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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Pflügers Archiv 408 (1987), S. 249-259 
    ISSN: 1432-2013
    Keywords: K-channel ; Ca-activation ; Leaky epithelia ; Patch-clamp ; Channel kinetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have studied a Ca2+-activated K+ channel in the ventricular membrane of the epithelium of choroid plexus by means of the patch-clamp technique, using excised inside-out patches. The channel was highly K+ selective. It had a conductance of ∼200 pS with 112 mM KCl on both sides of the membrane. The probability for the channel being open increased with intracellular Ca2+, pH and with membrane potential. The channel shows two gating modes. The primary gating mode has open and closed times which depend strongly on membrane potential, intracellular Ca2+ and pH. It accounts for the variation of the channel open probability. Lowering intracellular pH from 7.4 to 6.4 reduced the channel open probability mainly by increasing the channel closed time. It appears, that H+ can compete with Ca2+ in binding to the same site, thereby preventing channel opening. A second gating mode consisted of short-lived closures, or flickers. The open and closed time for this process were largely independent of membrane potential, intracellular Ca2+ and pH. The channel density was ∼0.4 μm−2 corresponding to a K+-permeability of 2.2 10−5 cm s−1 if the channels were fully open. In cell-attached patches we measured the open probability of the channel in the intact cell membrane. The channel is almost totally closed under normal cellular conditions. This type of channel is therefore not the membrane component that forms the electrodiffusive pathway for K+-ions.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2013
    Keywords: Electron microscopy Intracellular pH Monocarboxylate transport Pigment epithelium of eye Proton–lactate cotransport Retinal metabolism Sodium/proton exchange
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. Monolayer cultures of human fetal retinal pigment epithelial (RPE) cells were examined for ultrastructural characteristics and junctional integrity by means of electron microscopy. Intracellular pH (pHi) and cell volume changes were measured using the fluorescent dye BCECF. The EM studies indicate that the RPE cells preserve in vivo morphology before and after loading with BCECF. Monolayer cultures were placed in a perfusion chamber in which the solution facing the retinal cell membrane could be changed rapidly. Removal of Na+ or the addition of amiloride caused intracellular acidifications. pHi recovery from an NH4 +-induced acid load was blocked by sodium removal or amiloride addition. These results suggest the presence of a Na+–H+ exchange mechanism in the retinal cell membrane. When Cl- was replaced isotonically by lactate or pyruvate the cells acidified. The intracellular acidifications were saturable, reversibly reduced with the inhibitor probenecid (2 mM), and the lactate-induced acidifications were reversibly inhibited by equimolar concentrations of pyruvate. These results indicate the presence of a H+–lactate cotransport mechanism in the retinal membrane. When Cl– was replaced by lactate the cells not only acidified, they also swelled. The data are compatible with water transport induced by the H+–lactate cotransporter.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 39 (1978), S. 185-218 
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary InNecturus gallbladder epithelial cells the intracellular electrical potential, as recorded with microelectrodes, varied from −28 mV in the mucosal end to about −50 mV in the serosal end of the transporting cell. The Na+ activity varied concurrently from about 39mm to between 8 and 19mm. Thus, within the cell both the recorded electrical and chemical gradients caused Na+ to move towards the serosal end. Serosal addition of ouabain (5×10−4 m) caused the intracellular Na+ activity to attain electrochemical equilibrium within 30 min. However, the intracellular electrical potential gradient was only slowly affected. In cells from animals stored at 5°C, the Cl− activity varied from about 55mm in the mucosal end to 28mm in the serosal end, and the K+ activity from 50mm to between 95 and 131mm. Both ions were close to electrochemical equilibrium within the cytoplasm but were too concentrated to be in equilibrium with the mucosal solution. Bubbling CO2 through the mucosal solution caused the intracellular gradients to vanish. When Na+ in the bathing solutions was exchanged for K+, the intracellular electrical potential became roughly constant at about −5 mV. The Cl− activity became constant at 65mm, and the K+ activity became constant at 109mm, both close to equilibrium with the mucosal solution. The Na+ activity was reduced to about 1mm. The ratio of the cytoplasmic resistivities between cells bathed in K+-rich saline to cells bathed in Na+-rich saline was measured by means of triple-barreled electrodes and compared to the same ratio as assessed from the activity measurements. The two values were equal only if one assumes the mobility of Na+ inside the cell to be less than 1/10 of the mobility of K+ or Cl−. The same conclusion was reached by comparing the intracellular Na+ flux calculated from the gradient of electrochemical potential to that flux assessed from the net solute absorption. Animals kept at 15°C had lower intracellular Na+ activities, higher Cl− and K+ activities, and higher rates of absorption than animals stored at 5°C. Finally, the degree to which the intracellularly recorded electrical and chemical potentials could reflect an electrode artefact is discussed.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 76 (1983), S. 113-122 
