural characteristics of the epithelial cells in the frog bladder under the action of vasopressin and in a vasopressin-independent increase in permeability for water]. / Osobennosti ul'trastruktury kletok épiteliia mochevogo puzyria liagushki pri deistvii vazopressina i pri vazopressin-nezavisimom uvelichenii pronitsaemosti dlia vody.

In experiments on isolated frog urinary bladders it has been found that the low basal level of water permeability in the absence of arginine-vasopressin (AVP) could be significantly increased when the serosal solution was changed several times every 15 min for a fresh Ringer solution. The electron microscopic study of these cells by the freeze-fracture technique showed that the enhancement of water permeability by AVP-independent manner was related to the appearance of intramembranous particle aggregates in luminal membrane of granular cell, that are usually observed only under the action of AVP. The immunocytochemical experiments with monoclonal antibodies against actin revealed the similarity in intracellular actin distribution under the action of AVP and AVP-independent increase of water permeability.

An electron microscopic and X-ray diffraction study of osmium localization in membrane structures after fixation with OsO4 introduced to native and freeze-dried specimens.

Ultrathin sections of the sciatic nerve myelin fibres of the frog Rana temporaria and liquid crystals of egg lecithin fixed in OsO4 have been treated by 4% or 30% acetic acid. Following uranyl acetate contrasting of thus treated sections the myelin structure and that of lecithin crystals completely recover. However, in the case of preliminary treatment of the sections with lead tetraacetate the subsequent contrasting fails to recover lamellar structures. The data obtained how that during the fixation of myelin sheath and lecithin crystals with osmium tetroxide, the whole amount of osmium locates in charged regions. The data on the X-ray analysis of myelin with OsO4 introduced before or after freeze-drying are in agreement with these results. It is assumed that the stabilization of fatty acid tails of the membrane lipid molecules is accomplished via formation of hydrogen links induced by glycol hydroxyl groups.

Investigation of membrane complexes of myelin and gregarine pellicle by freeze-drying and freeze-etching methods.

By the changes introduced in the standard procedures of the freeze-drying method (fixation, transportation of specimens from a vacuum apparatus into glass capillary tubes, and embedding) we have managed to simplify the method and to increase the reliability of the results. Fixation of the tissue was carried out after freeze-drying in molecular beams of OsO4 or in an OsO4 solution in carbon tetrachloride. The samples were embedded in araldite through mixtures of carbon tetrachloride and araldite. Membranes of myelin and gregarine pellicle were investigated. The repeat period of myelin at fixation of nerves after freeze-drying in OsO4 molecular beams is 11.2 +/- 0.5 nm and in an OsO4 solution in carbon tetrachloride, 12 +/- 0.4 nm. A tetragonal reticulum was revealed on the plasma-membrane extracellular surface of gregarine which had been prepared by freeze-drying. This type of structure is never observed after the routine fixation in water, while an analogous structure is revealed after freeze-etching.

Tubular structures of Mycoplasma gallisepticum and their possible participation in cell motility.

In five strains of Mycoplasma gallisepticum, a protein with a molecular mass of about 40 kDa was detected by immunoblotting with anti-pig brain tubulin polyclonal and monoclonal antibodies. In eight other mycoplasma species similarly tested no reaction was observed. Thin serial sections of M. gallisepticum and Acholeplasma laidlawii cells examined by transmission electron microscopy revealed a submembrane system of tubules in M. gallisepticum but not in A. laidlawii. The intracellular spatial distribution of the tubular structures was reconstructed. Thin sections of M. gallisepticum treated with anti-tubulin antibodies and colloidal gold particles (immunogold labelling) revealed distinct labelling of the tubular system. Analysis of the tubular structures by high resolution electron microscopy and optical diffraction showed their helical organization to be: diameter 40 nm, helix pitch approximately 20 nm and electron-transparent core 10 nm in diameter. A possible involvement of the tubular system in mycoplasma motility is suggested.