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F V Sepúlveda

Publications and source records attributed to F V Sepúlveda.

At least 19 recordsLinked to original sources
PubMed1992

Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein.

The human multidrug resistance P-glycoprotein is an active transporter that pumps cytotoxic drugs out of cells. Expression of P-glycoprotein is also associated with a volume-activated chloride channel. Here we address the relationship between these two functions. Drug transport requires ATP hydrolysis while, in contrast, ATP binding is sufficient to enable activation of the chloride channel. The chloride channel and drug transport activities of P-glycoprotein appear to reflect two distinct functional states of the protein that can be interconverted by changes in tonicity. Transportable drugs prevent channel activation but have no effect on channel activity once it has been preactivated by hypotonicity. The transport and channel functions of P-glycoprotein have been separated by directed mutations in the nucleotide-binding domains of the protein. These data provide further evidence that P-glycoprotein is bifunctional with both transport and channel activities. Implications for the design of chemotherapeutic drugs and for the function of the related cystic fibrosis gene product, CFTR, are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein.

Expression of P-glycoprotein, the product of the MDR1 gene, confers multidrug resistance on cell lines and human tumours (reviewed in refs 1,2). P-glycoprotein (relative molecular mass 170,000) is an ATP-dependent, active transporter which pumps hydrophobic drugs out of cells, but its normal physiological role is unknown. It is a member of the ABC (ATP-binding cassette) superfamily of transporters, which includes many bacterial transport systems, the putative peptide transporter from the major histocompatibility locus, and the product of the cystic fibrosis gene (the cystic fibrosis transmembrane regulator, CFTR). CFTR is located in the apical membranes of many secretory epithelia and is associated with a cyclic AMP-regulated chloride channel. At least two other chloride channels are present in epithelial cells, regulated by cell volume and by intracellular Ca2+, respectively. Because of the structural and sequence similarities between P-glycoprotein and CFTR, and because P-glycoprotein is abundant in many secretory epithelia, we examined whether P-glycoprotein might be associated with one or other of these channels. We report here that expression of P-glycoprotein generates volume-regulated, ATP-dependent, chloride-selective channels, with properties similar to channels characterized previously in epithelial cells.

3T3 Cells↗
PubMed1992

The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression.

The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities. We have compared their cell-specific expression in the rat by in situ hybridization. In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions. Additionally, a switch in expression from one gene to the other was observed in certain tissues. For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary. A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy. These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume. The patterns of expression suggest that the regulation of these two genes is coordinately controlled.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Membrane conductance and cell volume changes evoked by vasoactive intestinal polypeptide and carbachol in small intestinal crypts.

We have used the perforated-patch whole-cell recording mode of the patch-clamp technique to monitor membrane potential and measured cell volume changes by image analysis, to determine the nature of the response to secretagogues of isolated whole guinea-pig small-intestinal crypts. Vasoactive intestinal polypeptide (VIP) produced a dose-dependent depolarisation (EC50 = 30 nM) and an increase in membrane conductance that could be potentiated by carbachol. Similar depolarisations were observed with forskolin. The depolarisation induced by 100 nM VIP was smaller when pipette [Cl-] was 60 mM than when it was 145 mM, suggesting an effect through Cl- conductance activation. Carbachol alone produced a hyperpolarisation (EC50 = 2 microM). The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) produced a small hyperpolarization. When VIP was added in the presence of NPPB, the depolarisation was observed instead, consistent with the parallel activation of a K+ conductance. Both carbachol (100 microM) and VIP (100 nM) induced a 25%-30% shrinkage of crypts, which was maximal 8 min after addition of the secretagogue. The induced shrinkage was sustained in the continued presence of agonist and was reversed upon washout. Shrinkage induced by the agonists was abolished by increasing extracellular K+ from 6 mM to 20 mM and was inhibited partially in the presence of 100 microM anthracene-9-carboxylic acid in the bath. The decrease in volume induced by 100 nM VIP was totally abolished in the presence of 100 microM NPPB. The results are consistent with the view that both VIP and carbachol induce secretion in small-intestinal crypts.

