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D Restrepo

Publications and source records attributed to D Restrepo.

At least 55 records · Page 3Linked to original sources
PubMed1990

Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish.

Olfactory transduction is thought to be mediated by a G protein-coupled increase in intracellular adenosine 3',5'-monophosphate (cAMP) that triggers the opening of cAMP-gated cation channels and results in depolarization of the plasma membrane of olfactory neurons. In olfactory neurons isolated from the channel catfish, Ictalurus punctatus, stimulation with olfactory stimuli (amino acids) elicits an influx of calcium that leads to a rapid increase in intracellular calcium. In addition, in a reconstitution assay a plasma membrane calcium channel has been identified that is gated by inositol-1,4,5-trisphosphate (IP3), which could mediate this calcium influx. Together with previous studies indicating that stimulation with olfactory stimuli leads to stimulation of phosphoinositide turnover in olfactory cilia, these data suggest that an influx of calcium triggered by odor stimulation of phosphoinositide turnover may be an alternate or additional mechanism of olfactory transduction.

Amino Acids↗
PubMed1990

Olfactory neurons exhibit heterogeneity in depolarization-induced calcium changes.

Olfactory neurons from the channel catfish, Ictalurus punctatus, were isolated by a brief (15 min) treatment with papain. After incubation with fura-2 acetoxymethyl ester (fura-2/AM) for 1 h, the isolated olfactory receptor cells are found to hydrolyze fura-2/AM to fura-2 free acid without detectable traces of intermediate products of hydrolysis. Intracellular calcium measured with fura-2 in single cells covers a wide range (from less than 2 to 100 nM with a median of 17.6 nM, n = 140 cells). Twenty-one percent of the cells respond to potassium-induced depolarization with an increase in intracellular calcium mediated by influx of extracellular calcium. The L-type calcium channel antagonist nimodipine inhibits the increase in intracellular calcium triggered by membrane depolarization and blocks small unitary barium currents displaying the characteristics of L-type calcium currents (unitary conductance of 29 +/- 5 pS in 55 mM BaCl2 and high selectivity for Ba2+ over Na+ and K+) recorded from azolectin bilayers at the tip of patch pipettes into which isolated olfactory cilia membrane vesicles had been incorporated. Olfactory neurons are found to be functionally heterogeneous in their response to membrane depolarization and can be separated into three groups: one in which the increase in intracellular calcium is rapid and transient, another in which calcium increases slowly, and a third group of cells in which depolarization causes no change in intracellular calcium.

Animals↗
PubMed1990

Essential activation of Na(+)-H+ exchange by [H+]i in HL-60 cells.

The intracellular pH (pHi) dependence of the rate of Na(+)-H+ exchange was determined in undifferentiated promyelocytic HL-60 cells by measuring alkalinization rates using the fluorescent pHi indicator 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). BCECF was calibrated in the pH range from 5 to 7 using the nigericin technique of Thomas and co-workers (J. A. Thomas, R. N. Buchsbaum, A. Zimniak, and E. Racker. Biochemistry 18: 2210-2218, 1979). Exchange rate increases as pHi is lowered below pH 7.00. At low pH (pH below 6.3), the dependence of Na(+)-H+ exchange rate on intracellular proton activity is well fitted by the Michaelis-Menten equation with a maximum exchange velocity of 33.7 +/- 2.4 mmol H(+).1 cell water-1.min-1 and a half-saturation constant of 1.35 +/- 0.28 microM (corresponding to a minus log of the Michaelis constant of 5.89). However, a Hill plot reveals that the Hill coefficient changes gradually from one to two when pH is changed from 5 to 7, ruling out Michaelian kinetics. The dependence of exchange flux on internal protons is well fit in the full pH range from 5 to 7 by a simple kinetic model (essential activation) with modifier and transport sites for internal proton binding. At low pH, failure to correct BCECF measurement of pHi for contribution to fluorescence signal from extracellular dye and for quenching of intracellular BCECF leads to an artifactual increase in the measured Hill coefficient. These two findings (increase in Hill coefficient as pHi is increased and artifactual increase in Hill coefficient because of methodological reasons) provide a good explanation for the wide range of Hill coefficients reported in the literature.

