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The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a two-year-old cow were investigated using EPR spin labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of six-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454–1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35°C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.
Cataract is one of the primary causes of vision loss in the elderly and blindness in ca. 25 million people worldwide. Epidemiological studies have shown that cataract is a multi-factorial disease involving genetic and environmental factors. Besides genetic factors, other factors that can cause cataract formation including age [1,2], hyperbaric oxygen treatment [3–7] and vitrectomy [8–10] are directly related to oxygen concentration within the lens interior and to formation of reactive oxygen species [2,3,11–13]. It is widely postulated that cataract formation results from any type of oxidative stress that perturbs the structure of lens fiber-cell membranes, disrupts the function of intrinsic proteins, and promotes the aggregation of cytosolic proteins, crystallins [1,4,14]. Aggregation of crystallins is an important feature of human cataract [15, 16] and cataract in animals . These observations indicate that, to protect the eye and prevent cataracts, oxygen concentration in the lens has to be maintained at a very low level.
Measurements of the fiber-cell membrane permeability coefficient for oxygen and localization of structural features that modulate oxygen transport into the lens interior should elucidate the mechanisms of cataract formation involving oxidative damage to lens lipids and proteins. McNulty et al. have proposed that the major function of the mitochondria in the lens cortex is not to generate ATP, but to maintain lens clarity by keeping the oxygen content very low and thus preventing protein and lipid oxidation [7,18]. A hypothetical high barrier to oxygen permeation located at the fiber-cell membrane would keep the oxygen concentration inside the eye lens at a very low level, even at the low oxygen consumption rate within the lens interior. However, literature reports on oxygen transport into the lens assume that membranes provide essentially no resistance to the diffusion of oxygen  even though direct support for this assumption is lacking.
Previously, we reported on the physical properties (including permeability for oxygen) of lipid bilayer membranes made of the total lipid extract from the fiber-cell membrane of bovine and porcine eyes from animals less than six months old [19–21]. Because these were young animals, we did not expect a significant difference in the composition of the fiber-cell membranes between the cortex and nucleus, and, thus, the lipid material from the entire lens was pooled. This difference is important only for older animals and manifests itself in changes in phospholipid composition [22–24] and, more significantly, in the cholesterol-to-phospholipid mole ratio [25,26]. In older cows, the PC/SM ratio changed from ~1.8 to ~0.2 , and the Chol/PL ratio from ~1 to ~2  when measured in the cortex and the nucleus, respectively. In young animals, the cholesterol-to-phospholipid mole ratio has been reported as less than or equal to 1 [29–32], and the reported differences in phospholipid composition are less pronounced . We also did not observe immiscible cholesterol crystalline domains (CCDs) in these systems [19,21], which is in agreement with earlier reports.
In the present study, we have utilized the lipid extracts of fiber-cell membranes isolated from two-year-old cow eye lens cortex and nucleus. This has allowed us to compare the behavior of the bulk phospholipid-cholesterol membranes from these two eye lens regions. In addition, we were able to discriminate and characterize the immiscible CCD within the bulk phospholipid-cholesterol membrane of the eye nucleus. This was possible because of the application of the saturation-recovery EPR discrimination by oxygen transport (DOT) method. This method has already been successfully used for detection and characterization of coexisting membrane domains in model phospholipid membranes [33–36] and in pig lens lipid membranes in which the CCD was induced by adding an excess of cholesterol.
In our studies, we used phospholipid-type spin labels and cholesterol analogue spin labels. Because of the overall similarity of the molecular structures of these spin labels with phospholipids and cholesterol, they should be distributed between different membrane domains similarly to the distribution of phospholipid and cholesterol molecules. Figure 1 is a schematic drawing illustrating the distribution of probe molecules in the reconstituted lipid bilayer membranes. This figure also forms the guideline for our experiments and interpretation of our data. In the case of membranes overloaded with cholesterol (cholesterol concentration >50 mol%), in which pure CCDs are formed, the distribution of lipid spin labels is unique (Fig. 1). The phospholipid-type spin labels should partition only into the bulk phospholipid-cholesterol domain (PCD) and the cholesterol analogues should distribute between both domains. Thus, only ASL and CSL can discriminate both coexisting domains. The unique distribution of the phospholipid-type spin labels allow additional information about the structure and dynamics of coexisting membrane domains to be obtained because profiles of the order parameter, hydrophobicity, and oxygen transport parameter obtained with the use of these spin labels should describe only properties of the bulk PCD, without “contamination” from the CCD.
