(Received for publication, January 29, 1996; and in revised form, March 28, 1996)
From the
Water rapidly crosses most membranes, but only slowly crosses
apical membranes of barrier epithelia such as bladder and kidney
collecting duct, a feature essential to barrier function. How apical
membrane structure reduces permeabilities remains unclear. Cell plasma
membranes contain two leaflets of distinct lipid composition; the role
of this bilayer asymmetry in membrane permeability is unclear. To
determine how asymmetry of leaflet composition affects membrane
permeability, effects on bilayer permeation of reducing single leaflet
permeability were determined using two approaches: formation of
asymmetric bilayers in an Ussing chamber, with only one of two leaflets
containing cholesterol sulfate, and stabilization of the external
leaflet of unilamellar vesicles with praeseodymium
(Pr). In both systems, permeability measurements
showed that each leaflet acts as an independent resistor of water
permeation. These results show that a single bilayer leaflet can act as
the barrier to permeation and provide direct evidence that segregation
of lipids to create a low permeability exofacial leaflet may act to
reduce the permeability of barrier epithelial apical membranes.
Epithelial cells generate and maintain apical membrane bilayers made up of leaflets of distinct composition by mechanisms involving asymmetric biosynthesis in the Golgi, oriented insertion into the plasma membrane, and the activity of ATP-driven phospholipid flippases (1, 2, 3, 4, 5, 6) . In several epithelia, such as those of the stomach, kidney collecting duct and thick ascending limb, and mammalian bladder, these apical membranes exhibit exceptionally low permeabilities to water, small nonelectrolytes, protons, and ammonia. These low permeabilities are critical to the barrier function of these epithelia(7, 8, 9, 10, 11) . The structural features responsible for the low permeabilities of these apical membranes remain poorly defined as is the physiological role of the bilayer asymmetry observed in these membranes. The present studies examine the role of bilayer asymmetry in governing the permeability of membranes to water by measuring permeabilities across artificial symmetric and asymmetric membranes. The results show that a single leaflet of a membrane bilayer can act as a barrier to water flux and provide strong evidence that each leaflet acts as an independent resistor to permeation.
where V(t) is the relative volume of the
vesicles at time t, P is osmotic water
permeability, SAV is the vesicle surface area-to-volume ratio, MVW is
the molar volume of water (18 cm
/mol), and C
and C
are the initial
concentrations of total solute inside and outside the vesicle,
respectively. Since the volume within the vesicle was small compared
with the volume outside, it was assumed that C
remained constant throughout the experiment. Parameters from the
exponential fit (amplitude and end point) were used to relate relative
fluorescence to relative volume using boundary assumptions that
relative fluorescence and volume are 1.0 at time zero and that relative
volume reaches a known value (if at time zero the osmolality outside is
double that inside, the relative volume reaches 0.5 at the end of the
experiment)(11, 15, 16, 17) .
To determine the effects of single leaflet structure on
bilayer permeability, P was measured using
tritiated water and values corrected for unstirred layer effects are
shown in Table 1. To correct for unstirred layer effects,
[
C]butanol fluxes were performed in a manner
identical with water fluxes. Because butanol is highly permeable across
membranes, its flux measures the thickness of the unstirred
layer(18) . P
of butanol averaged 18.2
± 1.8
10
cm/s (n =
3). Using this value and the known diffusion coefficient for butanol in
water of 1.0
10
cm
/s(18) , the unstirred layer thickness was
55 µm. The accuracy of the calculated permeability of DPhPC
bilayers using this correction for unstirred layers was determined by
measuring P
of DPhPC liposomes. Importantly, the P
values obtained at similar temperatures were
16.7
10
and 15.0
10
cm/s in two different liposome preparations, in good agreement
with the calculated P
from the chamber experiments
of 14.9 ± 1.7
10
cm/s. Since
artificial bilayers in chambers or liposomes do not contain water
channels, P
and P
are
expected to be equal. Therefore, the agreement of the P
values with the P
values corrected for
unstirred layers confirms the validity of the calculation of the
thickness of the unstirred layers used for all of Table 1.
