Acute and chronic changes in cholesterol modulate Na-Pi cotransport activity in - 《American Journal of Physiology》美国生理学杂志干细胞之家

干细胞之家 - 中国干细胞行业门户第一站 › 干细胞之家论坛 › 干细胞文献资源库 › 《American Journal of Physiology》美国生理学杂志 › Acute and chronic changes in cholesterol modulate Na ...



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 25 下一页

返回列表

查看: 427077\|回复: 246	go Acute and chronic changes in cholesterol modulate Na-Pi cotransport activity in [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 12:37 |只看该作者 |倒序浏览 |打印

Department of Medicine, Division of Renal Diseases and Hypertension, and Department of Physiology and Biophysics, University of Colorado Health Sciences Center and Denver Veterans Affairs Medical Center, Denver, Colorado
+ b5 o3 l( ?' j$ T4 G, {# K5 p
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas
6 J  B  B, g0 ~% f! a+ v
Department of Internal Medicine, University of Louisville, Louisville, Kentucky
7 M- F7 m7 G5 T
Department of Toxicology, University of Zaragoza, Zaragoza, Spain9 D  P6 j# A$ u' @
* L* [% X: s8 A- ]% l
ABSTRACT

We previously showed an inverse correlation between membrane cholesterol content and Na-Pi cotransport activity during the aging process and adaptation to alterations in dietary Pi in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85–F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-Pi cotransport activity and apical membrane Na-Pi protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with -methyl cyclodextrin (-MCD) resulted in a significant increase in Na-Pi cotransport activity accompanied by a moderate increase in apical membrane Na-Pi protein abundance and no alteration of total cellular Na-Pi protein abundance. Conversely, acute cholesterol enrichment achieved with -MCD/cholesterol resulted in a significant decrease in Na-Pi cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-Pi protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-Pi cotransport activity by diverse mechanisms that also include significant interactions of Na-Pi protein with lipid microdomains.

two-photon fluorescence microscopy; filipin; lipid microdomains; laurdan; opossum kidney cells
6 _6 L: P! l. ?. {
DISORDERS OF EXTRACELLULAR inorganic phosphate (Pi) concentration and impairments in renal and gastrointestinal Pi reabsorption are common clinical problems. Aging, diabetes mellitus, malignancy, alcoholism, transplantation, acquired immunodeficiency syndrome (AIDS), and several therapeutic drugs are well known to cause or to be associated with hypophosphatemia or hyperphosphatemia, mainly by affecting renal tubular Pi transport. The kidney plays a critical role in the regulation of Pi homeostasis. The evidence to date indicates that regulation of the overall renal tubular Pi transport by dietary, hormonal, or metabolic factors occurs mainly at the level of the proximal tubular apical brush-border membrane (BBM) Na-Pi cotransport system (36, 53). To date, three distinct families of renal Na-Pi cotransporters have been identified: type I, type II, and type III. These Na-Pi cotransporters are expressed in the proximal tubule of humans, rats, mice, rabbits. Experimental data suggest that the type IIa renal apical BBM Na-Pi cotransport system mediates the majority of the renal proximal tubular BBM Na-Pi transport (1, 2, 6, 19, 22, 26, 27, 29, 30, 35, 49, 56, 59).# |0 c7 n) g# r4 I; R

Studies to date have determined that dietary factors, hormones, metabolic factors, and the developmental and aging process regulate Na-Pi cotransport activity by diverse molecular and cellular mechanisms, including transcriptional control, translational control, and, most importantly, control via acute trafficking (endocytosis or exocytosis) of the type IIa Na-Pi cotransport protein to and from the apical membrane (36, 53). The net result is that with one known notable exception (61) Na-Pi cotransport activity is directly correlated with apical BBM type IIa Na-Pi cotransport protein abundance.

We have demonstrated that alterations in renal lipid composition, including BBM cholesterol, sphingomyelin, and glycosphingolipid content, play an important role in the regulation of renal Na-Pi cotransport activity. Specifically, adaptation to changes in dietary Pi as well as the aging process are associated with alterations in apical BBM cholesterol content, and there is an inverse relationship between BBM Na-Pi transport activity and BBM cholesterol content (25, 34). In addition, in diabetes, in dietary potassium deficiency, and following treatment with glucocorticoids, there is an inverse relationship between BBM Na-Pi transport activity and BBM sphingomyelin and glycosphingolipid (glucosylceramide and ganglioside GM3) content (27, 61). Furthermore, we have shown that inhibition of glucosylceramide and ganglioside GM3 synthesis results in modulation of Na-Pi transport activity (27, 61).+ T6 V6 C( e! o: @

While our previous studies do indicate that alterations in cholesterol and glycosphingolipid composition modulate renal Na-Pi cotransport activity, the mechanisms by which lipids modulate Na-Pi cotransport activity have not been determined and remain unknown., r& k# L( T$ I' W9 M$ ?/ L0 h& f$ s7 [
! G% [+ F- t0 l$ z/ I
The purpose of the present study was to determine the effects of alterations in cell cholesterol content on the regulation of Na-Pi cotransport activity and Na-Pi protein expression in opossum kidney (OK) cells, a cell line that expresses the fully functional type IIa Na-Pi cotransport system (3, 50)./ E3 u4 N4 y2 w' M' R( X) Y
1 b# Q7 V8 }- C3 Y
We have found that acute enrichment of OK cell cholesterol content causes a significant decrease in Na-Pi cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance in the absence of any changes in total cellular Na-Pi protein abundance. In contrast, chronic enrichment of OK cell cholesterol content causes parallel and significant decreases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance as well as decreases in apical membrane lipid fluidity and lipid dynamics. Acute depletion of OK cell cholesterol content causes increases in Na-Pi cotransport activity with a moderate increase in apical membrane Na-Pi protein abundance in the absence of any changes in total cellular Na-Pi protein abundance. In contrast, chronic depletion of OK cell cholesterol content causes parallel and significant increases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance as well as increases in apical membrane lipid fluidity and lipid dynamics. These results indicate that direct alterations in cell cholesterol content per se are an important modulator of Na-Pi transport activity and these effects of cholesterol are mediated by translational (chronic changes in cholesterol) as well as posttranslational (acute changes in cholesterol) mechanisms.
$ r4 N0 f6 d& A5 Y+ {- q: w$ b4 J
MATERIALS AND METHODS, |5 Z5 n- _! A, S, {
, L0 n8 u! ^- `
Materials. Filipin III, -methyl cyclodextrin (-MCD), -MCD/cholesterol complex, protease inhibitors and all other chemicals were obtained from Sigma (St. Louis, MO) except when noted. Cell culture media were also from Sigma. Sera and antibiotics were from Invitrogen (Carlsbad, CA), except for lipoprotein-deficient serum (LPDS), which was purchased from Intracel (Rockville, MD).0 n( P* k. U# ^' O. a9 O
$ N, u! T# i+ r  P% Y3 I
Cell culture. OK cells, a renal proximal tubular cell line derived from the opossum kidney (4), were grown in a humidified 5% CO2-95% air atmosphere in DMEM supplemented with 10% fetal calf serum (FCS), 100 IU penicillin G, and 100 μg/ml streptomycin. Cells were grown to confluence and then were rendered quiescent for 24 h by serum deprivation in Ham＊s F-12/DMEM (1:1, vol/vol) supplemented with 4 mM L-glutamine, pH 7.3. For transport studies, OK cells were grown in 24-well dishes. For isolation of cell membranes, fluorescence spectroscopy, and lipid composition measurements, the cells were grown in 10-cm-diameter tissue culture dishes. For fluorescence microscopic studies, cells were grown on coverslips.