1-palmitoyl-2-(n-doxylstearoyl)phosphatidylcholine spin labels (n-PC, n = 5, 7, 10, 12, 14, or 16), tempocholine-1-palmitoyl-2-oleoylphosphatidic acid ester (T-PC), and cholesterol were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). 9-doxylstearic acid spin label (9-SASL) and cholesterol analogues, androstane spin label (ASL) and cholestane spin label (CSL) were purchased from Molecular Probes (Eugene, OR). Other chemicals, of at least reagent grade, were purchased from Sigma-Aldrich (St. Louis, MO).
Fresh bovine eyes from two-year-old animals were obtained on the day of slaughter from Aurora Meat Packing Company (Aurora, IL). The eyes were dissected and the lenses from ca. 100 eyes collected. Each lens was frozen on a block of dry ice and, when hardened, the nucleus was removed with cork borer. Each of the ca. 1 mm ends of the bored out nucleus was sliced off with a razor and discarded. This method has been described . The total lipids either the nuclear plugs or the cortex samples were extracted separately based on minor modifications of the Folch procedure . The tissue samples were gently mashed in a 500 mL Erlenmeyer flask with the pestle from a tissue homogenizer to which ca. 200 mL of methanol/chloroform (2:1 v:v) mixture was added, and the slurry was stirred for 30 minutes. The sample was distributed to corex centrifuge tubes and centrifuged at 5000 rpm for 30 minutes. The supernatants were poured into a separatory funnel, and water and methanol were added so that the final ratio of methanol/chloroform/water was 2:1:1 (v/v). The chloroform layer was removed and the water layer was extracted two more times with chloroform. The chloroform layers were pooled, dried with MgSO4, filtered, and the solvent was removed. The resultant lipid samples were soft, white solids and were stored at −20°C.
The samples were sent to Avanti Polar Lipids (Alabaster, AL) for analysis of the total lipid extract. For HPLC analysis, the samples were dissolved in a chloroform/methanol mixture of approximately 10 mg/mL. Each sample was injected at three different volumes: 5, 30, and 60 μL. The standards and the samples were injected on a normal phase HPLC column and analyzed with an evaporative light-scattering detector. The molecular weights of the compounds in the HPLC standards (cholesterol: 386.654; PC: 786.15; and SM: 729.065) were used to calculate the Chol/PL and PC/SM molar ratios. The obtained results are as follows: for Chol/PL, 0.7 and 1.9, and for PC/SM, 2.0 and 0.5, for the cortex and nucleus, respectively.
The membranes used in this work were multilameller dispersions (multilamellar liposomes) made of the appropriate lipids (the lipid extract from fiber-cell membranes of bovine eye-lens cortex or nucleus) containing 1 mol% spin label. The membranes were prepared as described by us earlier . Multilamellar liposomes (multilamellar vesicles) are preferred in EPR investigations because the loose pellet after centrifugation contains a high amount of membranes (~20% lipids w/w), which significantly increases the signal-to-noise ratio. Dilution of the pellet by resuspension in 10 volumes of buffer did not change the EPR spectra .
The membranes (multilamellar liposomes) were centrifuged briefly, and the loose pellet was used for EPR measurements. The sample was placed in a 0.6 mm i.d. capillary made of gas-permeable methylpentene polymer, called TPX . The concentration of oxygen in the sample was controlled by equilibration with the same gas that was used for the temperature control (i.e., a controlled mixture of nitrogen and dry air adjusted with flowmeters (Matheson Gas Products model 7631H-604)) [39,40].
