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Vasa recta voltage-gated Na channel Na v 1.3 is regulated by calmodulin

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发表于 2009-4-22 09:47 |显示全部帖子
作者:Whaseon Lee-Kwon, Jae Hwan Goo, Zhong Zhang, Erik P. Silldorff, and Thomas L. Pallone,作者单位:1 Division of Nephrology, Department of Medicine, and 2 Department of Physiology, University of Maryland School of Medicine, Baltimore; and 3 Department of Biology, Towson University, Towson, Maryland
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$ |% e$ f" ?: v" V          【摘要】
, u- ]5 g7 ]9 F& Q( w3 n) M$ _      Rat descending vasa recta (DVR) express a tetrodotoxin (TTX)-sensitive voltage-operated Na   (Na V ) conductance. We examined expression of Na V isoforms in DVR and tested for regulation of Na V currents by calmodulin (CaM). RT-PCR in isolated permeabilized DVR using degenerate primers targeted to TTX-sensitive isoforms amplified a product whose sequence identified only Na V 1.3. Immunoblot of outer medullary homogenate verified Na V 1.3 expression, and fluorescent immunochemistry showed Na V 1.3 expression in isolated vessels. Immunochemistry in outer medullary serial sections confirmed that Na V 1.3 is confined to -smooth muscle actin-positive vascular bundles. Na V 1.3 possesses a COOH-terminal CaM binding motifs. Using pull-down assays and immunoprecipitation experiments, we verified that CaM binds to either full-length Na V 1.3 or a GST-Na V 1.3 COOH-terminal fusion protein. In patch-clamp experiments, Na V currents were suppressed by calmodulin inhibitory peptide (CIP; 100 nM) or the CaM inhibitor N -(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide hydrochloride (W7). Neither CIP nor W7 altered the voltage dependence of pericyte Na V currents; however, raising electrode free Ca 2  from 20 to 2,000 nM produced a depolarizing shift of activation. In vitro binding of CaM to GST-Na V 1.3C was not affected by Ca 2  concentration. We conclude that Na V 1.3 is expressed by DVR, binds to CaM, and is regulated by CaM and Ca 2 . Inhibition of CaM binding suppresses pericyte Na V currents.
: H4 R; Z% A, a6 n5 k2 Q          【关键词】 kidney medulla microcirculation patch clamp channel6 O0 s2 X( e* z' O( {$ h$ s
                  VOLTAGE - OPERATED Na   channels (Na V ) play a dominant role in the initiation and propagation of action potentials in neural tissue, cardiac myocytes, and skeletal muscle. Large pore-forming -subunits are encoded by 10 mammalian genes, Na V 1.1-Na V 1.9 and Na X. The majority of Na V subtypes are expressed in neurons, but Na V 1.4 and Na V 1.5 predominate in skeletal muscle and cardiac muscle, respectively ( 7, 30 ). In addition to those well-known distributions, Na V have also been identified in vascular, gastrointestinal, and uterine smooth muscle cells where they have been hypothesized to augment membrane depolarization, gate voltage-dependent Ca 2  channels, and/or provide a surrogate Ca 2  entry pathway via Na   /Ca 2  exchange ( 2, 4, 9, 20, 35 ). A calmodulin (CaM) IQ binding motif is present on the COOH termini of Na V isoforms implying probable association of CaM with the channels to modulate activity and/or voltage dependence of gating properties ( 17, 23, 31 ).
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7 H- }4 W; D3 T# ?: z! FDescending vasa recta (DVR) are 12- to 15-µm contractile microvessels that perfuse the renal medulla. They are surrounded by smooth muscle-like cells called pericytes that impart contractility ( 28, 29 ). Using whole cell patch-clamp methods, we previously identified a tetrodotoxin (TTX)-sensitive voltage-operated Na   conductance in DVR pericytes ( 46 ). In the current study, we investigated the isoform(s) of TTX-sensitive Na V expressed in DVR and determined whether CaM binds to and modulates their function. With the use of RT-PCR with degenerate primers for TTX-sensitive Na V channels and immunostaining with isoform-specific antibody, expression of Na V 1.3 was confirmed. We observed that CaM binds to glutathione- S -transferase (GST) COOH-terminal Na V 1.3 fusion constructs and that these two proteins coprecipitate from renal medullary homogenates. Finally, whole cell patch-clamp studies showed that DVR pericyte Na V inward currents are reduced by either a CaM-inhibitory peptide or the calmodulin inhibitor N -(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide hydrochloride (W7), while modulation of electrode free Ca 2  concentration by BAPTA chelation shifted the voltage dependence of activation. We conclude that DVR pericytes express Na V 1.3 and that pericyte Na V currents are regulated by CaM and Ca 2 .2 Z% r8 X) [: X. M) E5 P* ^