Acute modulation of OK cell cholesterol content. Confluent and quiescent OK cells grown in Ham＊s F-12/DMEM (1:1, vol/vol) supplemented with 4 mM L-glutamine, pH 7.3, were treated with 1) 10 mM -MCD for 30, 45, and 60 min to cause progressive depletion of cell cholesterol (see RESULTS) or 2) 10 mM -MCD/cholesterol for 30, 60, and 90 min to cause progressive enrichment of cell cholesterol (see RESULTS) (14).

To determine whether the effects of cholesterol modulation on Na-Pi cotransport activity are reversible, we performed add-back (repletion/depletion) experiments (58): 1) the cells were treated with either vehicle or 10 mM -MCD for 45 min to deplete cholesterol and then one-half of the cells were treated with 10 mM -MCD/cholesterol for 45 min to replace cholesterol; and 2) cells were treated with either vehicle or 10 mM -MCD/cholesterol for 45 min to enrich with cholesterol and then one-half of the cells were treated with 10 mM -MCD for 45 min to remove the cholesterol.

Chronic modulation of OK cell cholesterol content. Cells were seeded and incubated in DMEM in the presence of 10% FCS for 24 h. Cells were then either 1) grown in regular DMEM medium in the presence of 10% FCS, penicillin, and streptomycin; 2) grown in DMEM plus 5% LPDS for 2 days to deplete the cells of cholesterol; or 3) grown in DMEM plus 5% LPDS for 6 h, to upregulate their low-density lipoprotein (LDL) receptors, and then grown on DMEM plus 400 μg/ml of human LDL (Calbiochem, San Diego, CA) for 2 days (45).. r1 C0 v- Q7 {; q1 b' @2 d1 o& E

Measurement of transport activity. Before the uptake experiments, OK cell monolayers were rinsed once at 37°C with transport solution containing (in mM) 137 NaCl, 5.4 KCl, 2.8 CaCl2, 1.2 MgSO4, and 14 HEPES, pH 7.4 (3). To measure Na-Pi cotransport activity, the cell monolayers were then incubated with 200 μl of the above transport solution also containing 0.1 mM K2H32PO4. After 5 min, which represents the initial rate of linear uptake (3), transport was terminated by aspiration of the uptake solution and washing of the monolayers three times at 4°C. The cells were then scraped and analyzed for 1) 32PO4 counts and 2) protein content. Transport activity is expressed as nanomoles Pi per milligram protein per 5 minutes.
. x" I: l8 U& R: c+ O) \) k  X- z
To determine whether alterations in cholesterol modulate Na gradient-dependent Pi transport (active transport) vs. Na-independent Pi transport (diffusion), uptake was determined in parallel in the presence of 137 mM choline chloride.

To determine whether alterations in cholesterol modulate the activity of other transporters, we also measured Na gradient-dependent uptake of 1) methyl--glucopyranoside, 2) L-glutamate, and 3) sulfate, as described above.5 n# A" v* L4 u; v

To determine whether the effects of cholesterol modulation on Na-Pi cotransport activity was mediated by alterations in the maximal capacity/velocity (Vmax) of the Na-Pi cotransporter or the affinity for Pi (Km), we performed transport kinetic studies, measuring Na-Pi cotransport activity in the presence of 25–800 μM extracellular Pi. An equation containing saturable (Michaelis-Menten or transport) and nonsaturable (diffusion) components (50) was used to calculate the Km, Vmax, and Kd coefficients by iterative, nonlinear regression. The fits were accepted when two consecutive iterations changed the sum of squares by : L2 Q' G$ E8 V0 U2 @* x1 @  b

OK cell membrane preparation. Cells were rinsed three times with cold Tris-buffered saline (TBS) and scraped into 2 ml isolation buffer (5 mM HEPES-KOH, 4 mM EDTA, 1 mM PMSF, pH 7.4) and resuspended with a 22-gauge needle. The homogenate was centrifuged at 17,000 g for 60 min, and the pellet was resuspended in 400 μl of resuspension buffer (50 mM mannitol, 10 mM HEPES-Tris, pH 7.4). Protein measurement was performed by the Lowry assay (31), and equal amounts of total protein aliquots were set up for further processing (Western blotting, fluorescence spectroscopy, and measurement of lipid composition).
9 S( w+ y" r' L" E3 p! g
SDS-gel protein electrophoresis and Western blotting for Na-Pi protein. OK cell membrane samples were denatured for 2 min at 95°C in 2% SDS, 10% glycerol, 0.5 mM EDTA, and 95 mM Tris﹞HCl, pH 6.8. Ten micrograms of membrane protein/lane were separated on 10% polyacrylamide gels according to the method of Laemmli (20) and electrotransferred onto nitrocellulose membranes (55). After blockage with 5% fat-free milk powder plus 1% Triton X-100 in TBS (20 mM, pH 7.3), Western blotting was performed with antiserum against the COOH-terminal amino acid sequence of NaPi-4 at a dilution of 1:5,000 (22) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Western blots were developed using enhanced chemiluminescence (Pierce, Bradford, IL). The signals were quantified in a PhosphorImager with chemiluminescence detector and densitometry software (Bio-Rad, Richmond, CA).

