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D G Levitt

Publications and source records attributed to D G Levitt.

At least 55 records · Page 3Linked to original sources
PubMed1988

Electrostatic radius of the gramicidin channel determined from voltage dependence of H+ ion conductance.

The results of Decker and Levitt (1987) suggest that the conductance of H+ ion through the gramicidin channel is limited primarily by diffusion in the bulk solution at the channel mouth. It is assumed in this paper that the H+ conductance is 100% diffusion limited. This means that all the factors that influence the H+ flux are external to the channel and are presumed to be known. In particular, the diffusion coefficient of H+ in this region is assumed to be equal to the bulk solution value and the only force acting on the ion is that due to the applied voltage. A model of the H+ flux is derived, based on the Nernst-Planck equation. It has three adjustable parameters: the electrostatic radius, the capture distance, and the radius of the H+ ion. The acceptable range of the parameters was determined by comparing the predictions of the model with the experimental measurements of the H+ conductance at pH 3.75. The best fit was obtained for an electrostatic radius in the range 2.3-2.7 A. This is in good agreement with earlier predictions (2.5 A) based on the assumption that the dielectric constant of the channel water is equal to that of bulk water. The addition of 1 M choline Cl- (an impermeant) increases the H+ current at low voltage and decreases it at high voltage. The increase can be explained by the small surface charge that results from the separation of charge produced by exclusion of the large choline cation (relative to Cl-) from the membrane surface. The decrease at high voltages can be accounted for by the change in the profile of the applied potential produced by the increase in ionic strength.

Diffusion↗
PubMed1988

Physiological measurement of luminal stirring in perfused rat jejunum.

Poor stirring of intestinal contents yields a preepithelial diffusion barrier that is thought to be the rate-limiting step in absorption of many compounds. In many previous studies, the resistance of this barrier is equal to an unstirred water layer of 300-800 micron. Using three probes, CO, glucose, and [14C]warfarin, we measured the preepithelial resistance in a 30-cm segment in rats that were 1) conscious, 2) anesthetized with pentobarbital sodium, or 3) anesthetized and laparotomized. Measurements with each of the probes showed that the maximal preepithelial resistance in conscious rats was equivalent to an unstirred layer of only approximately 100 micron. Anesthesia roughly doubled this resistance, and anesthesia and laparotomy caused a sixfold increase (unstirred layer of approximately 600 micron). We conclude that the luminal stirring is much more efficient than previously has been appreciated. The very thick jejunal unstirred layers reported previously (300-800 micron) reflect the results of studies performed under nonphysiological conditions or studies employing inappropriate techniques to measure luminal stirring.

Anesthesia↗
PubMed1988

Use of laminar flow and unstirred layer models to predict intestinal absorption in the rat.

Carbon monoxide (CO) and [14C]warfarin were used to measure the preepithelial diffusion resistance resulting from poor luminal stirring (RL) in the constantly perfused rat jejunum at varying degrees of distension (0.05, 0.1, and 0.2 ml/cm). RL was much greater than epithelial cell resistance, indicating that poor stirring was the limiting factor in absorption and that an appropriate model of stirring should accurately predict absorption. A laminar flow model accurately predicted the absorption rate of both probes at all levels of gut distension, as well as the absorption of glucose when RL was the rate-limiting factor in absorption. In contrast, an unstirred layer model would not have predicted that gut distension would have little influence on absorption, and would have underestimated [14C]warfarin absorption relative to CO. We concluded that in the perfused rat jejunum, laminar flow accurately models luminal stirring and an unstirred layer should be considered to be a unit of resistance in laminar flow, rather than a model of luminal stirring.

Animals↗
PubMed1987

Potassium-selective ion channels in a transformed insulin-secreting cell line.

