1Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; and 2Department of Molecular Cell Physiology, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Submitted 20 January 2005 ; accepted in final form 23 February 2005
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ABSTRACT |
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aquaporin; urea; permeability; barrier; lipid composition
By contrast, Xenopus laevis oocytes have proven to be remarkably effective for expression of a wide variety of transporter proteins. In the case of ion channels, detailed functional studies of expressed proteins can be performed using two-electrode voltage-clamp or patch-clamp techniques. For proteins involved in electroneutral transport or flux of nonionic substances such as water or urea, electrode methods will not work. While isotopic fluxes or osmotically induced swelling in intact oocytes permits some measurement of function, the inability to control the composition of the interior of the oocyte limits the functional data that can be obtained. It is important to recognize that the intact oocyte is an imperfect system for making measurements of diffusive transport phenomena due to large unstirred layers on both sides of the limiting membrane. In addition, the internal milieu of the oocyte represents a viscous, heterogenous medium replete with diffusion barriers and areas that may be osmotically insensitive, e.g., the nucleus. Intracellular pH measurements related to CO2 or NH3 fluxes are also similarly compromised by multiple unmeasurable diffusion barriers within the cell.
Our aim in this study was to gain the advantages of the oocyte as an expression system, as well as the functional resolution of vesicle systems, by developing a method to isolate native X. laevis oocyte plasma membranes and plasma membranes expressing transporter proteins and determine whether these vesicles are suitable for the assessment of heterologously expressed proteins. We successfully demonstrate the utility of this method for the study of transporters by demonstrating many fold-increases in membrane permeability to water and to urea in oocytes expressing human aquaporin-1 (AQP1) and mouse urea transporter UT-A3.
One reason the oocyte has proven to be so useful for the study of water channels is that it has a remarkable capacity to resist volume expansion on osmotic challenge. When oocytes are placed in 10 mosmol/kgH2O buffer, 40% remain unburst after 2 h (14), demonstrating that the plasma membrane and potentially its associated structural components, i.e., the cytoskeleton and vitelline membrane, present an extremely effective barrier to the passive diffusion of water. Many investigators have measured the osmotic (Pf) water permeability of whole oocytes, often in the context of comparisons with aquaporin (AQP)-expressing cells, and values reached in all of these studies show good agreement in the range of 0.61.5 x 103 cm/s (1, 3, 4, 12, 15, 20, 24, 25). Isolation of the plasma membrane allowed us to test its permeability properties and confirm that its resistance to osmotic swelling was an intrinsic property of the membrane alone.
Low-permeability membranes are important in various tissues for the maintenance of osmotic and chemical gradients. In mammals, examples may be found in the urinary bladder, the stomach, and in specific segments of the nephron where impermeable epithelial apical membranes are required for the urine concentrating mechanism. These membranes not only lack AQP water channels but have unique lipids that appear to result in tighter packing or higher membrane order, thereby reducing defects through which water and solutes can enter and diffuse. How cells synthesize barrier membranes and how the presence or absence of particular lipids affects barrier function are largely unknown. We propose that the plasma membrane of the oocyte can also serve as a useful model for barrier membranes and that, having established a method for isolating it, we can take advantage of the oocyte's size and expression capabilities to explore barrier membrane biogenesis.
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METHODS |
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Harvesting oocytes. X. laevis frogs (Xenopus Express, Plant City, FL) were anesthetized in 1 liter of 0.5% (wt/vol) 3-aminobenzoic acid ethyl ester methanesulfonate salt (Tricaine) containing ice for 20 min. Oocytes were removed bilaterally from the abdominal cavity, and the egg mass was cut into small pieces and placed in calcium-free ND-96 buffer (in mM: 96 NaCl, 1 KCl, 1 MgCl2, 5 HEPES, pH 7.5).Oocytes were then defolliculated in 1.7% (wt/vol) collagenase, 0.17% (wt/vol) trypsin inhibitor in calcium-free ND-96 for 55 min with rotation on an Adams Nutator before being washed three times with hypotonic buffer [in mM: 100 K2HPO4, 0.1% (wt/vol) BSA, pH 6.5] and then allowed to incubate in hypotonic buffer for 10 min at room temperature. Oocytes were transferred to calcium-free ND-96 and then to modified Barth's solution [MBS; in mM: 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, 10 HEPES, pH 7.4] where they were maintained at 18°C. All animal procedures were performed under, and conformed with, an approved University of Pittsburgh IACUC protocol (#0308071A-1).