Surface membrane protein biotinylation. To determine the abundance of NaPi-4 in the apical membrane of OK cells, we used surface biotinylation (12, 15, 16, 39). OK cells grown in six-well plates were rinsed three times with PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS  ) at 4°C. The cells were then incubated with 0.5 ml biotinylation buffer (10 mM triethanolamine, pH 7.4, 2 mM CaCl2, 150 mM NaCl) containing 1.5 mg/ml sulfo-NHS-SS-biotin (EZ-Link Sulfo-NHS-SS-Biotin, Pierce) for 1 h at 4°C under horizontal agitation. Cells were washed with cold PBS and then with quenching solution (100 mM glycine in PBS  ) for 20 min at 4°C. After three additional washes with cold PBS  , the cells were lysed in 0.4 ml of a buffer containing 50 mM Tris﹞HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, and protease inhibitors for 15 min on ice, scraped, and homogenized by repeated pipetting. The lysates were cleared at 14,000 g for 5 min at 4°C, and the protein concentration of the supernatants was adjusted to 1 mg/ml. Biotinylated proteins were precipitated overnight by mixing 300 μl of cell lysate with 75 μl of streptavidin-agarose beads (ImmunoPure Immobilized Streptavidin, Pierce) in constant rotation at 4°C. The beads were collected by 2-min centrifugation at 2,500 g, washed twice in 1 ml buffer (50 mM Tris﹞HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl), then twice more in 20 mM Tris﹞HCl, pH 7.4, 500 mM NaCl, and once in 10 mM Tris﹞HCl, pH 7.4. The beads were resuspended in 100 μl of 2x Laemmli buffer containing 100 mM DTT, incubated at 42°C for 30 min, and centrifuged for 2 min at 2,500 g. Twenty-five microliters of supernatant were subjected to SDS-PAGE and blotted with anti-NaPi-4 antibodies as described above.

Measurement of cholesterol content. Lipids from OK cell membranes were extracted by the method of Bligh and Dyer (7), as we have previously described (24, 25, 27). For the acute cholesterol modulation experiments, free cholesterol was determined enzymatically using Amplex Red (Wako Chemicals, Richmond, VA). For the chronic cholesterol modulation experiments, free cholesterol content was determined by injection of an aliquot of the lipid extract into a 530-μm 50% phenylmethyl silicone column in a Hewlett-Packard model 5890 gas chromatograph with a flame ionization detector, run isothermally at 280°C, with coprostanol serving as an internal standard. Area ratios were completed with a Hewlett-Packard 3392A integrator.

Filipin staining for cholesterol imaging. Cells were grown on coverslips and treated with either -MCD or -MCD/cholesterol as described above. Cells were rinsed with TBS and fixed with 4% paraformaldehyde in TBS for 15 min. Unused fixative was quenched by incubation for 10 min with 50 mM NH4Cl. After being rinsed, cells were incubated with filipin III in TBS for 10 min, rinsed again, and mounted on a slide (42). Slides were viewed with two-photon excitation at 720 nm with a Zeiss laser-scanning confocal microscope (LSM 510, Thornwood, NY) equipped with a x40 water-immersion objective.
* {8 ]$ c9 U! m9 i+ Z2 K% R7 y
Cell transfection and GM1 staining. Cells on coverslips were grown to 80–90% confluency before transfection with enhanced green fluorescent protein (EGFP)-NaPi-2 plasmid (a gift from Prof. Heini Murer, University of Zürich, Zürich, Switzerland) using Lipofectamine 2000 (Invitrogen) according to the manufacturer＊s instructions. After 24 h, cells were rendered quiescent in serum-free Ham＊s F-12/DMEM for 24 h before treatment with either -MCD or -MCD/cholesterol as above. Cells were rinsed twice with PBS, put on ice, and incubated for 10 min with 1 μg/ml Alexa 555-conjugated cholera toxin B (Molecular Probes, Eugene, OR) for specific labeling of the ganglioside GM1, a well-described lipid raft marker (5, 13, 37). After three washes with PBS, cells were fixed with 4% paraformaldehyde in PBS, first for 15 min on ice, then for 15 min at room temperature. The fluorescence of EGFP and Alexa 555 was imaged using a Zeiss laser-scanning confocal microscope (LSM 510) equipped with a x40 water-immersion objective. EGFP was excited using a 488-nm argon ion laser and Alexa 555 using a 543-nm He/Ne laser.( c0 N) ~4 {/ Z1 {$ t% ?3 ^% H
: Y$ P, G8 E4 N1 o2 t0 M* E
Laurdan fluorescence spectroscopic and microscopic measurements. Laurdan is a lipophilic fluorescent dye responsive to the dipole moment of its solvent. For laurdan in the excited state, dipole relaxation of adjacent solvent molecules causes a red shift in emission. In nonpolar solvents, the emission peak can be as short as 380 nm whereas in polar solvents the emission maximum can be as long as 490 nm. When the hydrophobic tail of laurdan is inserted into a membrane, the excitation/emission dipole is located at the lipid head group and responds to the packing of the lipids (see Fig. 9A). In the more densely packed gel phase, laurdan emission peaks at 440 nm. In the liquid crystal phase, dipole relaxation of water molecules penetrating into the membrane causes a red shift to an emission peak at 490 nm. This shift is characterized by the generalized polarization (GP)$ V1 X+ _: R, p/ v0 ?5 V* O
) A0 I( g' _) P  W: z2 m7 w
(1)
; H2 \$ U5 T2 A1 f6 ~  I
Thus the laurdan GP value is inversely correlated with the lipid packing.* s9 X* F; @7 ?: N: G3 f) C6 ^/ D8 H

Laurdan fluorescence spectroscopy. Fluorescence emission spectra of laurdan-labeled OK cell membrane vesicles were obtained using a photon-counting spectrofluorometer (PC1, ISS, Champaign, IL) at 37°C with excitation at 340 nm. The excitation-generalized polarization (GP) values were calculated from the emission intensity at wavelengths of 440 or 490 nm (38) as in Eq. 1.

Two-photon excitation microscopic laurdan GP images. Laurdan labeling was performed directly on the coverslips in culture dishes by adding 1 μl of a 2 mM laurdan stock solution in DMSO/1 ml of the cell culture medium and incubating for 30 min in the dark. Cells were then gently washed with fresh medium, and the coverslips were mounted on a microscope hanging drop slide using fresh medium. The data-acquisition and image analyses for laurdan GP microscopic measurements were performed as previously described (38, 60). Briefly, a titanium-sapphire laser (Mira 900, Coherent, Palo Alto, CA) pumped by an argon-ion laser (Innova 310, Coherent) tuned at 770 nm was used as the excitation light source. The laser power was attenuated to 20 mW before entering the microscope, producing 2-mW laser power at the sample. The laser light was scanned across the sample using a galvanometer-driven x-y scanner (Cambridge Technology, Watertown, MA). A quarter-wave plate (CVI Laser, Albuquerque, NM) was placed after the polarizer to change the polarization of the laser light from linear to circular. Two optical band-pass filters (Ealing Electro-Optics, Holliston, MA) were used to collect fluorescence in the blue and red regions of the laurdan emission spectrum. A miniature photomultiplier (R5600-P, Hamamatsu, Bridgewater, NJ) amplified through an AD6 discriminator (Pacific, Concord, CA) was used for light detection in the photon-counting mode. A home-built card acquired the counts. Three images (256 x 256 pixels, 9 s/frame) were averaged for the GP calculation. Images were analyzed with Noesys 1.1 software (Fortner Software, Sterling, VA) for three-dimensional reconstruction.