Conventional EPR spectra were obtained with a Bruker EMX X-band spectrometer with temperature control accessories. For measurements of the order parameter the EPR spectra were recorded for the temperature range 15–45°C with modulation amplitude of 1.0 G and an incident microwave power of 5.0 mW. The values used for the calculation of the hydrocarbon chain order parameter, AII′ and A′ are measured directly from EPR spectra as indicated in Fig. 2 . To obtain hydrophobicity profiles across the membrane, the z-component of the hyperfine interaction tensor, AZ, for spin labels with the nitroxide moiety at different depths in the membrane was determined directly from EPR spectra for samples frozen at about −165°C and recorded with modulation amplitude of 2.0 G and an incident microwave power of 2.0 mW . This method is based on the dependence of unpaired electron spin density at the nitrogen nucleus on solvent polarity. With an increase in solvent polarity the z component of the hyperfine interaction, AZ, increases .
The spin-lattice relaxation times, T1s, of the spin-labels were determined by analyzing the saturation-recovery signal of the central line obtained by short-pulse saturation-recovery EPR at X-band [34,44,45]. Accumulations of the decay signals were carried out with 2048 data points on each decay. The saturation-recovery spectrometer used in these studies was described previously [45,46].
The bimolecular collision rate between oxygen and the free radical nitroxide moiety of spin label placed at specific locations in the membrane was evaluated in terms of an oxygen transport parameter, W(x), defined as:
where the T1s are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen, respectively [44,47]. W(x) is proportional to the product of the local translational diffusion coefficient D(x) and the local concentration C(x) of oxygen at a “depth” x in a lipid bilayer that is equilibrated in the atmospheric air:
where r0 (about 4.5 Å) is the interaction distance between oxygen and the nitroxide radical spin-label [48,49], and p is the probability that an observable event occurs when a collision does occur and is very close to 1 [40,50,51].
When located in two different membrane domains, the spin label alone most often cannot differentiate between these domains, giving very similar (indistinguishable) conventional EPR spectra and similar T1 values. However, even small differences in lipid packing in these domains will affect oxygen partitioning and oxygen diffusion, which can be easily detected by observing the different T1s from spin labels in these two locations in the presence of oxygen. In membranes equilibrated with air and consisting of two lipid environments with different oxygen transport rates—fast oxygen transport (FOT) domain and slow oxygen transport (SLOT) domain—the saturation-recovery signal is a simple double-exponential curve with time constants of T1−1(air, FOT) and T1−1(air, SLOT) [33,34].
Here “x” from Eq. 1 is changed to the two-membrane domain, FOT and SLOT, and the depth fixed (the same spin label is distributed between the FOT and SLOT domains). W(FOT) and W(SLOT) are oxygen transport parameters in each domain and represent the collision rates in samples equilibrated with air. For further detail and explanation of the DOT method, see Ref. .
EPR spectra were recorded at different temperatures (from 15 to 40°C) and different depths in the membranes (from the polar headgroup region with T-PC to the membrane center with 16-PC). Figure 2 shows a panel of conventional EPR spectra of 5-, 10-, and 16-PC in cortical and nuclear cow lens lipid membranes at 35°C. Because of the sharpness of the EPR lines and the method of measurements (see Fig. 2), AII′ and A′ values can be measured with the accuracy of ±0.1 G, and the order parameter can be evaluated with the accuracy of ±0.015. From the conventional EPR spectra of spin labels in samples frozen to −165°C, the z-component of the hyperfine interaction tensor of spin label AZ was determined and the hydrophobicity profiles across the membrane were constructed. Hydrophobicity profiles obtained for frozen solutions of membrane suspensions  are in agreement with profiles obtained at physiological temperatures as reported by Marsh et al. [52,53] where the isotropic hyperfine constant A0 was used as the hydrophobicity parameter. However, the method of A0 estimation must be changed for different n-SASLs or n-PCs, which makes the obtained hydrophobicity profiles less reliable. 2AZ values can be measured with the accuracy of ±0.25G.
