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F V Sepúlveda

Publications and source records attributed to F V Sepúlveda.

At least 55 records · Page 3Linked to original sources
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

Kinetics of voltage- and Ca2+ activation and Ba2+ blockade of a large-conductance K+ channel from Necturus enterocytes.

Potassium channels in membranes of isolated Necturus enterocytes were studied using the patch-clamp technique. The most frequent channel observed had a conductance of 170 pS and reversal potential of 0 mV in symmetrical potassium-rich solutions. Channels were highly K- selective. Channel activity was modulated by membrane potential and cytosolic Ca2+ concentration. Channel openings occurred in characteristic bursts separated by long closures. During bursts openings were interrupted by brief closures. Two gating modes controlled channel opening. The primary gate's sensitivity to intracellular Ca2+ concentration and membrane potential crucially determined long duration closures and bursting. In comparison, the second gate determining brief closures was largely insensitive to voltage and intracellular Ca2+ concentration. The channel was reversibly blocked by cytosolic barium exposure in a voltage-sensitive manner. Blockade reduced open-state probability without altering single-channel conductance and could be described, at relatively high Ca2+ concentration, by a three-state model where Ba2+ interacted with the open channel with a dissociation constant of about 10(-4) M at 0 mV.

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↗
PubMed1987

Changes in the apparent chloride permeability of Necturus enterocytes during the sodium-coupled transport of alanine.

The membrane potential and intracellular Cl- activity of Necturus enterocytes were measured with double-barrelled ion-selective microelectrodes and apparent permeability coefficients (PCl) for the apical membrane calculated from Cl(-)-replacement experiments. In the presence of L-alanine in the mucosal solution an increase in PCl took place. It is proposed that this might reflect the activation of a Cl- conductance during active substrate transport.

Alanine↗
PubMed1987

Inositol trisphosphate and calcium mobilisation in permeabilised enterocytes.

Saponin-permeabilised epithelial cells isolated by hyalurodinase incubation from chicken small intestine were used to study 45Ca uptake into intracellular stores. At low (6.7 X 10(-7) M) free Ca2+ concentration most of the Ca2+ appears to be taken up into non-mitochondrial stores, whilst the mitochondria seem to play a major role at high (2 X 10(-5) M) Ca2+ concentration. Addition of inositol trisphosphate (IP3) causes a rapid and reversible release of 45Ca from non-mitochondrial stores, with a half-maximal effect of approximately 1 microM.

Animals↗
PubMed1987

Calcium transport by permeabilised rabbit small intestinal epithelial cells.

Intestinal epithelial cells isolated from rabbit small intestine and whose plasma membrane had been rendered highly permeable were used to study the role of intracellular structures in Ca2+ buffering. Monitoring free Ca2+ with a selective electrode revealed that the cells could reduce Ca2+ concentration in the medium to a level of 3.6 X 10(-7) M independently, within a certain range, of the initial Ca2+ concentration or amount of cells used. Ca2+ buffering by permeabilised enterocytes was Mg2+- and ATP-dependent and was abolished in the presence of the Ca2+ ionophore A23187. The rapidity of Ca2+ buffering, but not the final Ca2+ level attained, was reduced in the combined presence of the mitochondrial inhibitors azide and oligomycin or in the presence of ruthenium red. Buffering of Ca2+ was abolished in the presence of vanadate, although some uptake was still observed. Complete blocking occurred in the presence of both vanadate and mitochondrial inhibitors. Measurement of initial rates of uptake with radioactive calcium revealed that mitochondrial uptake plays a role at relatively high Ca concentrations but that at the presumably physiological levels most of the uptake is into a non-mitochondrial compartment. Non-differentiated crypt cells seemed to handle intracellular Ca2+ in a similar way as mature villus cells, although they appeared to buffer at a level about 2 X 10(-7) M lower.

Adenosine Triphosphate↗
PubMed1986

Calcium movements accompanying the transport of sugar or amino acid by rabbit enterocytes.

Isolated rabbit enterocytes can be loaded with radioactive calcium most of which is presumably in intracellular stores. In the presence of sugar or amino acid there is a transient loss of calcium, followed by replenishment. It is suggested that this movement might be related in the signalling leading to increased potassium permeability observed in enterocytes transporting sugar and amino acids.

Amino Acids↗
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

Cationic amino acid transport by two renal epithelial cell lines: LLC-PK1 and MDCK cells.

LLC-PK1 and MDCK cells take up cationic amino acids (lysine and arginine) by a specific sodium independent transport system. Uptake is inhibited by ornithine in LLC-PK1 and MDCK cells either in the presence or absence of sodium and by glutamine or homoserine in MDCK cells in the presence of sodium. Trans-stimulation of uptake occurs in the presence of intracellular cationic amino acids. Experiments with valinomycin or with different extracellular potassium concentrations suggest that uptake is dependent on the membrane potential of these cells. These transport features are similar to those previously ascribed to a transport system denominated y+ in other cells. Further experiments suggested that this carrier system is localised to the basolateral membrane in each cell type.

Animals↗