The
presence of CS in both leaflets markedly reduced P. P
of asymmetrical bilayers
were determined by adding CS to one leaflet and not the other. Although
cholesterol rapidly flips from one leaflet to the other, CS exhibits a
half-time for flipping of 14 h or more, a value far higher than the 4 h
required for these measurements(19) . P
of
the asymmetric bilayer fell between the values obtained with symmetric
DPhPC and DPhPC-CS bilayers. This result suggests that the resistance
to permeation of a bilayer (the reciprocal of its permeability P
) is the sum of the resistances to permeation of
its two leaflets (the reciprocal of permeabilities of each leaflet, P
and P
): 1/P
= 1/P
+
1/P
, where P
is the measured
permeability of the bilayer, and P
and P
are the permeabilities of the individual
leaflets, A and B. In all artificial bilayer systems to date, both
leaflets have been identical(11, 20) , and P
= P
. If it is
assumed that a leaflet resists permeation to the same degree whether it
forms part of an asymmetric or a symmetric bilayer, then it is possible
to calculate (as shown in Table 1) the permeabilities of
individual single leaflets from symmetric bilayers. Using these, we
predict a permeability of the asymmetric bilayer of 7.9
10
cm/s, a value indistinguishable from the measured
value.
The measurements of P in bilayers
indicate that individual leaflets can act as independent resistors to
permeation in an asymmetric bilayer. To determine whether this finding
applies generally to asymmetric bilayers, an entirely different
asymmetric bilayer system was generated in large unilamellar vesicles
(LUV) using Pr
to reduce the fluidity of the external
leaflet. Pr
complexes with the phosphate head groups
of phospholipid molecules, reducing their mobility and that of the
adjacent hydrocarbon chains. This stabilization has been detected
previously as an increase in the phase transition temperature of the
outer leaflet of vesicles using NMR
measurements(21, 22, 23) . To determine
whether, in our experiments, Pr
was increasing the
phase transition temperature of the outer leaflet, we monitored
fluorescence anisotropy as an estimate of membrane fluidity. To examine
the fluidity of the outer leaflet, we introduced a phospholipid-bound
probe of anisotropy,
2-(3-(diphenylhexatrienyl)propanyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine
(DPH HPC) into the outer leaflet, and monitored fluorescence anisotropy
as a function of temperature(24, 25) . Because
phospholipids added to the outside of the liposomes enter the outer
leaflet rapidly but cross to the inner leaflet slowly, this probe
monitored exclusively the outer leaflet of the bilayer. As shown in Fig. 1A, addition of Pr
raised the
transition temperature for this probe by 2-3 °C, in good
agreement with the magnetic resonance
studies(21, 22, 23) .
Figure 1:
Effect of external Pr on phase transition temperature for outer leaflet fluidity and
for osmotic water permeability (P
) of
DPPC liposomes. A, fluidity of the outer leaflet was monitored
using DPH HPC (excitation 360 nm, emission 430 nm) at a probe:lipid
ratio of 1:400 at varying temperatures in the absence (filled
circles) and presence (filled squares) of 10 mM extravesicular Pr
on a SPEX spectrofluorimeter
using standard methods (24, 25) . B,
measurements of water flux at 44 °C in the absence (DPPC)
and presence (DPPC/Pr
) of 10 mM extravesicular Pr
. Data from 6-10 curves
were averaged and fitted as described; averaged data and fitted curves
are shown. C, effect of extravesicular Pr
on P
at varying temperatures. P
was calculated from curves similar to
those of B at varying temperatures as described in the absence
and presence of 10 mM Pr
. Data from 6
different experiments are shown. Values obtained with Pr
differ significantly from those obtained in the absence of
Pr
at 42-46 °C, by t test.
Fig. 1, B and C, shows the effect of Pr on water
permeability at varying temperatures. In the absence of
Pr
, P
rose abruptly at 42
°C, and leveled off at values of 0.03 cm/s, corresponding to the
known phase transition temperature of DPPC. Following addition of
Pr
to the same LUV, P
was
similar to control values at temperatures well below and above the
phase transition temperature. However, the phase transition temperature
for P
was raised by 2-3 °C, so that
Pr
markedly reduced P
at
temperatures in the vicinity of the transition temperature for control
LUV. These results indicate that reducing the fluidity of a single
leaflet reduces the permeability of water across the entire bilayer.