Isolation of OK cell membrane lipid raft fractions by nondetergent density gradient ultracentrifugation using OptiPrep (iodixanol). OK cell membranes were incubated in 100 μl of TNET buffer (50 mM Tris﹞HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and a complete protease inhibitor cocktail) at 4°C for 30 min. After Dounce homogenization on ice, the extract was adjusted to 50% OptiPrep, after which 600 μl were overlaid with 1 ml each of 40, 30, and 20% OptiPrep in TNET and then 400 μl of 10% OptiPrep in TNET (21, 22). Following centrifugation at 170,000 g at 4°C for 4 h, a continuous density gradient was formed. Fractions (8 x 500 μl) were collected from the top to the bottom of the gradient and analyzed for 1) total protein by BCA protein assay (Pierce), 2) Western blotting for Na-Pi, 3) cholesterol content using an enzymatic assay (Wako Chemicals), and 4) sphingomyelin content using an enzymatic assay as described (18).7 o9 D6 X  a+ N4 d
- P9 ^$ _1 a( x3 M8 T
Statistical analysis. The results are expressed as means ± SE. The statistical significance of differences was assessed by one-way ANOVA and the Tukey multiple comparison test, whereby P

RESULTS
0 R" `' c. u( D( {
Acute modulation of OK cell cholesterol content. Incubation of OK cells with 10 mM -MCD resulted in a time-dependent depletion of cell cholesterol, as determined both by a biochemical assay for free cholesterol (Fig. 1A) and by filipin staining and two-photon confocal microscopy (Fig. 1B). Conversely, incubation of OK cells with 10 mM -MCD/cholesterol resulted in a progressive enrichment of cell cholesterol (Fig. 1).9 ~9 P) h' m0 U3 \* o

Acute modulation of OK cell cholesterol content causes significant changes in Na-Pi cotransport activity. Acute increases in cholesterol content caused progressive and significant decreases in Na-Pi cotransport activity, whereas acute decreases in cholesterol content caused progressive and significant increases in Na-Pi cotransport activity (Fig. 2A). The effect of cholesterol on Pi transport activity was specific for Na gradient-dependent Pi transport activity (active Pi transport), as cholesterol had no effect on choline gradient-dependent Pi transport activity (passive Pi diffusion) (Fig. 2B). Correlation analysis between cell cholesterol and Na-Pi cotransport activity revealed an inverse relationship between cell cholesterol content and Na-Pi cotransport activity, with a slope of –0.006175 ± 0.0005841 and r = 0.8958 (Fig. 2C).

Effects of cholesterol on Na-Pi cotransport activity are reversible. Depletion of cholesterol in cholesterol-enriched cells and repletion of cholesterol in cholesterol-depleted cells showed that the effects of cholesterol on Na-Pi cotransport activity are totally reversible (Fig. 3).

Effects of alteration in cholesterol content on the activity of other transporters. We next determined whether the effects of alterations in cholesterol content are selective and specific for Na-Pi cotransport activity. We found that alterations in cholesterol content had no significant effects on Na--methyl-D-glucopyranoside (Fig. 4A) or Na-glutamate (Fig. 4B) cotransport activity. In contrast, alterations in cholesterol content had significant effects on Na-sulfate cotransport activity (Fig. 4C).* w! V1 K3 R5 o. O
8 Z7 A; n  ~: F
Acute modulation of OK cell cholesterol content causes significant changes in Vmax of Na-Pi cotransport activity. We performed kinetic studies to determine whether the effects of cholesterol on Na-Pi cotransport activity are mediated via changes in Vmax or Km. As shown in Fig. 5, A and B, and in Table 1, the kinetic analysis indicates that 1) cholesterol modulates Vmax of Na-Pi cotransport activity, such that a reduction of the cholesterol content with -MCD is accompanied by a 1.6-fold increase in Vmax; 2) there are no significant changes in the affinity (Km) of the Na-Pi cotransporter for Pi; and 3) there are also no significant changes in the diffusion constant (Kd) for Pi. This constant was similar in the absence or presence of Na  and corresponds to the slope of the diffusional, nonsaturable component, which also includes the unspecific binding or background of 32Pi during the uptake assay. The significance of the differences among the fits was obtained with an F-test, whereas the differences between kinetic constants were assayed with a t-test (Table 2).
3 e# ?  n% Q: N7 V% |2 l& d
View this table:% C: P* ~: e7 l' N/ f
! K8 |7 C( {1 {- w7 y& |2 [. H
View this table:8 S# d8 ~# B. p

Acute modulation of OK cell cholesterol content causes moderate changes in apical membrane Na-Pi protein abundance. Surface biotinylation studies showed that despite marked changes in the Vmax of Na-Pi cotransport activity, acute alterations in cholesterol caused only moderate changes in surface (apical membrane) Na-Pi protein abundance, whereas there were no changes in total cellular Na-Pi protein abundance (Fig. 6). The changes in Na-Pi protein abundance were significant only when cholesterol-depleted cells are compared with cholesterol-enriched cells. The correlation between the NaPi-4 protein abundance in the apical membrane and Pi transport under acute cholesterol modulation is further shown in Table 3.2 \3 z, O! _$ x& e
5 F, P9 W- m" u  S" P$ U. r. A7 }7 R7 C
View this table:' |9 \  e, v6 Q

Chronic modulation of OK cell cholesterol content causes significant changes in Na-Pi cotransport activity and Na-Pi cotransport protein abundance. We also examined the effects of chronic alterations in cell cholesterol content on Na-Pi cotransport activity. We found that when OK cells are grown in the presence of LPDS, there is a decrease in cell cholesterol content (Fig. 7A) that results in a significant increase in Na-Pi cotransport activity (Fig. 7B). However, when OK cells are grown in the presence of LPDS and then treated with LDL, there is an increase in cell cholesterol content (Fig. 7A) that results in a significant decrease in Na-Pi cotransport activity (Fig. 7B). In contrast to the effects of acute alterations in cell cholesterol content, which result only in moderate changes in apical membrane Na-Pi protein abundance in the absence of changes in total cellular Na-Pi protein abundance, chronic alterations in cell cholesterol content result in significant and marked changes in total cellular Na-Pi protein abundance as determined by Western blotting of total OK cell membranes (Figs. 7C and 8) as well as by surface biotinylation that measures apical membrane Na-Pi protein abundance (Fig. 8). The correlation between the NaPi-4 protein abundance in the OK cell apical membrane and Pi transport under chronic cholesterol modulation is summarized in Table 3.# `) F1 H2 t& T1 n8 K& e: x# L
* k, ]" m6 T- V9 g) p
Effect of OK cell cholesterol modulation on cell lipid dynamics. We determined the effects of cholesterol modulation on OK cell lipid dynamics by measuring the GP value of the fluorescent lipid probe laurdan (see MATERIALS AND METHODS and Fig. 9A). Because of fast laurdan photobleaching by one-photon UV excitation, we used two-photon excitation microscopy to obtain laurdan GP images in OK cells. Cholesterol depletion using LPDS resulted in a marked increase in low-GP domains, as seen in cross sections (x-y imaging) as well as in the apical membrane (x-z imaging) of OK cells (Figs. 9B and 10). Spectroscopic measurements using OK cell membranes similarly revealed that cholesterol depletion resulted in a decrease in GP or an increase in membrane lipid fluidity (results not shown). In contrast, cholesterol enrichment achieved by loading the cells grown in LPDS with LDL resulted in a marked increase in domains with high GP values, as seen in cross sections (x-y imaging) as well as in the apical membrane (x-z imaging) of OK cells (Figs. 9B and 10). In agreement, spectroscopic measurements using OK cell membranes showed that cholesterol enrichment resulted in higher GP values, i.e., a decrease in membrane lipid fluidity (results not shown).