There are two remarkable features of these spectra: first, the overall similarities for cortical and nuclear membranes and, second, the absence of two components in the EPR spectra at any temperature and at any depth in the membrane for all of the samples. The former feature indicates that the physical properties of both membranes should be very similar. The latter feature is consistent with the saturation-recovery EPR data (Sect. 3.2), in which all saturation-recovery curves recorded for phospholipid-type spin labels were single-exponential curves indicating the presence of a homogenous environment in cortical and nuclear membranes. It should be noted that these results were obtained with the phospholipid-type spin labels which are located solely in the bulk PCD (see Fig. 1). Thus, the above conclusions are valid only for this domain in cortical and nuclear membranes.
Figure 3 shows representative saturation-recovery curves for a phospholipid-type spin label 7-PC in cortical and nuclear cow lens lipid membranes at 35°C in the presence and absence of oxygen. The recovery curves were fitted by single and double exponentials and compared. The results indicate that for all of the recovery curves obtained in this work with the use of the phospholipid-type spin labels, no substantial improvement in the fitting was observed when the number of exponentials was increased from one, suggesting that these recovery curves can be analyzed as single exponentials. The decay time constants were determined from a minimum of three measurements within an accuracy of ±3%.
Saturation-recovery measurements were carried out systematically for all phospholipid-type spin labels as a function of the partial pressure of oxygen in the equilibrating gas mixture, the location of spin labels in the membrane, and within the temperature range of 15–40°C, and single exponential recovery was consistently observed. This indicates the presence of a single homogenous membrane when averaged over 0.4 μs (the shortest recovery time observed here), and that, in the bulk PCD of these membranes, the rates of lipid exchange among the purported membrane domains is greater than the spin-lattice relaxation rate (2.5 × 106 s−1, relaxation rate being the inverse of the spin-lattice relaxation time (see also Ref.  for more detail). Additionally, all measured spin-lattice relaxation times in both cortical and nuclear membranes were very similar confirming the conclusion based on the conventional EPR spectra that the physical properties of the bulk PCD in both membranes, including solubility and diffusion of oxygen should be very similar. From saturation-recovery signals, obtained in the absence and presence of oxygen, the oxygen transport parameter was evaluated (see Eq. 1). A minimum of three decay measurements were performed for each point with the accuracy of the evaluation of the oxygen transport parameter better than ±10%.
We also investigated whether or not the immiscible CCD is formed within the lipid bilayer made of the cortical and nuclear cow lens lipids. For these measurements, we used ASL and CSL (see Fig. 1). When ASL or CSL were added to the membranes made of the total lipid extract from cortical and nuclear fiber cells, only single exponential saturation recovery signals were observed in the absence of molecular oxygen. When samples were equilibrated with the air/nitrogen mixture, ASL and CSL also showed the single saturation-recovery signals for cortical membranes. However, for nuclear membranes the saturation-recovery signal for ASL, but not for CSL, was a double-exponential signal. Figure 4 shows saturation-recovery signals for ASL in cortical and nuclear membranes equilibrated with nitrogen and 50% air. The single-exponential fit is satisfactory for deoxygenated samples with very similar decay time constants in both membranes. In the presence of oxygen, the time constant obtained from single exponential fit in the cortical membrane is similar to the shorter time constant in the nuclear membrane obtained from the double saturation recovery fit, and these saturation-recovery signals were assigned to the bulk PCD. The longer time constant was assigned to the CCD (see also Sect. 3.4). The CCD is not formed in cortical membranes presumably because the cholesterol content is too low. The measured value of the cholesterol-to-phospholipid molar ratio in the total lipid extract was 0.7, which is in agreement with the values reported in the literature [25,28,29,31,54]. However, in nuclear membranes, where the measured value of the cholesterol-to-phospholipid molar ratio is 1.9 (values reported in the literature were close to 2 [25,28,54]), we expected formation of the CCD [55–61]. Data obtained using the probe ASL are consistent with this hypothesis.
