Biomedical subjects

J Randles

Publications and source records attributed to J Randles.

At least 19 recordsLinked to original sources

PubMed2003

Gut-focused hypnotherapy normalizes disordered rectal sensitivity in patients with irritable bowel syndrome.

BACKGROUND: We have previously shown that hypnotherapy alters rectal sensitivity in some patients with irritable bowel syndrome. However, this previous study used incremental volume distension of a latex balloon, which might be susceptible to subject response bias and might compromise the assessment of compliance. In addition, the study group was symptomatically rather than physiologically defined. AIM: To assess the effect of hypnotherapy on rectal sensitivity in hypersensitive, hyposensitive and normally sensitive irritable bowel syndrome patients using a distension technique (barostat) that addresses these technical issues. METHODS: Twenty-three irritable bowel syndrome (Rome I) patients (aged 24-72 years) were assessed before and after 12 weeks of hypnotherapy in terms of rectal sensitivity, symptomatology, anxiety and depression. Normal values for sensitivity were established in 17 healthy volunteers (aged 20-55 years). RESULTS: Compared with controls, 10 patients were hypersensitive, seven hyposensitive and six normally sensitive before treatment. Following hypnotherapy, the mean pain sensory threshold increased in the hypersensitive group (P = 0.04) and decreased in the hyposensitive group, although the latter failed to reach statistical significance (P = 0.19). Normal sensory perception was unchanged. Sensory improvement in the hypersensitive patients tended to correlate with a reduction in abdominal pain (r = 0.714, P = 0.07). CONCLUSION: Hypnotherapy improves abnormal sensory perception in irritable bowel syndrome, leaving normal sensation unchanged.

Abdominal Pain↗

PubMed2001

Characterization of Na+-coupled glutamate/aspartate transport by a rat brain astrocyte line expressing GLAST and EAAC1.

D-aspartate (D-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. A-asp influx is Na+-dependent with Km = 5 microm and Vmax = 0.7 nmoles x min(-1) x mg protein-1. Influx is sigmoidal as f[Na+] with Na+Km approximately 12 microm and Hill coefficient of 1.9. The cells establish steady-state D-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in Vmax, with no change in Km. At initial [D-Asp] = 10 microm, RBA take up more than 90% of total D-Asp, and extracellular levels are reduced to levels below 1 microm. Ionophores that dissipate the Delta(mu)Na+ inhibit gradient formation. Genistein (GEN, 100 microm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4alpha-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), L-Asp, and L-Glu, but not by D-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells.

ATP-Binding Cassette Transporters↗

PubMed1998

Na+-coupled alanine transport in LLC-PK1 cells: the relationship between the Km for Na+ at low [Alanine] and potential dependence for the system.

Analysis of the mechanistic basis by which sodium-coupled transport systems respond to changes in membrane potential is inherently complex. Algebraic expressions for the primary kinetic parameters (Km and Vmax) consist of multiple terms that encompass most rate constants in the transport cycle. Even for a relatively simple cotransport system such as the Na+/alanine cotransporter in LLC-PK1 cells (1:1 Na+ to substrate coupling, and an ordered binding sequence), the algebraic expressions for Km for either substrate includes ten of the twelve rate constants necessary for modeling the full transport cycle. We show here that the expression of Km of the first-bound substrate (Na+) simplifies markedly if the second-bound substrate (alanine) is held at a low concentration so that its' binding becomes the rate limiting step. Under these conditions, the expression for the KNam includes rate constants for only two steps in the full cycle: (i) binding/dissociation of Na+, and (ii) conformational 'translocation' of the substrate-free protein. The influence of imposed changes in membrane potential on the apparent KNam for the LLC-PK1 alanine cotransporter at low alanine thus provides insight to potential dependence at these sites. The data show no potential dependence for KNam at 5 micron alanine, despite marked potential dependence at 2 mm alanine when the full algebraic expression applies. The results suggest that neither translocation of the substrate-free form of the transporter nor binding/dissociation of extracellular sodium are potential dependent events for this transport system.

Adenosine Triphosphate↗

PubMed1996

A model for the kinetic mechanism of sodium-coupled L-alanine transport in LLC-PK1 cells.