OK cell Na-Pi protein is localized in lipid microdomains. In view of marked effects of acute and chronic alterations in cell cholesterol content on Na-Pi cotransport activity and apical membrane Na-Pi protein expression, we next determined whether Na-Pi protein is present in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched membrane microdomains (lipid rafts). Using a biochemical approach, OptiPrep gradient flotation in the absence of detergent, we found that a significant fraction of Na-Pi protein was present in the low-density fractions 1–3 (Fig. 11A), which are highly enriched by cholesterol and sphingomyelin (Fig. 11B). In addition, using fluorescence microscopy in OK cells with EGFP-tagged Na-Pi protein (rat NaPi-2), we found that a significant portion of Na-Pi protein localizes in GM1-enriched membrane microdomains (lipid rafts) (Fig. 12).
% E  ?7 H  _+ C3 z& D
DISCUSSION
' m% N2 ]+ b  P2 {3 R: ?
Previous studies have shown that during adaptation to alterations in dietary Pi intake and the aging process, there is an inverse correlation between apical brush-border membrane cholesterol content and renal proximal tubular Na-Pi cotransport activity (25, 34). Although these studies suggested a potential role for cholesterol in regulating Na-Pi cotransport activity, they did not establish a direct role for cholesterol in regulating Na-Pi cotransport activity. Also, these previous studies did not determine the mechanisms by which cholesterol may modulate Na-Pi cotransport activity.9 _3 ?; S2 q/ m/ r  U/ _% \$ p
5 r9 u' B& H3 L
In agreement with our earlier in vivo findings, the present study confirms that an increase in cell cholesterol content causes a decrease in Na-Pi cotransport activity whereas a decrease in cell cholesterol content causes an increase in Na-Pi cotransport activity (Figs. 2C and 7). The acute effects of alterations in cell cholesterol content on Na-Pi cotransport activity are fully reversible, as demonstrated by the depletion/repletion experiments (Fig. 3). Through transport kinetics measurements (Fig. 5), we determined that the effects of cholesterol on Na-Pi cotransport activity are mediated through alterations in the Vmax of Na-Pi cotransport, independently of changes in the affinity of the Na-Pi cotransporter for Pi or any changes in the diffusion of Pi (Tables 1 and 2). We also show that the effects of cholesterol are quite specific and selective for Na-Pi cotransport activity, as there are no changes in Na-glucose or Na-amino acid cotransport activities (Fig. 4, A and B). However, alterations in cholesterol do modulate Na-sulfate cotransport activity (Fig. 4C), a result that is in agreement with previous observations in Madin-Darby canine kidney cells transfected with the Na-sulfate cotransporter NaSi-1 (23).+ F0 v& n* g# p3 z

One of the new findings of our study is the fact that both acute (30–60 min) and chronic (24 h or longer) alterations in cell cholesterol content, well within the physiological changes that we have previously reported, modulate Na-Pi cotransport activity (Table 3). However, the mechanisms are potentially quite different, as acute alterations in cholesterol cause only moderate changes in apical membrane Na-Pi protein abundance and no changes in total cellular Na-Pi protein abundance (Fig. 6, Table 3). In contrast, chronic changes in cholesterol are associated with marked alterations in apical membrane as well as total cellular Na-Pi protein abundance (Fig. 8, Table 3).

There are several possible mechanisms by which alterations in cell cholesterol content can modulate Na-Pi cotransport activity independently of major changes in apical membrane Na-Pi cotransport protein abundance. One possible mechanism is modulation of Na-Pi cotransport protein activity at the level of the apical membrane. In support of this mechanism, we have previously shown that in isolated renal proximal tubular BBM in vitro enrichment with cholesterol, similar to the levels achieved in the present study, results in a decrease in Na-Pi cotransport activity (24). The in vitro enrichment with cholesterol was accompanied by changes in lipid dynamics that could have an important regulatory effect on the function of the Na-Pi cotransport protein.9 Z7 K4 n) c+ u% B# c6 y
+ G$ n6 q9 \# W' g  P
Cholesterol is known to associate with sphingomyelin and glycosphingolipids to form lipid microdomains or lipid rafts, which are thought to be associated with specific proteins while excluding others. The apical membranes of renal cells have a very high cholesterol and sphingomyelin content, and we have recently provided biophysical evidence for the presence of lipid rafts or lipid microdomains in these membranes (9, 10, 38). In this study, using the z-sectioning imaging of GP laurdan in the apical membranes of OK cells, we also show evidence for the presence of lipid microdomains (markedly differing GP values), and these domains are further modulated by alterations in OK cell membrane cholesterol content (Figs. 9B and 10). Increasing OK cell cholesterol content results in an increased fraction of apical membrane domains with higher GP values, which indicates an increased proportion of liquid-ordered domains that may slow the lateral diffusion and mobility of the Na-Pi cotransport protein within the apical membrane lipid bilayer. In contrast, decreasing OK cell cholesterol content results in an increased fraction of apical membrane domains with lower GP values, which indicates a decreased proportion of liquid-ordered domains that, in turn, may increase the lateral diffusion and mobility of the Na-Pi cotransport protein within the apical membrane lipid bilayer./ v7 W8 l, G0 p5 P7 I
: x2 Y$ `; ^0 U9 O& z8 `
In support of this possibility, many studies have similarly shown that the diffusion of several proteins can be modulated by lipids and/or their partitioning into lipid rafts (11, 32, 33, 43, 47, 51, 54, 57). Several but not all of these studies utilized the fluorescence recovery after photobleaching imaging technique to measure the mobile fraction and the lateral/translational diffusion of the proteins of interest. For example, one study showed that the lateral diffusion of GFP-labeled caveolin is highly restricted. Treatment of the cells with methyl--cyclodextrin to extract cholesterol and cytochalasin D to disrupt the actin cytoskeleton resulted in increased diffusion of the GFP-labeled caveolae (54). Similar results following the disruption of the actin cytoskeleton were obtained with GFP-labeled aquaporin-2 (57). Two studies using high-resolution single-particle tracking showed markedly decreased diffusion of raft-associated proteins compared with nonraft proteins and observation of a marked increase in the diffusion of the raft-associated proteins following lowering of the cell cholesterol content (11, 43).3 d5 M1 S; S+ W! g