  n8 z* b0 N) q+ P/ v6 z5 sMETHODS
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Isolation of DVR. Investigations involving animal use were performed according to protocols approved by the Institutional Animal Use and Care Committee of the University of Maryland. Sprague-Dawley rats (120-200 g) were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Under deep anesthesia, the abdomen was opened and the kidneys were excised. Euthanasia was induced by exsanguination under anesthesia, without the rats regaining conciousness. For patch-clamp studies, tissue slices were stored at 4°C in a physiological saline solution (PSS; in mmol/l: 155 NaCl, 5 KCl, 1 MgCl 2, 1 CaCl 2, 10 HEPES, and 10 glucose, pH 7.4). Small wedges of renal medulla were dissected and transferred to Blendzyme 1 (Roche) at 0.27 mg/ml in high-glucose DMEM media (Invitrogen), incubated at 37°C for 30 min, transferred to PSS, and stored at 4°C. At intervals, DVR were isolated from the enzyme-digested renal tissue by hand dissection and transferred to a perfusion chamber for patch-clamp recording. Gigaseals were directly formed on abluminal pericytes of intact vessels as previously illustrated ( 26 ).0 x5 ]) \' R) F* W8 ~+ S( R5 E
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RT-PCR. As previously described ( 21 ), kidneys were perfused with solution A containing (in mmol/l) 135 NaCl, 1 KCl, 0.1 Na 2 HPO 4, 0.12 Na 2 SO 4, 1.2 MgSO 4, 0.3 NaOAc, 5 HEPES, 2.5 CaCl 2, 1 DTT, and 5.5 glucose, pH adjusted to 7.4 with NaOH. Solutions for reverse transcriptase (RT)-PCR were prepared by adding either vanadyl ribonucleoside complex (VRC; 10 mM, solution B ) or RNase inhibitor (Clontech, 1 U/µl, solution C ) to solution A. Kidney slices were digested with collagenase type 1 (1 mg/ml), transferred to a petri dish, and maintained at 4°C. DVR and nephron segments were isolated by microdissection in solution B. Those structures were cleared of debris by transfer to a separate petri dish that contained solution C as a wash buffer. The microdissected structures were subsequently transferred from the wash buffer to an RT-PCR reaction tube. Each sample was centrifuged at 10,000 g for 10 min and then rinsed three times with 100 µl of ice-cold solution C. Samples of the wash buffer were also analyzed by RT-PCR to rule out contamination. Those controls were uniformly negative.
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Microdissected DVR segments were permeabilized in solution containing 2% Triton X-100, 5 mM DTT, and 1 U/µl RNase inhibitor. The mRNA from permeablized DVR was subjected to reverse transcription (RT) using Superscript III (RT-PCR kit, Invitrogen) in a 20-µl volume according to the manufacturer's instructions. DVR were screened for expression of TTX-sensitive Na V channel isoforms using degenerative forward (5'-GTCATGATCCTCACC/TGTG-3') and reverse (5'-GCCCAGCTGAAG/TGTGTCAAA-3') primers. Those primers were designed to yield a 367-bp product from TTX-sensitive Na V isoforms. An additional amplification step was performed using nested forward (5'-GTGTTCTGTCTCAGTGTC/GTTTGC-3') and reverse (5'-CCAGCC/TTTC/TACACAG/CATGTAT/ACC-3') primers to generate a 304-bp product. Both sets of primers were based on the published sequence of TTX-sensitive Na V isoforms corresponding to Genebank accession numbers: NM/200875 (Na V 1.1), NM/202647 (Na V 1.2), NM/203119 (Na V 1.3), and NM133289 (Na V 1.7). To avoid genomic DNA contamination, all primer combinations span introns. TTX-sensitive Na V isoform expression by the above steps was analyzed from multiple microdissected, permeabilized DVR derived from at least two rats. The PCR products were ethanol precipitated, separated by electrophoresis on 2% (wt/vol) agarose gels, stained with ethidium bromide, and photographed. The nested RT-PCR products were subcloned into pCRII-TOPO vector (Invitrogen) and sequenced to determine specific identity of Na V isoform(s).
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Plasmid constructs. COOH-terminal Na V 1.3 (Na V 1.3C, 250 amino acid) PCR products were generated from outer medullary total RNA by RT-PCR using the forward and reverse primers: 5'-TTTGTCAGCTACATCATCATATC-3' and 5'-TTTGTCAGCTACATCATCATATC-3', respectively. The RT-PCR products were ligated in frame into Bgl II and Xho I sites of pGEX-6P1 vector to produce the recombinant GST-Na V 1.3C construct. The open reading frame of rat CaM was generated as an RT-PCR product from outer medullary RNA and subcloned in frame into Bam HI and Xho I site of pGEX-6P1 or pET30 to produce the full-length recombinant GST-CaM or His-CaM constructs, respectively. The integrity of all subcloned RT-PCR products was confirmed by sequencing.. P, C$ {0 v2 a* T7 b

2 K% ~5 p) _4 K! f2 ~; `3 ]Expression and purification of GST and His-tagged fusion proteins. Na V 1.3C, in pGEX (pGEX-NaV1.3C), and CaM, in pGEX or pET30 (pGEX-CaM, pET30-CaM), were transformed into Escherichia coli BL21 and grown in culture. GST-tagged or His-tagged fusion proteins were induced by the addition of 1 mM isopropyl- D -thiogalactopyranoside for 4 h. Fusion proteins were purified using glutathione-sepharose 4B beads (Amersham) or nickel column (Qiagen), according to the manufacturer's protocol. To verify recombinant protein production, 5 µl of the eluted proteins were separated by SDS-PAGE, and the gel was stained with Coomassie blue.
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; y2 {  a6 X7 c5 |6 k6 dRenal tissue preparation and immunoblotting. The kidneys from Sprague-Dawley rats were divided into cortex, outer medulla, and inner medulla. The tissues were homogenized (Polytron) in chilled, buffered isolation solution containing 250 mmol/l sucrose, 10 mmol/l triethanolamine (Calbiochem), and protease inhibitor cocktail (Sigma), adjusted to pH 7.4. Protein concentrations of the homogenates were measured using a BCA Protein Assay Reagent kit (Bio-Rad). All samples were then diluted with isolation solution to a protein concentration of 1-3 mg/ml and solubilized at 80°C for 20 min in Laemmli sample buffer. Twenty micrograms of protein per sample were loaded onto individual lanes, separated by electrophoresis on 7-15% polyacrylamide gels, and electrophoretically transferred to nitrocellulose membranes. After being blocked with 5% milk for 30 min, membranes were probed overnight at 4°C with the appropriate primary antibody. To probe the blots, antibodies were diluted into a solution of 150 mmol/l NaCl, 50 mmol/l sodium phosphate, 0.03% sodium azide, 0.1% Tween 20, and 5% BSA (pH 7.5). The secondary antibody was conjugated to horseradish peroxidase (Jackson Laboratories, 0.1 mg/ml). Sites of antibody-antigen reaction were visualized by using luminol-based chemiluminescence (Amersham) and exposure to X-ray film.' X% f# A; i) [% a