    ISSN: 1432-1424
    Keywords: ion-selective microelectrodes ; lateral intercellular spaces ; osmolarity ; water transport ; epithelia ; cellular osmolarity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The ion activities in the lateral spaces of the unilateral preparation of the gallbladder ofRana catesbiana were measured by double-barrelled ion-selective microelectrodes. The bladders were bathed in a saline solution with a low osmolarity (62 mOsm) containing, inmm: 27 Na+, 27 Cl−, 2 K+, 1 Ca++, 4 HCO 3 − . Working at reduced osmolarities had the advantage of an increased volume transport and of widened intercellular spaces. The reference barrel recorded an electrical potential of +2.7 mV in the spaces; they contained a solution similar to the external solution. The electrodes recorded a Na+ concentration of 27mm, a K+ concentration of 1.7mm, a Ca++ concentration of 0.69mm and a Cl− concentration of 28.5mm. In the spaces there was a lower resistance between the tip of the electrode and the serosal bath than that recorded with the tip in the lumen, and injection of fluorescent dye (11 Å diameter) via the electrodes did not stain the cells. The concentrations in the secretion were similar to those in the spaces. The intracellular compartment had an apparent K+ concentration of 95mm, and the concentrations of Na+ and Cl− were both about 5mm. These data indicate that when the gallbladder is bathed with hypotonic solutions and is transporting fluid at approximately three or four times the normal rate, there are no significant osmotic gradients between the lumen and the lateral spaces. It is suggested that transcellular transport of water is implemented by a combination of high osmotic permeabilities across both mucosal and serosal cell membranes and low reflection coefficients (for K+ salts) at the serosal cell membranes.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 60 (1981), S. 105-128 
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Transport of potassium by bullfrog choroid plexus was studied using tracers and ion-selective microelectrodes. 1) Tracers: We measured unidirectional uptakes of42K across each surface of the plexus, efflux of42K from loaded tissues, intracellular potassium pools, and [3H]-ouabain binding.42K uptake across the brush border membrane was composed of diffusional and saturable components. The saturable component exhibited kinetics of a two-site, model withK m's of 0.3mm and aV max of 8 μmol cm−2 hr−1. Ouabain inhibited uptake across the brush border membrane with aK i of 1×10−7 m. Ethacrynic acid, phloretin, amiloride, and low sodium concentrations inhibited uptake, whereas bicarbonate ions increased transport up to 100%. The rate of ouabain-sensitive uptake across the serosal surface was only 6% of that across the ventricular surface. The efflux of potassium across the brush border membrane could account for most of the efflux from the epithelium. Potassium was accumulated within the plexus to a concentration in excess of 100mm.42K in the extracellular compartments exchanged with 40–55% of the intracellular potassium. Ouabain bound to the brush border membrane with aK m of 8×10−7 m, ak 1 of 3.8×104 mol−1 min−1 and ak −1 of 3×10−2 min−1. Ouabain binding was blocked by cymarin and gitoxigin. 2) Electrodes: Under control conditions the intracellular electrical potential,E vc, was −45 mV and the apparent intracellular K+-concentration, K c + , was 90mm. K+ ions appeared to be actively accumulated within the epithelium.E vc and K c + were followed under three experimental conditions: (i) treating the tissue with ouabain; (ii) varying the ventricular K+ concentration; and (iii) passing transmural currents. It is concluded that the permeability of the ventricular membrane to potassium (1–5 × 10−5 cm sec−1)_was much greater than the permeability of the serosal membrane and that the rate of K pumping into the epithelium was 0.35–0.55×10−9 mol cm−2 sec−1. 96% of the transmural current was paracellular, and the resistance of the serosal membrane was nine times greater than that of the ventricular membrane. Increasing K c + produced Nernstian changes inE vc andE K vc , but the cells only depolarized by 0.13 mV for each mV increase in the chemical potential of the ventricular solutions. Both types of experiments are consistent with a model of the epithelium where the cells are predominantly K+-permeable (at the ventricular membrane) and there is a large paracellular shunt. K+ is actively transported into the cells by a brush border Na/K-pump, only to leave the cell passively across the same membrane. The pump, which is electrogenic, transports two K ions into the cell for every three Na ions pumped out. There are about 10×106 pumps/cell, and each pump turns over 10 times/sec. The choroid epithelium behaves as predicted by the model proposed first by Koefoed-Johnsen, V., Ussing, H.H. (1958)Acta Physiol. Scand. 42:298.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-1424
    Keywords: furosemide ; affinity chromatography ; reconstitution ; purified K+ transport system ; cholate ; pore-gradient electrophoresis ; cotransport ; Ehrlich ascites tumor cells ; Ba2+
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K+ transport activity of these vesicles was assayed by measurement of86Rb+ uptake against a large opposing K+ gradient. The reconstituted system was found to contain a K+ transporting protein, which is sensitive to Ba2+ like the K+ channel previously demonstrated to be activated in intact cells after cell swelling.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-1424