The kinetics of sodium-dependent L-alanine transport were characterized in ATP-depleted LLC-PK1 cells, which allows experimental imposition of an interior negative diffusion potential across the plasma membrane. Under these conditions a wide range of sodium concentrations can be studied without altering the membrane potential. When Na+ is the variable substrate, the apparent maximal velocity (V max) for transport changes nearly fourfold for the five different alanine concentrations studied (0.05-2.0 mM). In contrast, at five different sodium concentrations, ranging from 10 to 135 mM, the apparent V max with variable alanine remains nearly constant at 5.3 +/- 1.2 nmol.min-1.mg cell protein-1. The ratio of the two primary kinetic parameters, Michaelis constant (Km)/V max, varies markedly no matter which solute is treated as the variable illustrate. These data are consistent with a simultaneous ordered transport mechanism in which sodium binds before alanine to the transport protein at the extracellular surface of the membrane. Alanine-dependent 22Na+ influx is more than five times faster if unlabeled intracellular sodium is present than in its absence. Sodium-dependent influx of [14C]alanine is more rapid than net alanine flux only if unlabeled Na+ and alanine are both present intracellularly. These results indicate that the cotransporter can function more rapidly in an exchange mode than when it catalyzes net solute uptake and that Na+ is the first solute to be released at the intracellular side of the membrane. A model is presented that can be used for further quantitative analysis of the kinetic and functional properties of the cotransport system.

Amino Acid Transport Systems↗

PubMed1994

Na(+)-coupled alanine transport in LLC-PK1 cells.

Transport of alanine (Ala) was characterized in LLC-PK1 renal epithelial cells. Transport capability for Ala falls by 75% in postconfluent cultures, while Na(+)-coupled alpha-methylglucoside (AMG) transport rises more than fourfold during the same interval. The kinetics of Ala transport were characterized in ATP-depleted cells to allow experimental imposition of changes in Na+ gradient and control of membrane potential across the plasma membrane. At 100 microM Ala and 135 mM Na+, > 98% of the unidirectional Ala influx is dependent on the presence of Na+ in cells from postconfluent cultures. Li+ is only 1% as effective as Na+, and other monovalent cations are ineffective in supporting Ala uptake. alpha-(Methylamino)isobutyric acid (MeAIB; 5 mM) causes only a small inhibition (approximately 10%) of 100 microM Ala influx. The low selectivity for Li+; low sensitivity to competition by MeAIB or aminoisobutyric acid; pronounced inhibition by serine, homoserine, cysteine, homocysteine and threonine; moderate inhibition by valine, isoleucine, proline and histidine; and lack of inhibition by lysine, arginine, and aspartate are more consistent with those characteristics reported for entry via the ASC amino acid transport system rather than those associated with the A system. Alanine influx exhibits a hyperbolic relationship with increasing Ala or Na+ concentration. Kinetic analysis suggests a single transport pathway with a Michaelis constant (Km) for alanine of 380 microM (when Na+ is 135 mM), apparent Km for Na+ of 32 mM (with 100 microM Ala), and a maximum velocity of 7 nmol.min-1.mg cell protein-1. An interior-negative diffusion potential induces a similar enhancement of [14C]alanine or [14C]tetraphenylphosphonium influx (approximately 40%). In contrast, AMG influx is enhanced by a factor of 2.2 under the same conditions. AMG uptake also shows a sigmoidal relationship with Na+ concentration. Hill coefficients are 1.56 for AMG and 1.0 for alanine. Direct measurement of Na(+)-Ala coupling stoichiometry yields a value of 1.01 +/- 0.07. Under the same conditions, Na(+)-AMG coupling stoichiometry is 2.1 +/- 0.25. The difference in coupling stoichiometries provides an explanation for differences in intensity of interaction between Na(+)-coupled transport systems for sugars and amino acids.

Alanine↗

PubMed1991

Sodium-dependent succinate transport by isolated chick intestinal cells.