K+ channels in inside-out patches from hamster insulin tumor (HIT) cells were studied using the patch-clamp technique. HIT cells provide a convenient system for the study of ion channels and insulin secretion. They are easy to culture, form gigaohm seals readily and secrete insulin in response to glucose. The properties of the cells changed with the passage number. For cell passage numbers 48 to 56, five different K+-selective channels ranging from 15 to 211 pS in symmetrical 140 mM KCl solutions were distinguished. The channels were characterized by the following features: a channel with a conductance (in symmetrical 140 mM KCl solutions) of 210 pS that was activated by noncyclic purine nucleotides and closed by H+ ions (pH = 6.8); a 211 pS channel that was Ca2+-activated and voltage dependent; a 185 pS channel that was blocked by TEA but was insensitive to quinine or nucleotides; a 130 pS channel that was activated by membrane hyperpolarization; and a small conductance (15 pS) channel that was not obviously affected by any manipulation. As determined by radioimmunoassay, cells from passage number 56 secreted 917 +/- 128 ng/mg cell protein/48 hr of insulin. In contrast, cells from passage number 77 revealed either no channel activity or an occasional nonselective channel, and secreted only 29.4 +/- 8.5 ng/mg cell protein/48 hr of insulin. The nonselective channel found in the passage 77 cells had a conductance of 25 pS in symmetrical 140 mM KCl solutions. Thus, there appears to be a correlation between the presence of functional K+ channels and insulin secretion.

Adenoma, Islet Cell↗
PubMed1987

Exact continuum solution for a channel that can be occupied by two ions.

The classical Nernst-Planck continuum equation is extended to the case where the channel can be occupied simultaneously by two ions. A two-dimensional partial differential equation is derived to describe the steady-state channel. This differential equation is of the form of the generalized Laplace equation, but it has the novel feature that the boundary conditions are periodic. The finite difference solution takes approximately 8 s on a large computer. The equations are solved for the special case of a cylindrical channel with a fixed charge in the center. It is assumed that the forces on the ions result entirely from the sum of the Born image potential, the fixed charge potential, the interaction potential between the two ions, and the applied voltage. Approximate simple analytical expressions are derived for these potential terms, based on the assumption that the electric field perpendicular to the channel wall is zero. The potentials include the contribution from a diffuse charge (Debye-Huckel) reaction field in the bulk solution for the monovalent cation flux was obtained for channels with a radius of 4 A and lengths of 16 and 32 A and a fixed charge valence of -1 and -1.5. For these channels, a significant fraction (up to 90%) of the total resistance is contributed by the bulk solution and results were obtained for the case where the "channel" included 8 A of bulk solution at each channel end. These results for the two-ion channel were compared with the analytical solution for a one-ion channel. The one-ion channel is a fair approximation to the two-ion channel for a fixed charge of -1, underestimating the flux at high concentrations by approximately 30%. However, for a fixed charge of -1.5, the one-ion model is a poor approximation, with the two-ion flux about seven times that of the one-ion model at high concentrations. The absolute conductance and concentration dependence of these channels (with a fixed charge of -1) mimic the behavior of the large conductance K+ channel and the acetylcholine receptor channel.

Electric Conductivity↗
PubMed1987

Quantitative assessment of luminal stirring in the perfused small intestine of the rat.

We measured the resistance (RL) to CO absorption that resulted from poor luminal stirring in the constantly perfused rat jejunum. RL or calculated unstirred layer thickness was greater for 30-cm than 10-cm long segments, indicating lack of a uniform thickness of unstirred layer. The possibility that laminar flow existed in the gut was first tested by calculating expected CO absorptions from fluid moving with laminar flow. These values agreed closely with observed absorption rates. Laminar flow also was supported by the observation that CO absorption was independent of perfusate viscosity. Lastly, after sudden addition of phenolsulfonphthalein (PSP) to the perfusate, PSP outflow concentration was similar in tygon tubing (which has laminar flow) and a gut segment of comparable dimension. We conclude that flow in the perfused gut is laminar and that this laminar flow has many implications for studies carried out with the constant-perfusion technique.

Animals↗
PubMed1986

Interpretation of biological ion channel flux data--reaction-rate versus continuum theory.

Although the reaction-rate theory may provide a useful mathematical description of the channel flux, it presents a misleading physical picture of the channel structure. There is a tendency to regard the barriers in the model as actual physical structures, whereas they are actually only mathematical artifacts that allow one to reduce a complicated differential equation with an infinite number of states to a finite difference equation with a minimum number of states. I argue that the energy profile in the permeation pathway of most biological channels should vary relatively smoothly with only a few localized energy barriers or wells. In these smoothly varying regions, the resistance to ion movement is similar to bulk diffusion and cannot be accurately modeled by one or two energy barriers. For the one-ion channel, the continuum approach is as general and at least as simple as the reaction-rate theory and may provide a more physical interpretation of the data. Thus for the SR K+ channel, the structure suggested by the reaction-rate theory seems inconsistent with some experimental data, while the continuum-theory model is not only consistent with, but complements, the structure suggested by other data. Multi-ion channels have such complicated kinetics that one can only expect the theories to provide a qualitative description of the experimental data. They can be modeled by either the reaction-rate model or a finite difference approximation to the continuum model.

