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  • 1
    ISSN: 1432-1424
    Keywords: acanthocytes ; bilayer ; cell shape ; chlorpromazine ; erythrocytes ; lipid ; McLeod ; membranes ; organization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary We have sought to elucidate the spiculated shape of McLeod erythrocytes. Red cells from a normal donor and from a McLeod patient were incubated in phosphate-buffered saline containing 0, 0.05, or 0.1mm chlorpromazine at 0°C for 5 min. then glutaraldehyde-fixed, and examined by scanning electron microscopy. The normal red cells were biconcave disks in which chlorpromazine induced inward (negative) curvature: deep cupping (stomatocytosis) and multiple invaginations. The McLeod cells were mostly spiculated. Chlorpromazine at lower concentration converted them into biconcave disks and, at higher concentration, into stomatocytes. These results support the hypothesis that the spiculation of McLeod cells is the result of an imbalance of surface area between the two lipid leaflets of the membrane; that is, a bilayer couple effect. We determined the numerical density of intramembrane particles (IMP) in replicas of both fracture faces of red cells subjected to freeze fracture and rotary shadowing. These values were as follows (expressed per μm2 of membrane ±sd): the normal protoplasmic fracture face had 2200±306 and the McLeod had 2300±250. The normal exoplasmic fracture face had 388±75 and the McLeod had 330±59. We conclude that there is no evidence for derangement of band 3, the principal protein in theIMP, in McLeod erythrocytes.
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  • 2
    ISSN: 1432-1424
    Keywords: cortical collecting tubule ; principal cell ; intercalated cell ; NaCl transport ; ouabain ; cell volume ; mineralocorticoids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Ouabain had no effect on the volume of intercalated cells of DOCA-stimulated rabbit cortical collecting tubules, but caused principal cells to swell rapidly at an initial rate of 67% min., Principal cells swelled 133% then activated regulatory volume decrease mechanisms and shrank at an initial rate of −3%/min to a new volume 13% above control. The initial rate of ouabain swelling was completely inhibited by perfusate Na+ removal or reduced 95% by luminal addition of 10−5 m amiloride. Luminal peritubular, or bilateral Cl− removal each caused cell shrinkages of 10% and reduced the rate of ouabain swelling by 70, 85, and 99%, respectively. The presence of an apical Cl− transport step in principal cells was confirmed by increasing luminal K+ from 5 to 53mm, which caused cell swelling of 22%. This volume increase was completely blocked by luminal Cl− removal, but was unaffected by peritubular Cl− substitution. Perfusion of CCT with 0.1mm acetazolomide, 0.1mm DPC or 0.5mm SITS caused principal cell shrinkages of 7–9% and reduced the rate of ouabain swelling by 60, 70, and 40%, respectively. The initial rate of ouabain swelling was inhibited 70% by bilateral CO2/HCO3 removal and 50% by whole animal acid loading. Taken together these results demounstrate that ouabain swelling is due to cellular NaCl accumulation and that Na+ enters the cell primarily through apical Na+ channels. Cellular Cl− entry occurs at least partially through the apical membrane and may be mediated by a Cl−/HCO 3 − exchanger. Brief (45–90 sec) exposure of principal cells to ouabain is associated with a rapid inhibition of Na+ and/or Cl− entry steps, whereas long-term (〉5min) ouabvain exposure completely blocks one or both of these transport pathways.