In our previous work , we have been able to demonstrate that the unique distribution of the phospholipid-type spin labels in membranes containing CCDs (see Fig. 1) allows for the comparison of properties such as the order, hydrophobicity and oxygen transport parameters, of the bulk PCD in different membranes independent of the presence and absence of the CCD.
Figure 5 shows profiles of the order parameter obtained at 35°C across the bulk PCD of cortical and nuclear bovine lens lipid membranes. In both membranes, values of the order parameter measured at the same depth are practically the same and are close to values reported for membranes from six-month-old calf lens lipids and equimolar POPC/cholesterol mixture . They are, however, significantly greater than those measured for the pure POPC membrane (Fig. 5), indicating that a saturating amount of cholesterol is responsible for lens-membrane rigidity. In all membranes, profiles have an inverted-bell shape and alkyl chain order gradually decreases with an increase in membrane depth. The observed weak dependence on temperature (data not shown) is characteristic for membranes saturated with cholesterol  and is in contrast with the dependence in membranes without cholesterol, where the change in the order parameter with temperature is significant .
Figure 6 shows hydrophobicity profiles across the bulk PCD of cortical and nuclear bovine lens lipid membranes. Here, the 2AZ data are presented as a function of the approximate position of the nitroxide moiety of the spin label within the lipid bilayer. Smaller 2AZ values (upward changes in the profiles) indicate higher hydrophobicity (for more details see Ref. ). In both membranes the hydrophobicity profiles show similar rectangular shape, with an abrupt increase of hydrophobicity between C9 and C10. The 2AZ values in the center of both membranes (positions of 10-, 12-, 14-, and 16-PC) indicate that hydrophobicity in this region can be compared to that of hexane and dipropylamine (ε = 2.0–2.9), and hydrophobicity near the membrane surface (positions of 5- and 7-PC) can be compared to that of methanol and ethanol (ε = 24.3–32.6), although this is still considerably less polar than the bulk aqueous phase (ε = 80). For brevity, we relate the local hydrophobicity as observed by 2AZ to the hydrophobicity (or ε) of the bulk organic solvent by referring to Fig. 2 in Ref. .
It is seen from Fig. 6 that the center of the bulk PCD of the nuclear membrane is slightly less hydrophobic than that of the cortical membrane. Similar differences are also observed close to the membrane surface (5- and 7-PC positions). For comparison, the hydrophobicity profile across the POPC bilayer (without cholesterol) is also presented in Fig. 6.
The profiles of the oxygen transport parameter obtained at 35°C across the bulk PCD of cortical and nuclear bovine lens lipid membranes have a rectangular shape with an abrupt increase of the oxygen transport parameter between the C9 and C10 positions (Fig. 7). The rectangular shape is maintained through all temperature regions (15–40°C) used in this work. This abrupt increase is as large as 2.5 times, and the overall change of the oxygen transport parameter across the membrane becomes as large as 5 times. The oxygen transport parameter from the membrane surface to the depth of the ninth carbon is as low as in gel-phase PC membranes, and at locations deeper than the ninth carbon, as high as in fluid-phase membranes [44,63]. Values measured in the membrane center for nuclear membranes are 5–10% smaller than those measured for cortical membranes. However, if we take into account the accuracy of the oxygen transport parameter measurements, evaluated as 10%, these profiles are practically identical. The profiles are very different from the bell-shaped profile across the POPC bilayer (without cholesterol) included in Fig. 7.
As was shown above, the conventional EPR spectra and saturation-recovery curves show that phospholipid-type spin labels detect a single homogenous environment in all membranes derived from cortical and nuclear bovine lens lipids. These suggest that the lipid exchange rates among possible domains within the bulk PCD, sensed by phospholipid-type spin labels, are faster than 10 ns, and/or these domains must be forming and dispersing rapidly on a time scale shorter than 0.3 μs.
