Correspondence to: Mark L. Zeidel, Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, A919 Scaife Hall, 3550 Terrace St., University of Pittsburgh, Pittsburgh, PA 15261. Fax:Fax: 412-647-6222; E-mail:zeidel{at}med1.dept-med.pitt.edu.
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Abstract |
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Bilayer asymmetry in the apical membrane may be important to the barrier function exhibited by epithelia in the stomach, kidney, and bladder. Previously, we showed that reduced fluidity of a single bilayer leaflet reduced water permeability of the bilayer, and in this study we examine the effect of bilayer asymmetry on permeation of nonelectrolytes, gases, and protons. Bilayer asymmetry was induced in dipalmitoylphosphatidylcholine liposomes by rigidifying the outer leaflet with the rare earth metal, praseodymium (Pr3+). Rigidification was demonstrated by fluorescence anisotropy over a range of temperatures from 24 to 50°C. Pr3+-treatment reduced membrane fluidity at temperatures above 40°C (the phase-transition temperature). Increased fluidity exhibited by dipalmitoylphosphatidylcholine liposomes at 40°C occurred at temperatures 13°C higher in Pr3+-treated liposomes, and for both control and Pr3+-treated liposomes permeability coefficients were approximately two orders of magnitude higher at 48° than at 24°C. Reduced fluidity of one leaflet correlated with significantly reduced permeabilities to urea, glycerol, formamide, acetamide, and NH3. Proton permeability of dipalmitoylphosphatidylcholine liposomes was only fourfold higher at 48° than at 24°C, indicating a weak dependence on membrane fluidity, and this increase was abolished by Pr3+. CO2 permeability was unaffected by temperature. We conclude: (a) that decreasing membrane fluidity in a single leaflet is sufficient to reduce overall membrane permeability to solutes and NH3, suggesting that leaflets in a bilayer offer independent resistances to permeation, (b) bilayer asymmetry is a mechanism by which barrier epithelia can reduce permeability, and (c) CO2 permeation through membranes occurs by a mechanism that is not dependent on fluidity.
Key Words: barrier function, epithelia, membrane fluidity, CO2, NH3
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Introduction |
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The epithelia that line the stomach, bladder, renal collecting duct, and thick ascending limb of the nephron limit the dissipation of large proton, solute, NH3, and CO2 gradients by creating and maintaining a barrier to diffusion (
Membrane permeability to a number of substances is clearly dependent on fluidity, and cells appear to limit fluidity by creating asymmetric lipid membranes at their apical pole. However, for mainly technical reasons, there have been virtually no studies undertaken to model the effects of bilayer asymmetry on permeability in artificial membranes of known composition. We have previously shown by the use of two independent methods that rigidifying a single leaflet in a bilayer reduced water permeability (
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Materials and Methods |
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Liposome Preparation
Powdered DPPC was obtained from Avanti Polar Lipids and suspended by vortexing (at 25 mg/ml) in buffer appropriate for the permeability to be measured. Buffers used were as follows (mM): for solutes, 150 NaCl, 10 HEPES, 20 carboxyfluorescein (CF), pH 7.5; for NH3 and protons, 150 NaCl, 30 KCl, 10 HEPES, 0.5 CF, pH 7.5; for CO2, 50 NaCl, 50 KCl, 20 HEPES, 0.5 CF, 0.5 mg/ml carbonic anhydrase, pH 7.4. Liposomes were prepared by probe sonication and after 90 min incubating on ice, extravesicular CF was removed by passing vesicles over a Sephadex G50 column (Sigma Chemical Co.). Vesicles were sized by quasi-elastic light scattering using a Nicomp model 270 submicron particle analyzer as described (
Solute Permeability Measurements
Permeability measurements were performed as described (
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(1) |
where Jz is the flux and Pz is the permeability of the permeant solute z, SA is the surface area of the vesicle, and C is the concentration difference of the permeant solute between the inside and outside of the vesicle. If
