Área mitocondrial relativa nos epitélios branquial e renal do baiacu estuarino tropical Sphoeroides testudineus após aclimatação a água do mar isosmótica

The gills of teleost fishes are responsible both for gas exchangein respiration and salt transport in osmoregulation (JOBLING, 1995; ZADUNAISKY, 1996; PERRY, 1997; VAN DER HEIJDEN et al., 1997; EVANS et al., 1999; EVANS et al., 2005). In marine teleosts the gill epithelium secretes salt mainly through chloride cells (JOBLING, 1995; ZADUNAISKY, 1996; PERRY, 1997; VAN DER HEIJDEN et al., 1997; FERNANDES et al., 1998; EVANS et al., 2005). These cells are typically located between secondary lamellae at their insertion in the gill filament (inter-lamellar region) or in the gill filament itself (LAURENT & DUNEL, 1980; PERRY, 1997; FERNANDES et al., 1998; EVANS et al., 2005). These cells are rounded, display abundant mitochondria, a tubular system of endomembranes, sub-apical vesicles, and extensive intercellular junctional complexes (LAURENT & DUNEL, 1980; JOBLING, 1995; ZADUNAISKY, 1996; PERRy, 1997; EVANS et al., 2005).

MAL regulates clathrin-mediated endocytosis at the apical surface of Madin–Darby canine kidney cells

MAL is an integral protein component of the machinery for apical transport in epithelial Madin–Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.

Hyperosmoregulation in the red freshwater crab Dilocarcinus pagei (Brachyura, Trichodactylidae): structural and functional asymmetries of the posterior gills

SUMMARY The osmotic and ionic status of the haemolymph and the structural and ion-transport characteristics of the posterior gills of Dilocarcinus pagei, a hololimnetic crab, were investigated. Haemolymph osmolality was 386±18 mosmol kg–1, while [Na+] and [Cl–] were 190±13 and 206±12 mmol l–1, respectively; [K+], [Ca2+] and [Mg2+] were 9.7±0.7, 10.2±0.5 and 2.8±0.4 mmol l–1, respectively (means ± s.e.m., N=12–17). The gill lamellae possess a central, osmiophilic area, which exhibits a marked structural asymmetry. The thick (18–20 μm) proximal epithelium is characterised by basal invaginations and a few apical vesicles, while the thin (3–10 μm) distal epithelium consists of apical pillar cell flanges populated by vesicles and membrane invaginations. Isolated gills, bathed and perfused with NaCl saline, spontaneously generate a negative transbranchial potential difference (Vte), which stabilises at positive or negative values. Ouabain shifts Vte to more positive values. When mounted in an Ussing chamber, distal split lamellae generate a negative, Cl–-dependent short-circuit current (Isc). Na+ substitution leads to more negative values of Isc. Internal ouabain is without effect, while diphenylamine-2-carboxylate and acetazolamide abolish Isc. Proximal split lamellae show a positive, Na+-dependent Isc, which decreases after internal application of ouabain. These data suggest that the thin epithelium actively absorbs Cl–, while the thick epithelium actively absorbs Na+.

Polarized localizations of annexins I, II, VI and XIII in epithelial cells of intestinal, hepatic and pancreatic tissues

The cellular and subcellular localizations of annexins I, II, VI and XIII in the rabbit intestine, liver and pancreas were studied by performing immunofluorescence labeling on thin frozen tissue sections using specific monoclonal antibodies. The expression of annexins was found to be finely regulated. Annexins XIII and I were expressed exclusively in the small intestine and the colon, respectively, whereas annexin II was present in all the tissues tested and annexin VI specifically in the liver and pancreas. These different annexins were concentrated in the basolateral domain of polarized cells, and some of them had an extra-apical localization: annexin XIII was concentrated in the lower 3/4 of enterocyte brush border microvilli; annexin II was present in the upper part of the terminal web in intestinal absorbent cells as well as in the bile canalicular area in hepatocytes, whereas annexin VI was detected on some apical vesicles concentrated around the bile canaliculi. In pancreatic acinar cells, the presence of annexin II on some zymogen granules provides further evidence that annexin II may be involved in exocytic events. In conclusion, this study shows that the basolateral domain of polarized cells appears to be the main site where annexins are located, and they may therefore be involved in the important cellular events occurring at this level.

Apical Vesicles Bearing Inositol 1,4,5-trisphosphate Receptors in the Ca2+Initiation Site of Ductal Epithelium of Submandibular Gland

In polarized epithelial cells, agonists trigger Ca2+ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools into specific regions. We have investigated the relationship between the distribution of IP3 receptors (IP3Rs) and the spatiotemporal pattern of Ca2+ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP3Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP3R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca2+ spike occurred in the apical region. Subsequently, repetitive Ca2+ spikes spread from the apical to the middle cytoplasm. These apical Ca2+ initiation sites were found only in some “pioneer cells,” rather than in all duct cells. We performed both Ca2+ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP3R2 in the Ca2+ initiation sites of the pioneer cells. The subcellular localization and expression level of IP3Rs correlated strongly with the spatiotemporal nature of the intracellular Ca2+ signal and distinct Ca2+ responses among the rat SMG duct cells.

The Coronavirus Transmissible Gastroenteritis Virus Causes Infection after Receptor-Mediated Endocytosis and Acid-Dependent Fusion with an Intracellular Compartment

ABSTRACT Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38°C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.

Effect of a lysine-enriched diet onl-lysine transport by the brush-border membrane of the chicken jejunum

The influx ofl-lysine into apical vesicles from the chicken jejunum occurs through two systems, one with low Michaelis constant ( K m) and features of system b0,+ and the other with relatively high K m forl-lysine and with properties of system y+. In the present study the effect of a lysine-enriched diet (Lys, containing 68 g l-lysine/kg dietary protein, control animals 48 g/kg) onl-lysine uptake through both transport systems was investigated. Results show that 1) lysine enrichment had no effect on either body weight or the efficiency of food utilization. 2) In Lys-fed animals, the mediatedl-lysine influx was best fitted to the two-system model with y+and b0,+ activity. 3) In the presence of an Na+ gradient, totall-lysine uptake is significantly higher in Lys-fed animals than in control birds (about 40% increase). 4) Lys diet increases K mb0,+6-fold (KSCN gradient) and 12-fold (NaSCN gradient) and maximum velocity ( V max) by 6- and 20-fold, respectively. The effects of Lys enrichment on the y+-like system are only observed on the V max and in the presence of a Na+ gradient (30% increase). 5) Na+ is involved in the activation of the transport process in the Lys-fed chickens, but there is no correlation between external Na+concentration and l-lysine influx. In conclusion, both b0,+- and y+-like transport systems are upregulated by dietary lysine but with different kinetic profiles; the high-capacity y+-like carrier shows a V maxincrease without changes in K m, whereas the low-capacity b0,+-like system shows an increase in V max as well as in the K m.

Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane.

The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.