Lipid microdomains or lipid rafts may also play a role in the regulation of Na-Pi cotransport activity and Na-Pi protein abundance by modulating the trafficking of the Na-Pi protein. Cholesterol has been shown to play an essential role in endocytosis and intracellular sorting (28, 37, 40, 41). For example, cholesterol has been shown to be necessary for the invagination of clathrin-coated pits, and cholesterol depletion has been shown to inhibit clathrin-dependent endocytosis (44, 52). Recent studies have shown that Na-Pi protein is internalized from the apical membrane in part by a clathrin-dependent mechanism (56). Therefore, cholesterol depletion may cause a decrease in clathrin-mediated endocytosis of Na-Pi, resulting in increased apical membrane abundance of Na-Pi protein and increased Na-Pi cotransport activity. In contrast, cholesterol enrichment may result in increased endocytosis and therefore a decreased apical membrane level of Na-Pi protein. Cholesterol also has been shown to modulate the structure of invaginated caveolae and inhibit caveolae-mediated endocytosis (17, 45, 46). At the present time, however, it is not known whether caveolae mediate Na-Pi protein endocytosis.
$ s5 M. p6 Y3 U1 {) \
Cholesterol and cholesterol-enriched microdomains also modulate intracellular trafficking and apical sorting of molecules (28, 37, 40, 41). In addition, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins that play an important role in the targeting of proteins from the trans-Golgi network to the apical membrane, as well as transcytosis of proteins from the basolateral membrane to the apical membrane, have recently been shown to be associated with lipid rafts or cholesterol-enriched lipid microdomains (8, 21). Therefore, in OK cells cholesterol may also modulate the apical membrane expression of Na-Pi proteins by altering the apical targeting of Na-Pi molecules./ k( D# |9 F, F2 M. Y

Another mechanism by which alterations in cholesterol-enriched lipid microdomains can regulate Na-Pi cotransport activity is through modulating signal transduction. The plasmalemmal caveolae and other lipid microdomains/rafts, which are highly enriched in cholesterol and glycosphingolipids, play an important role in this process (48). Signaling molecules enriched in caveolae include PDGF, EGF, and endothelin receptors, heterotrimeric G proteins, diacylglycerol, ceramide, PKC and IP3 receptors, adenylyl cyclase, nitric oxide, and MAP kinase (48). Interestingly, alterations in the activity of most of these signaling processes are known to regulate renal Na-Pi transport activity (22, 36, 53).. ~9 g2 J+ h$ l% f  h

In summary, we have demonstrated that in OK cells acute and chronic alteration in cholesterol content play a direct and important role in modulating lipid dynamics, lipid microdomains, and Na-Pi transport activity. Our data have established a significant inverse correlation between OK cell cholesterol content and Na-Pi cotransport activity (Fig. 2C). Acute alteration in cholesterol content modulates Na-Pi cotransport activity by modulating the apical membrane expression of Na-Pi protein without altering the total cellular Na-Pi protein content. This suggests posttranslational regulation via acute trafficking of the Na-Pi protein, similar to what we have previously described in acute regulation of Na-Pi protein and transport activity with parathyroid hormone and Pi adaptation (26, 29, 30). In contrast, chronic alteration in cholesterol content modulates Na-Pi cotransport activity by modulating both apical membrane and total cellular Na-Pi protein abundance, which indicate translational regulation of the Na-Pi cotransport protein. These studies are in agreement with our previous in vivo observations in aging rats and in rats adapted to dietary Pi where there is an inverse relationship between BBM cholesterol content and BBM Na-Pi cotransport activity (24, 25) and indicate that alterations in cholesterol per se is an important modulator of Na-Pi cotransport activity.
7 E4 E) p4 B' I6 D$ b: B! ^
GRANTS- {% y$ `% H/ l! a" Q% B
( W$ E+ ?1 W+ d4 }! X: l5 ]( f6 z0 ?
These studies were supported by grants to M. Levi [VA Medical Research Office Merit Review; National Institutes of Health (NIH) Grants 7RO3-AG-20361–2, 5RO1-DK-062209–02, RO1-DK-066029 and JDRF-1–2003-108]; E. Lederer (VA Medical Research Office Merit Review); H. K. Zajicek (NIH Grants NRSA 1 F32 DK-09689–01 and K08 DK-62220–01); V. Sorribas (MEC PR2003–0392); and S. Breusegem (American Heart Association, Pacific Mountain Affiliate Postdoctoral Fellowship 0520054Z). The laurdan GP imaging experiments reported in this study were performed at the Laboratory for Fluorescence Dynamics (LFD) in the Department of Physics, University of Illinois at Urbana-Champaign (UIUC). The LFD is funded by the NIH (RR-03155) and by UIUC.
$ q4 f: n# _5 X& O" A7 L. [
FOOTNOTES
) B+ S- m6 U/ i/ }3 j/ x" S3 I* e
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.
4 f) I0 n4 Z% Q2 {5 j$ B
REFERENCES

Alcalde AI, Sarasa M, Raldua D, Aramayona J, Morales R, Biber J, Murer H, Levi M, and Sorribas V. Role of thyroid hormone in regulation of renal phosphate transport in young and aged rats. Endocrinology 140: 1544–1551, 1999.( H- m9 n) V5 A  X
! v. N  ]7 D* P5 @$ Q
Ambuhl PM, Zajicek HK, Wang H, Puttaparthi K, and Levi M. Regulation of renal phosphate transport by acute and chronic metabolic acidosis in the rat. Kidney Int 53: 1288–1298, 1998.4 T; c+ {. l9 T" x# x2 m: ]
. l9 U; S( L+ |( Y8 `) d" Q! F4 b# q
Arar M, Baum M, Biber J, Murer H, and Levi M. Epidermal growth factor inhibits Na-Pi cotransport and mRNA in OK cells. Am J Physiol Renal Fluid Electrolyte Physiol 268: F309–F314, 1995.7 b1 M" c, T( t2 v6 k$ h8 \( P( ~  ~

Arar M, Zajicek HK, Elshihabi I, and Levi M. Epidermal growth factor inhibits Na-Pi cotransport in weaned and suckling rats. Am J Physiol Renal Physiol 276: F72–F78, 1999.5 G9 z* h' [5 p8 ?) s2 S

Bacia K, Schuette C, Kahya N, Jahn R, and Schwille P. SNAREs prefer liquid-disordered over "raft" (liquid-ordered) domains when reconstituted into giant unilamellar vesicles. J Biol Chem. In press.9 k4 i4 d5 l- r; B( w7 q
9 c; U( O2 z0 M1 M
Beck L, Karaplis AC, Amizuka N, Hewson AS, Ozawa H, and Tenenhouse HS. Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities. Proc Natl Acad Sci USA 95: 5372–5377, 1998.