+ P3 l7 F  l6 u3 T3 i, A: xPull-down assay. To determine binding of GST-Na V 1.3C to CaM or GST-CaM to Na V 1.3, the tissue lysates from OM and IM (2 mg) were incubated with either GST-Na V 1.3C or GST-CaM (3 µg) bead-bound fusion proteins. Bead-bound GST served as negative control. After being washed four times with binding buffer (in mmol/l: 20 Tris·HCl, 120 NaCl, 1 EDTA, protease inhibitor cocktail, pH 7.4) containing 0.1% Triton X-100, the bound proteins were resolved by SDS-PAGE. Na V 1.3 and CaM proteins were detected by immunoblotting with anti-CaM (Zymogen) or anti-Na V 1.3 antibodies (Alomone). To verify the specificity of the Na V 1.3 antibody, immunoblotting was also performed after preabsorption with antigenic peptide.( i; i" M1 C: e9 r" g* M7 S- o
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Immunoprecipitation. Immunoprecipitation was performed with antibody directed against CaM. Tissue samples were homogenized in lysis buffer (10 mmol/l triethanolamine, 250 mmol/l sucrose, protease inhibitor cocktail, pH 7.4) using a Polytron tissue grinder following which the tissue lysates were incubated on ice for 30 min after adding 1% Triton X-100. Cellular debris was removed by centrifugation (20 min, 1,000 g ). Lysates were mixed with anti-CaM antibody and rotated at 4°C for 2 h. Immune complexes were precipitated by adding 50 µl of protein A/G agarose beads (Roche Diagnostics) preequilibrated in the lysis buffer. After rocking overnight at 4°C, the beads were recovered and washed four times with lysis buffer containing 0.1% Triton X-100. The immune complexes were eluted with 50 µl of Laemmli buffer and fractionated by SDS-PAGE.! u9 u) ?) b8 l$ x

