[FUNCTIONAL LINK BETWEEN TRANSPORT PARAMETERS OF MDCK1 CELLS MONOLAYER AND THEIR ACTIN CYTOSKELETON ORGANIZATION].

On MDCK1 cell monolayer, dynamics of the spatial organization of actin cytoskeleton and dynamics of trans-epithelial electrical resistance (TEER) have been studied upon exposure of arginine-vasopressin (AVP) and protein-kinase A (PKA) activator forskolin. It has been found that exposure to these physiologically active compounds causes fibrillary actin depolymerization (both in apical and in basal cytoplasm) and, simultaneously, significant decrease in the cell monolayer trans-epithelial electrical resistance. TEER decrease indicates the stimulation of ions and water flow across the cell monolayer. In order to clarify pathways of movement of ions and water across MDCK monolayer, we have carried out an immunofluoresence study of claudin 1 and 2 localization in the tight junctions of MDCK ATCC cells (low TEER) and MDCK1 cells (high TEER). We have demonstrated that in the tight junctions of MDCK ATCC cells both claudin 1 and claudin 2 are present. In MDCK1 cells tight junctions, claudin 1 is localized and pore-forming claudin 2 is completely lacking. Under forskolin exposure to MDCK1 cells, no alterations in studied claudins distribution has been found. These data indicate that forskolin-induced TEER decrease is linked with alterations in trans-cellular, not in para-cellular, permeability of monolayer.

Giant vacuoles arising during ADH-induced transcellular bulk water flow across the epithelium of the frog urinary bladder.

Structural changes of the cytoplasm of urinary bladder granular cells after an antidiuretic hormone (ADH) stimulation of water transport were studied using standard and cryogenic methods of electron microscopy. Numerous changes occurred in these cells, the cytoplasm of the granular cells becoming swollen, and the intercellular spaces enlarged. Most granules become fused with the apical membrane. Under maximal ADH action, giant vacuoles appear in the cytoplasm of granular cells, in association with microfilaments and microtubules. Analysis of ultrastructure of the granular cells has established the origin of giant vacuoles from the cis -cisterna of the Golgi complex. A hypothesis based on the morphofunctional homology of giant vacuoles in granular cells with the contractile vacuoles of Protozoa is proposed in which the giant vacuoles ('contractile-like' vacuoles) are seen as operating a osmoregulatory role in these cells. It is also proposed that microtubules and microfilaments participate in giant vacuole migration through the cytoplasm.

Structural correlates of the transepithelial water transport.

Transepithelial permeability is one of the fundamental problems in cell biology. Epithelial cell layers protect the organism from its environment and form a selective barrier to the exchange of molecules between the lumen of an organ and an underlying tissue. This chapter discusses some problems and analyzes the participation of intercellular junctions in the paracellular transport of water, migration of intramembrane particles in the apical membrane during its permeability changes for isotonic fluid in cells of leaky epithelia, insertion of water channels into the apical membrane and their cytoplasmic sources in cells of tight epithelia under ADH (antidiuretic hormone)-induced water flows, the osmoregulating function of giant vacuoles in the transcellular fluxes of hypotonic fluid across tight epithelia, and the role of actin filaments and microtubules in the transcellular transport of water across epithelia.

Immunogold visualization of actin near the coated pits at the apical surface of human mammary epithelial cells.

This ultrastructural study of both the normal human breast tissue and differentiated mammary carcinoma (NOS) epithelial cells has revealed pictures demonstrating luminal receptor-mediated endocytosis. By application of immunogold anti-actin labeling, actin surrounding the fusion ring of coated pits was visualized. However, the coated membrane was not actin labeled. We suggest that association of the actin with coated pits may evidence for its participation in pinching off of the coated vesicles.

Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor.

Here we report identification of a novel member of the thiol protease superfamily in the yellow fever mosquito, Aedes aegypti. It is synthesized and secreted as a latent proenzyme in a sex-, stage-, and tissue-specific manner by the fat body, an insect metabolic tissue, of female mosquitoes during vitellogenesis in response to blood feeding. The secreted, hemolymph form of the enzyme is a large molecule, likely a hexamer, consisting of 44-kDa subunits. The deduced amino acid sequence of this 44-kDa precursor shares high similarity with cathepsin B but not with other mammalian cathepsins. We have named this mosquito enzyme vitellogenic cathepsin B (VCB). VCB decreases to 42 kDa after internalization by oocytes. In mature yolk bodies, VCB is located in the matrix surrounding the crystalline yolk protein, vitellin. At the onset of embryogenesis, VCB is further processed to 33 kDa. The embryo extract containing the 33-kDa VCB is active toward benzoyloxycarbonyl-Arg-Arg-para-nitroanilide, a cathepsin B-specific substrate, and degrades vitellogenin, the vitellin precursor. Both of these enzymatic activities are prevented by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a thiol protease inhibitor. Furthermore, addition of the anti-VCB antibody to the embryonic extract prevented cleavage of vitellogenin, strongly indicating that the activated VCB is involved in embryonic degradation of vitellin.

