Immunocytochemical detection of mitochondria-rich cells in the brood pouch epithelium of the pipefish, Syngnathus schlegeli : structural comparison with mitochondria-rich cells in the gills and larval epidermis

The brood pouch of the male pipefish (Syngnathus schlegeli) is a ventral organ located on the tail, with the anterior region closely associated with the genital pore. The embryos in the pouch are attached to highly vascularized placenta-like tissue which seals the pouch folds from inside during incubation. The epithelium of the placenta-like tissue consists of mitochondria-rich cells (MRCs) and pavement cells. Differences in MRC morphology in the brood pouch epithelium, the gills and the larval epidermis of the pipefish were examined by light and electron microscopy. Transmission electron microscopy revealed that the MRCs in the brood pouch and the gills shared common characteristics: the presence of numerous mitochondria packed among a well-developed tubular system and the close association of the basal parts with the capillaries running underneath the epithelia. The size of the apical opening of the elongate, flask-shaped brood pouch MRC was about one-tenth that of the apical pit of the gill MRC. The gill and larval epidermal MRCs formed a multicellular complex, in contrast to solitary brood pouch MRCs. The brood pouch MRCs were intensively stained by immunocytochemistry with an antiserum specific for Na⁺,K⁺-ATPase. The Na⁺ concentrations in the brood pouch were maintained near those in the serum rather than seawater during incubation. We conclude that the brood pouch MRCs function as an ion-transporting cell, absorbing ions from the brood pouch lumen, perhaps to protect the embryos from the hyperosmotic environment.     

Prolonged apoptosis in mitochondria-rich cells of tilapia (<Emphasis Type="Italic">Oreochromis mossambicus</Emphasis>) exposed to elevated salinity

The time-course of programmed cell death (apoptosis) during reorganization of gill epithelium in salinity-stressed tilapia was analyzed using a recently developed method based on laser scanning cytometry (LSC) of dissociated gill cells. Apoptosis in mitochondria-rich cells (MRC) was distinguished from that in other cell types using Na⁺/K⁺ ATPase (NKA) as a cell-specific marker. Caspase 3/7 activity in MRC, assessed using LSC and microplate assays, increased significantly starting at 6 h of salinity stress and remained elevated for at least 5 days. This time-course of apoptosis in MRC during acute salinity stress was reflected in elevated apoptotic DNA fragmentation. In parallel to induction of apoptosis, MRC showed a pronounced shift to G2 phase of the cell cycle, which is indicative of G2/M cell cycle arrest, and an increase in NKA abundance per MRC. Unlike in MRC, apoptosis was not significantly increased in other gill cell types, although there was a small transient increase in DNA fragmentation at 6 h. G2 arrest was also observed. Overall, we interpret our data as evidence for a significant role of apoptosis in the extensive reorganization of MRC populations that takes place during salinity acclimation, perhaps similar to its well-established role during organismal development.     

The interaction of human erythrocyte band 3 with cytoskeletal components

Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of ³⁵SO₄. was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 n NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 n NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.     

Activation of Apical K<Superscript>+</Superscript> Conductances by Muscarinic Receptor Stimulation in Rat Distal Colon: Fast and Slow Components

In the epithelium of rat distal colon the acetylcholine analogue carbachol induces a transient increase of short-circuit current (I_sc) via stimulation of cellular K⁺ conductances. Inhibition of the turnover of inositol-1,4,5-trisphosphate (IP₃) by LiCl significantly reduced both the amplitude and the duration of this response. When the apical membrane was permeabilized with nystatin, LiCl nearly abolished the carbachol-induced activation of basolateral K⁺ conductances. In contrast, in epithelia, in which the basolateral membrane was bypassed by a basolateral depolarization, carbachol induced a biphasic increase in the K⁺ current across the apical membrane consisting of an early component carried by charybdotoxin- and tetraethylammonium-sensitive K⁺ channels followed by a sustained plateau carried by channels insensitive against these blockers. Only the latter was sensitive against LiCl or inhibition of protein kinases. In contrast, the stimulation of the early apical K⁺ conductance by carbachol proved to be resistant against inhibition of phospholipase C or protein kinases. However, apical dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, or a Ca²⁺-free mucosal buffer solution significantly reduced the early component of the carbachol-induced apical K⁺ current. The presence of an apically localized Na⁺/Ca²⁺-exchanger was proven immunohistochemically. Taken together these experiments reveal divergent regulatory mechanisms for the stimulation of apical Ca²⁺-dependent K⁺ channels in this secretory epithelium, part of them being activated by an inflow of Ca²⁺ across the apical membrane.