Animals↗
PubMed1985

Strong electrolyte continuum theory solution for equilibrium profiles, diffusion limitation, and conductance in charged ion channels.

The solution for the ion flux through a membrane channel that incorporates the electrolyte nature of the aqueous solution is a difficult theoretical problem that, until now, has not been properly formulated. The difficulty arises from the complicated electrostatic problem presented by a high dielectric aqueous channel piercing a low dielectric lipid membrane. The problem is greatly simplified by assuming that the ratio of the dielectric constant of the water to that of the lipid is infinite. It is shown that this is a good approximation for most channels of biological interest. This assumption allows one to derive simple analytical expressions for the Born image potential and the potential from a fixed charge in the channel, and it leads to a differential equation for the potential from the background electrolyte. This leads to a rigorous solution for the ion flux or the equilibrium potential based on a combination of the Nernst-Planck equation and strong electrolyte theory (i.e., Gouy-Chapman or Debye-Huckel). This approach is illustrated by solving the system of equations for the specific case of a large channel containing fixed negative charges. The following characteristics of this channels are discussed: anion and mono- and divalent cation conductance, saturation of current with increasing concentration, current-voltage relationship, influence of location and valence of fixed charge, and interaction between ions. The qualitative behavior of this channel is similar to that of the acetylcholine receptor channel.

Animals↗
PubMed1984

Use of carbon monoxide to measure luminal stirring in the rat gut.

We used carbon monoxide (CO) as a probe to quantitatively measure intestinal unstirred water layers in vivo. CO has several features that make it uniquely well suited to measure the unstirred layer in that its tight binding to hemoglobin makes uptake diffusion limited, and its relatively high lipid solubility renders membrane resistance negligible relative to the water barriers of the unstirred layer and epithelial cell. The unique application of CO was the measurement of the absorption rate of CO both from the gas phase as well as a solute dissolved in saline. Several lines of evidence showed that a gut stripped free of saline and then filled with gas contained a negligible unstirred layer. Thus, absorption of CO from the gas phase measured resistance of just the epithelial cell. Subtraction of this value from the resistance of CO absorption from saline provided a direct measure of unstirred layer resistance. Studies in the rat showed for a 3-min absorption period that the conventionally calculated apparent unstirred layer for the jejunum was 411 micron and for the colon was 240 micron. However, this conventionally calculated unstirred layer resistance did not truly depict the situation in the rat gut, since there was a continuing depletion of CO from outer surfaces of luminal contents throughout the experiment period. This produced a continually increasing diffusion barrier with time. Calculation of expected absorption rate from unstirred cylinders with the dimensions of the rat gut indicated that there was virtually no stirring in the small intestine and minimal stirring in the colon. The technique described in this paper appears to be simpler and to require fewer assumptions for validity than other techniques previously used to measure unstirred layers in vivo.

Animals↗
PubMed1983

Comparison of the gramicidin A potassium/sodium permeability and single channel conductance ratio.

If the ion concentration is low enough that most channels are unoccupied, then the 'independence relations' should be satisfied and the permeability ratio should equal the conductance ratio. It has been previously reported that for the gramicidin A channel these ratios for Na+ and K+ were not equal at concentrations as low as 10 mM. However, these ratios were not measured at the same applied potential, as is required by the theory. Instead, the conductance ratio was measured at 100 mV and corrected using calculated current-voltage relations. In this report the comparison between permeability and conductance ratios is reexamined using data obtained at the correct potential. There is no significant difference in the ratios at 10 mM when they are measured at the same voltage. This implies that most channels are not occupied by sodium or potassium ions at 10 mM.

Cell Membrane Permeability↗
PubMed1983

Osmotic water permeability of the human red cell. Dependence on direction of water flow and cell volume.