Animals↗
PubMed1992

A large conductance K(+)-selective channel of guinea pig villus enterocytes is Ca2+ independent.

The presence of K(+)-selective channels has been probed in enterocytes isolated from guinea pig small intestinal villi by the patch-clamp technique. A channel with a single-channel conductance of approximately 130 pS was observed in excised inside-out patches bathed in symmetrical K+. A change in the K+ concentration in the intracellular aspect of the membrane altered the current-voltage relationship as expected from the constant-field equation when it is assumed that K+ is the only permeant ion. A change in Cl- concentration was without effect. Neither the activity of the channel nor its conductance was altered by addition of ATP or Ba2+ to the intracellular side of the patches. Changes in the free Ca2+ concentration were also without effect. The channel's open probability showed no voltage dependence and appeared only occasionally active in cell-attached patches where it had a linear current-voltage relation. The K+ channel described, which cannot be readily classified in any of the known classes of K+ channels, might provide an exit pathway for K+ recycling in guinea pig villus enterocytes.

Animals↗
PubMed1991

Regulatory volume decrease in small intestinal crypts is inhibited by K+ and Cl- channel blockers.

Total crypt volume has been estimated by analysis of photographic images of intact viable crypts isolated from guinea-pig small intestine. Exposing these crypts to a hypotonic medium, led to transient swelling followed by regulatory volume decrease (RVD) in 12-20 min. RVD was blocked by inhibitors of K+ and Cl- conductance, suggesting that it occurs by activation of K+ and Cl- permeability pathways and loss of these ions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

A basolateral K+ conductance modulated by carbachol dominates the membrane potential of small intestinal crypts.

The contribution of possible ionic conductances to the membrane potential (Em) of cells in guinea-pig small intestinal crypts has been studied using the nystatin "perforated-patch" approach in current-clamp experiments. Changes in extracellular K+ produced shifts in Em, with a 37 mV change in potential per ten-fold increase in extracellular K+ concentration. Reduction of extracellular Cl- by 130 mM led to a 7 mV hyperpolarisation while Na+ replacement was without effect on Em. The muscarinic agonist carbachol produced a hyperpolarisation which could be ascribed to an increase in basolateral K+ conductance. This effect was sustained in the presence of extracellular Ca2+ but was transient in its absence. We conclude that the conductance of the basolateral membrane of small intestinal crypts is mainly K+ selective and can be reversibly increased by muscarinic activation.

Animals↗
PubMed1991

Two types of potassium currents seen in isolated Necturus enterocytes with the single-electrode voltage-clamp technique.

1. The ionic permeability of Necturus maculosus small intestine epithelial cells was investigated using intracellular microelectrodes to measure membrane potential in intact tissue or by the single-electrode voltage-clamp technique in isolated cells. 2. The basolateral membrane of enterocytes appears to be K+ selective as demonstrated by the dependence of membrane potential and fractional serosal resistance measured in the intact epithelium on serosal K+ concentration. 3. Isolated cells had membrane potentials similar to those measured in the intact tissue. Voltage-clamp experiments in a physiological Ringer solution showed the presence of both large outward and inward currents. 4. Removal of Cl- from the bathing medium, linear subtraction or the use of a Cl- channel blocker revealed outwardly rectifying currents. The quasi-linear component was also revealed following K+ channel inhibition; it reversed near ECl, suggesting that the charge carrier was Cl-. 5. Outwardly rectifying currents could be kinetically resolved into two components. A fast component (tau for activation < 4 ms) accounted for 60-80% of the total current at positive potentials. A slowly activating component appeared at voltages positive to 50 mV with tau for activation of > 25 ms. 6. The slow outward current showed strong voltage dependence of both activation and relaxation, which were faster at more depolarized potentials. 7. Both fast and slow outward currents seem to be carried by K+ as they were blocked by Ba2+ and tetraethylammonium (TEA). Tail current analysis of the slow component indicated a reversal potential very similar to EK. 8. Fast outward currents were half-activated at about -40 mV whereas slow outward currents were only apparent at more positive potentials. It is proposed that the fast outward K+ current plays a role, together with Cl- currents, in determining the resting membrane potential of Necturus enterocytes.