7 m, D. `9 r7 t/ r- \6 m0 X# HImmunofluorescent labeling of isolated DVR. With the use of methods previously described in detail ( 6, 21 ), immunofluorescent labeling was performed to verify expression of Na V 1.3. Labeling of -smooth muscle actin (SMA) was performed to identify pericytes. Microdissected DVR were transferred onto slides and fixed with 2% paraformaldehyde in 100 mmol/l cacodylate buffer, pH 7.4. The fixed vessels were incubated overnight at 4°C in PBS containing 5% BSA, 0.1% Triton X-100 (PBS/Triton solution), polyclonal rabbit anti-Na V 1.3 channel antibody (Alomone Labs, 1:50), and monoclonal mouse anti-SMA (Sigma, 1:500). After four washes with 1 x PBS containing 0.1% Triton X-100, the vessels were incubated with Alexa Flour 488 goat anti-rabbit IgG (1:200) and Alexa Flour 568 goat anti-mouse IgG (1:200, Molecular Probes, Eugene, OR) for 1 h at room temperature. After several washes with PBS/Triton, coverslips were mounted with Vectorshield (Vector Laboratories, Burlingame, CA). To verify the specificity of Na V 1.3 and SMA antibodies, negative controls were performed in which those primary antibodies were omitted. Fluorescent images were obtained using a Zeiss LSM410 confocal microscope.
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* y7 u& W/ R7 UImmunostaining of kidney sections. Rat kidneys were fixed by perfusing them for 2 min in PBS, 5 min in 2% paraformaldehyde, and 2 min in cryoprotectant (10% EDTA, 0.1 mol/l Tris). After the perfusion fixation, kidneys were removed, postfixed overnight in 2% paraformaldehyde in PBS, and embedded in paraffin. Labeling of 50-µm-thick sections was carried out by the indirect immunoperoxidase method (DAKO Cytomation, Carpinteria, CA). The sections were dewaxed and rehydrated. Endogenous peroxidase was blocked by 3% H 2 O 2 for 30 min at room temperature. Nonspecific binding of IgG was prevented by incubating in 50 mmol/l NH 4 Cl for 30 min, followed by blocking with 5% BSA, 0.05% saponin, and 0.2% gelatin in PBS. Sections were subsequently incubated overnight at 4°C with primary antibody diluted in PBS supplemented with 5% BSA, 0.1% Triton X-100. After being rinsed three times (PBS, 5% BSA, 0.1% Triton X-100, 10 min), the sections were incubated in horseradish peroxidase-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse IgG (DAKO K609). To detect horseradish peroxidase, sections were incubated in either 0.1% 3,3-diaminobenzidine (DAB, brown color) or Vector SG (blue color, Vector Laboratories). After being washed, the sections were dehydrated in a graded series of ethanol solutions and embedded in permount (Sigma).
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+ y. S6 u. J# k& r( h- \6 `/ aWhole cell patch-clamp recording. Patch pipettes were made from borosilicate glass capillaries (PG52151-4, external diameter 1.5 mm, internal diameter 1.0 mm; World Precision Instruments, Sarasota, FL), using a two-stage vertical pipette puller (Narshige PP-830) and heat polished to a final resistance of 4-8 M. Whole cell-ruptured patch recording was performed with a CV201AU headstage and Axopatch 200 amplifier (Axon Instruments, Foster City, CA) at room temperature as previously described ( 26, 46 ). The following solution designed to achieve intracellular free Ca 2  concentration of 50 nM was used in the electrode (in mmol/l): 115 Csmethanesulfonate, 18 CsCl, 10 NaCl, 2.35 MgATP, 1 CaCl 2, 5 EGTA, 10 HEPES, pH 7.2 adjusted with CsOH. In experiments designed to evaluate the effect of electrode free Ca 2  on Na V current, BAPTA [1,2-bis(o-aminophenoxy)ethane- N, N, N ', N '-tetraacetic acid] was used as a chelator in place of EGTA. BAPTA concentration was 5 mmol/l, and total CaCl 2 concentration was either 0.63 mmol/l ( 20 nmol/l free Ca 2  ) or 4.7 mmol/l ( 2,000 nmol/l free Ca 2  ), respectively. In some experiments, calmodulin inhibitory peptide (CIP; 100 nmol/l, Ac-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu-NH2, Calbiochem) was included in the electrode. The extracellular buffer was PSS. To inhibit K   channels and Cl - channels, respectively, all measurements were performed in PSS containing ANG II (10 nmol/l) and niflumic acid (100 µmol/l). ANG II suppresses K   currents but does not augment Na V currents in DVR pericytes ( 46 ). Results have been corrected for junction potentials ( 26 ).
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) g3 H. O% b5 J/ B% D) CStatistics. Curve fits were performed with Clampfit 9.2 (Axon Instruments) using Levenberg-Marquardt algorithms. Data are reported as means ± SE. Significance of effects was evaluated with SigmaStat 3.11 (Systat Software) using parametric or nonparametric tests as appropriate. Group comparisons were performed with Student's t -test (paired or unpaired, as appropriate) or the rank sum test (nonparametric). Comparisons between multiple groups were with one-way ANOVA, repeated-measures ANOVA, or repeated-measures ANOVA on ranks (nonparametric). Post hoc comparisons were performed using Tukey's or Holm-Sidak tests. P
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RESULTS
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1 i1 ?3 E* a8 ?& k  g3 gIdentification of Na V 1.3 in DVR. We previously showed that the voltage-gated Na   current in DVR pericytes is exquisitely sensitive to TTX ( K d = 2.2 nmol/l) ( 46 ). To determine which Na V isoform(s) are expressed in rat DVR, we designed degenerate, nested primers that yield an expected 304-bp product from the cDNA that codes for the four known TTX-sensitive Na V isoforms (Na V 1.1, 1.2, 1.3, and 1.7). Based on sequence alignment and comparison of the -subunits, we selected conserved regions that provide appropriate targets for amplification while spanning introns to avoid amplification of genomic DNA. Hand-dissected DVR were permeabilized and subjected to RT-PCR. As shown in Fig. 1 A, lanes 5 and 6, the nested, degenerate primers yielded the anticipated 304-bp DNA fragment(s). PCR products from four rats (3-4 vessels each) were pooled and subcloned. Seven of the subclones were selected for sequencing, all of which identified expression of Na V 1.3. Assuming similar efficiency of amplification for the TTX-sensitive Na V isoforms, these findings imply predominance of Na V 1.3 message in DVR.
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Fig. 1. Expression of voltage-gated sodium channel 1.3 (Na V 1.3). A : nested primers for tetrodotoxin (TTX)-sensitive NaV channels amplified a product at the expected size (304 bp) from microdissected permeabilized descending vasa recta (DVR; lanes 5 and 6 ) but not from thin descending limb ( lane 4 ). No bands were detected in dissection buffer ( lane 1 ) or RT negative control ( lane 2 ). Sequence analysis of cDNA fragments from RT-PCR products identified only the Na V 1.3 isoform. Results are similar to 3 independent experiments. B : to verify Na V 1.3 protein expression in renal outer and inner medulla, lysates prepared from rat outer and inner medulla were subjected to immunoblot analysis. Anti-Na V 1.3 antibody recognized 3 bands near the expected size ( 250 kDa). The results are representative of 4 experiments.
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To confirm Na V 1.3 isoform expression in the kidney, tissue lysates from rat outer and inner medulla were subjected to immunoblot analysis. An affinity-purified rabbit polyclonal antibody directed against Na V 1.3 recognized three distinct bands near 250 kDa ( Fig. 1 B ). To confirm expression of Na V 1.3 in isolated DVR and determine its distribution to pericytes and endothelium, hand-dissected vessels were immunostained for Na V 1.3 and the pericyte marker -SMA. Figure 2 provides an example of individual and merged confocal fluorescent images. Figure 3 shows additional examples of SMA and Na V 1.3 staining along with corresponding white-light differential interference contrast (DIC) photomicrographs. In both panels, the arrowheads point to pericytes and asterisks label endothelial cells. Unexpectedly, Na V 1.3 immunostaining was present in both endothelium and pericytes. To determine the distribution of Na V 1.3 in the renal medulla, immunostaining was also performed on serial tissue sections. Both SMA and Na V 1.3 were confined to DVR in vascular bundles ( Fig. 4 ).
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Fig. 2. Immunocytochemical detection of Na V 1.3 in isolated DVR. Hand-dissected DVR were subjected to immunofluorescence staining. The presence of Na V 1.3 protein and the pericyte marker -smooth muscle actin (SMA) was determined using specific antibodies. Left : SMA. Middle : Na V 1.3. Right : merged images. Pericytes (white arrowheads) showed positive staining for SMA and Na V 1.3. SMA-negative cells (*) were also positive for Na V 1.3. The results are representative of 9 experiments.
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! ]) \( q: {! X1 qFig. 3. Immunocytochemical detection of Na V 1.3 in pericytes and endothelium. Hand-dissected DVR were subjected to immunofluorescence staining. Figure shows high-magnification confocal images of SMA (red) and Na V 1.3 (green) fluorescence. Left and right, bottom : white light/differential interference contrast (DIC) images of the same vessels. Endothelial cells (*) that underlie pericytes (arrowheads) show expression of Na V 1.3 but not SMA. The results are representative of 7 experiments.
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# j7 n: M6 A6 [: ?, l6 F5 ]4 O* [Fig. 4. Localization of Na V 1.3 expression in the renal outer medulla. Light micrographs of 50-µm-thick sections illustrate Na V 1.3 immunostaining in the renal outer medulla (brown). A and B : positive immunostaining for Na V 1.3 protein in outer medullary vascular bundles (brown). C and D : sequential sections corresponding to panels stained for Na V 1.3 were positive for -SMA in vascular bundles (blue). Immunostaining was not detected when the primary antibody was omitted (not shown). The results are similar to sections from 3 rats.& L6 G, ]+ K4 ~7 R