The osmotic permeability coefficient (Pf) was measured with a stopped-flow light-scattering technique. There is an artifactual light-scattering signal produced by the initial mixing that decays with a half-time of approximately 0.2 s. This seriously interferes with the measurement of the osmotically induced change in cell volume, which has a similar half-time. This "injection artifact" is associated with the biconcave shape of the cells. It is negligible for cells that have been made nearly spherical by swelling them in 160 mosmol. The dependence of this artifact on the cell volume may explain the previously observed dependence of Pf on the cell volume. When cells are made echinocytic (and therefore spherically symmetric), this injection artifact becomes negligible at all cell volumes and Pf can be accurately measured. The Pf of echinocytic cells was nearly constant, varying by less than 10% with the direction of flow and the medium osmolarity (160-360 mosmol). The average value of Pf was 2.0 X 10(-2) cm/s (T = 23 degrees C).

Cell Membrane Permeability↗
PubMed1983

Urea and ethylene glycol-facilitated transport systems in the human red cell membrane. Saturation, competition, and asymmetry.

The equilibrium exchange of [14C]urea and ethylene glycol was measured using a new type of fast flow system. Approximately equal volumes of saline and air were mixed to form a segmented fluid stream into which 14C-loaded red cells are injected. The stream flows through three filter chambers which allow sampling of the 14C in the extracellular fluid at three time points. The chambers are designed so that they do not disrupt the segmented bubble pattern. The alternating air and saline segments prevent laminar dispersion in the flowing stream and ensure good mixing at the injection and sampling sites. The equilibrium exchange of both urea and ethylene glycol showed saturation kinetics. The maximum permeability (Po) measured in the limit of zero solute concentration is 1.6 X 10(-3) cm/s for urea and 4.8 X 10(-4) cm/s for ethylene glycol (T = 23 degrees C). The apparent dissociation constant (Km) was 218 mM for urea and 175 mM for ethylene glycol. The Po for thiourea is 2.3 X 10(-6) cm/s and the Km is 19 mM. Urea and thiourea inhibit the transport of each other and the inhibition constant (KI) is approximately equal to the Km for both compounds. 53 other analogues of urea were screened for their inhibition of urea or thiourea transport. Several analogues [e.g., 1-(3,4-dichloro-phenyl)-2-thiourea] had a KI in the range of 0.03 mM. The affinity of the inhibitor increased as it was made more hydrophobic. The urea analogues did not significantly inhibit the ethylene glycol or osmotic permeability. Glycerol inhibited ethylene glycol permeability with a KI of 1,200 mM.

Biological Transport↗
PubMed1983

Reflection coefficient and permeability of urea and ethylene glycol in the human red cell membrane.

The reflection coefficient (sigma) and permeability (P) of urea and ethylene glycol were determined by fitting the equations of Kedem and Katchalsky (1958) to the change in light scattering produced by adding a permeable solute to a red cell suspension. The measurements incorporated three important modifications: (a) the injection artifact was eliminated by using echinocyte cells; (b) the use of an additional adjustable parameter (Km), the effective dissociation constant at the inner side of the membrane; (c) the light scattering is not directly proportional to cell volume (as is usually assumed) because refractive index and scattering properties of the cell depend on the intracellular permeable solute concentration. This necessitates calibrating for known changes in refractive index (by the addition of dextran) and cell volume (by varying the NaCl concentration). The best fit was for sigma = 0.95, Po = 8.3 X 10(-4) cm/s, and Km = 100 mM for urea and sigma = 1.0, Po = 3.9 X 10(-4) cm/s, and Km = 30 mM for ethylene glycol. The effects of the inhibitors copper, phloretin, p-chloromercuriphenylsulfonate, and 5,5'-dithiobis (2-nitro) benzoic acid on the urea, ethylene glycol, and water permeability were determined. The results suggest that there are three separate, independent transport systems: one for water, one for urea and related compounds, and one for ethylene glycol and glycerol.

Calibration↗
PubMed1983

Regulation of bile acid synthesis in man. Presence of a diurnal rhythm.

