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T Zeuthen

Publications and source records attributed to T Zeuthen.

At least 37 records · Page 2Linked to original sources
PubMed1997

Antibodies against a furosemide binding protein from Ehrlich ascites tumour cells inhibit Na+, K+, Cl- co-transport in frog retinal pigment epithelium.

PURPOSE: To investigate whether antibodies against a 100 kDa protein purified by furosemide affinity chromatography from Ehrlich ascites tumour cells could inhibit Na+, K+, Cl- co-transport in the isolated frog retinal pigment epithelium. METHODS: The rate of Na+, K+, Cl- co-transport across the retinal membrane in the isolated frog RPE preparation was measured as the rate of decrease in the intracellular Cl- activity observed after administration of furosemide in the apical bath. The intracellular Cl- activity was measured with double barrelled Cl- sensitive microelectrodes. RESULTS: Incubation of frog retinal pigment epithelium for 30 min with antibodies reduced the rate of Na+, K+, Cl- co-transport by 43%, while leaving all other measured electrophysiological parameters intact. CONCLUSION: The antibodies inhibit Na+,K+,Cl- co-transport in the frog retinal pigment epithelium. This could be due to binding of the antibodies to the co-transporter itself or to a regulatory protein.

Animals↗
PubMed1996

Cotransport of H+, lactate and H2O by membrane proteins in retinal pigment epithelium of bullfrog.

1. The interaction between H+, lactate and H2O fluxes in the retinal membrane of the pigment epithelium from bullfrog Rana catesbiana was studied by means of ion-selective micro-electrodes. 2. Changes in intracellular pH and cell volume were recorded in response to abrupt changes in retinal solution concentration and/or osmolarity. 3. Two parallel pathways for water transport were identified across the retinal membrane, an osmotic one with a hydraulic water permeability of 3.2 x 10(-4) cm s-1 (osmol l-1)-1 and one which depended on the presence of lactate. 4. Addition of sodium lactate to the retinal solution caused cell shrinkages that were small compared with those produced by mannitol. The reflection coefficient for sodium lactate was 0.25. 5. Isosmotic replacement of Cl- with lactate caused an influx of water. Simultaneous acidification of the retinal solution from pH 7.4 to 6.4 enhanced the effect. The influx of water could proceed against osmotic gradients elicited by mannitol. 6. The interdependence of the fluxes of H+, lactate and H2O can be described as cotransport: the fluxes had a fixed ratio of about 109 mmol of lactic acid per litre of water, the flux of one species was able to energize the flux of the other two, and the fluxes exhibited saturation for increasing driving forces. 7. The Gibbs equation gives an accurate quantitative description of these coupled fluxes.

Animals↗
PubMed1996

Cotransport of water by the Na+/glucose cotransporter.

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na+/glucose cotransporter. The Na+/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na+/CI-/gamma-aminobutyric acid, Na+/iodide and H+/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.

Animals↗
PubMed1996

Regulation of Ca2+-activated K+ channel from rabbit distal colon epithelium by phosphorylation and dephosphorylation.

The Ca2+-activated maxi K+ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na+ reabsorption from the intestinal lumen. Previous measurements of these basolateral K+ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca2+ with a K0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca2+. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K+ channels lost their high sensitivity to Ca2+, yet they could still be activated by high concentrations of Ca2+ (10 micron). Furthermore, the high sensitivity to Ca2+ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30-120 sec for dephosphorylation and of 10-280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca2+ of the maxi K+ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca2+ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca2+ sensitivities for maxi K+ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems.

Animals↗
PubMed1995

Molecular mechanisms for passive and active transport of water.

Water crosses cell membranes by passive transport and by secondary active cotransport along with ions. While the first concept is well established, the second is new. The two modes of transport allow cellular H2O homeostasis to be viewed as a balance between H2O leaks and H2O pumps. Consequently, cells can be hyperosmolar relative to their surroundings during steady states. Under physiological conditions, cells from leaky epithelia may be hyperosmolar by roughly 5 mosm liter-1, under dilute conditions, hyperosmolarities up to 40 mosm liter-1 have been recorded. Most intracellular H2O is free to serve as solvent for small inorganic ions. The mechanism of transport across the membrane depends on how H2O interacts with the proteinaceous or lipoid pathways. Osmotic transport of H2O through specific H2O channels such as CHIP 28 is hydraulic if the pore is impermeable to the solute and diffusive if the pore is permeable. Cotransport of ions and H2O can be a result of conformational changes in proteins, which in addition to ion transport also translocate H2O bound to or occlude in the protein. A cellular model of a leaky epithelium based on H2O leaks and H2O pumps quantitatively predicts a number of so-far unexplained observations of H2O transport.