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(2) |
where V0 is the initial volume of the vesicle and Vrel and V(t) are the relative and absolute volumes, respectively, at time t, then for our experimental conditions:
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(3) |
and
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(4) |
therefore,
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(5) |
By use of parameters from the single exponential curve fit to the data, Psolute was solved using commercially available MathCad software (
Proton Permeability
Proton permeabilities were measured using pH-dependent quenching of fluorescence as described (
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(6) |
where JH+ is the flux of protons, C is the initial difference in concentration of protons between the inside and outside of the vesicle,
pH is the change in pH when time equals
, the time constant of the single exponential curve describing the initial change in fluorescence as a function of time, and BCV is the buffer capacity of an individual vesicle (
NH3 Permeability
NH3 permeability was determined using stopped-flow fluorimetry by monitoring the pH-sensitive increase in fluorescence when vesicles equilibrated to pH 6.8 were rapidly mixed with the same buffer containing 20 mM NH4Cl as described (
CO2 Permeability
CO2 fluxes were determined by monitoring the pH-sensitive decrease in fluorescence when vesicles were mixed with a 100 mM NaHCO3/CO2, 20 mM HEPES, pH 7.4 buffer. CO2 gas in the bicarbonate solution diffuses into the liposomes, whereupon it is converted to HCO3- and H+ by the entrapped carbonic anhydrase. By combining values for the initial rate of change of fluorescence, the final pH, and the buffer capacity of the vesicles, PCO2 was calculated as described (
Fluorescence Anisotropy
Membrane fluidity measurements were performed by incubating DPPC liposomes in 1 mM DPH-HPC (Molecular Probes), and then measuring anisotropy using excitation/emission wavelengths of 360 nm/430 nm on a SPEX Fluorolog 1680 double spectrometer according to standard methods (
Statistics
For all comparisons, n = 46 liposome preparations. Groups were compared using unpaired t tests. P < 0.05 was considered significant.
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Results |
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Before determining the effect of bilayer asymmetry on solute fluxes, it was necessary to ensure that the solute was not altering the ability of Pr3+ to rigidify the outer leaflet of the liposome. We therefore examined the effect of Pr3+ on anisotropy of the external leaflet of liposomes in the presence of 200 mM of solutes being examined. DPPC liposomes were incubated with the fluorescent phospholipid analogue and anisotropy probe, DPH-HPC [2-(3-(diphyenylhexatrienyl)propanyl)-1-hexadecanyl-sn-glycero-3-phosphocholine] to "label" the outer (exofacial) leaflet of the membrane for fluidity measurements (
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Glycerol (Mr = 92.09) has a relatively large molecular volume. Therefore, we tested three other uncharged solutes of varying molecular weight to ascertain whether inducing bilayer asymmetry with Pr3+ would result in reduced permeability for other nonelectrolytes. In panel A, Figure 2 Figure 3 Figure 4, it can be noted that addition of Pr3+ to formamide (Mr = 45.04), acetamide (Mr = 59.07), and urea (Mr = 60.06) equilibrated liposomes, respectively, resulted in a reduction in fluidity of the exofacial leaflet in a manner similar to what is observed in the presence of glycerol. Therefore, the nature of the solute did not alter the interaction with Pr3+ or its effect on the membrane. Panel B, Figure 2 Figure 3 Figure 4, shows representative tracings of stopped-flow experiments carried out above 40°C in the presence or absence of Pr3+. These demonstrate a reduction in solute permeability as judged by the initial rate of shrinkage for each solute. Combined permeability data for each solute is shown as a function of temperature (Panel C, Figure 2 Figure 3 Figure 4). Each shows a significantly lowered permeability above Tc when fluidity is reduced by Pr3+. It is clear that reducing acyl chain fluidity in a single leaflet is sufficient to alter the permeability of the entire membrane to multiple small nonelectrolytes.