Carrier Proteins↗
PubMed1989

Synthetic diacylglycerols trigger an increase of intracellular free calcium in promyelocytic HL60 cells.

Phosphoinositide turnover is known to play an important role in intracellular free calcium homeostasis through the inositol trisphophate-mediated release of calcium from intracellular stores. We find that the other product of phosphoinositide turnover, 1,2-diacylglycerol, elicits an increase in intracellular free calcium in HL60 cells which is due, at least in part, to release of calcium from intracellular stores. This effect is specific for calcium, since intracellular sodium and potassium levels and cellular volume were unaffected. Concomitant with the intracellular calcium increase, we find an increase in cellular inositol trisphosphate levels, suggesting that the effect of diacylglycerol on calcium may be mediated by inositol trisphosphate. Diacylglycerols also stimulate calcium efflux. This stimulation is not simply due to the increase in intracellular calcium. These effects appear not to be mediated through stimulation of a phorbol ester-activatable protein kinase C (Ca2+/phospholipid-dependent enzyme) since phorbol esters do not elicit an increase in cytoplasmic free calcium or an increase in calcium efflux.

Benzofurans↗
PubMed1989

Cl-Cl exchange in promyelocytic HL-60 cells follows simultaneous rather than ping-pong kinetics.

The intra- and extracellular chloride concentration dependencies of the rate of Cl-Cl exchange in human promyelocytic leukemic HL-60 cells were studied by means of radioactive isotope (36Cl) efflux measurements. Efflux of isotope from cells follows an exponential time course. The Cl-Cl exchange flux follows Michaelis-Menten kinetics as a function of both intra- and extracellular chloride concentrations. The ratio of the maximum exchange velocity to the apparent Michaelis constant for both extracellular and intracellular substrate increases as a function of trans Cl concentration, indicating that Cl-Cl exchange in the HL-60 cell does not follow ping-pong kinetics. A kinetic scheme in which extracellular and intracellular chloride ions bind in random order to the transporter and are then translocated simultaneously can adequately model the experimental data.

Anion Exchange Resins↗
PubMed1988

pH homeostasis in promyelocytic leukemic HL60 cells.

By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Conversion of esterified fura-2 and indo-1 to Ca2+-sensitive forms by mitochondria.

Rat liver mitochondria are shown to convert the acetoxymethyl ester forms of fura-2 and indo-1 into Ca2+-dependent forms of these indicators. The excitation spectrum of the Ca2+-dependent conversion product of fura-2 acetoxymethyl ester is shown to be similar to that of the pentacarboxylic acid form of fura-2. A systematic investigation of the ionic strength and pH dependences of the fluorescence of the pentacarboxylic acid forms of these indicators shows small changes within the ranges thought to obtain within the mitochondrial matrix after Ca2+ uptake. Intramitochondrial free Ca2+ levels are studied both before and after Ca2+ sequestration by mitochondria, and a rough estimate is made of the mitochondrial contribution to the Ca2+-dependent fura-2 fluorescence of a hepatocyte suspension.

Animals↗
PubMed1987

Thromboembolic and other events following valve replacement in a pediatric population treated with antiplatelet agents.