    Keywords: Maxi K+ channel ; Calcium ; pH ; Charybdotoxin ; Rabbit distal colon
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca2+-activated K+ channels from the basolateral plasma membrane of the surface cells in the distal colon have been characterized by single channel analysis after fusion of vesicles with planar lipid bilayers. A Ca2+-activated K+ channel with a single channel conductance of 275 pS was predominant. The sensitivity to Ca2+ was strongly dependent on the membrane potential and on the pH. At a neutral pH, the K 0.5 for Ca2+ was raised from 20nm at a potential of 0 mV to 300nm at −40 mV. A decrease in pH at the cytoplasmic face of the K+ channel reduced the Ca2+ sensitivity dramatically. A loss of the high sensitivity to Ca2+ was also observed after incubation with MgCl2, possibly a result of dephosphorylation of the channels by endogenous phosphatases. Modification of the channel protein may thus explain the variation in Ca2+ sensitivity between studies on K+ channels from the same tissue. High affinity inhibition (K 0.5=10nm) by charybdotoxin of the Ca2+-activated K+ channel from the extracellular face could be lifted by an outward flux of K+ through the channel. However, at the ion gradients and potentials found in the intact epithelium, charybdotoxin should be a useful tool for examination of the role of maxi K+ channels. The high sensitivity for Ca2+ and the properties of the activator site are in agreement with an important regulatory role for the high conductance K+ channel in the epithelial cells.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1432-1424
    Keywords: gallbladder ; numerical model ; effects of ouabain ; steady states ; effects of transmural currents
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary A simple numerical model for theNecturus gallbladder epithelium is presented. K+, Na+ and Cl− cross the mucosal and serosal membranes as well as the junctions by means of electrodiffusion; furthermore the mucosal membrane contains a neutral entry mechanism for NaCl and the serosal membrane contains an active pump for K+ and Na+. The values which have been used for the model are taken from the literature. The model can only attain steady states if the resistance of the serosal membrane is lower than 1000ω cm2. Values reported in the literature for the resistance of this membrane vary from about 3000 to about 100ω cm2. We shall argue, however, that the higher estimates are in error because they are derived from a model of the tissue in which each membrane and the junction are modeled by a resistor; this procedure is invalid because the resistance of the lateral intercellular space relative to the resistance of the tight junctions is neglected and consequently the resistance of the serosal membrane is overestimated by a factor of about four. Apart from predicting a realistic steady state at normal external concentrations the model can predict quantitatively several experimental results obtained from the living epithelium. We have focused on the experiments which test the permeabilities of the serosal membrane and the properties of the pump:i) Replacement of serosal Cl− by an impermeant ion.ii) Replacement of serosal K+ by Na+.iii) Inhibiting the (Na+, K+)-pump. The best correspondence between model and experiments is obtained when the pump is assumed to be electrogenic (or rheogenic) with a ratio of coupling between Na+ and K+ of 3∶2. In this case both model and direct experiments (also presented in this paper) show an initial abrupt depolarization of 6 to 7 mV. The model also shows that it cannot be concluded fromi andii that the Cl− permeability of the serosal membrane is low. The model explains, even with high passive Cl− permeabilities, why the intracellular Cl− concentration is relatively unaffected by paracellular currents, a fact which in other epithelia has been taken as an implication of a low Cl− permeability of the serosal membranes.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 66 (1982), S. 109-121 
    ISSN: 1432-1424
    Keywords: intracellular ion activities ; intracellular osmolarity ; ion-selective microelectrodes ; water permeability of cell membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in theNecturus gallbladder. The intracellular activities of K+ and Cl− in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K+, Cl− and some unidentified anions. Surprisingly, the apparent K+ concentration remained high: at an external osmolartity of 64 mOsm the intracellular K+ concentration averaged 95mm. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na+, K+ and Cl− were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na+ and Cl− were equal. The data were consistent with Na+ and Cl− ions initially remaining inside the cell and a cell membraneL p of 10−3 cm sec−1 osm−1, which is close to the values determine by Spring and co-workers (K.R. Spring, A. Hope & B.-E. Persson, 1981.In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190–200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K+ was only 0.1–0.2 times the change observed in Na+ and Cl− activities, and suggests that K+ ions leave the cell during the osmotically induced H2O efflux and enter with an induced H2O influx. The coupling is between 98 and 102 mmoles liter−1. Various explanations for the anomalous behavior of intracellular K+ ions are considered. A discussion of the apparent coupling between K+ and H2O, observed in nonsteady states, and its effects on the distribution of K+ and H2O across the cell membrane in the steady states, is presented.
    Type of Medium: Electronic Resource
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