Ultrastructural correlates of the antidiuretic hormone-dependent and antidiuretic hormone-independent increase of osmotic water permeability in the frog urinary bladder epithelium.

Electron and confocal microscopy, using immunocytochemical methods, was employed to assess osmotic water permeability of the frog (Rana temporaria) urinary bladder during transcellular water transport, induced by antidiuretic hormone (ADH) or by wash-out of autacoids from serosal, ADH-free Ringer solution. The increase of osmotic water permeability of the urinary bladder was accompanied by relevant ultrastructural changes, the most remarkable being: (1) the appearance of aggregates of intramembranous particles in the apical membrane of granular cells, and the extent of the membrane area covered by the aggregates proportional to that of the water flow; (2) redistribution of actin filaments in the cytoplasm of granular cells; judging from the anti-actin label density, the number of actin filaments in the apical region of cytoplasm was reduced by 2.5-4 times compared with normal; (3) a decrease in the total electron density of the cytoplasm due to the increased water content of granular cells.

Prostaglandin-dependent osmotic water permeability of the frog and trout urinary bladder.

Washout of autacoids from serosal Ringer solution, using a repeated change of the solution of the frog and trout urinary bladder, was accompanied by a pronounced rise in the osmotic water permeability: the water transport in the frog rose from 0.05 +/- 0.02 to 1.21 +/- 0.26 microliter min-1.cm-2, in the trout, from 0.041 +/- 0.011 to 0.26 +/- 0.034 microliter min-1.cm-2. Such an increase in the osmotic water permeability in the trout and frog urinary bladder occurred in the background of a decrease in the prostaglandin E2 concentration in the serosal Ringer solution. This permeability increase was accompanied by the formation of aggregates of intramembranous particles in the apical plasma membrane of the trout and frog urinary bladder. A decrease in the osmotic water permeability was achieved by the addition to the serosal Ringer solution of 10-8 M prostaglandin. Experiments on the frog urinary bladder have shown that prostaglandins E1, I2 and F2 alpha also decrease the osmotic water permeability. Vasotocin increased the osmotic water permeability in the frog urinary bladder but did not affect the osmotic water permeability of the trout urinary bladder. The data obtained indicates a role of the endogenous prostaglandin production in maintaining the low osmotic water permeability in the frog and trout urinary bladder. A suggestion is made that in the vertebrate evolution, colonisation of the fresh-water was connected with the maintenance of the low osmotic water permeability via participation of prostaglandins, whereas the vasotocin hydroosmotic effect developed in the vertebrate evolution later and provided for the possibility of the water absorption, osmotic homeostasis and animal migration from fresh-water to the land.

Mosquito clathrin heavy chain: analysis of protein structure and developmental expression in the ovary during vitellogenesis.

We have deduced the amino acid sequences of clathrin heavy chain (CHC) polypeptides based on cDNA and genomic clones from the mosquito, Aedes aegypti. Two isoforms which differ in the very beginning of the N-terminal domain, ovary-specific AaCHCa and somatic-specific AaCHCb, were identified, characterized and compared to one another as well as to CHC polypeptides from different species. The 1682 amino acid sequence of the AaCHCa isoform predicts a molecular mass (M[r]) of 191,743 daltons and an isoelectric point of 5.80, whereas the 1674 amino acid sequence of the AaCHCb isoform predicts a M(r) of 191,033 daltons and an isoelectric point of 5.71. Both mosquito AaCHC isoforms are highly conserved, with full-sequence identities of 88% to Drosophila melanogaster, 81% to mammal (rat, cow and human), 71% to C. elegans, 58% to Dictyostelium discoideum, and 49% to yeast CHC polypeptides. The highest degree of conservation is in the middle portion of the mosquito CHC molecule which includes the linker region and extended triskelion arm, with decreasing conservation through the N-terminal domain, trimerization domain, and the relatively diverged C-terminal region. The protein domains do not directly correspond to specific exons of the mosquito AaCHC gene, with the exception of exon 6 which encodes the C-terminal domain of the CHC polypeptide. Polyclonal antibodies raised against a bacteria-expressed AaCHC fusion protein recognized one major band of about 180 kDa in vitellogenic ovary whole-lysate. Immunogold labelling of the AaCHC polypeptide localized it to the coat of coated pits and coated vesicles in oocytes from vitellogenic follicles. Northern blot and in situ hybridization analyses suggest that regulation of AaCHC gene expression in the ovary is complex, and it likely involves both developmental and hormonal signals.

Secretory and internalization pathways of mosquito yolk protein precursors.