Isolation of oocyte plasma membrane.
Between 400 and 1,000 oocytes were homogenized in oocyte homogenization buffer (OHB; 250 sucrose, 5 MgCl2, 10 HEPES, pH 7.4; 10 µl/oocyte) containing protease inhibitors (Complete Mini, Roche Diagnostics, Mannheim, Germany) using a Maxima homogenizer (Fisher Scientific, Pittsburgh, PA) on setting 7 (400 rpm) and 15 strokes of a Teflon-coated pestle in a glass Dounce. When membranes were intended for permeability measurements, 20 mM 5,6-carboxyfluorescein (CF) was added to the OHB. Homogenates were centrifuged at 500 g for 5 min, and the supernatant was recovered. The pellet was resuspended in the same volume of OHB and rehomogenized as above before centrifugation at 500 g for 5 min. The supernatant was recovered and combined with the first. The crude membranes (3 ml) were then overlaid on top of a discontinuous sucrose gradient composed of 50% sucrose/OHB (6 ml) and 20% sucrose/OHB (6 ml) and centrifuged at 30,000 g for 1 h in a swinging bucket rotor. Two major membrane bands were visible, the lower at the interface between 20 and 50% sucrose (designated heavy or H-membranes) and the other
1 cm from the top (designated light or L-membranes). Lipid floating on the top of the gradient was removed with a tap aspirator and Pasteur pipette, each band was then collected and diluted threefold in OHB, and total membranes were pelleted at 100,000 g for 30 min.
Marker enzyme assays. Alkaline phosphodiesterase (PDE) activity (plasma membrane marker) was measured by adding 50-µl sample membranes to 200 µl reaction buffer containing 40 mM Na2CO3/NaHCO3, 0.1% (vol/vol) Triton X-100, and 2 mM thymidine-5'-monophospho-p-nitrophenyl ester, pH 10.5. Tubes were incubated at 37°C for 1 h, the reaction was terminated by addition of 250 µl 10% (wt/vol) trichloroacetic acid, and then absorbance at 400 nm was read after addition of 1.5 ml 2 M NaOH. Assay standards were prepared from 0.25 mM p-nitrophenol in 5% trichloroacetic acid.
Succinate dehydrogenase (mitochondrial marker) was measured by reduction of the artificial acceptor p-iodonitrotetrazolium violet (Sigma) as described elsewhere (9) and -N-acetylglucosaminidase (NAG; lysosomal marker) by cleavage of p-nitrophenyl N-acetyl-
-D-glucosaminide (Sigma) as described elsewhere (13, 23).
Expression of AQP1. The human AQP1 X. laevis expression vector (no. 99538) was acquired from ATCC (Manassas, VA). Capped cRNA was prepared with an mMessage mMachine kit from Ambion (Austin, TX). Oocytes were injected with 10 ng RNA and incubated at 18°C for 3 days. Expression was confirmed by placing oocytes in distilled water and monitoring the swelling and bursting times before membrane preparation.
Expression of mouse UT-A3. Mouse UT-A3 (GenBank accession no. AF258602) (7) in pT7TS transcription vector was linearized using EcoR1, and cRNA was prepared using the T7 mMessage mMachine kit. Defolliculated oocytes were injected with 30 ng of cRNA or deionized H2O and incubated for 3 days at 18°C. Expression was validated by performing 14C urea flux experiments as previously described (7).
Assessment of swelling kinetics. Oocytes were imaged on a Nikon inverted microscope using a x2 objective, and images were captured with a Hamamatsu CCD camera and Simple PCI software. Images were acquired every 15 s, and image stacks were thresholded to black and white and analyzed for oocyte cross-sectional area using ImageJ software.