# W, `3 B2 `' z$ S  n$ tInteraction between CaM and Na V 1.3. As with other isoforms, Na V 1.3 has a COOH-terminal IQ motif, available for binding and interaction with CaM ( 17, 23, 31 ). To explore that interaction, several studies were performed. First, GST fusion proteins that include 250 amino acid COOH-terminal Na V 1.3 (GST-Na V 1.3C) or full-length CaM (GST-CaM) were generated. GST-CaM, but not GST alone, yielded three bands from medullary homogenate near the expected size, 250 kDa ( Fig. 5 A ). Those bands were reduced by preabsorption with antigenic peptide ( Fig. 5 A, lane 2 vs. lane 4 ). Figure 5 B shows that GST-CaM but not GST yielded bands from outer and inner medullary homogenates. The GST-Na V 1.3C construct, but not GST alone, bound CaM to yield a single band of the expected size ( 17 kDa) from outer and inner medullary homogenates ( Fig. 5 C ). Finally, coimmunoprecipitation of CaM and native Na V 1.3 was also performed to verify their association in vivo. In that case, immunoblot with anti-Na V 1.3 identified two bands from the immunoprecipitates of anti-CaM antibody ( Fig. 5 D ).8 j! f/ ^5 @5 a8 J
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Fig. 5. Binding between Na V 1.3 and CaM. A : tissues lysates of rat outer medulla were incubated with GST or GST-CaM immobilized on glutathione beads and a pull-down assay was performed. Immunoblot using antibody against Na V 1.3 identified a primary band near 250 kDa and 2 discrete bands of lower molecular weight. These were present from GST-CaM ( lane 4 ) but not GST ( lanes 1 and 3 ). Bands were suppressed when the antibody was preabsorbed with antigenic peptide ( lane 2 vs. lane 4 ). B : tissue lysates prepared from rat inner and outer medulla were incubated with immobilized GST or GST-CaM fusion protein and a pull-down assay was performed. Immunoblot using antibody against Na V 1.3 identified bands near 250 kDa from outer medullary lysate ( lane 1 ) and GST-CaM ( lane 5 ) but not GST ( lanes 2 and 4 ). C : reverse pull-down assay was performed with immobilized GST or GST-Na V 1.3C fusion protein. Immunoblot with antibody directed against CaM identified a band near 17 kDa from outer medullary and inner medullary lysate ( lanes 1 and 2 ), GST-Na V 1.3C ( lanes 4 and 6 ), but not GST ( lanes 3 and 5 ). The results are representative of 3 experiments. D : immunoprecipitation was performed with anti-CaM antibody using outer and inner medullary tissue lysates (IP lane). The precipitated proteins were separated by SDS-PAGE and probed with anti-Na V 1.3 ( top ) and anti-CaM antibodies ( bottom ). Two bands were recovered near 250 kDa from outer and inner medullary lysates ( lanes 1 and 2 ) and from anti-CaM immunoprecipitates ( lanes 3 and 4 ). Inner medullary homogenate yielded greater coprecipitate than outer medullary homogenate. CaM was identified near the expected size near 17 kDa. The results are similar to 4 experiments.
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Modulation of pericyte Na V current by CaM. Current-voltage characteristics of fast Na   currents ( I Na ) in DVR pericytes were studied using the pulse protocol defined by the inset in Fig. 6 A. Periytes were depolarized from the holding potential of -100 mV to values between -70 and 40 mV in 10-mV increments. Rapidly activating and inactivating inward currents were observed the electrophysiological characteristics of which have been previously analyzed ( 46 ). Inclusion of CIP (100 nmol/l) in the electrode significantly reduced peak inward currents over a range of pulse potentials ( Fig. 6 B ). The voltage dependence of inactivation was investigated using the protocol defined in Fig. 6 C, inset. Pericytes were held at various conditioning potentials ranging from -110 to 10 mV for 2 s and then depolarized to -10 mV. Conditioning potentials greater than about -90 mV led to decreases in peak I Na. Despite the general reduction of inward current amplitude by CIP, there was no shift in its voltage dependence. Fit of the data to the Boltzman equation, y = 1/{1   exp[s( V m - V 1/2 )/ K ]}, where V m is membrane potential, s = -1 or  1 for activation or inactivation, respectively, and V 1/2 and K are constants, is shown in Fig. 6 D. A Levenberg-Marquardt parameter search yielded V 1/2 and slope factor, K of -72.1 and 8.64 mV for inactivation, respectively, and -19.2 and 4.91 mV for activation, respectively.  @- a8 c; f8 {7 v9 b% Y7 ~