Animals↗
PubMed1991

Potassium currents of isolated Necturus enterocytes: a whole-cell patch-clamp study.

1. The whole-cell recording mode of the patch-clamp technique was used to investigate the ionic currents of enterocytes isolated from the small intestine epithelium of Necturus maculosus. 2. When enterocytes were bathed in a physiological Ringer solution and dialysed with a K(+)-rich intracellular solution containing 1.5 x 10(-7) M intracellular Ca2+, strongly outwardly rectifying currents were observed. These currents were absent from enterocytes internally dialysed with K(+)-free solutions. 3. The kinetics of activation of the outwardly rectifying current was monoexponential with rate constants decreasing with depolarization from 160 ms at 20 mV to 40 ms at 60 mV. Similar voltage dependence of the relaxations after activation were observed. 4. Strongly buffering intracellular Ca2+ with EGTA inhibited outward currents, while increasing Ca2+ increased both their magnitude and rate of activation. 5. Bath application of the K+ channel inhibitors Ba2+ and TEA greatly attenuated outwardly rectifying currents. This observation plus the fact that tail currents reverse near EK points to K+ as the charge carrier in these currents. 6. Outside-out patches showed maxi K+ channels and lower conductance K+ channels. Averaging fluctuations of the maxi K+ channels gave a kinetic behaviour similar to the whole-cell currents.

Animals↗
PubMed1991

K+ and Cl- currents in enterocytes isolated from guinea-pig small intestinal villi.

1. The whole-cell configuration of the patch-clamp technique has been used to investigate the conductance properties of villus enterocytes isolated from guinea-pig small intestinal epithelium. 2. With near physiological ionic gradients inward and outward rectification was observed in the hyperpolarizing and depolarizing voltage domains respectively. 3. Replacement of intra- and extracellular K+ with N-methyl-D-glucamine (NMG) eliminated inward rectification but did not alter outward currents. In symmetrical low Cl- solutions outward currents were reduced but inward rectification was not affected. Under these conditions increases in extracellular K+ shifted both the current-voltage relation and the extrapolated reversal potential as expected for a K(+)-selective current. 4. The inwardly rectifying nature of the K+ current observed here remained unaltered after chelation of internal Mg2+ with ATP or EDTA. 5. Extracellular application of 5 mM-Ba2+ or 50 micrograms ml-1 of the venom of the scorpion Leiurus quinquestriatus abolished the inward K+ current, while 5 mM-extracellular tetraethylammonium (TEA) had little effect. 6. The current remaining in the presence of symmetrical Cl- solutions and in the complete absence of K+ rectified outwardly and reversed at 0 mV. The anionic nature of this current was confirmed by replacing Cl- with different anions. SCN- and Br- carried more current than Cl-, while F- and gluconate were less permeant. 7. Anionic currents of villus guinea-pig enterocytes were not stimulated by cyclic AMP and were strongly and reversibly inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 10(-5) M). 8. The inwardly rectifying K+ current described here shares some, but not all, characteristics with others previously described. It is postulated that this conductance might function to couple K+ permeability and the Na(+)-K+ pump rate in enterocytes. Absorption of chloride may be mediated by the Cl- channels.

Animals↗
PubMed1990

Possible involvement of GTP-binding proteins in the deactivation of an inwardly rectifying K+ current in enterocytes isolated from guinea-pig small intestine.

The possible regulation of guinea pig enterocyte ion channels by GTP-binding proteins has been investigated by using the whole-cell recording mode of the patch-clamp technique and non-hydrolysable analogues of GTP. The main K+ currents in these cells are mediated by inwardly-rectifying K+ channels. Intracellular dialysis with hydrolysis-resistant GTP analogues leads to the deactivation of the inward K+ currents. Non-hydrolysable analogues of ATP or GDP were without effect. The effect occurs after a lag phase of 2 to 7 min, suggesting a multistep mechanism. Cl currents were not affected by any of the nucleotide analogues. It is suggested that inwardly-rectifying K+ currents are deactivated by a G-protein-dependent process.