Permeability assays. Permeabilities were measured with stopped-flow fluorometry (10, 19). Water permeability was measured from the rate of shrinkage after exposure of membrane vesicles to a hyperosmotic solution with double the osmolality. Urea transport was measured in membrane vesicles preequilibrated for 2030 min in OHB containing 1 M urea. Vesicles were then rapidly exposed to an osmotically balanced solution containing 500 mM urea. Urea efflux in response to the chemical gradient leads to vesicle shrinking. In some experiments, UT-A3 was inhibited by preincubation with 0.5 mM phloretin for 2030 min.
Analysis of lipids by mass spectrometry. Lipids were extracted in chloroform:methanol (2:1, vol/vol) containing 0.2 mg/ml BHT overnight at 4°C under a nitrogen atmosphere. Three hundred microliters of 0.15 M NaCl were added to affect phase separation. The chloroform layer was removed and dried under a stream of nitrogen. Lipids were analyzed by electrospray ionization tandem mass spectrometry either by direct infusion or after chromatographic enrichment on a Micromass Quattro II triple quadrupole mass spectrometer (Micromass, Manchester, UK). Sheath flow was adjusted to 5 µl/min and consisted of methanol:chloroform (2:1, vol/vol). The electrospray probe was operated at a voltage differential of 3.5 keV in either the positive or negative ion mode. Mass spectra were obtained by scanning in the range of 400950 m/z every 1.6 s and summed. Source temperature was maintained at 70°C. CID spectra were obtained by selecting the ion of interest and performing daughter ion scanning in Q3 at 400 amu/s using Ar gas in the collision chamber. The spectrometer was operated at unit resolution. A combination of daughter, parent, and neutral loss ion-scanning techniques was used to identify and quantitate the various lipid species.
Immunoblotting. Membrane samples were run on SDS-PAGE using 10% precast minigels (Gradipore, French's Forest, NSW, Australia) and then electrotransferred to Immun-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA) at 250 mA for 1 h. Antibodies to AQP1 (1:1,000), calnexin (1:2,000), Rap1 (1:200), MnSOD (1:500), and PDI (1:200) were incubated with electrotransferred samples and detected by ECL (Amersham Biosciences, Piscataway, NJ) using standard methods.
Cholesterol assay. Cholesterol was assayed on membranes extracted with hexane/isopropanol (3:2) and dried under nitrogen, using a Wako Cholesterol E kit (Wako Chemicals, Richmond, VA) according to the manufacturer's instructions.
Phospholipid quantitation. Lipids were extracted from membranes with chloroform:methanol (1:2), and then the solvent was evaporated in a boiling water bath. Lipids to which 0.65 ml 70% perchloric acid had been added were heated to over 350°C in a glass-bead sterilizer for 30 min, and then inorganic phosphate was assayed by the method of Fiske and Subbarow (8). Briefly, samples were mixed with 3.3 ml water, 500 µl of 2.5% (wt/vol) ammonium molybdate, and 500 µl 10% (wt/vol) ascorbic acid. Standards (050 µl, 439 µg/ml KH2PO4) were run in parallel. Tubes were incubated in a boiling water bath for 5 min, and then absorbance was read at 800 nm.
Electron microscopy. Freshly prepared oocyte membranes were mixed with 2x fixative containing 2.0% (vol/vol) glutaraldehyde and 2.0% (wt/vol) paraformaldehyde in 200 mM Na cacodylate, pH 7.4, and centrifuged in a RP45A rotor at 100,000 g in a RC M120EX ultracentrifuge (Sorvall) for 30 min at 4°C. The fixed pellet was removed from the bottom of the centrifuge tube, rinsed with 100 mM Na cacodylate, pH 7.4, buffer, cut into small pieces, and treated with 1% (wt/vol) OsO4 in 100 mM Na cacodylate, pH 7.4, buffer for 60 min at 4°C. After several water rinses, the samples were stained en bloc overnight with 0.5% uranyl acetate in water. Samples were dehydrated in a graded series of ethanol, embedded in the epoxy resin LX-112 (Ladd), and sectioned with a diamond knife (Diatome). Sections, silver to pale gold in color, were mounted on Butvar-coated copper grids, contrasted with uranyl acetate and lead citrate, and viewed at 80 kV in a Jeol 100 CX electron microscope. Images were captured on film, scanned on a Linotype-Hell Saphir Ultra II scanner (Eschborn), and contrast adjusted in Photoshop 7.0 (Adobe).