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  • 3
    ISSN: 1432-1424
    Keywords: epithelial cells ; cell polarity ; plasma membrane proteins ; sulfo-NHS-biotin ; streptavidin ; Triton X-114
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Selective biotinylation of the apical or basolateral domains of confluent MDCK monolayers grown on polycarbonate filters with a water soluble biotin analog, sulfo-NHS-biotin, was employed to reveal strikingly distinct patterns of endogenous “peripheral” and “integral” membrane proteins. “Peripheral” proteins were found to be approximately fivefold more abundant with this procedure than “integral” membrane proteins, both on the apical and on the basolateral surface. The distinct apical and basal patterns were shown to depend upon the integrity of the monolayer; when the tight junctions were disrupted by preincubation in calcium-depleted medium, the patterns appeared practically indistinguishable. Two-dimensional gel electrophoresis demonstrated that only a very small percentage of the biotinylated proteins were found in similar amounts on both apical and basolateral domains. These results indicate that the sorting mechanisms that segregate apical and basolateral epithelial proteins are very strict. The simple procedure described here has clear advantages over other methods available to label apical and basal epithelial surface domains, namely, higher accessibility of the biotin probe to the basolateral membrane, possibility of purifying biotinylated proteins via immobilized streptavidin and minimal exposure of the researcher to isotopes. It should be very useful in characterizing the apical and basolateral protein compositions of other epithelial cells and in studies on the development of epithelial cell polarity.
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  • 4
    ISSN: 1432-1424
    Keywords: trout ; intestine ; brush-border membrane ; vesicle ; Cl− conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Experiments were performed to determine the presence of a Cl−−OH− exchange (Cl−−H+ cotransport) in the brush-border membranes isolated from the intestinal epithelium of freshwater trout. Determinations of alkaline phosphatase activities have shown that vesicle suspensions had an enrichment factor of about 17 in this enzyme indicating a high degree of purification of the brush-border membrane preparation. Cl− uptake by vesicles in the presence of a proton gradient occurs against a concentration gradient with an overshoot ratio of about 2 and is inhibited by SITS. Several lines of evidence suggest that the mechanism involved is electrical in nature: (i) Cl− uptake is increased when the proton gradient is increased, but there is a linear relationship between the Cl− uptake and the Nernst potential of protons. (ii) Cl− uptake is increased when a proton ionophore is added at low concentration and inhibited at high concentration, suggesting that a proton conductance is involved in the Cl− uptake. (iii) there is a linear relationship between the initial speed of the uptake of increasing Cl− concentrations and the Cl− concentration. (iv) Cl− uptake can be modulated by different potassium gradients with or without valinomycin. It is concluded that the enterocyte of the freshwater trout is not equipped with a Cl−−OH− exchange and the Cl− uptake by vesicles is realized by a Cl− conductance.
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  • 5
    ISSN: 1432-1424
    Keywords: rat colon ; Cl− channels ; anthracene-9-carboxylic acid ; apical membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Chloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl− channels with a conductance of 35–40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mm). In excised patches from single enterocytes, isolated by calcium removal, the Cl− channels were studied in more detail. TheI–V relation was linear between ±80 mV. The selectivity was I−〉Br−〉Cl−=NO 3 − 〉F−=HCO 3 − . Thirty pS Cl− channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. TheI–V curve of these Cl− channels was also linear. The results provide direct evidence for the existence of Cl− channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.
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  • 6
    ISSN: 1432-1424
    Keywords: barium current ; internal perfusion ; patch clamp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Isolated nerve cells fromLymnaea stagnalis were studied using the internal-perfusion and patch-clamp techniques. Patch excision frequently activated a voltage-independent Ba2+-permeable channel with a slope conductance of 27 pS at negative potentials (50mm Ba2+). This channel is not seen in patches on healthy cells and, unlike the voltage-dependent Ca channel, is not labile in isolated patches. The activity of the channel in inside-out patches is unaffected by intracellular ATP, Ca2+ below 1mm or the catalytic subunit of cAMP-dependent protein kinase but is reversibly blocked by millimolar intracellular Ca2+ or Ba2+. The channel can be activated in on-cell patches by either internal perfusion with high Ca2+ or the long-term internal perfusion of low Ca2+ solutions not containing ATP. These channels may carry the inward Ca2+ current which causes a regenerative increase in intracellular Ca+ when snail neurons are perfused with high Ca2+ solutions. High internal Ca2+, or long periods of internal perfusion with ATP-free solutions, induces an increase in a resting (−50 mV) whole-cell Ba2+ conductance. This conductance can be turned off by returning the intracellular perfusate to a low Ca2+ solution containing ATP and Mg2+. The activity of this channel appears to have an opposite dependence on intracellular conditions to that of the voltage-dependent Ca channel.