. G$ u0 Q2 I" u6 J/ {1 S. s! bFig. 6. Inhibition of Na V currents by calmodulin inhibitory peptide. A : example of rapidly inactivating Na   inward currents elicited from DVR pericytes held at -100 mV and depolarized to levels between -70 and 40 mV (protocol, inset ). B : maximum inward current vs. pulse potential in the absence ( n = 11) or presence ( n = 10) of calmodulin inhibitory peptide (CIP; 100 nM) in the electrode (* P 4 B+ w- Y( `! E3 a7 U5 E/ I6 B
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We also tested the effects of the membrane permeant CaM inhibitor W7 on I Na elicited by sequential pulses from -100 to -10 mV at 5-s intervals. During pulsation, W7 was introduced into the bath at 1, 10, and 100 µmol/l. Inhibition of peak inward current was achieved at 10 and 100 µmol/l ( Fig. 7, A and B ). Fit of the data to the equation I Na / I Na0 = 1/(1   [W7]/ K d ) yielded a best fit for K d = 8.04 µmol/l. The ability of W7 to inhibit I Na was slowly reversible. An example of reversibility is provided as a concatenated display of sequential inward currents in Fig. 7 C. I Na was nearly eliminated by W7 (100 µM) and slow reversal occurred over several minutes during washout. To investigate the effect of W7 on the voltage dependence of activation and inactivation, we used W7 near its K d, at 10 µmol/l. Higher W7 concentrations eliminated I Na to a degree that analysis of voltage dependence was impossible. As with CIP ( Fig. 6 ), W7 reduced I Na without shifting the voltage dependence of activation or inactivation ( Fig. 8 ).2 t  l0 L$ h1 P: J
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Fig. 7. Inhibition of Na V currents by calmodulin inhibitor N -(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide hydrochloride (W7). A : DVR pericytes were held at -100 mV and pulsed to -10 mV for 100 ms at 5-s intervals. Example shows superimposed currents elicited by W7 added to the bath at 0, 1, 10, and 100 µmol/l. B : averaged maximal inward currents elicited during pulses from -100 to -10 mV ( A ) at 0, 1, 10, and 100 µmol/l W7 ( n = 33, 6, 21, 15, respectively). Significant inhibition of inward Na   currents was achieved at W7 concentrations of 10 and 100 µmol/l (* P
/ g; ~3 X1 V+ k. m  [+ ?
% @1 d6 K2 d& q  j# U: w. T! bFig. 8. Inhibition of Na V currents by calmodulin inhibitor, W7. A and B : examples of I - V currents elicited by pulse depolarizations using the protocol defined by Fig. 7 A, inset. Protocols were executed in the absence ( A ) and presence ( B ) of W7 (10 µmol/l). C : summary of means ± SE of maximal inward currents elicited in the absence ( n = 8) and presence ( n = 8) of W7 (10 µmol/l). * P 4 |5 p) u! e: t& _) X! M

2 R' z1 |$ S; ^1 a* i) oIn addition to the COOH-terminal motifs that bind CaM, Na V isoforms possess EF-hand domains that might bind Ca 2 . Thus cytoplasmic Ca 2  might modify conductance or voltage dependence of gating through EF-hand or CaM interaction. To test whether cytoplasmic Ca 2  affects Na V current, we measured I Na in a series of cells, alternating between electrode buffer with low ( 20 nmol/l) or high ( 2,000 nmol/l) free Ca 2 . In contrast to the effects of interfering with CaM binding ( Fig. 6 ), raising cytoplasmic Ca 2  shifted the voltage dependence of activation ( Fig. 9, A and B ). In vitro binding of His-CaM to GST-Na V 1.3C was Ca 2  dependent ( Fig. 9 C ) so that it is unlikely that effects of Ca 2  on activation of I Na involve dissociation of CaM from the COOH terminus./ P2 u- m' ~3 h. `3 i: z* t
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Fig. 9. Modulation of Na V currents and CaM binding by free Ca 2 . A : maximum inward current vs. pulse potential using BAPTA-chelated free Ca 2  ([Ca 2  ] f ) in the electrode buffer of 20 nM ( ) or 2,000 nM (; n = 6 each; * P ( p9 S( U! W) l# [: {+ Y

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DVR supply blood flow to the medulla of the kidney. They arise from juxtamedullary glomerular efferent arterioles in the outer stripe of the outer medulla. In the inner stripe, DVR coalesce to join vascular bundles wherein they closely associate with ascending vasa recta and descending thin limbs of short looped nephrons ( 22 ). In addition to transmural transport functions that accommodate countercurrent trapping of NaCl and urea, DVR are contractile ( 28, 29 ). Pericytes surround the DVR endothelial monolayer and respond to a variety of agents with vasoconstriction and dilation ( 27 ). It has been inferred from the anatomical arrangement of DVR in vascular bundles that they regulate both total blood flow to the medulla and its distribution between the outer and inner medulla of the kidney.  q$ ]4 O: t! o
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In the past, the presence of voltage-gated cation channels in the efferent circulation of the kidney was controversial. More recently, it has been recognized that voltage-gated Ca 2  channels (Ca V ) are present in the juxtamedullary efferent arterioles and DVR ( 15, 16, 48 ). In a recent communication, we reported the surprising finding that voltage clamp depolarizations of DVR pericytes, designed to elicit Ca V currents, yielded a rapidly inactivating, TTX-sensitive Na V current. The expected L-type Ca V currents were only observed when pericytes were treated with the agonist, FPL-64176 ( 46 ). In this study, we sought to identify expression of molecular isoforms of Na V in DVR. RT-PCR amplification of RNA from isolated, permeabilized DVR with degenerate primers targeted to TTX-sensitive Na V isoforms yielded only Na V 1.3 sequences from hand-dissected vessels. Immunoblot of medullary homogenate ( Fig. 1 B ), immunochemistry in isolated vessels ( Figs. 2 and 3 ), and immunochemistry in tissue sections ( Fig. 4 ) verified expression of Na V 1.3, confined to DVR of outer medullary vascular bundles. To firmly establish that Na V 1.3 carries the DVR pericyte fast Na   currents, it would be necessary to silence its expression. Measurements of Na V currents in DVR pericytes have been performed on acutely isolated vessels that are not amenable to long-term incubations required for mRNA silencing. An appropriate cell culture model of the DVR pericyte does not exist. Finally, a murine knockout of Na V 1.3 is unavailable and would seem unlikely to be viable.
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Beginning with the report of TTX-resistant Na V currents in the vasculature by Sturek and Hermsmeyer ( 37 ), smooth muscle Na V have been intermittently described. Mesenteric myocytes have been shown to have a TTX-sensitive Na V conductance ( 2 ), while the TTX-resistant skeletal muscle isoform Na V 1.5 may be prevalent in gastrointestinal smooth muscle ( 24, 36, 44 ). Ten isoforms of voltage-operated Na   channels have been identified, nine of which are reasonably placed into a single family (Na V 1.1-Na V 1.9), while one other (Na x ) has more divergent structure and properties ( 7, 8, 30 ). Six of the Na V isoforms are TTX sensitive while Na V 1.5, 1.8, 1.9, and Na X are resistant ( 25 ). A recent report of Na V 1.6, Na V 1.7, and Na X expression in portal vein myocytes has been provided ( 32 ). The DVR pericyte Na   conductance is exquisitely TTX sensitive ( K d = 2.2 nM), and its putative identification as Na V 1.3 is consistent with that characteristic. It seems likely that Na V 1.3 is the predominant isoform in DVR because screening of several PCR products yielded only Na V 1.3 sequences. None the less, it remains possible that other Na V isoforms are expressed at lower levels. Significant expression of TTX-insensitive channels in DVR pericytes seems unlikely because 10 nM TTX eliminated inward Na   currents in patch-clamp studies ( 46 ). Due to their luminal location, DVR endothelium cannot be easily accessed for patch clamp to test for fast Na   currents and examine TTX sensitivity. Appropriate cell culture models of either DVR pericytes or endothelium do not exist to facilitate electrophysiological studies. Since the degenerate primers we used would not identify TTX-resistant isoforms, it remains possible that the DVR endothelium expresses Na V 1.5, 1.8, 1.9, or Na X.( K8 J$ v  w" C' @9 c2 O1 x$ r