To determine whether the resistance equation applies as well to the
LUV exposed to Pr, leaflet A (DPPC without
Pr
) permeabilities were calculated from the values
for control LUV at temperatures above the phase transition. Leaflet B
(DPPC + Pr
) permeabilities (P
) were estimated by taking the anisotropy value
obtained with Pr
and applying that value to a
standard curve relating permeability to anisotropy in control LUV.
Because we have previously shown that DPH anisotropy is directly
related to measured water permeability(11) , this approach
should give an accurate estimate of the water permeability of leaflet
B. Using these P
and P
values, we calculated expected values for P
, and these expected values are shown in Fig. 1C (as filled triangles) in comparison
with the measured values (filled squares). It is apparent that
this equation applies equally well to diffusive water permeability in
bilayers made asymmetric with CS and to osmotic water permeability in
liposomes made asymmetric with Pr
.
Previous studies had provided evidence that individual leaflets of membrane bilayers could alter their physical properties independent of the other leaflet(23, 24) . Our results demonstrate for the first time that individual leaflets of the membrane bilayer can independently regulate permeation. If the permeabilities of both leaflets are similar, then both will contribute similarly to membrane permeability. By contrast, if one of the leaflets has a very low permeability, the permeability of this leaflet will predominate, so that the permeability of the entire bilayer will be close to that of the low permeability leaflet. This conclusion has several important implications for our current understanding of the role of bilayer asymmetry in epithelial cell biology.
We have previously reconstituted lipids quantitatively extracted from gastric apical membrane vesicles and shown that the reconstituted lipids newly arranged in artificial liposomes do not reconstitute low water permeability(15) . The failure of the extracted lipids when reconstituted into artificial liposomes to duplicate the low permeabilities of the intact membrane may be due to the influence on permeability of two factors in the intact membrane: the presence of integral membrane proteins or the arrangement of the lipid components of the membrane into asymmetrical leaflets(15) . Our new results show directly that segregation of lipid of low fluidity in a single leaflet of the bilayer reduces the permeability of the entire bilayer. Therefore, we can anticipate that cells create apical membranes of low permeability by segregating phospholipid molecules with long saturated hydrocarbon chains and cholesterol in the outer leaflet of the bilayer. Indeed, where the composition of individual bilayer leaflets has been examined, this segregation has been observed(26, 27, 28) . Such segregation of lipids is important in determining the low permeability properties of the apical as opposed to the basolateral membrane domain, because, in epithelial cells, lipids of the exofacial but not cytoplasmic leaflet of the apical membrane are prevented from mixing with those of the corresponding basolateral membrane leaflets by the tight junctions (3, 6) . These considerations plus the evidence from the current study indicate that the lower permeabilities of apical as compared with basolateral membrane domains are due to the lipid structure of the exofacial leaflet.
Cells invest a great deal of effort to generate and maintain bilayers with distinct leaflet compositions. Membrane biosynthesis in the Golgi apparatus occurs in an asymmetric fashion, with distinct lipids going into the different membrane leaflets(1, 2, 3) . Moreover, cells maintain phospholipid ``flippases,'' in their plasma membranes(4, 5) . These transporters couple ATP degradation to the movement of phospholipid molecules from one leaflet to the other, a process which is otherwise energetically unfavorable and occurs at a very slow rate. Our studies provide direct biophysical evidence that the generation and maintenance of asymmetric apical membranes can result in effective barrier function.
The present studies emphasize the importance of segregation of low fluidity lipids in the exofacial leaflet for maintenance of low permeability. Any disease process which disrupts the ability of cells to create and maintain membranes with distinct cytoplasmic and exofacial leaflets may result in failure of apical membrane barrier function, with resulting damage to subepithelial structures or loss of homeostatic function. Candidate diseases which may disrupt the generation and maintenance of asymmetric bilayer structure include ulcer disease in the stomach, cystitis in the bladder, and renal tubular acidosis and inability to concentrate or dilute the urine in the kidney collecting duct.