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  • 7
    ISSN: 1432-1424
    Keywords: proton flux ; lipid bilayers ; membrane permeability ; gramicidin ; ion channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 8
    ISSN: 1432-1424
    Keywords: cardiac gap junctions ; thiol/disulfide exchanges
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary SDS-polyacrylamide gel electrophoresis and immunoblotting were used to investigate inter- and intramolecular disulfide bonds to connexin 43 (the cardiac gap junctional protein) in isolated rat heart gap junctions and in whole heart fractions. In gap junctions isolated in the absence of alkylating agent, connexin 43 molecules are cross-linked by disulfide bonds. The use of iodoacetamide (100mm) for the first steps of isolation procedure prevents the formation of these artifactual linkages. Investigation of connexin 43 in whole heart fractions by means of antibodies confirms the results obtained with isolated gap junctions; that is, connexin 43 molecules are not interconnected with disulfide bridges. In whole heart fractions treated with alkylating agents, a 38 kD protein, immunologically related to connexin 43, and containing intramolecular disulfide bonds is detected. It is hypothesized that this protein might be a folded form of connexin 43, a precursory form of the molecules embedded in the gap junctions.
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  • 9
    ISSN: 1432-1424
    Keywords: monensin ; cell ATP ; cell ions ; water ; liver ; lung ; fetal liver ; fetal lung
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Effects of the proton-alkali cation-exchanging ionophore, monensin, on aspects of cellular metabolism and ionic exchanges have been studied in rat tissues in vitro. Incubation of liver slices at 38°C with 0.1 μm monensin induced timedependent vesiculation, initially in the Golgi region, reduction of ATP content and of protein synthesis. At 1 νm, monensin also reduced net, active movements of K+, Na+, Cl− and water in liver slices and inhibited state 3 respiration in isolated mitochondria. The respiratory inhibitor, amytal, similarly reduced ATP content and protein synthesis at concentrations lower than those inhibiting ion transport in slices. Low concentrations of monensin (0.1–1.0 μm) had similar effects on ATP and ion transport in slices of adult lung. By contrast, late-fetal liver and lung were much less sensitive to monensin; in these tissues, glycolysis sustained substantial levels of ATP. Monensin also induced vesiculation of the Golgi apparatus in fetal lung cells. It is concluded that by lowering ATP levels, monensin can markedly alter various metabolic activities in those cells which depend primarily on oxidative phosphorylation for their metabolic energy.
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  • 10
    ISSN: 1432-1424
    Keywords: electrical coupling ; gap junction ; double whole cell patch clamp ; cAMP ; protein kinase C ; OAG
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Gap junctional coupling was studied in pairs of murine pancreatic acinar cells using the double whole-cell patch-clamp technique. During stable electrical coupling, addition of OAG (1-oleoyl-2-acetyl-sn-glycerol) induced a progressive reduction of the junctional conductance to the detectable limit (∼3 pS). Prior to complete electrical uncoupling, varius discrete single channel conductances between 20 and 100 pS could be observed. Polymyxin B, a potent inhibitor of the protein kinase C (PKC) system, completely suppressed OAG-stimulated electrical uncoupling. Dialysis of cell pairs with solutions containing PKC. isolated from rat brain, also caused electrical uncoupling. The presence of 0.1mm dibutyryl cyclic AMP and 5mm ATP in the pipette solution, which serves to stabilize the junctional conductance, did not suppress the effects of OAG or isolated PKC. We conclude that an increase of protein kinase C activity leads to the closure of gap junction channels, presumably via a PKC-dependent phosphorylation of the junctional peptide, and that this mechanism is dominant over cAMP-dependent upregulatory effects in the experimental time range (≤1 hr). A correlation of the observed single channel conductances with the appearance of channel subconductance states or various channel populations is discussed.
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