5 U) ?; K: w; Z& h7 [The isoform-specific Na V 1.3 antibody used in these studies recognized three protein bands, either from medullary homogenate ( Fig. 1 B ), or isolated with GST-CaM binding studies ( Fig. 5, A and B ). The specificity of antibody recognition was verified though preabsorption with the immunizing antigen. The associated epitope of the commercial antibody corresponds to amino acids 511-524 of the intracellular loop between domains I and II (HLEGNHRADGDRFP, acquisition P08104 ; Fig. 5 A ). Na V are large molecules comprised of four homologous domains (I-IV) each characterized by six (S1-S6) -helical membrane-spanning regions. Splice variants of Na V 1.3 alter the intracellular loop between domains I and II through alternate splicing of exon 12. The known splice variants do not affect the antibody binding site. Three and four splice variants of Na V 1.3 have been described in rat and human, respectively ( 14, 33, 38 ). The three rat variants differ from the full-length Na V 1.3 described in humans (2,000 amino acids, acquisition number NM_006922 .2) by deletion of 51, 138, or 147 nucleotides ( 38 ). Those splice variants are expected to yield proteins of 1951, 1954, or 1983 amino acids, respectively. Given that the former two should comigrate during SDS-PAGE, it is unlikely that they correspond to the two lower molecular weight bands in Figs. 1 and 5 A. It is possible that the middle band is a previously unknown splice variant that exists in the rat kidney or that a posttranslational modification of a known Na V 1.3 splice variant accounts for its presence. The coprecipitation of CaM ( Fig. 5 D ) yielded two bands recognized by the antibody. The reason for the absence of the middle band in the CaM coprecipitation is uncertain, although expression of the known splice variants of Na V 1.3 in the rat could account for the pattern in Fig. 5 D. The apparent increase in association of Na V 1.3 with CaM in inner vs. outer medullary homogenate shown in Fig. 5 D is also of interest, suggesting enhanced association of these proteins through stabilized interaction in the vasculature of the inner medulla compared with the outer medulla. Given that the function of Na V 1.3 in pericytes and endothelium remains to be fully elucidated, we cannot provide a sound hypothesis for the purpose of that difference.- s% Z3 a9 T# `; L
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The COOH terminus of voltage-gated Na   channels express consensus IQ and Baa motifs for interaction with CaM. Mori et al. ( 23 ) showed that binding of CaM to those sites was Ca 2  independent and dependent, respectively. We examined the presence of CaM binding and its modulation of DVR Na V currents. GST fusion constructs of Na V 1.3 COOH terminus (GST-Na V 1.3C) and CaM were generated to perform pull-down assays. Those experiments verified that GST-Na V 1.3C binds to CaM ( Fig. 5 C ). Conversely, GST-CaM binds to native Na V 1.3 from medullary lysates ( Fig. 5, A and B ). Finally, antibody directed against CaM coprecipitates Na V 1.3 ( Fig. 5 D ). Studies by Herzog and colleagues ( 17 ) examined the effect of COOH-terminal deletions from the TTX-sensitive isoform, Na V 1.4. In their hands, deletions that included the IQ motif virtually eliminated currents from transfected cells. Similarly, mutations of the IQ motif of Na V 1.6 led to marked reduction of current density. Those results mirror our findings that blockade of CaM interactions using CIP or W7 reduced native Na V currents in DVR pericytes ( Figs. 6 - 8 ). The Ca 2  independence of CaM binding to GST-Na V 1.3C ( Fig. 9 C ), combined with the inhibitory effects of CIP support the interpretation that CaM exerts permissive effects on DVR Na V 1.3 channel activity through Ca 2  -independent binding to the IQ motif. Moreover, as with mutation of the IQ motif of Na V 1.4 ( 17 ), the voltage dependence of activation and inactivation of I Na in DVR pericytes was not affected by CIP or W7 ( Figs. 6 and 8 ).1 ~2 h- D8 x+ C% P/ D' @

