Howard Kutchai

Asymmetric transport of a fluorescent glucose analogue by human erythrocytes

A fluorescent glucose analogue, 6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose (NBDG), was synthesized and its interactions with the hexose transport system of the human red blood cell were investigated. NBDG entry is inhibited by increasing concentrations of D-glucose (Ki = 2 mM). However, NBDG exit is unaffected by D-glucose in red blood cells. Cytochalasin B was found to inhibit both NBDG entry and exit. NBDG accumulates in the red blood cell above the theoretical equilibrium concentration. Accumulation of NBDG is temperature-sensitive and is due to the binding of NBDG to some intracellular substance. The binding of NBDG to purified hemoglobin suggests that accumulation of NBDG by erythrocytes is due to the intracellular binding of NBDG to hemoglobin. NBDG does not accumulate in pink erythrocyte ghosts, while its rate of uptake is still inhibited by D-glucose and cytochalasin B. Although there was no apparent D-glucose inhibition of NBDG exit by intact red blood cells, D-glucose was able to inhibit NBDG exit by pink erythrocyte ghosts. The differing properties of NBDG influx and efflux support the interpretation that the hexose transport system of the human red blood cell appears asymmetric although it may be intrinsically symmetric.

Effects of cholesterol on acyl chain dynamics in multilamellar vesicles of various phosphatidylcholines

Phase modulation fluorescence spectroscopy was used to investigate the influence of cholesterol (0 to 50 mol%) on acyl chain dynamics in multilamellar vesicles of phosphatidylcholine. Four different phosphatidylcholines (DPPC, DOPC, POPC, and egg PC) and six different fluorescent probes (diphenylhexatriene and five anthroyloxy fatty acids) were employed. We found that: (1) Increased cholesterol content had only slight effects on fluorescence lifetimes of the six probes. (2) Increased cholesterol content increased the steady-state fluorescence anisotropy (r) of all the probes except 16-anthroyloxy palmitate (16-AP) in each of the four phosphatidylcholines. (3) Added cholesterol tended to limit the extent of probe rotation (as reflected by r infinity, the infinite-time anisotropy) to a much greater extent than it altered the rate of probe rotation. (4) The tendency for cholesterol to order the structure of the bilayer was greatest in the proximal half of the acyl chains and diminished toward the center of the bilayer. (5) In some phosphatidylcholines the rotations rates of probes located near the bilayer center (diphenylhexatriene and 16-AP) were apparently increased by increasing levels of cholesterol. (6) In several respects dipalmitoylphosphatidylcholine (DPPC) vesicles responded differently to increased cholesterol than vesicles of the other three phosphatidylcholines. (7) A single second-order equation described the relationship between r infinity and r for the five anthroyloxy fatty acid probes in the four different phosphatidylcholines over a wide range of cholesterol content. The data for diphenylhexatriene in the different phosphatidylcholines could not be fit by a single equation.

Nonequilibrium-Facilitated Oxygen Transport in Hemoglobin Solution

We have used the quasi-linearization method to obtain numerical solutions to the equations which describe steady-state diffusion of oxygen through layers of hemoglobin solution. The numerical solutions show how the facilitated flux of oxygen depends upon the layer thickness, reaction-rate coefficients, and other parameters of the system. The results indicate that steady-state oxygen diffusion in layers of hemoglobin solution, similar to those studied by Scholander, should be adequately described by the models which assume chemical equilibrium exists throughout the layer, but for layers of concentrated hemoglobin solution about the thickness of a human erythrocyte, the facilitation of oxygen diffusion should be much less than the equilibrium models predict.

The effect of the red cell membrane and a diffusion boundary layer on the rate of oxygen uptake by human erythrocytes

1. This paper deals with the contributions of the red cell membrane and an external diffusion boundary layer (‘unstirred layer’) to the resistance to O2 entry into the red cell. Bovine serum albumin (BSA) was added to the extracellular fluid to enhance the effect of the diffusion boundary layer by diminishing both the solubility and the diffusivity of O2. The rate of O2 uptake by human red cells at various extracellular BSA concentrations was determined with a stopped‐flow rapid‐reaction apparatus.

Developmental changes in plasma membrane fluidity in chick embryo heart

1. Decreases in the rate of transport of sugars (facilitated transport), amino acids (active transport), and urea (simple diffusion) occur in chick embryo heart during development. This work considers the possibility that changes in the plasma membrane fluidity during development contribute to the observed changes in transport activities. 2. Technics were developed for subcellar fractionation of chick embryos and adult chickens. 3. The depolarization of the fluorescence of 1,6-diphenylhexatriene was used to estimate the fluidity of the lipid portion of plasma membrane enriched fractions of hearts from chick embryos at various stages of development and from adult hearts. 4. There is a pattern of decreasing membrane viscosity as development proceeds. Between 5-6 days and 10 days of embryonic life a 20% decrease in viscosity of the plasma membrane-enriched fraction occurs. Between 10 and 20 days of embryonic life there is no significant change in viscosity. Between 20 days of development (1 day before hatching) and adulthood there is a further 55% decrease in plasma membrane viscosity. 5. It is proposed that the changes in membrane fluidity observed may contribute to developmental changes in membrane transport activities, but other factors must also be involved.