To determine if anticoagulation therapy is necessary after valve replacement with the St. Jude Medical prosthesis in young subjects, 30 consecutive patients were studied. They ranged in age from 4 to 20 years, and each had undergone valve replacement some time between February, 1982, and June, 1984. There was 1 hospital death (3.3%; 70% confidence limits [CL] = 0.4% to 10.9%). The 29 hospital survivors were treated with aspirin and dipyridamole. All patients were followed up. There were 5 late deaths (17.2%; 70% CL = 9% to 27%), at a mean of 8 months postoperatively. Follow-up revealed that 7 thromboembolic events occurred in 7 of the hospital survivors (24.1%; 70% CL = 15% to 35%). Hemiparesis occurred in 1 patient, and documented valve thrombosis and death occurred in another. Transient sensorimotor deficits were found in the 5 other patients. Prosthetic valve endocarditis developed in 1 hospital survivor (3.5%; 70% CL = 0.4% to 11%) and resulted in late death. There were no reoperations in the hospital survivors. We conclude that the St. Jude Medical prosthesis is useful for valve replacement in the young because of its low profile, durability, and hemodynamic characteristics. However, we recommend that these patients receive full anticoagulation therapy.

Adolescent↗
PubMed1987

Changes in Na+-H+ exchange regulation upon granulocytic differentiation of HL60 cells.

The changes in activation of Na+-H+ exchange on granulocytic differentiation of human leukemic promyelocytic HL60 cells have been studied by measuring changes in intracellular pH with the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). It was found that the Na+-H+ exchanger is activated by stimulation of protein kinase C in the dimethyl sulfoxide (DMSO)-differentiated (neutrophil-like) HL60 cell, but not in the undifferentiated (promyelocyte-like) cell. In contrast, osmotic shrinkage of the cells triggers the exchanger in the undifferentiated cells, but not in the DMSO-differentiated cells. The data suggest that activation of Na+-H+ exchange by osmotic shock does not occur exclusively through stimulation of kinase C.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Phlorizin binding to isolated enterocytes: membrane potential and sodium dependence.

Phlorizin binding is studied in isolated intestinal epithelial cells of the chick. Cells are ATP depleted to allow extensive manipulation of ionic gradients and membrane potential (delta psi). Phlorizin binding is assayed at steady state. Carrier specific phlorizin binding is defined as D-glucose (90 mM) inhibitable binding. Specific binding displays simple Michaelian kinetics as a function of phlorizin, indicating the presence of a single homogeneous binding site. Sodium concentrations and delta psi modify the apparent binding affinity but not the maximum number of binding sites. In contrast, the activation curve as a function of sodium concentrations is sigmoid and the apparent maximum number of binding sites at saturating sodium is phlorizin dependent. The rate of phlorizin association is both delta psi and sodium-concentration dependent. Dissociation is sodium-concentration dependent but not delta psi dependent. Theoretical analysis indicates binding order of substrates is random. In addition, data suggests that the phlorizin/sodium stoichiometry is 2:1. The delta psi dependence can be explained by two models: either translocation is the delta psi-dependent step and the free carrier is anionic, or sodium binding is the delta psi-dependent step.

Animals↗
PubMed1985

The mechanistic nature of the membrane potential dependence of sodium-sugar cotransport in small intestine.

Methods are described which demonstrate the use of unidirectional influx of 14C-tetraphenylphosphonium (14C-TPP+) into isolated intestinal epithelial cells as a quantitative sensor of the magnitude of membrane potentials created by experimentally imposed ion gradients. Using this technique the quantitative relationship between membrane potential (delta psi) and Na+-dependent sugar influx was determined for these cells at various Na+ and alpha-methylglucoside (alpha-MG) concentrations. The results show a high degree of delta psi dependence for the transport Michaelis constant but a maximum velocity for transport which is independent of delta psi. No transinhibition by intracellular sugar (40 mM) can be detected. Sugar influx in the absence of Na+ is insensitive to 1.3 mM phlorizin and independent of delta psi. The mechanistic implications of these results were evaluated using the quality of fit between calculated and experimentally observed kinetic constants for rate equations derived from several transport models. The analysis shows that for models in which translocation is the potential-dependent step the free carrier cannot be neutral. If it is anionic, the transporter must be functionally asymmetric. A model in which Na+ binding is the potential-dependent step (Na+ well concept) also provides an appropriate kinetic fit to the experimental data, and must be considered as a possible mechanistic basis for function of the system.

Animals↗
PubMed1985

The potential dependence of the intestinal Na+-dependent sugar transporter.