Isolated chick intestinal epithelial cells take up succinate by a Na(+)-coupled transport system similar in some characteristics to those described for renal epithelium. The transport system exhibits a hyperbolic dependence on succinate concentration but a sigmoidal dependence on Na+ concentration. Best nonlinear fit of the Na+ dependence data to the Hill equation indicates a Michaelis constant for half-maximal transport rate (Km) for Na+ of approximately 20 mM, a maximal transport rate (Vmax) of 1.1 nmol succinate.min-1.mg protein-1, and a Hill coefficient of 2.5. Nearly equivalent fit was obtained with trial Hill coefficients down to 2.0. The data for succinate dependence indicated a Km of 25 microM and Vmax of 1.05 nmol.min-1.mg protein-1. The kinetic parameters indicate a higher affinity, lower capacity system than for succinate transport in the renal brush-border system. Thiocyanate-induced diffusion potentials cause no change in Na(+)-dependent succinate influx despite pronounced effects on the influx of tetraphenylphosphonium and on Na(+)-dependent alpha-methylglucoside (AMG) and alanine uptake. Several other dicarboxylic and tricarboxylic metabolic intermediates (but not the dicarboxylic amino acids) compete with succinate for uptake via the transport system. The data are consistent with the likelihood that these cells have a succinate transport system with a 2Na+:1succinate stoichiometry per transport cycle. The system catalyzes no net charge transfer and is therefore different from the potential-responsive succinate transporter described for renal tissue.

Alanine↗

PubMed1990

Whole cell recording of sugar-induced currents in LLC-PK1 cells.

Gigaohm-seal whole cell recording techniques were used to monitor function of the Na(+)-coupled sugar transport system in LLC-PK1 cells. The currents coupled to sugar transport were identified as those that are induced by the presence of 10 mM alpha-methylglucoside (AMG) in either the extracellular or intracellular compartment and were inhibited by addition of 320-800 microM phlorizin to the extracellular bathing medium. The sugar-induced currents are small, 15-20 pA, but of the expected magnitude as determined from the known kinetic parameters for Na(+)-coupled sugar transport in LLC-PK1 cells. The phlorizin-sensitive currents are Na+ dependent and can be studied under conditions in which the net Na+ and sugar flux (and consequently the Na+ electrical current) is in either the inward or outward direction. The reversal potential of the sugar-induced currents measured under conditions with high Na+ and AMG concentrations inside the cell is close to values predicted from thermodynamic principles, assuming a coupling stoichiometry of 2 Na+: 1 sugar for the transport system. The reversal potential of the sugar-induced currents with high extracellular Na+ and AMG is not equal to the predicted value, but it is of the polarity expected for inward-imposed solute gradients. Reasons for the observed discrepancy between observed and calculated values are discussed.

Animals↗

PubMed1989

Effect of saccharin on the ATP-induced increase in Na+ permeability in isolated chicken intestinal epithelial cells.

When isolated intestinal cells from 3-wk-old chickens are treated with exogenous ATP they undergo a dramatic increase in permeability towards Na+. The increase occurs instantaneously and maximum cell loading with Na+ occurs within 2 min. The response is dose dependent (0.1-1.0 mM-ATP) and results in as much as a 10-fold increase in unidirectional influx of 22Na+ into the cells. The resting cellular Na+ gradient and membrane potential are partially dissipated and consequently Na+-dependent transport of sugars and amino acids is inhibited. Sodium saccharin (20 mM), added at the same time as ATP, completely blocks the effect of ATP on Na+ permeability and preserves the functional capacity of the cells for Na+-dependent sugar or amino acid transport. Partial protection is afforded by 10 mM-saccharin. Saccharin added 2 min after ATP will reverse the enhanced Na+ permeability that has already been induced. In cells that have not been treated with ATP, saccharin induces enhanced sugar and amino acid gradients (P less than 0.05 in paired comparisons from the same cell preparation), indicating that it may also inhibit Na+ permeability of the unperturbed membrane and allow cells to establish higher Na+ gradients and/or membrane potentials. The effect of saccharin in blocking ATP-induced Na+ permeability occurs within 10 sec and at a much lower dose than that required for blockade of facilitated diffusional sugar transfer in these cells.

Adenosine Triphosphate↗

PubMed1988

Na+-coupled sugar transport: membrane potential-dependent Km and Ki for Na+.