Animals↗
PubMed1989

Characterization of a phosphorylation-activated Cl-selective channel in isolated Necturus enterocytes.

1. The cell-attached and excised inside-out configurations of the patch-clamp technique were employed to probe isolated enterocytes of Necturus maculosus for the presence of Cl(-)-selective channels. 2. Chloride-selective channels were rarely observed unless cells were previously stimulated by agonists that raise cyclic AMP. In cell-attached patches forskolin (20 microM) or dibutyryl cyclic AMP 2 mM) evoked single-channel activity that reversed, depending on the cell, between 9 and 27 mV positive to the spontaneous membrane potential. This is close to the Cl- equilibrium potential in those cells; the single-channel current-voltage relationship was linear with a unitary slope conductance between 17 and 25 pS (pipettes filled with 100 mM-NaCl). 3. Large depolarizing voltage steps also activated Cl- channels in excised inside-out membrane patches that were previously quiescent. This mode of activation produced a distinctive single-channel current-voltage relationship with strong outward rectification at depolarizing membrane potentials. Single-channel cord conductance at negative potentials was 15-18 pS and increased to 45 pS at + 100 mV. 4. Altering the Cl- concentration in the bathing solution of excised inside-out patches displaced the observed reversal potential (Erev) to values predicted for Cl- equilibrium potential. Replacement of K+ for Na+ was without effect. 5. The effect of different anions upon Erev was used to determine the channel anion selectivity in excised inside-out patches. The permeability sequence was SCN- greater than I- greater than Br- greater than Cl- greater than F- greater than HCO3- greater than gluconate which corresponds to Eisenman's sequence 1. Neither ionic size nor diffusion rates determine the permeation of ions through the channel. 6. In channels activated by depolarization the open probability (Po) was insensitive to changes in the Ca2+ concentration (less than 10(-8)-10(-3) M) bathing the cytoplasmic face of excised inside-out patches. Depolarization was also without marked effect on Po. 7. Chloride channels in excised inside-out patches were inhibited by stilbene and diphenylamine-2-carboxylate derivatives. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS, 5 x 10(-5) M) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 1 x 10(-5) M) caused an irreversible 'flickery' blockade without altering single-channel current. 3'5-Dichlorodiphenylamine-2-carboxylic acid (DDPC, 5 x 10(-5) M) reduced the currents at every voltage without apparent effects on gating properties of the channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

K+ channels activated by L-alanine transport in isolated Necturus enterocytes.

Using the patch-clamp technique, we demonstrate here the opening of K+ channels evoked by the actively transported amino acid L-alanine in isolated Necturus enterocytes. These channels had a conductance of about 30 pS and their activation was dependent on transmembrane electrical potential and cytosolic Ca2+.

Alanine↗
PubMed1988

Activation of basolateral membrane K+ permeability by bradykinin in MDCK cells.

We study bradykinin-stimulated K+ efflux in Madin-Darby canine kidney (MDCK) cells using 86Rb as an isotopic tracer. Bradykinin brings about a rapid increase in the permeability of MDCK cells to K+, the effect is dose-dependent with a plateau at 10(-6) M. The effect seems to be mediated by Ca2+-activated K+ channels, localised at the basolateral aspect of the epithelium. Unlike alpha-receptors, which mediate a similar effect of adrenalin in these cells, bradykinin receptors seem to be present at both sides of the epithelium. Bradykinin increases the labelling of IP3, and bradykinin-stimulated K+ efflux persists even in cells which are bathed in Ca2+-free medium, suggesting that the effects seen in the present work are probably due to Ca2+ release from intracellular stores. Some extracellular Ca2+ also might be involved in the bradykinin effect, consistent with the kinin-increasing membrane permeability to Ca2+.