Regulation of bile acid synthesis in man is incompletely understood, in part because of difficulty in making measurements over short time periods when the enterohepatic circulation is intact. We investigated the possibility of a diurnal rhythm of bile acid synthesis in three human subjects given [26-14C]cholesterol. When this isotope of cholesterol, which is randomly labeled in the 26 and 27 positions, is converted to bile acid, the 14C is released as propionic acid randomly labeled in the 1 and 3 positions. The labeled propionic acid is then oxidized to 14CO2, output of which is a function of bile acid synthesis. However, delays in transit of the 14C through propionic acid and CO2-HCO-3 pools would shift the phase and dampen the amplitude of 14CO2 output relative to an existing diurnal rhythm of bile acid synthesis. Therefore, using constant infusion methods, we determined the turnover constants for conversion to 14CO2 of [1-14C]propionic acid and [3-14C]propionic acid to be 0.36-0.59 h-1 and 0.14-0.16 h-1, respectively. Using these constants and modeling the diurnal rhythm as a cosine function, we determined that amplitude of 14CO2 output from [26-14C]cholesterol was reduced 35% and acrophase was delayed 2.4-3.0 h relative to the diurnal rhythm of bile acid synthesis. None of the diurnal rhythm in 14CO2 output from [26-14C]cholesterol resulted from diurnal variation in propionic acid or CO2-HCO-3 metabolism since constant infusion of [1-14C]propionic acid and [3-14C]propionic acid for 30 h revealed no diurnal variation in output of 14CO2. These studies demonstrate for the first time that humans with an intact enterohepatic circulation have a diurnal rhythm of bile acid synthesis with an amplitude of +/- 35-55% around mean synthesis, and an acrophase at about 9 a.m.

Adult↗
PubMed1982

Comparison of Nernst-Planck and reaction rate models for multiply occupied channels.

The Nernst-Planck continuum equation for a channel that can be occupied by at most two ions is solved for two different physical cases. The first case is for the assumption that the water and ion cannot get around each other anywhere in the channel, so that if there are two ions in the channel the distance between them is fixed by the number of water molecules between them. The second case is for the assumption that there are regions at he ends of the channel where the ions and water can get around each other. For these two cases, the validity of the simple two-site reaction-rate approximation when there is a continuously varying central energy barrier was evaluated by comparing it with the exact Nernst-Planck solution. For the first continuum case, the kinetics for the continuum and reaction-rate models are nearly identical. For the second case, the agreement depends on the strength of the ion-ion interaction energy. For a low interaction energy (large channel diameter) a high ion concentrations, there is a large difference in the flux as a function of voltage for the two models-with the continuum flux becoming more than four times larger at 250 mV. Simple analytical expressions are derived for the two-ion continuum channel for the case where the ends are in equilibrium with the bulk solution and for the case where ion mobility becomes zero when there are two ions in the channel. The implications of these results for biological channels are discussed.

Ion Channels↗
PubMed1981

Binding constants of Li+, K+, and Tl+ in the gramicidin channel determined from water permeability measurements.

In an open circuit there can be no net cation flux through membranes containing only cation-selective channels, because electroneutrality must be maintained. If the channels are so narrow that water and cations cannot pass by each other, then the net water flux through those "single-file" channels that contain a cation is zero. It is therefore possible to determine the cation binding constants from the decrease in the average water permeability per channel as the cation concentration in the solution is increased. Three different methods were used to determine the osmotic water permeability of gramicidin channels in lipid bilayer membranes. The osmotic water permeability coefficient per gramicidin channel in the absence of cations was found to be 6 x 10(-14) cm3/s. As the cation concentration was raised, the water permeability decreased and a binding constant was determined from a quantitative fit to the data. When the data were fitted assuming a maximum of one ion per channel, the dissociation constant was 115 mM for Li+, 69 mM for K+, and 2 mM for Tl+.

Gramicidin↗
PubMed1981

Water transport and ion-water interaction in the gramicidin channel.

The diffuse permeability and the diffusion coefficient of water (Dw) in the gramicidin channel is determined from the osmotic water permeability of the channel and "single file" pore theory. Dw is about 7% of the self-diffusion coefficient of bulk water. The diffusion coefficient of a single water molecule alone in the channel is also determined and is about equal to the value in bulk water. This provides an estimate of the mobility of water on the channel walls in the absence of water-water interaction. Since the gramicidin channel walls should be representative of uncharged polar protein surfaces, this result provides direct evidence that the presence of a cation in the channel reduces the hydraulic water permeability by a factor ranging from 60 for Tl+ to 5 for Na+. The diffusion coefficient of a cation (Dc) in the channel is estimated and compared with Dw. For Na+ it is found that Dc approximately equal to Dw, which implies that the movement of the row of water molecules through the channel determines the local mobility of Na+. Thus, it seems that short range ion-wall interactions are not important in determining the channel conductance for Na+. In contrast, for Li+, local ion-wall interactions probably do limit the conductance.

Gramicidin↗