Biological Transport, Active↗
PubMed1994

Cotransport of K+, Cl- and H2O by membrane proteins from choroid plexus epithelium of Necturus maculosus.

1. The interaction between K+, Cl- and H2O fluxes was studied in the ventricular membrane of the choroid plexus epithelium from Necturus maculosus by means of ion-selective microelectrodes. 2. Three experimental strategies were adopted: the osmolarity of the ventricular solution was increased abruptly by addition of (i) mannitol or (ii) KCl; (iii) Na+ in the ventricular solution was replaced isosmotically by K+. 3. The mannitol experiments showed that H2O had two pathways across the ventricular membrane. One was purely passive, with a water permeability, L'p, of 0.64 x 10(-4) cm s-1 (osmol l-1)-1. This operated in parallel with an ion-dependent pathway of similar magnitude which was abolished in Cl(-)-free solutions. 4. When KCl was added there was a flow of H2O into the cell. Surprisingly, this took place despite the osmotic gradient which favoured an efflux of H2O. The effect was blocked by frusemide (furosemide), in which case KCl had the same effects as applications of NaCl or mannitol. 5. Replacement of Na+ with K+ caused an influx of H2O. This flux could proceed against osmotic gradients implemented by mannitol. 6. The present data and those of earlier publications show that the interdependence of the fluxes of K+, Cl- and H2O in the exit membrane can be described as cotransport. The fluxes have a fixed stoichiometry of 1:1:500, the flux of one species is able to energize the flux of the two others, and the transport exhibits saturation and is specific for K+ and Cl-. 7. A molecular model based upon a mobile barrier in a membrane spanning protein gives an accurate quantitative description of the data.

Animals↗
PubMed1993

Osmotic properties of the frog retinal pigment epithelium.

The water permeabilities of the membranes of the frog retinal pigment epithelial cells (RPE cells) were studied by means of double-barrelled, ion-selective microelectrodes. The cells were loaded with tetramethylammonium ion (TMA+) or with choline ions (Ch+) which served as intracellular volume markers. The intracellular activities of tetramethylammonium ions (TMAi+) and choline ions (Chi+) were measured with K+ selective intracellular microelectrodes. Intracellular activities of K+ and Cl- were also measured, and these ions were investigated as intracellular volume markers. The osmolarity on retinal side of the epithelium was changed abruptly. The rates of change in TMAi+ were linearly related to the changes in extracellular osmolarity in the range between (-46)-(+46) mosmol l-1. The osmotic water permeability of the retinal membrane, Lp,r, estimated from these experiments was 3.8 x 10(-4) cm sec-1 (osmol l-1)-1. For an osmotic gradient of 23 mosmol l-1 there was no difference between Lp,r determined with Ch+, TMA+ and Cl- as intracellular volume markers. This rules out any specific biological artifacts of the volume measurement. Intracellular K+ was not suitable as intracellular volume marker, since the initial rates of change in the intracellular K+ activity were about 30% lower than the rates of change in TMAi+ when osmotic gradients less than +/- 46 mosmol l-1 were applied. Among the exogenous volume markers, TMA+ was found more reliable than Ch+. For osmotic gradients larger than 23 mosmol l-1 the volume change recorded with Ch+ was smaller than that recorded with TMA+ as volume marker. Since the membrane permeability for Ch+ was 23 times larger than for TMA+, this suggests that Ch+ left the cells during higher rates of shrinkage. The cells acted almost as ideal osmometers, since the steady-state changes in intracellular osmolarity closely reflected the retinal extracellular osmolarity changes. It is concluded that the osmotic water permeability of the retinal membrane is larger than that of the choroidal membrane. This suggests that the rate limiting step for osmotic water movements between the eye and the blood is the choroidal membrane of the retinal pigment epithelium.

Animals↗
PubMed1993

Ca2+ activation and pH dependence of a maxi K+ channel from rabbit distal colon epithelium.

To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca(2+)-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 Ca(2+)-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 K0.5 for Ca2+ was raised from 20 nM at a potential of 0 mV to 300 nM 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 (K0.5 = 10 nM) by charybdotoxin of the Ca(2+)-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.

Animals↗
PubMed1991

Water permeability of ventricular cell membrane in choroid plexus epithelium from Necturus maculosus.

1. The osmotic water permeability Lp and the relations between the flows of H2O, K+ and Cl- were studied in the ventricular membrane of the epithelium from the choroid plexus of Necturus maculosus. 2. The flows were induced by abrupt changes in external osmolarity of the ventricular solution. Solution changes were convective and no effects of unstirred layers could be detected on measured parameters. 3. The initial rate of change in intracellular concentrations of K+ and Cl- was monitored by double-barrelled ion-selective microelectrodes. 4. The initial rate of flux of H2O could be monitored as the changes in the concentration of intracellular choline ions (Ch+i). When 0.5 mmol l-1 of choline chloride was added to the external solutions, Ch+i attained values of 1-5 mmol l-1. The dilution or concentration of Ch+i could be recorded by K+ electrodes since the sensitivity of these to Ch+ is more than 50 times greater than to K+. 5. The Lp of the ventricular membrane of the epithelium was 1.4-2.1 x 10(-4) cm s-1 (osmol l-1)-1 and independent of the direction of the induced water flux. Lp was unchanged in tissues adapted to osmolarities of half the physiological value. 6. The efflux of H2O induced by mannitol was associated with an instantaneous efflux of K+ which was inhibited by furosemide. The fluxes had a ratio of 40 mmol l-1. The influx of H2O induced by the removal of NaCl from the ventricular solution was associated with an instantaneous influx of K+. The H2O influx had a ratio to the flux of K+ of 70 mmol l-1. 7. The efflux of H2O induced by mannitol was associated with an efflux of Cl- which was inhibited by furosemide. The ratio of the two fluxes was in the range 15-44 mmol l-1. 8. The conclusion is that the Ch+ method gives a reliable measure of the movement of H2O across the ventricular membrane. The magnitude of the Lp and its relevance to transepithelial transport are discussed. The osmotically induced H2O movement is accompanied by furosemide-sensitive fluxes of K+ and Cl- of the same magnitude. This suggests that co-transport between H2O and KCl can take place in the membrane.

Animals↗
PubMed1991

Secondary active transport of water across ventricular cell membrane of choroid plexus epithelium of Necturus maculosus.

1. The interaction between Cl-, K+ and H2O fluxes were studied in the ventricular membrane of the choroid plexus epithelium from Necturus maculosus by means of ion-selective microelectrodes. The flux of H2O was measured by means of K+ electrodes as the dilution or concentration of intracellular choline ions, Ch+i. 2. In one series of experiments Cl- was readministered to the ventricular solution of tissues incubated in media with low Cl- concentrations. The resulting influx of Cl- was associated with an instantaneous influx of K+ and H2O. 3. Both the Cl- and the K+ influxes were reduced by the diuretic furosemide but were unaffected by inhibitors of Na+, K(+)-ATPase or changes in membrane potentials induced by Ba2+. Since the influx of K+ proceeds against its electrochemical gradient and is unaffected by changes in membrane potentials, the membrane exhibits secondary active, electroneutral transport of K+. 4. The influx of water, initiated simultaneously with the influx of K+ and Cl-, commenced before these ions had changed the osmolarity of the intracellular solution significantly. The influx of H2O could proceed against an osmotic gradient. The influx stopped when 100 mmol l-1 of mannitol was added to the ventricular solution at the same time as the Cl- ions. The influx of H2O was inhibited by K+ removal, furosemide or high external Ba2+ (10 mmol l-1), but not by strophanthidin, ouabain or low concentrations of Ba2+ (0.5 mmol l-1). The influx could not continue with other permeable anions, NO3-, acetate- or SCN-, replacing Cl-. 5. In another series of experiments Cl- was removed from the ventricular solution of tissues bathed in saline solutions with normal concentrations of Cl-. The resulting efflux of Cl- was associated with an instantaneous efflux of K+ and H2O. This efflux of H2O could proceed against an osmotic gradient of up to 70 mosmol l-1. This effect was inhibited by furosemide, in which case the water fluxes were entirely dependent on the osmotic gradients and the osmotic water permeability Lp of the ventricular membrane. 6. The data suggest that there is a coupling between the flux of KCl and of water in the ventricular membrane, which implies that the reflection coefficient sigma for KCl under the given circumstances is less than one. I suggest that the ability of leaky epithelia to transport against osmotic gradients depends on such a coupling, which derives from the properties of the proteins through which K+, Cl- and H2O leave the cell.

Animals↗
PubMed1989

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells.

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, non-denaturing 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 of 86Rb+ 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.

Animals↗
PubMed1988

Characterization of a purified co-transporting protein.

1. A protein of mol weight 280,000 D was isolated and purified by means of a furosemide affinity gel. 2. Binding of 3H-bumetanide suggests that the protein is identical to the Na-K-2Cl co-transporter. 3. If the protein was reconstituted into a planer lipid bilayer, a Cl- -channel of 12 pS and a K+-channel of about 130 pS was observed. 4. Whether these channel activities represent a co-purification of channel proteins or whether the channel activity originates from the purified and reconstituted co-transporting protein itself was discussed.

Animals↗
PubMed1987

Maxi K+ channels in leaky epithelia are regulated by intracellular Ca2+, pH and membrane potential.

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 approximately 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 approximately 0.4 micron-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.

Animals↗
PubMed1987

The mechanism of electrodiffusive K+ transport in leaky epithelia and some of its consequences for anion transport.

The ventricular membrane of the epithelium from the choroid plexus of Necturus maculosus was probed with double-barrelled ion-selective microelectrodes while the ventricular bathing solution was changed abruptly. The transient states induced by increasing the external concentration of K+ or by the application of ouabain showed that the passive movements of K+ across the ventricular membrane were electro-diffusive and could be described by the Goldmann equation and one constant permeability (PK) of 24 X 10(-6) cm s-1. The passive efflux was balanced by a ouabain-sensitive influx. PK was different in different steady states; when the cell was acidified by half a pH unit by increasing CO2 in the bathing solutions from 1 to 5%, then PK decreased to 13 X 10(-6) cm s-1. Removal of Cl- from the bathing solution increased PK by 50% and reduction of Cl- transport by furosemide did not alter PK, consequently major movements of K+ were independent of Cl- movements. Depolarizations of the cell caused an increase in the cellular HCO-3 concentration due to an electrodiffusive permeability (PHCO3), the value of which was estimated to 17 X 10(-6) cm s-1.

Animals↗
PubMed1987

The effects of chloride ions on electrodiffusion in the membrane of a leaky epithelium. Studies of intact tissue by microelectrodes.

The electrodiffusive permeability for Cl-, its dependence on low extracellular Cl--concentrations and the interaction between the movements of Cl- and K+ were investigated in the ventricular membrane of epithelial cells from the choroid plexus of Necturus maculosus. Cells were probed with ion-selective microelectrodes sensitive to Cl-, K+ and H+. The initial effects of abrupt changes in the Cl--concentration (Cl-v) and/or the K+-concentration (K+v) of the ventricular solution were investigated. The effect of changing the membrane potential by changing K+v was twofold: It caused an electrodiffusive flux of Cl- via a permeability of 1.3 X 10(-6) cm s-1. This permeability together with the K+-permeability of the ventricular membrane (24 X 10(-6) cm s-1) determined the membrane potential in the given steady state within a few mV. The other effect of the depolarization was an increase in the intracellular concentration of HCO-3 which in turn caused an influx of Cl- via electroneutral Cl-/HCO-3 exchange. The Cl--permeability was reduced by more than 60% and the neutral exchange by more than 90% by furosemide. The effect of decreases in Clv was a tenfold increase of the electrodiffusive Cl--permeability of the ventricular membrane to 12.2 X 10(-6) cm s-1 and also a tenfold increase in the permeability to K+. This activation was reduced by two thirds by furosemide, and by depolarizations of the cell by high K+v. In the given steady state the HCO-3/Cl- exchanger at the ventricular membrane transports at a rate of 300 pmol cm-2 s-1 and moves Cl- into the cell and HCO-3 into the ventricular solution. Thus the epithelium alkalinizes the cerebrospinal fluid at a rate which is about three times faster than the net transport rate of Na+.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