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Water and solutes appear to obey the solubility-diffusion model for permeation across a bilayer. However, less information is available on the mechanisms of permeation of gases and protons. Figure 5 shows the results of proton permeability experiments on DPPC liposomes over a range of temperatures. Figure 5 A shows two experiments in which liposomes were rapidly exposed to a pH gradient (pH 7.50 inside/7.06 outside). The pH-dependent quenching of entrapped CF illustrates the reduction in acidification rate at 48°C when Pr3+ is present. Figure 5 B shows H+ permeability as a function of temperature. There is a noticeable increase in proton permeability above Tc; however, at 48°C it is only fourfold higher than baseline levels. This is compared with 129 ± 36-fold (SEM) increases at 48°C for the four small nonelectrolytes tested in this study. Therefore, dramatically increasing bilayer fluidity at temperatures above Tc only results in modest increases in the ability of protons to cross the bilayer. Rigidification of the exofacial leaflet with Pr3+ completely abolishes the phase-transitioninduced permeability increase seen in native vesicles. These data argue that proton permeability is only weakly correlated with membrane fluidity.
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NH3 and CO2 diffuse rapidly across cell membranes, and NH3 permeation is thought to occur by solubility diffusion. We examined the transport properties of both gases across DPPC liposomes and examined the influence of temperature and bilayer asymmetry on the process. Figure 6A and Figure B, shows the permeation of NH3 into DPPC liposomes at 42°C in the presence and absence, respectively, of Pr3+. The rate of permeation is slower when the exofacial leaflet is rigidified (Figure 6 A). Of note is the extreme rapidity of this process, which is complete within 4 ms. The temperature dependence of NH3 permeation is shown in Figure 6 C. At 25°C, the permeability coefficient is ~0.4 cm/s, compared with 4 x 10-6 cm/s for formamide (the fastest of the solutes). Permeability rapidly increased above 40°C, indicating that fluidity is a major determinant in the rate of NH3 permeation across phospholipid bilayers. At temperatures higher than 42°C, NH3 permeation in response to the applied NH3 gradient was complete within the dead time of the instrument (~0.7 ms), and therefore unmeasurable. Rigidification of the exofacial leaflet resulted in a marked reduction in the permeability of the membrane to NH3 at temperatures above Tc. This suggests that NH3, like water and solutes, crosses biological membranes by a solubility-diffusion mechanism.
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A CO2 permeability assay recently developed in our laboratory measures the acidification occurring within liposomes after exposure to a CO2 gradient that is supplied in the form of a CO2/HCO3- solution (
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Discussion |
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The rare earth metal Pr3+ has been used as a nuclear magnetic resonance shift reagent to discriminate between the inner and outer choline methyl resonances of dimyristoylphosphatidylcholine liposomes (
DPPC has been used extensively as a model lipid to explore the behavior of phospholipid bilayers and is therefore well characterized. The advantages of using DPPC in these experiments were the phosphorylcholine head groups necessary for Pr3+ binding, and its high Tc, which allows an exploration of permeant behavior in both the gel and liquid-crystal states. As most PCs have a very low Tc and exist in cell membranes only in the liquid-crystal phase, they don't allow an exploration of membrane permeant behavior in asymmetric bilayers at temperatures above and below Tc.
In a series of solute flux experiments, we initially sought to determine whether Pr3+ would reduce the fluidity of the outer leaflet in liposomes that were loaded with high concentrations of solute (200 mM). Panel A, Figure 1 Figure 2 Figure 3 Figure 4, demonstrates that there was no observable difference on membrane fluidity in the presence or absence of Pr3+ as measured by fluorescence polarization anisotropy between 24° and 35°C. The control liposomes exhibited a steep decrease in anisotropy when temperatures were raised higher than 39°C, which indicated dramatic increases in membrane fluidity as a result of the membrane phase transition from a gel to liquid-crystalline state. When Pr3+ was added, the thermotropic phase-transition occurred 1°3°C higher. This demonstrated that high concentrations of solute were not affecting Pr3+ binding or its influence as a stabilizing reagent on the outer leaflet. The permeability of DPPC membranes to glycerol, formamide, acetamide, and urea were tested over a range of temperatures (panel C, Figure 1 Figure 2 Figure 3 Figure 4) and the effect of membrane phase transition on permeability was striking, with permeabilities above baseline (i.e., 24°C) of 235-, 99-, 74-, and 106-fold for glycerol, formamide, acetamide, and urea, respectively. This is consistent with the high degree of disorder that prevails in the liquid-crystal state. Phase transition is associated with a conformational change in the acyl chains from a predominantly straight (trans) conformation to the gauche conformation, which occurs due to CC bond rotation. This results in an expansion of the area occupied by the chains and a concomitant reduction in the thickness of the bilayer (
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(7) |
where Pab is the permeability of the membrane, Pa is the permeability of leaflet a and Pb is the permeability of leaflet b. To test whether this relationship accurately predicts the solute permeability behavior of Pr3+-rigidified liposomes, we calculated asymmetric bilayer permeabilities. At temperatures above Tc, we know the temperatures at which anisotropies of native and Pr3+-treated liposomes are the same (panel A, Figure 1 Figure 2 Figure 3 Figure 4); e.g., in Figure 3 A the anisotropy of the Pr3+-treated leaflet at 44°C has the same value as the control leaflet at 41°C. For identical fluidities, we assume identical permeabilities (
We now calculate a DPPC leaflet permeability at 44°C from the experimentally determined value for control liposomes.