Bligh EG and Dyer WJ. A rapid method of total lipid extraction and purification. Can J Med Sci 37: 911–917, 1959.
; O7 _. F/ i. M5 g2 I  s4 z
Chamberlain LH, Burgoyne RD, and Gould GW. SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis. Proc Natl Acad Sci USA 98: 5619–5624, 2001.: i# F. w3 h7 L3 F

Dietrich C, Bagatolli LA, Volovyk ZN, Thompson NL, Levi M, Jacobson K, and Gratton E. Lipid rafts reconstituted in model membranes. Biophys J 80: 1417–1428, 2001.0 d8 e$ T+ F9 ?$ Q, m" L4 e" J
' l1 v9 P2 V2 b2 N+ m
Dietrich C, Volovyk ZN, Levi M, Thompson NL, and Jacobson K. Partitioning of Thy-1, GM1, and cross-linked phospholipid analogs into lipid rafts reconstituted in supported model membrane monolayers. Proc Natl Acad Sci USA 98: 10642–10647, 2001.

Dietrich C, Yang B, Fujiwara T, Kusumi A, and Jacobson K. Relationship of lipid rafts to transient confinement zones detected by single particle tracking. Biophys J 82: 274–284, 2002.
3 E: ^/ e8 A* v9 I: ]
Duffield A, Kamsteeg EJ, Brown AN, Pagel P, and Caplan MJ. The tetraspanin CD63 enhances the internalization of the H,K-ATPase -subunit. Proc Natl Acad Sci USA 100: 15560–15565, 2003.
+ _2 _3 F# V+ Z2 \# |, A0 W
Edidin M. The state of lipid rafts: from model membranes to cells. Annu Rev Biophys Biomol Struct 32: 257–283, 2003.
6 T: @  ?/ b# m  T7 r
Gimpl G, Klein U, Reilander H, and Fahrenholz F. Expression of the human oxytocin receptor in baculovirus-infected insect cells: high-affinity binding is induced by a cholesterol-cyclodextrin complex. Biochemistry 34: 13794–13801, 1995.( S: ?. y& t; ?: V2 t0 j! p
" V5 v) z( K  Y4 d
Gonin S, Deschenes G, Roger F, Bens M, Martin PY, Carpentier JL, Vandewalle A, Doucet A, and Feraille E. Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells. Mol Biol Cell 12: 255–264, 2001.8 [+ U2 ]$ T" f

Gottardi CJ, Dunbar LA, and Caplan MJ. Biotinylation and assessment of membrane polarity: caveats and methodological concerns. Am J Physiol Renal Fluid Electrolyte Physiol 268: F285–F295, 1995.

Hailstones D, Sleer LS, Parton RG, and Stanley KK. Regulation of caveolin and caveolae by cholesterol in MDCK cells. J Lipid Res 39: 369–379, 1998.+ [; }7 G+ n4 M- ?

Inoue M, Digman MA, Cheng M, Breusegem SY, Halaihel N, Sorribas V, Mantulin WW, Gratton E, Barry NP, and Levi M. Partitioning of NaPi cotransporter in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched membrane domains modulates NaPi protein diffusion, clustering and activity. J Biol Chem 279: 49160–49171, 2004./ v' p/ B* l* g( o0 d/ Q) Q5 P

Kempson SA, Lotscher M, Kaissling B, Biber J, Murer H, and Levi M. Parathyroid hormone action on phosphate transporter mRNA and protein in rat renal proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 268: F784–F791, 1995.( m( A8 m3 r: V) R! S, [

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.$ W  K  ?! W. X5 {" s$ Z

Lang T, Bruns D, Wenzel D, Riedel D, Holroyd P, Thiele C, and Jahn R. SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis. EMBO J 20: 2202–2213, 2001.2 k% Y2 C3 }% t/ S) _! B; U

Lederer ED, Sohi SS, Mathiesen JM, and Klein JB. Regulation of expression of type II sodium-phosphate cotransporters by protein kinases A and C. Am J Physiol Renal Physiol 275: F270–F277, 1998.  X2 D3 p. y$ c5 {
* M; f" s0 g' I7 ]4 W$ F
Lee HJ, Balasubramanian SV, Murer H, Biber J, and Morris ME. Modulation of sulfate renal transport by alterations in cell membrane fluidity. J Pharm Sci 88: 976–980, 1999." l; n4 j  O3 x1 Y) o
% ^# C: P# y4 I9 l4 p# l; y' }
Levi M, Baird BM, and Wilson PV. Cholesterol modulates rat renal brush border membrane phosphate transport. J Clin Invest 85: 231–237, 1990.6 Y8 i$ e; v3 k; t2 M( {, d8 d
& ?) b8 N3 v0 z
Levi M, Jameson DM, and van der Meer BW. Role of BBM lipid composition and fluidity in impaired renal Pi transport in aged rat. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85–F94, 1989." Y: w2 l( p; N

Levi M, Lotscher M, Sorribas V, Custer M, Arar M, Kaissling B, Murer H, and Biber J. Cellular mechanisms of acute and chronic adaptation of rat renal Pi transporter to alterations in dietary Pi. Am J Physiol Renal Fluid Electrolyte Physiol 267: F900–F908, 1994.- v( e, C. m1 C2 h" X6 p' ~
" B7 x- G6 ?; L4 m' r3 O* @0 t
Levi M, Shayman JA, Abe A, Gross SK, McCluer RH, Biber J, Murer H, Lotscher M, and Cronin RE. Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycosphingolipid composition. J Clin Invest 96: 207–216, 1995.

Li X, Galli T, Leu S, Wade JB, Weinman EJ, Leung G, Cheong A, Louvard D, and Donowitz M. Na -H  exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3. J Physiol 537: 537–552, 2001.
7 T! \- B7 Z. ~( m" I- ^# h0 G( P
Lotscher M, Kaissling B, Biber J, Murer H, and Levi M. Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content. J Clin Invest 99: 1302–1312, 1997.