Mitochondria-rich cells and carbonic anhydrase content of toad skin epithelium

Summary The density and carbonic anhydrase (CA) content of the mitochondria-rich cells (MRCs) in the skin epithelium of the toad, Bufo viridis, were studied under conditions of acclimation to various chlorinities. Long-term (days to weeks) acclimation to chloride-free solutions induced a great increase in the MRC density and the area occupied by the apical portion of these cells on the surface of the epithelium. The CA content of the epithelium, and individual MR cells, showed a 5- to 10-fold reduction after acclimation to solutions containing high chloride levels. The MRC density and their relative apical surface area correlated with the chloride permeability of the skin in acclimated (long-term) toads. It is concluded that the MRCs are the principal site of chloride permeability across the amphibian skin, and they respond in an adaptive manner to long-term changes in environmental chloride levels.This study was partially supported by the J. and A. Taub Fund for Biological Research at The Technion, and by the basic research fund of the Israel Academy of Sciences     

H2O2-Induced Oxidative Stress Affects SO4= Transport in Human Erythrocytes

The aim of the present investigation was to verify the effect of H₂O₂-induced oxidative stress on SO₄⁼ uptake through Band 3 protein, responsible for Cl^-/HCO₃^- as well as for cell membrane deformability, due to its cross link with cytoskeletal proteins. The role of cytoplasmic proteins binding to Band 3 protein has been also considered by assaying H₂O₂ effects on hemoglobin-free resealed ghosts of erythrocytes. Oxidative conditions were induced by 30 min exposure of human erythrocytes to different H₂O₂ concentrations (10 to 300 μM), with or without GSH (glutathione, 2 mM) or curcumin (10 μM), compounds with proved antioxidant properties. Since SO₄⁼ influx through Band 3 protein is slower and better controllable than Cl^- or HCO₃^- exchange, the rate constant for SO₄⁼ uptake was measured to prove anion transport efficiency, while MDA (malondialdehyde) levels and –SH groups were estimated to quantify the effect of oxidative stress. H₂O₂ induced a significant decrease in rate constant for SO₄⁼ uptake at both 100 and 300 μM H₂O₂. This reduction, observed in erythrocytes but not in resealed ghosts and associated to increase in neither MDA levels nor in –SH groups, was impaired by both curcumin and GSH, whereas only curcumin effectively restored H₂O₂-induced changes in erythrocytes shape. Our results show that: i) 30 min exposure to 300 μM H₂O₂ reduced SO₄⁼ uptake in human erythrocytes; ii) oxidative damage was revealed by the reduction in rate constant for SO₄⁼ uptake, but not by MDA or –SH groups levels; iii) the damage was produced via cytoplasmic components which cross link with Band 3 protein; iv) the natural antioxidant curcumin may be useful in protecting erythrocytes from oxidative injury; v) SO₄⁼ uptake through Band 3 protein may be reasonably suggested as a tool to monitor erythrocytes function under oxidative conditions possibly deriving from alcohol consumption, use of drugs, radiographic contrast media administration, hyperglicemia or neurodegenerative diseases.     

Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat

We used cytochrome oxidase (CytOx) staining intensity, which is correlated with neuronal functional activity, to evaluate maturity and functionality of newborn rat olfactory epithelium (OE) and olfactory receptor neurons (ORNs). Nasal olfactory tissue of neonatal rats was stained with CytOx and analyzed qualitatively and quantitatively. Results revealed that newborn OE shows six differentially stained horizontal bands. Bands run parallel to the OE surface and were categorized as very light, medium or darkly stained. A narrow and pale Band 1 overlapped with horizontal basal cells. Next, a wide and lightly stained Band 2 was observed that coincides with the globose basal cell layer and immature ORNs, deep in OE. Next apically, a medium-staining Band 3 overlapped with ORN perikarya. Closer to the surface, a medium to light Band 4 was discerned where dendrites of mature ORNs normally occur. This band was interrupted with lighter areas due to the presence of supporting cells nuclei. Next, a superficial but dark Band 5 occurred, populated by the apical portions of ORN dendrites and their ciliated knobs and by supporting cell apices; mitochondria in apices of supporting cells contribute predominantly to dense staining of this Band 5. Apical to Band 5, a thin and fairly light Band 6 was observed which overlaps with the mucus layer that contains part of the ORN knobs, their cilia and supporting cell microvilli. Along the length of ORN dendrites, apical segments just below the ORN knobs, and wide basal segments showed a darker staining than the middle segments implying “microzones” of higher neural activity within the most apical and basal regions of dendrites. Our findings agree with ultrastructural studies showing a presence of mitochondria in knobs, basal portions of ORN dendrites and in OE supporting cell apices, suggesting that apical regions of both olfactory and supporting cells near the surfaces are metabolically most active, in odorant detection, signal processing, and detoxification, the latter for supporting cells.     

Middle ear cell line that maintains vectorial electrolyte transport

The middle ear epithelium plays a major role in keeping the temporal bone cavities fluid-free and air-filled, which is a mandatory condition to allow optimum transmission of the sound vibrations from the tympanic membrane to the inner ear. Previous works have recently established the absorptive function of the middle ear epithelium, using primary cultures derived from Mongolian gerbil (Meriones unguiculatus). Because of the paucity of cells as obtained by enzymatic digestion, we developed a middle ear cell line (MESV) using wild-type SV40 infection of primary culture of Mongolian gerbil's middle ear epithelial cells. Transformation was attested by nuclear expression of SV40 large T antigen, prolonged in vitro passages (presently beyond 50 passages), and tumor-inducing ability when subcutaneously injected in athymic mice. Transport properties were evaluated after the fifteenth passage. MESV cells retained most cardinal properties of the original middle ear epithelial cells: cell polarization was evidenced by the presence of mature junctional complexes that separate the cell membrane in two distinct domains, with apical microvilli at the luminal side, and by vectorial sodium transport responsible for the transepithelial lumen-negative potential difference (?9.3 ± 0.14 mV in culture conditions (n=9), ?2.1 ± 0.25 mV after overnight growth factors and serum deprivation). Short-circuit current was, like in primary cultures, mainly related to a sodium transport occuring through amiloride-sensitive apical sodium channels, since apical addition of amiloride (10^?5 M) reduced I_sc from 7.0 = 1.4 to 0.6 ± 0.1 μA/cm² (P < 0.01, n = 6). Cellular cAMP content was increased by isoproterenol and prostaglandin E2 from 40.5 ± 5.6 to 258.5 ± 17.3 and 55.6 ± 6.2 pmol/mg protein per 5 min, respectively (P < 0.05, n = 10). Isoproterenol and prostaglandin E2 increased I_sc with very similar maximal effects: isoproterenol (10^?4 M) increased I_sc from 5.73 ± 0.31 to 12.77 ± 0.39 μA/cm², while prostaglandin E2 increased I_sc from 5.47 ± 0.21 to 12.87 ± 0.42 (n = 3). Since amiloride (10^?5 M) abolished this stimulation, this may be related to an increase of the electrogenic sodium transepithelial transport. The MESV cell line could provide an interesting tool as a model of middle ear epithelial cells for the study of pathophysiological modulations of ion transport. © 1993 Wiley-Liss, Inc.     

Stimulus-induced translocation of the protein kinase A catalytic subunit to the apical membrane in blowfly salivary glands

Secretion in blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT), which activates the InsP₃/Ca²⁺ pathway and the cAMP/protein kinase A (PKA) pathway in the secretory cells. The latter signaling cascade induces the activation of a vacuolar H⁺-ATPase on the apical membrane. Here, we have determined the distribution of PKA by using antibodies against the PKA regulatory subunit-II (PKA-RII) and the PKA catalytic subunit (PKA-C) of Drosophila. PKA is present in high concentrations within the secretory cells. PKA-RII and PKA-C co-distribute in non-stimulated glands, being enriched in the basal portion of the secretory cells. Exposure to 8-CPT-cAMP or 5-HT induces the translocation of PKA-C to the apical membrane, whereas the PKA-RII distribution remains unchanged. The recruitment of PKA-C to the apical membrane corroborates our hypothesis that vacuolar H⁺-ATPase, which is enriched in this membrane domain, is a target protein for PKA. This work was supported by grants Wa463/9–5 and GRK837 from the Deutsche Forschungsgemeinschaft.     

Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO₃⁻/CO₂ stimulation to increase ciliary beat frequency (CBF). Because apical HCO₃⁻ exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO₃⁻/CO₂ exposure in part because of greater intracellular acidification from unbalanced CO₂ influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO₃⁻/CO₂ perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR_inh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO₃⁻/CO₂ rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.     

Involvement of Protein Tyrosine Kinase in Osmoregulation of Na+ Transport and Membrane Capacitance in Renal A6 Cells

Renal A6 cells have been reported in which hyposmolality stimulates Na⁺ transport by increasing the number of conducting amiloride-sensitive 4-pS Na⁺ channels at the apical membrane. To study a possible role of protein tyrosine kinase (PTK) in the hyposmolality-induced signaling, we investigated effects of PTK inhibitors on the hyposmolality-induced Na⁺ transport in A6 cells. Tyrphostin A23 (a PTK inhibitor) blocked the stimulatory action of hyposmolality on a number of the conducting Na⁺ channels. Tyrphostin A23 also abolished macroscopic Na⁺ currents (amiloride-sensitive short-circuit current, I _Na ) by decreasing the elevating rate of the hyposmolality-increased I _Na . Genistein (another type of PTK inhibitor) also showed an effect similar to tyrphostin A23. Brefeldin A (BFA), which is an inhibitor of intracellular translocation of protein, blocked the action of hyposmolality on I _Na by diminishing the elevating rate of the hyposmolality-increased I _Na , mimicking the inhibitory action of PTK inhibitor. Further, hyposmolality increased the activity of PTK. These observations suggest that hyposmolality would stimulate Na⁺ transport by translocating the Na⁺ channel protein (or regulatory protein) to the apical membrane via a PTK-dependent pathway. Further, hyposmolality also caused an increase in the plasma (apical) membrane capacitance, which was remarkably blocked by treatment with tyrphostin A23 or BFA. These observations also suggest that a PTK-dependent pathway would be involved in the hyposmolality-stimulated membrane fusion in A6 cells. Received: 6 October 1999/Revised: 4 February 2000     

Adenovirus Entry From the Apical Surface of Polarized Epithelia Is Facilitated by the Host Innate Immune Response

Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAR^Ex8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAR^Ex8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAR^Ex8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAR^Ex8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAR^Ex8, to allow the initial infection the intact epithelium. In addition, CAR^Ex8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.     

Proteolytic cleavages of cytochalasin B binding components of Band 4.5 proteins of the human red blood cell membrane

The putative hexose transport component of Band 4.5 protein of the human erythrocyte membrane was covalently photolabelled with [³H]cytochalasin B. Its transmembrane topology was investigated by electrophoretically monitoring the effect of proteinases applied to intact erythrocytes, unsealed ghosts, and a reconstituted system. Band 4.5 was resistant to proteolytic digestion at the extracellular face of the membrane in intact cells at both high and low ionic strengths. Proteolysis at the cytoplasmic face of the membrane in ghosts or reconstituted vesicles resulted in cleave of the transporter into two membrane-bound fragments, a peptide of about 30 kDa that contained its carbohydrate moiety, and a 20 000 kDa nonglycosylated peptide that bore the cytochalasin B label. Because it is produced by a cleavage at the cytoplasmic face and because the carbohydrate moiety is known to be exposed to the outside, the larger fragment must cross the bilayer. It has been reported that the Band 4.5 sugar transporter may be derived from Band 3 peptides by endogenous proteolysis, but the cleavage pattern found in the present study differs markedly from that previously reported for Band 3. Minimization of endogenous proteolysis by use of fresh cells, proteinase inhibitors, immediate use of ghosts and omission of the alkaline wash resulted in no change in the incorporation of [³H]cytochalasin B into Band 4.5, and no labelling of Band 3 polypeptides. These results suggest that the cytochalasin B binding component of Band 4.5 is not the product of proteolytic degradation of a Band 3 component.     