5 X8 {5 i- p4 X4 VIn contrast to inhibition of CaM binding by CIP ( Fig. 6 ), modulating cytoplasmic Ca 2  concentration of pericytes through BAPTA chelation within the patch-clamp electrode induced a significant change in the voltage dependence of Na V current activation. Compared with 20 nM free Ca 2 , 2,000 nM free Ca 2  caused a depolarizing shift ( Fig. 9, A and B ). Parallel in vitro binding studies of the effects of free Ca 2  on the binding of CaM to GST-Na V 1.3C showed no effect, strongly suggesting that the depolarizing shift does not require dissociation of CaM from the Na V 1.3 COOH terminus ( Fig. 9 C ). CaM has a bilobed structure with two EF-hand domains at each of its NH 2 and COOH termini. Association of CaM with its binding partners may be Ca 2  dependent or independent. The modulatory effects of Ca 2  on the function of interacting proteins can also be dependent on or independent of Ca 2  binding to the EF-hand domains of CaM ( 18 ). In addition to effects exerted through CaM binding, regulation of Na V isoforms by intracellular Ca 2  might also occur via direct interaction with the EF-hand domain that exists on COOH termini ( 34, 43 ). Given the complex array of possible interactions, we cannot be certain whether the Ca 2  -dependent shift in voltage dependence of activation ( Fig. 9, A and B ) is mediated through CaM. It seems unlikely, however, that the mechanism involves dissociation of CaM from Na V 1.3 ( Fig. 9 C ).
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5 ?$ P6 l0 Q$ {( r/ T' d* ]! g- tThe immunochemical finding that Na V 1.3 is expressed by the DVR endothelium ( Figs. 2 and 3 ) is unexpected but not unique ( 12, 13, 39, 41 ). Gordienko and Tsukahara ( 12 ) observed TTX-resistant Na V currents in cultured endothelia from rat interlobar arteries and human umbilical vein. Similarly, TTX-resistant currents were identified in cultured human saphenous vein endothelia. In that study, RT-PCR amplified a product from the 3'-untranslated region of hH1 (Na V 1.5). Expression level was dependent on the source of human serum but immunochemistry identified clear staining in sections from native tissue, mitigating against an artifact related to cell culture ( 13 ). In addition to TTX-resistant isoforms, TTX-sensitive Na V have been identified in cultured microvascular endothelial cells from cardiac ventricle ( 41 ) and bovine aorta ( 39 ). Interestingly, the latter study found that elimination of Na   influx by TTX or by extracellular Na   replacement with N -methyl- D -glucamine inhibited extracellular signal related kinase (ERK1/2) activation by shear stress. The manner in which voltage-gated Na   influx participates in ERK1/2 activation is unknown. We have shown that stretch-related responses occur in DVR endothelium ( 47 ) so that a role for endothelial Na V 1.3 expression in stretch-activated signal transduction will be a topic worthy of future exploration.! t6 U7 {9 U, G- f) x
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The functional role(s) of Na V 1.3 expression in DVR is uncertain. Similarly, the role of Na V expression in various types of smooth muscle is controversial. In neurons, coordinated opening of Na V channels provides a depolarizing Na   current that facilitates transmission of neural impulses and release of neurotransmitters. DVR endothelia are an electrical syncytium connected through gap junctions ( 45 ). Based on that finding, a possibility is that Na V participate in the conduction of depolarizing waves along the vessel axis. It has been proposed that Na   entry into smooth muscle increases the rate of depolarization to stimulate opening of voltage-gated Ca 2  channels. Such activity might serve to shape the spatial or temporal profile of cytoplasmic Ca 2  concentration changes within the cell that confer specificity to Ca 2  signaling ( 3, 5 ). An alternate possibility is that resting membrane potential lies within the range at which Na V are neither completely activated nor inactivated. The associated small Na V "window current" might provide a steady trickle of Na   ions into the cell the magnitude of which is regulated through subtle variation of membrane potential. Similarly, the intracellular loop between domains I and II has consensus sites for PKA, PKC, and casein kinase II so that regulation of a putative window current by kinase cascades is possible. Entry of Na   into cells is energetically costly unless it is utilized as a surrogate Ca 2  entry mechanism via Na   /Ca 2  exchange ( 1, 4 ). If Na V and Na   /Ca 2  exchangers exist in sequestered cellular "microdomains," substantial near membrane elevations of Ca 2  concentration might occur to influence nearby signaling events or to load Ca 2  into cellular stores. Experimentally, such near membrane cytoplasmic Ca 2  changes might be difficult to observe with conventional fluorescent probes that have high Ca 2  affinity and distribute diffusely into the cytoplasm. Given the fact that Na V expression has been observed in nonexcitable cells as diverse as chondrocytes ( 11, 40 ) and corneal epithelium ( 10, 42 ), it seems likely that some roles of Na V are unknown. The beta subunits associated with Na V behave as cell adhesion molecules so that a role of Na V in cell matrix interactions has been proposed ( 19 ).  b2 X0 c7 J! F# A) ~% P: C% D
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In summary, we used RT-PCR with degenerate primers for TTX-sensitive voltage-operated Na   channels to amplify a product from DVR whose sequence identified Na V 1.3 expression. Immunochemistry showed that both DVR pericytes and endothelium express Na V 1.3. Reciprocal pull-down assays with GST fusion proteins of COOH-terminal Na V 1.3 or CaM verified their ability to, respectively, bind CaM or Na V 1.3 from inner and outer medullary homogenates. Inhibition of CaM binding with an interfering peptide or block of CaM with the membrane permeant inhibitor W7 reduced DVR pericyte Na V currents. We conclude that DVR express Na V 1.3, which binds CaM and that CaM, is essential for pericyte Na V currents. We speculate that Na V currents in DVR may serve to shape the spatial and temporal profile of Ca 2  signaling events by stimulating Ca V current or through Na   /Ca 2  exchange./ \4 F' R2 X+ W% y$ w* f
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ACKNOWLEDGMENTS
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4 m$ F, t0 a& S8 ?Studies in the laboratory were supported by National Institutes of Health Grants DK-42495, DK-68492, DK-67621, and HL-78870.. `* `6 U" S* l: [0 G( D4 R9 J
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赚点分不容易啊  

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希瑞干细胞
偶啥时才能熬出头啊.  

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干细胞之家微信公众号
转基因动物

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每天早上起床都要看一遍“福布斯”富翁排行榜,如果上面没有我的名字,我就去上班……  

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就为赚分嘛  

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一个子 没看懂  

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几头雾水…  

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孜孜不倦, 吾等楷模 …………  

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