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• W2016516475 abstract "The present experiments using primary cultures of renal proximal tubule cells derived from wild-type and NHERF-1 knockout animals examines the regulation of NHE3 by phenylthiohydantoin (PTH) and the regulation of phosphate transport in response to alterations in the media content of phosphate. Forskolin (34.8 ± 6.2%) and PTH (29.7 ± 1.8%) inhibited NHE3 activity in wild-type proximal tubule cells but neither forskolin (-3.2 ± 3.3%) nor PTH (-16.6 ± 8.1%) inhibited NHE3 activity in NHERF-1-/- cells. Using adenovirus-mediated gene transfer, expression of NHERF-1 in NHERF-1-/- proximal tubule cells restored the inhibitory response to forskolin (28.2 ± 3.0%) and PTH (33.2 ± 3.9%). Compared with high phosphate media, incubation of wild-type cells in low phosphate media resulted in a 36.0 ± 6.3% higher rate of sodium-dependent phosphate transport and a significant increase in the abundance of Npt2a and PDZK1. NHERF-1-/- cells, on the other hand, had lower rates of sodium-dependent phosphate uptake and low phosphate media did not stimulate phosphate transport. Npt2a expression was not affected by the phosphate content of the media in NHERF-1 null cells although low phosphate media up-regulated PDZK1 abundance. Primary cultures of mice proximal tubule cells retain selected regulatory pathways observed in intact kidneys. NHERF-1-/- proximal tubule cells demonstrate defective regulation of NHE3 by PTH and indicate that reintroduction of NHERF-1 repairs this defect. NHERF-1-/- cells also do not adapt to alterations in the phosphate content of the media indicating that the defect resides within the cells of the proximal tubule and is not dependent on systemic factors. The present experiments using primary cultures of renal proximal tubule cells derived from wild-type and NHERF-1 knockout animals examines the regulation of NHE3 by phenylthiohydantoin (PTH) and the regulation of phosphate transport in response to alterations in the media content of phosphate. Forskolin (34.8 ± 6.2%) and PTH (29.7 ± 1.8%) inhibited NHE3 activity in wild-type proximal tubule cells but neither forskolin (-3.2 ± 3.3%) nor PTH (-16.6 ± 8.1%) inhibited NHE3 activity in NHERF-1-/- cells. Using adenovirus-mediated gene transfer, expression of NHERF-1 in NHERF-1-/- proximal tubule cells restored the inhibitory response to forskolin (28.2 ± 3.0%) and PTH (33.2 ± 3.9%). Compared with high phosphate media, incubation of wild-type cells in low phosphate media resulted in a 36.0 ± 6.3% higher rate of sodium-dependent phosphate transport and a significant increase in the abundance of Npt2a and PDZK1. NHERF-1-/- cells, on the other hand, had lower rates of sodium-dependent phosphate uptake and low phosphate media did not stimulate phosphate transport. Npt2a expression was not affected by the phosphate content of the media in NHERF-1 null cells although low phosphate media up-regulated PDZK1 abundance. Primary cultures of mice proximal tubule cells retain selected regulatory pathways observed in intact kidneys. NHERF-1-/- proximal tubule cells demonstrate defective regulation of NHE3 by PTH and indicate that reintroduction of NHERF-1 repairs this defect. NHERF-1-/- cells also do not adapt to alterations in the phosphate content of the media indicating that the defect resides within the cells of the proximal tubule and is not dependent on systemic factors. There is growing recognition that transporters, ion channels, receptors, and signaling proteins exist in cells as multiprotein complexes (1Fanning A.S. Anderson J.M. J. Clin. Investig. 1999; 103: 767-772Crossref PubMed Scopus (396) Google Scholar, 2Dunbar L.A. Caplan M.J. Eur. J. Cell Biol. 2000; 79: 557-563Crossref PubMed Scopus (34) Google Scholar, 3Jelen F. Aradiusz O. Smietana K. Otlewski J. Acta Biochim. Pol. 2003; 50: 985-1017Crossref PubMed Scopus (133) Google Scholar, 4van Ham M. Hendriks W. Mol. Biol. Rep. 2003; 30: 69-82Crossref PubMed Scopus (117) Google Scholar, 5Gomperts S.N. Cell. 1996; 84: 659-662Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 6Minkoff C. Shenolikar S. Weinman E.J. Curr. Opin. Nephrol. Hypertens. 1999; 8: 603-608Crossref PubMed Scopus (32) Google Scholar, 7Weinman E.J. Minkoff C. Shenolikar S. Am. J. Physiol. 