Apical CFTR Expression in Human Nasal Epithelium Correlates with Lung Disease in Cystic Fibrosis

Introduction

Although most individuals with cystic fibrosis (CF) develop progressive obstructive lung disease, disease severity is highly variable, even for individuals with similar CFTR mutations. Measurements of chloride transport as expression of CFTR function in nasal epithelial cells correlate with pulmonary function and suggest that F508del-CFTR is expressed at the apical membrane. However, an association between quantitative apical CFTR expression in nasal epithelium and CF disease severity is still missing.

Methods and Materials

Nasal epithelial cells from healthy individuals and individuals with CF between 12–18 years were obtained by nasal brushing. Apical CFTR expression was measured by confocal microscopy using CFTR mAb 596. Expression was compared between both groups and expression in CF nasal epithelial cells was associated with standardized pulmonary function (FEV₁%).

Results

The proportion of cells expressing apical CFTR in columnar epithelium is lower in CF compared to non-CF. The apical CFTR expression level was significantly correlated with FEV₁% in F508del homozygous subjects (r = 0.63, p = 0.012).

Conclusion

CFTR expression in nasal epithelial cells is lower in subjects with CF compared to healthy subjects. The proportion of cells expressing F508del-CFTR at the apical membrane is variable between subjects and is positively correlated with FEV₁% in F508del-CFTR homozygous subjects.     

The H+ Pump in Frog Skin (Rana esculenta): Identification and Localization of a V-ATPase

We here report on studies on the frog skin epithelium to identify the nature of its excretory H⁺ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry using V-ATPase-directed antibodies. Bafilomycin A₁ (10 μm) blocked H⁺ excretion (69 ± 8% inhibition) and therefore Na⁺ absorption (61 ± 17% inhibition after 60 min application, n= 6) in open-circuited skins bathed on their apical side with a 1 mm Na₂SO₄ solution, ``low-Na<sup>+</sup> conditions' under which H<sup>+</sup> and Na<sup>+</sup> fluxes are coupled 1:1. The electrogenic outward H<sup>+</sup> current measured in absence of Na<sup>+</sup> transport (in the presence of 50 μm amiloride) was also blocked by 10 μm bafilomycin A<sub>1</sub> or 5 μm concanamycin A. In contrast, no effects were found on the large and dominant Na<sup>+</sup> transport (short-circuit current), which develops with apical solutions containing 115 mm Na<sup>+</sup> (``high-Na⁺ conditions'), demonstrating a specific action on H⁺ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations now deliver conclusive evidence that H⁺ excretion is mediated by a V-ATPase being the electrogenic H⁺ pump in frog skin. Received: 21 May 1996/Revised: 24 December 1996     

Stimulation of Transepithelial Na<Superscript>+</Superscript> Current by Extracellular Gd<Superscript>3+</Superscript> in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Alveolar Epithelium

In the present study we investigated the effect of extracellular gadolinium on amiloride-sensitive Na⁺ current across Xenopus alveolar epithelium by Ussing chamber experiments and studied its direct effect on epithelial Na⁺ channels with the patch-clamp method. As observed in various epithelia, the short-circuit current (I _sc) and the amiloride-sensitive Na⁺ current (I _ami) across Xenopus alveolar epithelium was downregulated by high apical Na⁺ concentrations. Apical application of gadolinium (Gd³⁺) increased I _sc in a dose-dependent manner (EC ₅₀ = 23.5 µM). The effect of Gd³⁺ was sensitive to amiloride, which indicated the amiloride-sensitive transcellular Na⁺ transport to be upregulated. Benz-imidazolyl-guanidin (BIG) and p-hydroxy-mercuribenzonic-acid (PHMB) probably release apical Na⁺ channels from Na⁺-dependent autoregulating mechanisms. BIG did not stimulate transepithelial Na⁺ currents across Xenopus lung epithelium but, interestingly, it prevented the stimulating effect of Gd³⁺ on transepithelial Na⁺ transport. PHMB increased I _sc and this stimulation was similar to the effect of Gd³⁺. Co-application of PHMB and Gd³⁺ had no additive effects on I _sc. In cell-attached patches on Xenopus oocytes extracellular Gd³⁺ increased the open probability (NP _o) of Xenopus epithelial sodium channels (ENaC) from 0.72 to 1.79 and decreased the single-channel conductance from 5.5 to 4.6 pS. Our data indicate that Xenopus alveolar epithelium exhibits Na⁺-dependent non-hormonal control of transepithelial Na⁺ transport and that the earth metal gadolinium interferes with these mechanisms. The patch-clamp experiments indicate that Gd³⁺ directly modulates the activity of ENaCs.     