Restrictions for the distribution of lipid spin labels in membranes containing CCD (see Fig. 1), indicate that only spin-labeled cholesterol analogues could discriminate this domain. Indeed, in both cortical and nuclear membranes, saturation-recovery signals for n-PCs, 9-SASL, and T-PC were single-exponential signals (see Fig. 3). The only observed two-exponential saturation-recovery signals (Fig. 4) were with the probe, ASL, and air/nitrogen mixtures, which indicated the presence of two environments. We assign the two environments to the bulk phospholipid-cholesterol bilayer and to the pure CCD. Comparison of the oxygen transport parameter values obtained with ASL and those obtained with phospholipid-type spin labels in the bulk PCD allow us to conclude that the domain with greater oxygen transport parameter is the bulk PCD. The domain with smaller oxygen transport parameter is the pure CCD. In cortical membranes, ASL, in the presence and absence of oxygen, always showed single-exponential saturation-recovery signal indicating homogenous environment detected by this spin label. Thus, cortical membranes consist of bulk PCD without detectable sign of CCD. The oxygen transport parameter value detected by ASL in cortical membranes was practically the same as that detected by 10-PC (data not shown). This confirms that the nitroxide moiety of ASL is located at the same depth as the nitroxide moiety of 10-PC. The CSL in both cortical and nuclear membranes did not detect two environments but instead consistently exhibited single-exponential saturation-recovery signals. Thus, the oxygen transport parameter values detected in polar headgroup region with CSL located in the bulk PCD and in CCD (see Fig. 1) are very similar and cannot be distinguished by the DOT method. For clarity, only values of the oxygen transport parameter measured with CSL and ASL in CCD are presented in Fig 7. These data have been used to draw the approximate profile of the oxygen transport parameter across the CCD coexisting with the bulk PCD in the nuclear membranes. The CCD was observed only in the membrane made from nuclear lens lipids. In cortical membranes the CCD was not detected.
Profiles across the bulk PCD of the cortical and nuclear membranes, as shown in Figs. 5, ,6,6, and and7,7, are very similar to those across the bulk phospholipid-cholesterol membranes of the six-month-old calf [20,21] and the six-month-old pig  lens lipids, as well as those across the liquid-ordered-phase membranes made from equimolar mixtures of phosphatidylcholines (PCs) and cholesterol [21,36,42,64]. They are, however, very different from profiles across pure PC membranes (without cholesterol) [36,42,44,62–65]. These data indicate that a saturating amount of cholesterol is responsible for creating these unique physical properties of lens-lipid membranes. Surprisingly, the observed physical properties of these membranes are nearly independent of their phospholipid composition and are mainly determined by the saturating amount of cholesterol. Measured values of the PC/SM molar ratio for cortical and nuclear membranes were 2.0 and 0.5, respectively, which was in agreement with trends observed in older animals. Reported values of the PC/SM molar ratio for different regions of the cortical and nuclear membranes of the two-year-old cow are 0.66 to 1.8 and 0.21 to 0.24, respectively . These differences are less pronounced for younger animals .
The small differences in the hydrophobicity profiles shown in Fig. 6 suggest that the bulk PCD of the cortical membrane is not yet saturated with cholesterol, while the bulk PCD of the nuclear membrane is saturated. This conclusion is based on the observation that in model PC membranes cholesterol causes a significant increase in the hydrophobicity of the membrane center when its concentration increases to 30 mol%; this is then followed by a moderate decrease in hydrophobicity when the cholesterol concentration increases further to 50 mol% . Addition of cholesterol (from 0 to 50 mol%) monotonically decreases hydrophobicity in the region closest to the membrane surface .
To analyze the oxygen transport within the cortical and nuclear membrane, we constructed Fig. 8 in which W(x)−1, which is a measure of resistance to oxygen permeation , is plotted as a function of the distance from the membrane center. It can be seen that in the bulk phospholipid-cholesterol cortical and nuclear membrane, a rather high permeability barrier for oxygen transport is located in the polar headgroup region and in the hydrocarbon region to the depth of the ninth carbon, which is approximately where the rigid steroid-ring structure of cholesterol reaches into the membrane . The resistance to oxygen permeation in this region is much higher than the resistance in the water phase, as indicated by the broken line in Fig. 8. However, resistance to oxygen permeation in the membrane center becomes much less than in the water phase and is comparable to the resistance in the pure PC membranes (see profile for POPC bilayer included in Fig. 8). Figure 8 also includes the profile of the resistance to oxygen permeation across the CCD of the nuclear membrane obtained with ASL and CSL. The resistance in this domain is higher than that in water and the bulk PCD at all depths (the difference in the membrane center is as great as 7 times).