The unidirectional influx of the lipophilic cation tetraphenylphosphonium (TPP+) into isolated intestinal epithelial cells exhibits a marked dependence on the membrane potential (delta psi) maintained by or experimentally imposed on these cells. By taking advantage of this fact, we have described a "crossover" procedure that allows the relative permeability of a cation and anion pair to be determined. Measurements of such relative permeabilities permits diffusion potentials of defined magnitude to be imposed across the plasma membrane of ATP-depleted cells. This in turn allows description of the relationship between [14C]TPP+ influx and delta psi. We have determined that the flux-potential relationship is that predicted by the Goldman flux equation. Using this relationship as a calibration tool for delta psi, we then determined the quantitative relationship between membrane potential and the Na+-dependent influx of an actively transported sugar, alpha-methylglucoside (alpha-MG). The influx of [14C]alpha-MG also shows an exponential dependence on delta psi although it is more sharply potential dependent than that shown by TPP+. The specific relationship is consistent with that expected for a system with 2:1 Na+ stoichiometry which obeys the potential dependence predicted by Eyring rate theory with a single energy barrier occurring near the midpoint of the membrane. Over the range of potentials from +33 to -61 mV, we find no evidence for a minimum or threshold potential necessary to support transport and no evidence for an optimal potential that can maximize sugar transport. The data raise the possibility for using either [14C]TPP+ or [14C]alpha-MG influx as the basis for a new noninvasive procedure for measurement of delta psi.

Animals↗
PubMed1985

A new method for determination of relative ion permeabilities in isolated cells.

The unidirectional influx of the lipophilic cation tetraphenylphosphonium (TPP+) into isolated epithelial cells is a function of the membrane potential that exists across the cellular plasma membrane. Because of the potential dependence, [14C]TPP+ influx can be used as a qualitative sensor of changes in the membrane potential induced by diffusion of ions after the experimental imposition of transmembrane ion gradients. This report describes a "crossover" procedure in which the influx of [14C]TPP+ during systematic changes in the ionic composition of incubation media is used to identify conditions in which no change in membrane potential occurs. The ion ratio at the crossover provides a measure of the relative permeabilities of the two test ions being compared. By using this approach, the ion permeabilities for intestinal epithelial cells prepared from White Rock chickens can be ranked relative to the permeability of Na+ (PNa), i.e., when PNa is equal to 1.0. The permeability sequence and relative values for ion permeability in this system are tris(hydroxymethyl)aminomethane-gluconate (less than 0.1) less than Li+ (0.3) less than Na+ (1.0) less than Cl- (2.0) less than K+ (6.0) = NO3- (6.0) less than SCN- (18) less than K+ + valinomycin (40). The procedure is general enough in principle to be of broad application to a wide variety of cell or membrane vesicle preparations.

Animals↗
PubMed1985

Kinetic analysis of mechanism of intestinal Na+-dependent sugar transport.

Zero-trans kinetics of Na+-sugar cotransport were investigated. Sugar influx was measured at various sodium and sugar concentrations in K+-loaded cells treated with rotenone and valinomycin. Sugar influx follows Michaelis-Menten kinetics as a function of sugar concentration but not as a function of Na+ concentration. Nine models with 1:1 or 2:1 sodium:sugar stoichiometry were considered. The flux equations for these models were solved assuming steady-state distribution of carrier forms and that translocation across the membrane is rate limiting. Classical enzyme kinetic methods and a least-squares fit of flux equations to the experimental data were used to assess the fit of the different models. Four models can be discarded on this basis. Of the remaining models, we discard two on the basis of the trans sodium dependence and the coupling stoichiometry [G. A. Kimmich and J. Randles, Am. J. Physiol. 247 (Cell Physiol. 16): C74-C82, 1984]. The remaining models are terter ordered mechanisms with sodium debinding first at the trans side. If transfer across the membrane is rate limiting, the binding order can be determined to be sodium:sugar:sodium.

Animals↗