Having now derived values for the acetamide permeability of a DPPC leaflet and a Pr3+-DPPC leaflet at 44°C, we can add their reciprocals to arrive at a predicted Pr3+-treated membrane permeability of 1.78 x 10-4 cm/s. This compares favorably with the experimentally measured value of 2.12 ± 0.06 x 10-4 cm/s.
Predicted values compared with those measured for the other solutes were 1.08 x 10-5 vs. 1.26 x 10-5 cm/s for glycerol, 2.21 x 10-4 vs. 2.78 x 10-4 cm/s for formamide, and 8.58 x 10-6 vs. 9.55 x 10-6 cm/s for urea. This close concordance of measured and predicted permeabilities for asymmetric membranes strongly supports the model of leaflets offering independent resistances to solute permeation. Bilayer asymmetry is therefore a plausible mechanism by which epithelial cells may limit the permeation of solutes such as urea.
The permeability of water, solutes, and NH3 have been found to correlate strongly with membrane fluidity; however, proton permeability correlates only weakly (
NH3 is a neutral lipophilic molecule that is freely diffusible across most cell membranes. Notable exceptions have been described, however, in the medullary thick ascending limb of Henle (
The system we have used to create bilayer asymmetry allows us to demonstrate the effect of a single leaflet rigidification on the permeation behavior of a range of biologically relevant molecules. These features are applicable to real cell membranes that, it should be noted, exist physiologically in the liquid-crystal rather than the gel state; i.e., in a state analogous to DPPC membranes at temperatures above 41°C. These results clearly demonstrate that epithelial cells with a requirement to restrict diffusional processes; e.g., in the thick ascending limb or collecting duct of the kidney, can do so by means of erecting apical membranes with asymmetric leaflet fluidities.
Gastric glands, which contain parietal and chief cells, are the only epithelia described that possess a barrier to CO2 permeation (
These results demonstrate that for molecules that permeate across membranes by a solubility-diffusion mechanism, reducing the fluidity of a single leaflet of the bilayer is sufficient to reduce permeability. This finding has implications for our understanding of permeation processes in that it allows us to treat the resistance to permeation offered by each leaflet as an independent parameter. Therefore, the permeability properties of the bilayer are not some amalgam or synergy of the activities of each leaflet, but are independent and additive in their own right. The bilayer can be considered, much like an electrical circuit, as a pair of resistors in series for the permeation of solutes, NH3 and water (
CO2 permeability was shown not to occur by a solubility-diffusion pathway as its rate of passage across the liposomal membrane was completely independent of temperature and membrane fluidity. It is likely that these unusual properties are due to its molecular linearity and lack of any permanent dipole moment.
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Footnotes |
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Dr. Rivers died on 19 December 1998.
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Acknowledgements |
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We thank Dr. Dexi Liu for the use of the submicron particle analyzer and Dr. Fred Lanni for the use of the SPEX Fluorolog double spectrometer.
This work was supported by grant DK43955 from the National Institutes of Health.
Submitted: May 4, 1999; Revised: July 13, 1999; Accepted: July 14, 1999.
1used in this paper: CF, carboxyfluorescein; DPPC, dipalmitoylphosphatidylcholine
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