2000; 279: F393-F399Crossref PubMed Google Scholar). These complexes link together critical pathways thereby conferring selectivity to the cellular responses to second messengers that regulate the activity and/or trafficking of these proteins. Adaptor proteins appear central to the assembly and stability of these protein complexes. In the kidney, the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1, 1The abbreviations used are: NHERF, sodium-hydrogen exchanger regulatory factor; NHE3, Na+-H+ exchanger isoform 3; BCECF, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein; TMA-Cl, (tetramethylammonium chloride; EIPA, ethyl isopropylamiloride; MES, 4-morpho-lineethanesulfonic acid; PTH, phenylthiohydantoin; PKC, protein kinase C; GFP, green fluorescent protein. 1The abbreviations used are: NHERF, sodium-hydrogen exchanger regulatory factor; NHE3, Na+-H+ exchanger isoform 3; BCECF, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein; TMA-Cl, (tetramethylammonium chloride; EIPA, ethyl isopropylamiloride; MES, 4-morpho-lineethanesulfonic acid; PTH, phenylthiohydantoin; PKC, protein kinase C; GFP, green fluorescent protein. also called EBP50) and NHERF-2 (also called E3KARP, TKA1) were initially identified as proteins required for cAMP-associated phosphorylation and inhibition of the renal brush border membrane Na+-H+ exchanger isoform 3 (NHE3) (8Weinman E.J. Steplock D. Shenolikar S. J. Clin. Investig. 1993; 92: 1781-1786Crossref PubMed Scopus (110) Google Scholar, 9Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Investig. 1995; 95: 2143-2149Crossref PubMed Scopus (301) Google Scholar, 10Yun C. Oh S. Zizak M. Steplock D. Tsao S. Tse C. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (396) Google Scholar). NHERF-1 and NHERF-2 are adaptor proteins that share a common modular structure including two tandem PDZ (PSD-95/Dlg/ZO-1) protein interactive domains and a C-terminal ezrin-radixin-moesin-merlin (MERM) binding domain. Since their discovery, nearly 50 proteins have been identified that bind to the NHERF isoforms (11Voltz J.W. Weinman E.J. Shenolikar S. Oncogene. 2001; 20: 6309-6314Crossref PubMed Scopus (139) Google Scholar). More recently, other PDZ proteins such as PDZK1 (NaPi-Cap1) have been found to be present in renal tubules and to interact with many of the same proteins that bind NHERF-1 and NHERF-2 (12Custer M. Spindler B. Verrey F. Murer H. Biber J. Am. J. Physiol. 1997; 273: F801-F806PubMed Google Scholar). NHERF-1, NHERF-2, and PDZK1 have the capacity not only to self-associate but also to heterodimerize with one another (13Shenolikar S. Minkoff C.M. Steplock D. Chuckeree C. Liu M-Z. Weinman E.J. FEBS Lett. 2001; 489: 233-236Crossref PubMed Scopus (68) Google Scholar, 14Fouassier L. Yun C.C. Fittz J.G. Doctor R.B. J. Biol. Chem. 2000; 275: 25039-25045Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 15Wade J.B. Liu J. Coleman R.A. Cunningham R. Steplock D. Lee-Kwon W. Pallone T.L. Shenolikar S. Weinman E.J. Am. J. Physiol. 2003; 285: C1494-C1504Crossref PubMed Scopus (98) Google Scholar, 16Gisler S.M. Pribanic S. Bacic D. Forrer P. Gantenbein A. Sabourin L.A. Tsuji A. Zhao Z.S. Manser E. Biber J. Murer H. Kidney Int. 2003; 64: 1733-1745Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Given their location in the renal proximal convoluted tubule, the concept emerged that these adaptor proteins form an apical/subapical mesh or scaffold that regulates the activity and trafficking of target proteins. We, as well as others, have speculated that NHERF-1, NHERF-2, and PDZK1 may represent a redundant physiologic control mechanism for regulation of renal ion transport and receptor activity based on studies in heterologous expression systems (10Yun C. Oh S. Zizak M. Steplock D. Tsao S. Tse C. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (396) Google Scholar, 11Voltz J.W. Weinman E.J. Shenolikar S. Oncogene. 2001; 20: 6309-6314Crossref PubMed Scopus (139) Google Scholar, 16Gisler S.M. Pribanic S. Bacic D. Forrer P. Gantenbein A. Sabourin L.A. Tsuji A. Zhao Z.S. Manser E. Biber J. Murer H. Kidney Int. 2003; 64: 1733-1745Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Evidence has begun to emerge, however, suggesting that there are unique specificities for some target proteins with individual adaptor proteins. Initially, it was reported that calcium-mediated endocytosis of NHE3 in OK cells, a renal proximal tubule cell line, required NHERF-2 and that the presence of endogenous NHERF-1 was not sufficient (17Kim J.H. Lee-Kwon W. Park J.B. Ryu S.H. