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  • 1
    Electronic Resource
    Electronic Resource
    Transmitter-induced changes of the membrane voltage of HT29 cells (1992)
    Springer
    Pflügers Archiv 421 (1992), S. 224-229 
    Details
    ISSN: 1432-2013
    Keywords: Cl− conductance ; HT29 ; P2 receptor ; Colon ; Cl− secretion ; cAMP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The colonic carcinoma cell line HT29 was used to examine the influence of agonists increasing cytosolic cAMP and Ca2+ activity on the conductances and the cell membrane voltage (V m). HT29 cells were grown on glass cover-slips. Cells were impaled by microelectrodes 4–10 days after seeding, when they had formed large plaques. In 181 impalements V m was −51±1 mV. An increase in bath K+ concentration from 3.6 mmol/l to 18.6 mmol/l or 0.5 mmol/l Ba2+ depolarized the cells by 10±1 mV (n=49) or by 9±2 mV (n=3), respectively. A decrease of bath Cl− concentration from 145 to 30 mmol/l depolarized the cells by 11±1 mV (n=24). Agents increasing intracellular cAMP such as isobutylmethylxanthine (0.1 mmol/l), forskolin (10 μmol/l) or isoprenaline (10 μmol/l) depolarized the cells by 6±1 (n=13), 15±3 (n=5) and 6±2 (n=3) mV, respectively. In hypoosmolar solutions (225 mosmol/l) cells depolarized by 9±1 mV (n=6). Purine and pyrimidine nucleotides depolarized the cells dose-dependently with the following potency sequence: UTP 〉 ATP 〉 ITP 〉 GTP 〉 TIP 〉 CTP = 0. The depolarization by ATP was stronger than that by ADP and adenosine. The muscarinic agonist carbachol led to a sustained depolarization by 27±6 mV (n=5) at 0.1 mmol/l, and to a transient depolarization by 12±4 mV (n=5) at 10 μmol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l. The depolarization induced by nucleotides and neurotensin was transient and followed by a hyperpolarization. We confirm that HT29 cells possess Cl−- and K+-conductive pathways. The Cl− conductance is regulated by intracellular cAMP level, cytosolic Ca2+ activity, and cell swelling. The K+ conductance in HT29 cells is regulated by intracellular Ca2+ activity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374831
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  • 2
    Electronic Resource
    Electronic Resource
    Forskolin and PMA pretreatment of HT29 cells alters their chloride conductance induced by cAMP, Ca2+ and hypotonic cell swelling (1994)
    Springer
    Pflügers Archiv 428 (1994), S. 76-83 
    Details
    ISSN: 1432-2013
    Keywords: HT29 ; CFPAC-1 ; Cl− secretion ; CFTR ; CF ; Cl− conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract HT29 cells were preincubated with forskolin (10−5 mol/l, FORHT) or phorbol 12-myristate 13-acetate (PMA) (10−7 mol/l, PMAHT) for 20 h, which has been shown previously and is also shown here, to upregulate and downregulate, respectively, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). CFPAC-1 cells underwent the same protocols. HT29 cells were examined by slow (SWC) and fast (FWC) whole-cell patch-clamp techniques. The results of SWC and FWC were indistinguishable and were pooled. CFPAC-1 cells were examined with FWC. The membrane voltage (V) of FORHT was -41.8±1.4 mV (n=77) and that of PMAHT was -43.6±2.4 mV (n=76). The conductance (G) of FORHT (9.4 ±0.9 nS, n=77) was significantly larger than that of PMAHT (3.7±0.4 nS, n=76). Acute application of forskolin (10−5 mol/l, FOR) plus 0.5 mmol/l 8-(4-chlorophenylthio)-cAMP (cAMP) depolarized V by 12 (FORHT) and 8 (PMAHT) mV, respectively. The acute increase of G by FOR plus cAMP was by 7.6±1.9 nS for FORHT (n=22) and only 2.2±1 nS for PMAHT (n=13). ATP (10−4 mol/l) depolarized V in both types of cells. It enhanced G by 16.7±4.1 nS in FORHT (n=14) and significantly less (by 5.5±1.2nS, n=14) in PMAHT. Also the G increase lasted longer in FORHT. Neurotensin (NT, 10−8 mol/l) also had a stronger and longer lasting effect in FORHT. Exposure to hypotonic bath solution (160 mosmol/l) depolarized V in both types of cells. The increase in G was by 15±2.2 nS in FORHT (n=18) and by 11±1.3 nS in PMAHT (n=23). After being returned to the normotonic media, the decline of G to the control value was delayed in FORHT when compared to PMAHT. Ionomycin (10−7 mol/l) increased G significantly more (to 47±8.5 nS, n=13) in FORHT when compared to PMAHT (to 28±4 nS, n=13). The present data indicate that a 20-h exposure of HT29 cells to FOR versus PMA alters markedly the CFTR concentration. The cells with high CFTR (FORHT) when compared to those with low CFTR (PMAHT) differ not only in their acute G response to cAMP, but also in that to ATP, NT, hypotonic cell swelling, and ionomycin. In contrast, the same pretreatment of CFPAC-1 cells did not alter the G changes induced by ionomycin or hypotonic cell swelling. These results indicate that changes in CFTR expression correlate with the Cl− conductances induced by cAMP, Ca2+ and hypotonic cell swelling.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374754
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  • 3
    Electronic Resource
    Electronic Resource
    Piretanide-dextran and piretanide-polyethylene glycol interact with high affinity with the Na+ 2 Cl− K+ cotransporter in the thick ascending limb of the loop of Henle (1989)
    Springer
    Pflügers Archiv 413 (1989), S. 559-561 
    Details
    ISSN: 1432-2013
    Keywords: Isolated perfused tubule ; cTAL ; Na+ 2Cl− K+ cotransporter ; piretanide ; macromolecular probe
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Piretanide blocks the Na+ 2Cl− K+ cotransporter protein in the thick ascending limb (TAL) of the loop of Henle reversibly. When tested from the luminal side in isolated perfused cTAL segments it leads to a half maximal inhibition (IC50) of the equivalent short circuit current (Isc) at a concentration of 10−6 mol/l. From the basolateral side it has no effect on Isc up to 10−4 mol/l. The present study was designed to search for high affinity blockers of the Na+ 2Cl− K+ cotransporter with large molecular weight in an attempt to use these macromolecules for antibody-labelling or affinity separation of this transport-protein. Amino-ethyl-dextran or amino-ethyl-polyethylene glycol (M.W. 5kd) were coupled to isothiocyanato-piretanide (ISO-PIR) at room temperature in DMSO. The resulting compounds dextran-sulfonylurea-piretanide (PIR-DEX) and polyethylene glycol-sulfonylurea-piretanide (PIR-PEG) (M.W. 5.38kd) were purified and tested in isolated perfused cTAL segments. IC50 values for ISO-PIR, PIR-DEX and PIR-PEG were estimated from dose response curves after their addition to the lumen or bath perfusate, respectively. ISO-PIR, PIR-DEX and PIR-PEG acted from the lumen side at 3·10−6, 6·10−6 and 2·10−6 mol/l. The inhibitory effect was easily reversible. From the basolateral side no effect for any compound was seen at up to 10−4 mol/l. In clearance experiments PIR-DEX was given to female Wistar rats as an i.v. bolus (25 μmol/kg) and the diuretic urine was collected. After dialysis (exclusion limit 2.5kd) the dialysed urine and the dialysate were tested in isolated perfused cTAL segments. The dialysates had no effect on Isc, but the dialysed urine inhibited Isc by 35% from the luminal side. The present data show: High molecular derivatives of piretanide with dextran or polyethylene glycol moieties block the Na+ 2Cl− K+ cotransporter in cTAL segments at roughly the same low concentration as piretanide itself. Our data exclude a metabolism of these piretanide compounds in the kidney. Since these macromolecular probes can probably not enter the cell their inhibitory effect indicates that the binding site for piretanide diuretics on the Na+ 2Cl− K+ cotransporter is exposed on the surface of the luminal cell membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00594189
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  • 4
    Electronic Resource
    Electronic Resource
    Nucleoside transport in mammalian cell membranes (1978)
    Springer
    The journal of membrane biology 39 (1978), S. 133-157 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Transport of the nucleoside analog cytosine-arabinoside (CAR) in transformed hamster cells in culture has been studied in conditions of minimal metabolic conversion. Uptake (zero-trans in) properties at 20°C over a limited range of CAR concentrations were characterized by aK m of 350 μm and a maximal velocity (V) of 780 μm·min−1 (V/K m =2.28 min−1). Equilibrium exchange at 20°C over a wider range of concentrations was best described by a saturable component with aK m of 500 μm and av of 1230 μm·min−1 (V/K m =2.26 min−1) and either a saturable component of highK m or a nonsaturable component ofk=0.3 min−1. For the saturable component, thev/K m values were similar in both procedures. CAR transport was inhibited by various metabolizable nucleosides. Uptake of some of these nucleosides was inhibited by CAR. CAR transport and uridine uptake were inhibited in a reversible but partially competitive fashion by high affinity probes like S-(p-nitrobenzyl-6-mercaptoinosine (NBMI) (K i 〈0.5nm) and in an irreversible fashion by SH reagents such as N-ethylmaleiimide (NEM). The organomercurialp-hydroxymercuribenzene sulfonate (pMBS) markedly stimulated transport of these nucleosides, but also markedly potentiated the inhibitory effects of either NBMI or NEM. These effects are interpreted either in terms of models which invoke allosteric properties or in terms of two transport systems which display distinct chemical susceptibilities to externally added probes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870329
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  • 5
    Electronic Resource
    Electronic Resource
    Nucleoside transport in mammalian cell membranes (1978)
    Springer
    The journal of membrane biology 39 (1978), S. 159-183 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Organomercurials form stable stoichiometric complexes with thiolated nucleosides. The complexes inhibited uptake of ribonucleosides and cytosine arabinoside (CAR) in various types of normal and transformed cells. The inhibition was competitive and reversible (K i =3–6 μm). The interaction between complexes and transport system displayed a 1∶1 stoichiometry. Chemical factors which contributed to the inhibitory power were evaluated with a series of S-alkylated derivatives and S−Hg−R complexes of mercaptonucleosides. The inhibitory potency was not determined exclusively by the hydrophobic nature of either the S-alkylated or the S−Hg−R moieties. Chemical modification of cells with penetrating and nonpenetrating organomercurials lead to stimulation of nucleoside uptake and to an increase in its susceptibility to inhibition by S−Hg−R complexes or S-alkylated derivatives of mercaptopurine ribosides. The kinetic and chemical data obtained with nucleoside analogs and with chemical modifiers suggested complex features of nucleoside transport systems. Four distinct classes of sites were implied: (i) a substrate binding site susceptible directly to competitive inhibition by organomercurial-mercaptonucleoside complexes, (ii) an additional site susceptible either to S-arylalkylated or S-mercuriated derivatives of 6-mercaptopurine ribosides, (iii) SH-containing modifier sites whichstimulate uridine uptake upon binding of organomercurials, and (iv) SH-containing modifier sites whichinhibit the function upon binding of organomercurials. From the observation that only SH sites related to stimulation were susceptible to modification by macromolecular-SH modifier probes, some conclusions can be drawn regarding the disposition of the various sites in the cell membrane in general and among membrane components in particular.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870330
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  • 6
    Electronic Resource
    Electronic Resource
    The nature of the membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives (1972)
    Springer
    The journal of membrane biology 10 (1972), S. 311-330 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The disulfonic acid stilbene derivative SITS reported to be covalently bonded to the membrane of the red blood cell, was found to be largely reversibly bound. Reversal of its specific inhibitory effect on anion permeability was attained by washing the cells with buffer containing albumin. The small fraction of covalently bonded SITS could be increased by prolonging the time of exposure of the cells or by multiple exposures. A series of other disulfonic stilbene derivatives was synthesized. All of them specifically inhibited anion permeability whether or not they are capable of forming covalent bonds. Their inhibitory effectiveness, however, varied over a 5,000-fold range, allowing certain conclusions to be made concerning the chemical architecture of the binding site. Certain of the compounds were almost entirely covalently bonded. One of them was labeled with125I and used to determine to which membrane proteins the compound is bound. Over 90% was found in a protein band on acrylamide gels of 95,000 mol wt. The most effective compound against sulfate permeability was equally effective against chloride permeability, producing a maximum inhibition of over 95%. The residual anion fluxes respond differently to pH and temperature than do the fluxes of unmodified cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01867863
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  • 7
    Electronic Resource
    Electronic Resource
    Membrane proteins related to anion permeability of human red blood cells (1974)
    Springer
    The journal of membrane biology 15 (1974), S. 227-248 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The proteolytic enzymes, pronase, chymotrypsin and trypsin, release a small fraction of covalently bonded 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonate or (3H)DIDS, a specific inhibitor of anion permeability, from intact human red cells. The rate of release is parallel to the digestion of the sialoglycoprotein, indicating that the released (3H)DIDS was bound to that component. Most of the label is associated with a protein that behaves on SDS acrylamide gel electrophoresis as though its molecular weight was 95,000 Daltons (95K). Trypsin has no effect on this protein, but after pronase or chymotrypsin treatment of the cells, the label is found in three peaks of 95, 65 and 35K in proportions of 5, 85 and 10%. In parallel, the enzyme treatment results in a shift of most of the 95K protein to 65 and 35K. The digestion of the glycoprotein and splitting of the 95K protein can occur without any appreciable effects of the enzymes on anion permeability or on the inhibitory effects of DIDS treatment either before or after proteolytic attack. It is suggested that the sialoglycoprotein and the 35K segment of the 95K protein are not involved directly in anion permeation. The most likely location of the inhibitory site is in a portion of the 65K segment, exposed to the outside surface. Some additional observations are presented concerning the shielding effects of the negatively charged sialoglycoprotein and the arrangement of the 95K protein in the membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870089
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  • 8
    Electronic Resource
    Electronic Resource
    Membrane proteins related to anion permeability of human red blood cells (1974)
    Springer
    The journal of membrane biology 15 (1974), S. 207-226 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary (3H)DIDS (4,4′-diisothiocyano-2,2′-ditritiostilbene-disulfonate) was used as a convalent label for membrane sites involved in anion permeability. The label binds to a small, superficially located population of sites, about 300,000 per cell, resulting in almost complete inhibition of anion exchange. The relationship of biding to inhibition is linear suggesting that binding renders each site nonfunctional. In the inhibitory range less than 1% of the label is associated with lipids but at higher concentrations of DIDS, the fraction may be as high as 4%. In ghosts, however, treatment with (3H)DIDS results in extensive labeling of lipids. In cells, a protein fraction that behavens on SDS acrylamide gels as thought its molecular weight is 95,000 daltons (95K) is predominatly labeled by (3H)DIDS. The only other labeled protein is the major sialoglycoprotein which contains less than, 5% of the total bound (3H)DIDS. Because of the linear relationship of binding to inhibition and the unique architecture of the site, it is suggested that the (3H)DIDS-binding site of the 95K protein is the substrate binding site of the anion transport system. The 95K protein is asymmetrically arranged in the membrane with the sites arranged on the outer face accessible to agent in the medium. In “leaky” ghost, only a few additional binding sites can be reached from the inside of the membrane in the 95K protein, in contrast to the extensive labeling of other membrane proteins in ghosts as compared to cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870088
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  • 9
    Electronic Resource
    Electronic Resource
    The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: I. Kinetic properties of NBD-taurine transfer in symmetric conditions (1983)
    Springer
    The journal of membrane biology 71 (1983), S. 141-148 
    Details
    ISSN: 1432-1424
    Keywords: anion exchange ; fluorescent studies ; membrane permeability ; red blood cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The molecular mechanism of anion exchange across the human red blood cell membrane was assessed with the fluorescent substrate analog NBD-taurine and the method of continuous monitoring of transport by fluorescence. The efflux of NBD-taurine was studied under a variety of experimental conditions such as temperature, pH and anion composition of cells and media. The temperature profile of NBD-taurine transfer from Cl-loaded cells into Cl media resembled that of Cl self-exchange, whereas that of NBD-taurine transfer from sulfate-loaded cells into sulfate media resembled that of sulfate self-exchange. Although the pH profiles of NBD-taurine transfer from Cl-loaded cells into Cl media and that of Cl self-exchange resembled each other, the analogous transfer with sulfate replacing Cl was markedly different. These and other data were analyzed and found to be consistent with a model which comprises the following: (a) a H+-titratable group in the carrier mechanism; (b) alteration of transport sites between the two sides of the membrane (i.e., ping-pong kinetics); and (c) transmembrane distribution of transport sites which is modulated by pH. It is shown that NBD-taurine transfer represents a tracer flux of a fluorescent substrate which gives a measure for the presence of monovalent transport sites at the inner surface of the membrane. The latter is markedly affected by the relative concentrations of anions and H+ on both sides of the red blood cell membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870682
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  • 10
    Electronic Resource
    Electronic Resource
    The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: II. Kinetic properties of NBD-taurine transfer in asymmetric conditions (1983)
    Springer
    The journal of membrane biology 71 (1983), S. 149-161 
    Details
    ISSN: 1432-1424
    Keywords: anion exchange ; fluorescent studies ; membrane permeability ; red blood cells ; conformational states
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The transport of inorganic anions across human red blood cell membranes is accomplished by a carrier-like mechanism which involves an electroneutral and obligatory one-for-one anion exchange. The transport kinetics were described by models that involve alternation of single transport sites between the two membrane surfaces. These models predict that each carrier shows either an inward-facingE i or an outward-facingE o, conformation, each capable of binding either a monovalent anion or a divalent anion+a proton, to yield an electroneutral translocating complex. Unidirectional transport rates provide, therefore, a measure for the relative concentration of carriers at a given membrane surface. In the present work we assessed how modulation of the transmembrane distribution of carriers by the anion composition of cells and media, and by pH, affect the anion transport system. We have set the system in asymmetric conditions with respect to anions, so that a fast transportable anion (e.g., chloride) was present in one side of the membrane and slow transportable anions (e.g., sulfate, phosphate, oxalate, isethionate, gluconate, HEPES) were present on the other side of the membrane. The skewed distribution of carriers induced in these conditions were assessed by two methods: 1) NBD-taurine transfer which provided a measure for [E i], the monovalent inward-facing form of the carrier, and 2) inhibition of NBD-taurine transfer by the specific impermeant and competitive inhibitor 4,4′-dinitro-2,2′-stilbene disulfonic acid (DNDS), which provided a measure for the availability of the carrier at the outer membrane surface. In the various symmetric and asymmetric conditions, we found marked differences in transport rates and transport profiles as well as in the susceptibility of the system to inhibition by DNDS. Direct binding studies of DNDS to cells in the various asymmetric conditions supported the conclusion derived from transport studies that transport sites can be recruited towards the membrane surface facing the slow transportable anions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870683
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