Determination of membrane vesicle diameters. Size distributions were determined by quasi-elastic light scattering using a DynaPro LSR particle sizer and DYNAMICS data collection and analysis software (Protein Solutions, Bucks, UK).
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RESULTS |
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We characterized the permeability properties of the L- and H-membrane fractions, because we were also interested in defining the biophysical properties of the oocyte plasma membrane in detail. Figure 3 shows the results of stopped-flow fluorometry experiments in which vesicles were rapidly exposed to either an osmotic or outward urea gradient. Under the conditions chosen, vesicles shrink. The kinetics of water and urea flux are reported by entrapped intravesicular CF, which self-quenches on vesicle shrinkage. Water flux kinetics (Fig. 3A) for H- and L-membranes reveal that L-membranes have a lower water permeability than H-membranes. The heavy fraction exhibits two distinct exponential decay rates (appropriate single and double exponential curves have been computer fitted to the raw data). This likely reflects the presence of at least two populations of membrane vesicles with very different permeability coefficients. Pf values from five separate oocyte membrane preparations are shown in the inset. Permeabilities were assigned to both the fast and slow component of the H-membrane curve. L-membranes have Pf = 8.07 x 104 cm/s, whereas the fast and slow flux rates of H-membranes yielded values of 2.83 x 102 and 8.58 x 104 cm/s, respectively.
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A long-standing interest of our laboratory has been to understand the nature of the barrier function exhibited by some naturally occurring membranes and, more specifically, whether certain lipids or lipid combinations are responsible for reducing membrane permeability. Accordingly, we analyzed the lipid and acyl chain composition of L-membranes by mass spectrometry. The results, not including cholesterol, are shown in Table 2 and reveal that the oocyte plasma membrane is extremely rich in phosphatidylcholine (PC) and sphingomyelin (SM) species, which together account for 61.6% of total phospholipid. Separate assays of cholesterol and total phospholipid showed that cholesterol is present at mole percentages in L- and H-membranes of 20.6 ± 1.2 and 12.9 ± 1.9, respectively (means ± SE, n = 5 frogs). This is rather lower than is seen in the red blood cell, where membrane cholesterol constitutes 46 mole percent (16). Perhaps the most surprising result is the high level of phosphatidylinositol (PI).
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Ooyctes were also injected with 30 ng of RNA from UT-A3, the phloretin-inhibitable mouse urea transporter (7). Membranes prepared from these oocytes exhibited markedly elevated urea flux kinetics compared with water-injected control membranes (Fig. 6). Like AQP-expressing membranes, the kinetics of urea flux were best fitted by a double exponential (labeled in the inset bar graph as UT-fast and UT-slow). The fast rate was 7.5-fold higher than the control (Fig. 6, inset), and the slow rate was not different from control and therefore consistent with passive urea diffusion through the membrane. Despite the apparent variability in urea transport rate (Fig. 6, inset), the differences in two experiments between UT- and water-injected membranes were consistent at 7.7- and 6.9-fold, respectively. Phloretin effectively inhibited urea transport activity by 75%, demonstrating the utility of this approach for studying transporter kinetics and the pharmacological profile. Pf of UT-A3-expressing membranes was the same as control membranes, demonstrating no significant water permeability associated with this transporter (not shown).
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DISCUSSION |
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The second application lies in the ability to study the kinetics and regulation of transporters like aquaporins and urea transporters in a system which, while not as compositionally defined as proteoliposomes, offers significant improvements on studies performed in intact oocytes.
The results presented here collectively demonstrate that L-membranes are highly enriched for plasma membrane and exhibit permeability coefficients consistent with, and toward the lower end of, those measured for intact oocytes (1, 3, 4, 12, 15, 20, 24, 25). This is an important confirmation of oocyte membrane permeabilities reported on the basis of osmotic swelling experiments and implies that the membrane itself has an intrinsic permeability of 8.1 x 104 cm/s, a low value that places it in the category of a barrier membrane.