Animals↗
PubMed1988

A chloride conductance activated by adenosine 3',5'-cyclic monophosphate in the apical membrane of Necturus enterocytes.

1. Intracellular potentials, Cl- activity and membrane resistances were measured in Necturus small intestinal epithelium during Cl- replacement experiments using conventional or Cl- -selective double-barrelled microelectrodes. A Cl- conductance, located in the apical membrane and activated by cyclic nucleotides is demonstrated by ion-substitution experiments. 2. The mean mucosal membrane potential (Em) was -35.5 mV. Removal of Cl- from the mucosal medium by replacement with gluconate, evoked a sudden depolarization of Em and an immediate increase in the fractional resistance of the mucosal membrane (f(Rm)). The size of the change in Em varied between 3 and 65 mV, corresponding to Cl- to K+ permeability ratios between 0.2 and 20. It was inversely related to the initial f(Rm), which ranged from 0.04 to 0.50. 3. Prolonged incubation in low-Cl- solutions led to a reversal of the initial depolarization and to a sustained hyperpolarization accompanied by a marked increase in f(Rm). The new value of Em was close to the K+ equilibrium potential, consistent with a depletion of cellular Cl- and the preponderance of a K+ membrane permeability in the absence of Cl-. This emphasizes the role of Cl- in establishing Em. 4. Removal of mucosal Cl- produced a fast decrease in intracellular Cl-, as measured with Cl- -selective microelectrodes. The efflux was consistent with electrodiffusion across the mucosal membrane. Changes in Em paralleled changes in intracellular Cl- activity, indicating the presence of a large Cl- conductance. 5. Dibutyryl cyclic AMP or forskolin produced a slow depolarization, a decrease in f(Rm) and an increased change in intracellular potential in low mucosal Cl- which on average corresponds to an approximately 15-fold increase in the relative Cl- permeability. These results are consistent with an activation of apical Cl- conductance. 6. The selectivity of Cl- channels of Necturus enterocytes to different anions was obtained from potential measurements. The sequence of permeabilities was SCN- greater than I- greater than or equal to Br- greater than NO3- greater than Cl- much greater than HCO3- greater than gluconate. This is consistent with a model involving a weak interaction of the anions with the selectivity filter. 7. The selectivity of the anion conductance was maintained after activation with cyclic nucleotides, suggesting a single channel for the permeation of the different anions tested, rather than parallel channels. 8. Derivatives of 9-anthracene which are potent inhibitors of Cl- channels in other systems failed to block the apical Cl- conductance of Necturus enterocytes. Chloride conductance was also insensitive to furosemide.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Single channel recordings obtained from basolateral membranes of isolated rabbit enterocytes.

Epithelial cells isolated by hyaluronidase incubation from rabbit small intestine were used to explore the presence of ionic channels by the patch-clamp method. Recordings were made from cell-attached or excised patches of basolateral membrane. Evidence was obtained for the presence of at least two kinds of channels conducting potassium currents. One of these can be shown to be activated by Ca2+.

Animals↗
PubMed1985

A rabbit jejunal isolated enterocyte preparation suitable for transport studies.

A method is described for isolating viable enterocytes from rabbit jejunum. Estimates of sucrase and gamma-glutamyl transferase activities in cells isolated by this method suggest that they originate from the upper villus only. Isolated cells accumulate both alpha-methyl-D-glucoside and alanine, maintaining high intracellular concentrations for at least 60 and 40 min respectively. Accumulation of alpha-methyl-D-glucoside is inhibited by the presence of phloridzin. The cells accumulate 42K and 86Rb in an identical manner. This uptake, which is maintained for at least 60 min, is inhibited in the presence of ouabain. Passive efflux of 42K and 86Rb occurs with rate constants which are virtually identical. The efflux follows a single exponential suggesting that it originates from only one intracellular compartment. It is suggested that the preparation can be used to study the effect of sugars and amino acids on K efflux. The advantages of using such a preparation are discussed.

Alanine↗