The vitellogenic female fat body of the mosquito Aedes aegypti produces three yolk protein precursors that are deposited in the yolk bodies of developing oocytes: vitellogenin, vitellogenic carboxypeptidase (VCP), and 44-kDa protein (44KP). We have used gold immunocytochemistry to investigate the pathways of their secretion in fat body trophocytes and their internalization by oocytes. In fat body trophocytes, all three yolk protein precursors are co-localized in the Golgi complex and secretory granules, indicating that they proceed simultaneously through the secretory pathway. The lysosomal system plays an important role in the termination of vitellogenesis in mosquito trophocytes, by degrading biosynthetic organelles and secretory granules. At this time, VCP and 44KP are found together with vitellogenin in trophocyte autophagolysosomes, suggesting that all three yolk protein precusors are redirected from the secretory to the lysosomal degradative pathway. Localization of VCP and 44KP in developing mosquito oocytes clearly shows that the internalization of these yolk protein precursors by oocytes occurs via the same endocytotic route as vitellogenin: all three yolk protein precursors are found on the oocyte microvillus membrane, in coated vesicles, and early endosomes. They are observed intermixed with one another in the late endosomes or in transitional yolk bodies. In mature yolk bodies, however, 44KP and VCP are segregated from vitellin, the crystallized storage form of vitellogenin; 44KP and VCP reside in the non-crystalline cortex, surrounding the vitellin core in nature yolk bodies.

Internalization and recycling of vitellogenin receptor in the mosquito oocyte.

The major yolk protein precursor in mosquito oocytes, vitellogenin (Vg), is internalized by a 205-kDa membrane-bound receptor (VgR). Recently, VgR has been isolated permitting the production of polyclonal anti-VgR antibodies. To elucidate the pathway of VgR internalization and recycling in mosquito oocytes during Vg uptake, we carried out an immunogold electron-microscopic study, labeling both Vg and VgR in ultrathin frozen sections of ovarian tissue. VgR immunolabeling demonstrated that the oocyte plasma membrane was subdivided into microdomains, with VgR being located between and at the lower portions of the oocyte microvilli. During the early stages of internalization, Vg and VgR were observed together in coated pits, coated vesicles, and early endosomes. Fusion of early endosomes created transitional yolk bodies (TYB) in which Vg and VgR became segregated. VgR label was present in the numerous tubular compartments that protruded from the TYBs. These tubular organelles extended to and fused with the plasma membrane, suggesting that they represented the vehicle for VgR recycling. Vg label was not observed in the tubular compartments. Instead, Vg accumulated in the core of the TYB, a region free of VgR label. Mature yolk bodies (MYB) were heavily labeled for Vg, but completely lacked any VgR label, indicating that MYB are storage compartments that do not participate in receptor recycling. Thus, our immunocytochemical data clearly visualize the steps in Vg/VgR internalization, dissociation, sorting, and recycling of the receptor to the plasma membrane.

AVP-independent high osmotic water permeability of frog urinary bladder and autacoids.

In the isolated frog urinary bladder a 20- to 50-fold increase of the osmotic water permeability has been revealed in the absence of arginine vasopressin (AVP) as a result of several successive changes of the serosal Ringer solution. This increase of the osmotic water permeability was of the same magnitude as that of the effect of 1 nM AVP. Similarly to the effect of AVP, the amount of adenosine 3',5'-cyclic monophosphate (cAMP) in the cells rose, and aggregates of intramembraneous particles were formed in the apical plasma membrane of granular cells (as shown by the freeze-fracture method). Immunocytochemical studies using anti-actin monoclonal antibodies indicated depolymerization of F-actin following the AVP-independent change in water permeability. It was possible to decrease the high level of osmotic permeability to the initial level if 10 microl/ml of frog blood serum or a lipid extract of this blood serum, or 1 microM arachidonic acid or 1 nM prostaglandin E2 was added to the serosal Ringer solution. The rapid restoration of the osmotic water impermeability of the epithelium after the AVP- evoked effect was achieved by the addition to the serosal Ringer solution of Ringer solution in which intact frog urinary bladders had been previously incubated for 1 h. The data obtained indicate that the maintenance of the impermeability to water of the osmoregulating epithelium and the restoration of the initial low level of the osmotic permeability after the effect of AVP are due to participation of prostaglandin E2 and other autacoids as well as, probably, some physiologically active substances of a lipid nature that are present in the blood serum.

Structural and functional analysis of glucose absorption mechanisms in the rat small intestine in vivo.