Lotscher M, Scarpetta Y, Levi M, Halaihel N, Wang H, Zajicek HK, Biber J, Murer H, and Kaissling B. Rapid downregulation of rat renal Na/Pi cotransporter in response to parathyroid hormone involves microtubule rearrangement. J Clin Invest 104: 483–494, 1999.- \5 g/ V1 g7 k; `' M/ i% \  l. Y
7 [9 O" {( F0 c, m: O( {- M2 ~2 v
Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951., b6 ^( p" `! q7 p, I$ i( m$ b

Magnani F, Tate C, Wynne S, Williams C, and Haase J. Partitioning of the serotonin transporter into lipid microdomains modulates transport of serotonin. J Biol Chem 279: 38770–38778, 2004.( Y, U/ X! n5 Y) V! U) p

Milhiet PE, Giocondi MC, Baghdadi O, Ronzon F, Roux B, and Le Grimellec C. Spontaneous insertion and partitioning of alkaline phosphatase into model lipid rafts. EMBO Rep 3: 485–490, 2002.( K0 a0 p& c" {, e) k$ B: t
9 F2 [. v. E, h9 V+ e' o
Molitoris BA, Alfrey AC, Harris RA, and Simon FR. Renal apical membrane cholesterol and fluidity in regulation of phosphate transport. Am J Physiol Renal Fluid Electrolyte Physiol 249: F12–F19, 1985.9 C, c6 d  {7 X. D: @( U

Mulroney SE, Woda CB, Halaihel N, Louie B, McDonnell K, Schulkin J, Haramati A, and Levi M. Central control of renal sodium-phosphate (NaPi-2) transporters. Am J Physiol Renal Physiol 286: F647–F652, 2004.

Murer H, Hernando N, Forster I, and Biber J. Regulation of Na/Pi transporter in the proximal tubule. Annu Rev Physiol 65: 531–542, 2003.
7 S3 \& w7 f' Q; I
Nichols BJ, Kenworthy AK, Polishchuk RS, Lodge R, Roberts TH, Hirschberg K, Phair RD, and Lippincott-Schwartz J. Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J Cell Biol 153: 529–541, 2001.

Parasassi T, Gratton E, Yu WM, Wilson P, and Levi M. Two-photon fluorescence microscopy of Laurdan generalized polarization domains in model and natural membranes. Biophys J 72: 2413–2429, 1997.
7 d/ X: z3 b& W+ L% n7 H
Peng Y, Amemiya M, Yang X, Fan L, Moe OW, Yin H, Preisig PA, Yanagisawa M, and Alpern RJ. ETB receptor activation causes exocytic insertion of NHE3 in OKP cells. Am J Physiol Renal Physiol 280: F34–F42, 2001.
( R2 {4 N/ Y0 R3 b+ k% m
Pike LJ. Lipid rafts: heterogeneity on the high seas. Biochem J 378: 281–292, 2004.
8 l  x6 q# ^8 f  `
Pike LJ and Casey L. Cholesterol levels modulate EGF receptor-mediated signaling by altering receptor function and trafficking. Biochemistry 41: 10315–10322, 2002.
# L: \! @9 N% Q0 k& R: M; y
Porn MI and Slotte JP. Localization of cholesterol in sphingomyelinase-treated fibroblasts. Biochem J 308: 269–274, 1995.1 @& l4 q1 I, ^* V' |) _
9 L1 ^/ i7 P0 Y
Pralle A, Keller P, Florin EL, Simons K, and Horber JK. Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J Cell Biol 148: 997–1008, 2000.

Rodal SK, Skretting G, Garred O, Vilhardt F, van Deurs B, and Sandvig K. Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell 10: 961–974, 1999.

Rothberg KG, Ying YS, Kamen BA, and Anderson RG. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J Cell Biol 111: 2931–2938, 1990.

Schnitzer JE, Oh P, Pinney E, and Allard J. Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J Cell Biol 127: 1217–1232, 1994.
, V, t/ s5 H. r# k. @. W2 v3 n
Sharma P, Varma R, Sarasij RC, Ira Gousset K, Krishnamoorthy G, Rao M, and Mayor S. Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell 116: 577–589, 2004.! E/ z# D! O9 T9 I& j( l% f

Simons K and Toomre D. Lipid rafts and signal transduction. Nature Rev Mol Cell Biol 1: 31–40, 2000.' R( t0 K7 w: t% M& L# A* _8 |

Sorribas V, Lotscher M, Loffing J, Biber J, Kaissling B, Murer H, and Levi M. Cellular mechanisms of the age-related decrease in renal phosphate reabsorption. Kidney Int 50: 855–863, 1996.

Sorribas V, Markovich D, Hayes G, Stange G, Forgo J, Biber J, and Murer H. Cloning of a Na/Pi cotransporter from opossum kidney cells. J Biol Chem 269: 6615–6621, 1994.% e% S" @8 P, Y) B
2 W$ V' d8 i& n& R. ?& E; j* C
Sprong H, van der Sluijs P, and van Meer G. How proteins move lipids and lipids move proteins. Nat Rev Mol Cell Biol 2: 504–513, 2001., w, {' a# X% ^1 H$ C& G
) \6 Q2 q8 R6 Z  j9 j' k6 e
Subtil A, Gaidarov I, Kobylarz K, Lampson MA, Keen JH, and McGraw TE. Acute cholesterol depletion inhibits clathrin-coated pit budding. Proc Natl Acad Sci USA 96: 6775–6780, 1999.
9 W; u4 K$ [* `$ [4 i6 c% Q& `
Tenenhouse HS and Murer H. Disorders of renal tubular phosphate transport. J Am Soc Nephrol 14: 240–248, 2003./ `# s6 L/ n' C  s. P/ D/ ]

Thomsen P, Roepstorff K, Stahlhut M, and van Deurs B. Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol Biol Cell 13: 238–250, 2002.

Towbin H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979.
( p4 i! w" w  V) V
Traebert M, Roth J, Biber J, Murer H, and Kaissling B. Internalization of proximal tubular type II Na-Pi cotransporter by PTH: immunogold electron microscopy. Am J Physiol Renal Physiol 278: F148–F154, 2000.! L9 u& ]9 d1 W" e

Umenishi F, Verbavatz JM, and Verkman AS. cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera. Biophys J 78: 1024–1035, 2000.% n* e% f% I# C) p
2 o7 ~* B0 z$ b" I  b5 e& n
Westover EJ, Covey DF, Brockman HL, Brown RE, and Pike LJ. Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. Studies with cholesterol enantiomers. J Biol Chem 278: 51125–51133, 2003.

Woda CB, Halaihel N, Wilson PV, Haramati A, Levi M, and Mulroney SE. Regulation of renal NaPi-2 expression and tubular phosphate reabsorption by growth hormone in the juvenile rat. Am J Physiol Renal Physiol 287: F117–F123, 2004.6 T8 d: x' O7 H- d, ~: F% X
; a7 Q7 d( V8 ^3 N6 V/ t3 @: ]( \
Yu W, So PT, French T, and Gratton E. Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach. Biophys J 70: 626–636, 1996.

Zajicek HK, Wang H, Puttaparthi K, Halaihel N, Markovich D, Shayman J, Beliveau R, Wilson P, Rogers T, and Levi M. Glycosphingolipids modulate renal phosphate transport in potassium deficiency. Kidney Int 60: 694–704, 2001.(Sophia Y. Breusegem, Nabi)

收藏0 分享0 顶0 踩0