Comparisons with human red cell plasma membrane (2) show the oocyte has a higher concentration of PC (35.8 vs. 29.3), the same amount of SM, lower concentrations of phosphatidylethanolamine (PE; 21.8 vs. 27.6), and phosphatidylserine (5.3 vs. 14.9) but much higher concentrations of PI (6.8 vs. 0.6). Compared with rabbit intestinal brush-border membrane, there is the same amount of PC, less PE, and about the same PI. One very noticeable difference between oocytes and brush-border membrane is a much higher concentration of SM in the oocyte (25.8 vs. 10.3%). Calculation of the unsaturation index (UI), which sums the proportion of each fatty acid multiplied by the number of double bonds, results in a UI for the oocyte plasma membrane of 123. This places it midrange for the membranes from a number of different cell types, including L-cell plasma membrane (UI = 71) and skin fibroblasts (UI = 147). Therefore, the lipids in this membrane do not appear to be unusually high in unsaturated acyl chains (22). In addition, the concentration of cholesterol appears to be relatively modest compared with many cell types. Therefore, there are no obvious clues to explain its low permeability. This compositional analysis, however, does not allow us to draw conclusions as to the distribution of lipids in each leaflet. It is well known that cell membranes asymmetrically distribute particular lipids in the outer and cytoplasmic leaflets (21). It is conceivable that with high concentrations of PC and SM and lower levels of PE, the oocyte maintains an inner leaflet that somewhat resembles the outer leaflet of epithelial cells. We have previously shown that barrier function resides in the outer leaflet (11), so by constructing a membrane with higher than normal concentrations of PC and highly saturated SM in the inner leaflet, permeabilities would be lower. In addition, PC, SM, and cholesterol are thought to form highly ordered domains through hydrogen bonding interactions and for reasons of optimal acyl chain packing (5). The fact that two membrane fractions were isolated with different buoyant densities and that the majority of the plasma membrane was found in the light membranes lends circumstantial support to the presence of membrane microdomains. The presence of all three lipids may explain the low permeability of these membranes to water and urea.
AQP1 and UT-A3 expressed heterologously in oocytes were able to be functionally measured in L-membranes using stopped-flow fluorometry. However, there were two exponential components to the flux curves. That the slow rate was identical to the rate of passive diffusion in control membranes strongly suggests that it is due to passive diffusion rather than some transporter-mediated phenomenon. Fast and slow water fluxes in AQP-expressing L-membranes probably indicate the presence of a population of vesicles without water channels. These may derive from intracellular membrane contaminants that vesiculated and thereby entrapped CF. Contaminating membrane fragments that are not vesiculated do not report any changes in the stopped-flow assay.
UT-A3 expressing membranes also exhibited fast and slow shrinkage rates. Characteristically, phloretin inhibited UT-A3-mediated urea transport. UT-A3 is predominantly expressed on the basolateral membrane of the inner medullary collecting duct and is assumed to mediate urea efflux. Therefore, our data are analogous to and agree with observations made using in vitro perfused inner medullary collecting duct (6). There is clearly sufficient discrimination between control membranes and urea transporter-containing membranes to demonstrate regulation of channel activity. The almost complete elimination of the fast component on treatment of membranes with phloretin demonstrates convincingly that we can study transporter pharmacology, regulation, and kinetics using this system.
In conclusion, we believe that an improved isolation procedure for the plasma membrane of X. laevis oocytes will allow a range of transporters to be studied with much greater precision, control, and reliability than is currently available in the intact oocyte. The removal of unstirred layers from consideration will simplify interpretation and allow a much greater range of experimental manipulations to be performed. The use of vesicles also allows a greater number of transporter substrates and analogs to be tested. This preparation will also be useful for studies of barrier membrane synthesis and may lead to a greater understanding of how coordinate regulation of lipid synthesis and trafficking results in the creation of low-permeability membranes.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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