The absorption of glucose (free, and released from membrane hydrolysis of maltose) and water in the isolated loop of the rat small intestine was studied in chronic experiments. Even at maximum glucose (75 mmol/l) or maltose (37.5 mmol/l) concentrations the rate of glucose transfer by solvent drag and by diffusion did not exceed 13% and 25%, respectively, of the total rate of glucose absorption. Electron microscopic and immunocytochemical analysis revealed a significant widening of intercellular gaps in the basal epithelium region and an increase of actin density in the vicinity of the tight junctions and between the apical root filaments in enterocytes after glucose or glycine load in chronic and acute experiments. However, very rarely (in 1% of all cases), and only in chronic experiments, structural changes in the tight junctions such as "blisters" and dilatations were also recognised. It is concluded that under normal physiological conditions the absorption of glucose (free, and released from maltose hydrolysis) mainly uses active transport across the apical membrane of the enterocytes.

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.

A novel type of microtubules in the frog urinary bladder epithelium stimulated by vasopressin.

Changes in the cytoskeleton of granular cells of the frog urinary bladder under conditions of low and vasopressin-induced water permeability were investigated. Using a conventional electron microscopical preparation with glutaraldehyde/osmium fixation we found only the usual type of microtubules in granular cells of both resting and stimulated bladders. These microtubules, 20 nm in diameter, were associated with centrioles and Golgi complexes. Under the high water permeability conditions the large vacuoles appear in granular cells. The 20-nm microtubules and microfilaments were connected to the membrane of these vacuoles. The second type of 'thick' microtubules was found in granular cells of the stimulated bladder after the fixation with glutaraldehyde without osmium postfixation or freeze-substitution. The tubulin nature of these 'thick' microtubules was identified immunocytochemically. In addition, tubulin was present in the granules with crystalline content.

Specific vacuolation of frog urinary bladder granular cell after ADH stimulation of water transport.

The present study deals with an analysis of specific traits of cell vacuolation induced by water flow and ADH. During incubation of frog urinary bladders in Ringer's solution diluted 2-fold, the water content of the bladder wall increased by an average of 19%. In case of ADH-stimulated water flow the water content increased by an average of 15.7%. Cell swelling induced by hypotonic conditions on the serosal side resulted in a drastic decrease of the response to the hydroosmotic action of ADH. Electron microscopy revealed significant differences between cells hydrated in the above conditions. Two-fold hypotonicity of the serosal solution caused a slight swelling of all types of cells accompanied by a narrowing of intercellular spaces. With ADH stimulation of water transport (at maximal water movement) granular cells were characterized by the presence of irregularly shaped giant vacuoles with processes. The limiting membranes of the vacuoles were closely connected with microtubules and microfilaments. The electron microscopic study of these cells by the freeze-substitution method revealed, in addition to giant vacuoles, a highly complex system of microtubules 35-40 nm in diameter. A morphological similarity was observed between the vacuolar systems of these granular cells and the contractile vacuole complex of protozoans. Possible mechanisms for the participation of giant vacuoles, electron-dense canaliculi, microtubules and microfilaments in transcellular water flow across epithelium are discussed.

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.

Interaction of vibrio cholerae El Tor and gut mucosa in ligated rabbit ileal loop experiment.

Light and electron microscope studies in ligated rabbit gut loops showed that cholera vibrios El Tor attached themselves to the enterocytes and multiplied, most probably on the intestinal epithelium surface. But they did not penetrate deeper and were unable to cause destruction of the epithelium or marked inflammation. Ultrastructural manifestations of both apocrine and merocrine secretion of the enterocytes were observed. In addition, the enterocytes showed signs of generally enhanced activity. Some other changes and alterations in the lamina propria, presumably induced by excessive transport of water, are also described. The enterotoxin-induced epithelial hypersecretion with fluid accumulation in the gut loops resulted in the attached vibrios being cleared off the epithelium lining into the gut lumen. NAG vibrios were mostly incapable of inducing the above changes. We discuss the importance of cholera vibrio attachment to and its multiplication on the intestinal epithelium in the pathogenesis of cholera.

Pathogenic effect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea.

Macroscopic, light and electron microscopic alterations in ligated rabbit intestinal loops challenged with five standard enterotoxigenic Escherichia coli (ETEC) and twenty-three enteropathogenic E. coli (EEC-I) strains, freshly isolated from infantile enteritis cases, were investigated. Only two O26 : K60 : H11 strains produced enterotoxin. Their living cultures, sterile filtrates of the fluid medium and ultrasonic lysates of the bacteria resulted in pronounced hypersecretion of the intestinal epithelium followed by fluid accumulation and loop dilatation. These two E. coli strains, similarly as the other loop-negative EEC-I strains, were able to penetrate into the intestinal epithelium. In contrast to the standard ETEC strains, the EEC-I bacteria, adhering to the brush border, intruded into the microvilli, multiplied on the outer epithelial cell membrane making close contact with it and, causing, shedding of microvilli, penetrated into enterocytes becoming enclosed in membrane-bound phagosome-like vacuoles, appeared in the lamina propria and elicited mild focal polymorphonuclear infiltration.