Kinetic analysis of the characteristics of phlorizin binding and of the Na+, sugar, and potential dependence of alpha-methylglucoside (alpha-MG) influx into isolated avian intestinal cells has pointed toward two alternative models for the transport mechanism (D. Restrepo and G. A. Kimmich, J. Membr. Biol. 89: 269-280, 1986). One of these models envisions a potential-dependent Na+ binding event (Na+ well concept) as a part of the molecular mechanism. The data reported here show that the apparent Km for Na+ for sugar transport is sharply dependent on the magnitude of the membrane potential. When intracellular Na+ is absent, the maximal velocity (Vmax) achieved for sugar influx is the same with or without a potential, although Vmax is obtained at a lower Na+ concentration when a potential is imposed (interior negative). Intracellular Na+ severely inhibits the influx of sugar in the absence of a potential, but this effect is largely overcome when a potential is present. The Vmax obtained when intracellular Na+ is present is a function of the potential. These results are consistent with a transport model in which Na+ binding to the Na+-dependent sugar carrier at the extracellular surface of the membrane and debinding at the inner surface of the membrane are both potential-dependent events.

Animals↗

PubMed1987

SITS-sensitive Cl- conductance pathway in chick intestinal cells.

The unidirectional influx of 36Cl- into isolated chick epithelial cells is 30% inhibited by 300 microM SITS. Characteristics of the SITS-sensitive flux pathway were examined in terms of sensitivity to changes in membrane potential and intracellular pH. Potential dependence was evaluated using unidirectional influx of [14C]tetraphenylphosphonium ([14C]-TPP+) as a qualitative sensor of diffusion potentials created by experimentally imposed gradients of Cl-. Steady-state distribution of [14C]methylamine ([14C]MA) was used to examine for Cl(-)-dependent changes in intracellular pH. Imposed Na+ gradients, but not Cl- gradients, induce changes in [14C]MA distribution. SITS does not alter the [14C]MA distribution observed in cells with imposed gradients of Na+ and Cl-. Both results suggest that inhibition of Cl(-)-OH- exchange system is not the basis for the SITS effect on Cl- influx. However, if relative permeabilities for ion pairs via conductance pathways are compared, it can be shown that SITS causes a marked reduction of Pcl relative to either PNa or PK. SITS also inhibits electrically induced influx of [14C]TPP+ or [14C] alpha-methylglucoside driven by imposed Cl- gradients. Conversely, electrically driven Cl- influx can be blocked by SITS. These observations are all consistent with a SITS-sensitive Cl- conductance pathway associated with the plasma membrane of chick intestinal cells. No Cl(-)-OH- exchange capability can be detected for chick intestinal cells.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

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↗

PubMed1984

Sodium-sugar coupling stoichiometry in chick intestinal cells.

Sodium-dependent sugar transport systems involve the function of membrane components that couple the transmembrane flow of Na+ to the concomitant flow of certain sugar molecules. The coupling stoichiometry between Na+ and sugar fluxes via these systems must be measured under conditions in which the membrane potential does not change due to the induction of transport or during the interval of flux measurement. This can be accomplished by utilizing gradients of highly permeant ions (NO-3 and K+ plus valinomycin) to create diffusion potentials of sufficient magnitude that the sugar-induced Na+ flux does not introduce an appreciable change in the imposed potential. Under these conditions, the coupling stoichiometry for chicken intestinal cells proves to be 2 Na+:1 sugar as reported earlier for studies performed in the absence of a membrane potential. When control of the potential is not maintained, a coupling ratio of 1:1 is observed. The stoichiometry does not change as a function of Na+ concentration, which suggests that carrier forms with only one Na+ bound do not contribute to the carrier-mediated Na+ or sugar fluxes. When no potential is present, the stoichiometry is modified by the level of intracellular Na+ and sugar in a manner indicative of a transport mechanism in which Na+ must dissociate from the "loaded" carrier at the inward facing membrane surface before the sugar molecule dissociates.

Adenosine Triphosphate↗

PubMed1982

An ATP- and Ca2+-regulated Na+ channel in isolated intestinal epithelial cells.