The permeability coefficients for oxygen, PMs, across the membrane domains were estimated using previously described procedures [20,44] and are presented in Table 1. At all temperatures, the PM for the bulk PCD is practically the same in cortical and nuclear membranes. The PM obtained for the CCD is, however, lower than that in the bulk PCD and the difference is greater at higher temperatures. It should be pointed out here that the CCD is significantly thinner than the PCD which may compensate the greater difference in resistance to oxygen permeation between these two membranes (see Fig. 8). The effect of the membrane thickness is clearly seen when PM is compared with the permeability coefficient for oxygen across a water layer of the same thickness as the membrane (Table 1). At all temperatures oxygen permeation across the CCD is about 2.5 to 3 times smaller than across a water layer of the same thickness.
We have previously reported  values of the PM across the CCD induced in the porcine lens membrane by the addition of excess cholesterol to the total lipid extract. In the present work, we provided values of the PM across the CCD which occurs in membranes made of the natural lipid mixture isolated from the nuclear fiber cells of the bovine eye lens for which the cholesterol-to-phospholipid molar ratio is reported to be ~2 [25,28,54]. In both cases, the evaluation provides the upper limit of the PM across this domain. These data strongly suggest that the CCD forms a barrier to oxygen transport, especially in the lens nucleus where the cholesterol concentration is much higher. In cortical membranes, the CCD was not observed, presumably because the cholesterol-to-phospholipid ratio was too low (measured and reported values are smaller than 1 [25,28,54]).
Values of the PM across the membrane region from the membrane surface to the depth of the ninth carbon, values of the membrane center (between the tenth carbons in each leaflet), and values of the activation energy for oxygen diffusion at different depths are presented in Tables 2 and and3.3. These values clearly demonstrate that the center of the bulk PCD, which has a much higher oxygen permeability than water, in both the cortical and the nuclear membranes can serve as a channel for oxygen transport (parallel to the membrane surface), while high barriers for oxygen permeation exist at both sides of the membrane. Similar channels were observed in six-month-old-calf and six-month-old-pig lens lipid membranes and in the POPC/cholesterol (1:1 molar ratio) membrane [19–21].
The data presented here provide strong evidence for the crucial role of CCDs in the maintenance of the physical properties of the lens fiber cell membrane. First, the CCD raises the barrier for oxygen transport across the fiber cell membrane. This helps to maintain a low oxygen concentration in the lens interior, especially in the nucleus. Second, the presence of CCDs ensures that the surrounding phospholipid bilayer is saturated with cholesterol, which helps to maintain membrane homeostasis. We hypothesize that this high cholesterol content keeps the bulk physical properties of the membrane relatively constant even with age-related changes in phospholipid composition.
A goal of the present study was to locate structural features of the fiber cell membrane that modulate oxygen transport in the lens interior. We have shown that the major permeability barrier in the membrane is located at the CCD, which should occupy a significant part of the membrane surface, especially in the human-lens nucleus where the cholesterol-to-phospholipid mole ratio is as high as 4 [68–70]. This indicates that the fiber cell membrane’s resistance to oxygen permeability should increase with age and should be greater in the nucleus than in the cortex when coupled with the reported changes in lens lipid composition with age [68,70–75]. These interactions ensure maximal protection against oxidative damage and, thus, cataract formation. They are also likely to contribute to lens transparency.
This work was supported by grants EY015526, TW008052, EB002052, and EB001980 of the National Institutes of Health and Research to Prevent Blindness (JPD). J.W. thanks Lena Widomska for her constant support during the preparation of this manuscript.
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