Effects of n-Alkanols on the membrane fluidity of chick embryo heart microsomes

The n-alkanols from butanol through octanol are membrane perturbing agents that fluidize the microsomal membranes of 20-day-old chick embryo hearts as measured by the fluorescence depolarization of 1,6-diphenylhexatriene. In terms of the aqueous concentrations of n-alkanols the fluidizing effect increases with increasing number of carbons per n-alkanol. In terms of the membrane concentrations of n-alkanols the fluidizing effect is roughly equivalent for all the n-alkanols studied.

Erythrocyte water permeability. The effects of anesthetic alcohols and alterations in the level of membrane cholesterol

1. Treatment of human erythrocytes with anesthetic n-alkanols (pentanol, hexanol and hepatanol) results in a decrease in the osmotic water permeability of the red cell membrane. 2. The alcohol-induced changes in osmotic water permeability are proportional to the alcohol concentration and roughly parallel diphenylhexatriene that are induced by the alcohols. 3. Enrichment of the red cell membrane in cholesterol also results in a decrease in the osmotic water permeability. 4. Moderate depletion (9% or 40%) of membrane cholesterol is without effect on the osmotic water permeability, even though this treatment results in a significant increase in the rotational mobility of diphenylhexatriene in the membrane lipids.

Effect of diffusion boundary layers on the initial uptake of O2 by red cells. Theory versus experiment

We have applied theories of mass transfer, in laminar and turbulent flow, to red cells in the stopped-flow apparatus in order to estimate the effect of extracellular diffusion boundary layers on the initial rate of O2 uptake. We compared the theoretical predictions with the results of our previous stopped-flow experiments with suspensions of red cells to which bovine serum albumin (BSA) had been added to decrease O2 solubility and diffusivity (Huxley and Kutchai, 1981). Models of red cells in laminar flow (Friedlander, 1957, 1961; Harriott, 1962) predict a significant retardation of the rate of O2 entry into red cells by diffusion boundary layers. Mixing in the stopped-flow apparatus occurs by convective and turbulent mechanisms in addition to simple molecular diffusion. The more complex theory of mass transfer to particles in turbulent flow (Levich, 1962) shows, because the red cells are highly entrained in the flow, that mixing is not complete on the time scale of initial O2 uptake. The O2 permeability of the diffusion boundary layer, predicted by both laminar and turbulent flow theories, approximates the experimentally obtained value. The theories of mass transfer to particles in laminar flow predict the dependence of the rate of O2 uptake on red cell size observed by other investigators. This suggests that these experimental results may be primarily due to the effect of diffusion boundary layers. Our studies are consistent with the interpretation that red cells in rapid mixing devices may be mixed incompletely with the suspending fluid; thus O2 transfer is limited by molecular diffusion in the immediate vicinity of the erythrocyte. We conclude that the rates of respiratory gas exchange by red cells in flowing systems may be partly limited by diffusion boundary layers.

Role of the red cell membrane in oxygen uptake

The rate at which red blood cells take up O2 or CO as measured in a rapid reaction apparatus is considerably less than predicted from solution of the equations for diffusion and chemical reaction in a layer of hemoglobin solution about the same thickness as the red cell. Nicolson and Roughton (1951) showed that this discrepancy could be accounted for by postulating that the red cell membrane is an important barrier to gas uptake. Sinha (1969) measured the rate of O2 uptake by single red cells located near a gas-plasma interface. The equations for diffusion and chemical reaction of O2 in a membraneless layer of hemoglobin solution for conditions that correspond to Sinhas experiments are solved. The calculated time course of O2 uptake fits the experimental data sufficiently well to suggest that the resistance of the red cell membrane to O2 diffusion is not an important limiting factor. Also analyzed in this way is the data of Carlson and Comroe (1958). The author finds that calculations predict a faster rate of CO uptake by biconcave disc shaped red cells than was observed experimentally, but that calculations for sphered red cells agree well enough with experimental data that membrane CO permeability may not be primary in limiting CO uptake by spherocytes.

Effects of anesthetic alcohols on membrane transport processes in human erythrocytes.

1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent Km and V values for the equilibrium exchange of uridine. 4. The facilitated transport of galactose was only slightly inhibited by hexanol. 5. Hexanol was without effect on the passive and active fluxes of Na+ and K+ in red cells with altered cation contents. Cells that were slightly depleted of K+ and cells that were highly K+ -depleted were both insensitive to hexanol.