When isolated intestinal epithelial cells are treated with 2 mM ATP, the unidirectional influx of Na+ to those cells increases from values near 50 to rates over 200 nmol . min-1 . mg protein-1. Calcium influx increases from 1 to 40 nmol . min-1 . mg protein-1. Within 2 min, the total cell Na+ increases two- to threefold, and total Ca+ increases about fivefold. The cells lose a major part of their capability for accumulating sugars during this interval. About 2 min after the time of ATP addition the normal permeability for Na+ and Ca2+ is restored, at which time the previously accumulated ions are rapidly extruded on a net basis until control levels are attained and the cells regain their usual sugar transport capability. The "repair" process requires Ca2+ in the incubation medium and is dependent on cellular uptake of Ca2+. Chlorpromazine (0.5 mM) blocks the Ca2+ entry route and the restoration of normal Na+ permeability. The Na+ entry route is selectively blocked by 4-acetamido-4'-isocyanostilbene-2,2'-disulfonic acid. The data show that ATP induces the influx of Na+ and Ca2+ by two different routes, which can be selectively inhibited. These ion flux routes may be involved in the events that allow intestinal tissue to convert from an absorptive state to a state in which net ion secretion occurs.

Adenosine Triphosphate↗

PubMed1982

Classification of several tobamoviruses isolated in China on the basis of the amino acid composition of their virion proteins.

The amino acid compositions of the virion proteins of several tobamoviruses isolated in China were determined. A classification computed from these and published data of other tobamoviruses was compared with published data of their relatedness assessed using the cDNA:RNA molecular hybridization technique. The classifications are clearly congruent; that based on amino acid composition appears to be best for determining the hierarchical relationships of members within a taxonomic group, rather than for distinguishing between closely related members, whereas nucleic acid hybridization unequivocally places a virus into the appropriate subgroup but is less useful for determining distant relationships.

Amino Acids↗

PubMed1981

alpha-Methylglucoside satisfies only Na+-dependent transport system of intestinal epithelium.

The unidirectional influx of alpha-methylglucoside (alpha-MG) by isolated chicken intestinal epithelial cells is 98% inhibited by phlorizin. The remaining 2% of the total influx occurs in the absence of Na+, is not sensitive to phloretin, and is equal to the diffusional entry rate for 2-deoxyglucose. The glucoside is much more strongly accumulated (75-fold) than 3-O-methylglucose (3-OMG) (10-fold). Inhibitors of the serosal sugar carrier (phloretin, cytochalasin B, theophylline, and flavanoids) do not enhance alpha-MG accumulation. It is concluded that the glycoside is not a substrate for the intestinal serosal transport system. Steady-state gradients of the sugar can be represented accurately by a concentrative, phlorizin-sensitive system that is opposed by a diffusional efflux process.

3-O-Methylglucose↗

PubMed1980

Evidence for an intestinal Na+:sugar transport coupling stoichiometry of 2.0.

Membrane potentials maintained by normally-energized intestinal epithelium interfere with an accurate determination of the Na+:sugar coupling stoichiometry associated with Na+-dependent transport systems. The interference is due to the fact that basal Na+ influx is itself a potential-dependent event, and sugar transport induces a membrane depolarization which therefore modifies basal Na+ entry. New information obtained under circumstances in which the membrane potential is maintained near 0 indicates that the true coupling stoichiometry is 2:1 rather than the commonly-accepted value of 1:1. A 2:1 stoichiometry means that cellular electrochemical Na+ gradients are adequate to account for recently observed 70-fold sugar gradients maintained by these cells under certain conditions.

Animals↗

PubMed1980

Regulation of Na+-dependent sugar transport in intestinal epithelial cells by exogenous ATP.

Exogenous ATP (1 mM) exerts a dramatic biphasic effect on the accumulation of 100 micro M 3-O-methylglucose by isolated intestinal epithelial cells. The initial effect ensues approximately 15 s after exposure and inhibits 80% of the undirectional sugar influx. Cellular phosphatases totally degrade the added ATP within a period of 20 min leading to a reactivation of transport capability. The cells exposed to ATP ultimately establish a concentration gradient of sugar about twice that observed for control cells. Pyrophosphate (10 mM) delays the degradation of added ATP and prolongs the interval of transport inhibition. The late effect of gradient enhancement is still observed. No other nucleoside triphosphate induces the early inhibition of transport, but ADP is approximately two-thirds as effective as ATP. AMP and other molecules containing the adenine ring system can cause the late effect of gradient enhancement without causing an early transport inhibition. Because rotenone-treated ATP-depleted cells also show an ATP-induced inhibition of sugar influx, it seems likely that the early effect represents a direct modification of carrier capability rather than an effect mediated via an alteration of cellular energetics.

Adenosine Triphosphate↗