Erythrocytes anion transport and oxidative change in β‐thalassaemias

β‐Thalassaemia is characterized by a decrease in globin β‐chain synthesis and an excess in free α‐globin chains. This induces alterations in membrane lipids and proteins resulting from a reduction in spectrin/band 3 ratio, partial oxidation of band 4.1 and clustering of band 3. The membrane injury provokes hyperhaemolysis and bone marrow hyperplasia. The pathophysiology of thalassaemia is associated with iron overload that generates oxygen free radicals and oxidative tissue injury with ocular vessel alterations. The aim of this research is to investigate the influence of oxidative stress on band 3 efficiency, which is an integral membrane protein of RBCs (red blood cells). Band 3 protein, of which there are more than 1 million copies per cell, is the most abundant membrane protein in human RBCs. It mediates the anion exchange and acid–base equilibrium through the RBC membrane. Some experiments were performed on thalassaemic cells and β‐thalassaemia‐like cells and tested for sulfate uptake. To test the antioxidant effect of Mg²⁺, other experiments were performed using normal and pathological cells in the presence of Mg²⁺. The oxidant status in thalassaemic cells was verified by increased K⁺ efflux, by lower GSH levels and by increased G6PDH (glucose‐6‐phosphate dehydrogenase) activity. The rate constant of SO₄ ^2? uptake decreases in thalassaemic cells as well as in β‐thalassaemia‐like cells when compared with normal cells. It increases when both cells are incubated with Mg²⁺. Our data show that oxidative stress plays a relevant role in band 3 function of thalassaemic cells and that antioxidant treatment with Mg²⁺ could reduce oxidative damage to the RBC membrane and improve the anion transport efficiency regulated by band 3 protein.     

Control of Na+ and H+ transports by exocytosis/endocytosis phenomena in a tight epithelium

The relationship linking Na⁺ and H⁺ transports and exocytosis/endocytosis located in the apical membranes of the frog skin epithelium was investigated under various conditions of ion transport stimulation. The exocytosis process, indicating insertion of intracellular vesicles, which were preloaded with fluorescent FITC-dextran (FD), was measured by following the FD efflux in the apical bathing solution.Na⁺ transport stimulators such as serosal hypotonic shock (replacement of serosal Ringer solution by half-Ringer or 4/5-Ringer), apical PCMPS (10^–3 m) and amphotericin-B (20 g/ml), were also found to stimulate the exocytotic rates of FD. Acidification of the epithelium by CO₂ or post NH₄ load, conditions which increase the proton secretion also stimulated the FD release in the apical bathing solution. On the other hand, alkalization of the epithelial cells increased the endocytosis rate. Hypotonic shock, acid load and PCMPS induced an increase in cell calcium which is probably the signal within the cell for exocytosis. In addition, quantitative spectrofluorimetric measurements of F-actin content after rhodamine-phalloidin staining, indicated a decrease in the F-actin content as a result of cell acidosis, hypotonic conditions and amphotericin additions. It is proposed that the insertion/retrieval of intracytoplasmic vesicles containing H⁺ pumps plays a key role in the regulation of proton secretion in tight epithelia. In addition, it is suggested that cytoskeleton depolymerization of F-actin filaments facilitates H⁺ pump insertion. A comparable working hypothesis for the control of Na⁺ transport is proposed.This work was supported by grants from the Commissariat à l'Energie Atomique and The Centre National de la Recherche Scientifique UA 638.We would like to thank Dr. R.M. Hays and Dr. J. Condeelis (Albert Einstein College of Medicine, New York) for stimulating discussions. The confocal microscope observations were done through the courtesy of Dr. C. Sardet and C. Rouvière (Station Marine de Villefranche/mer France).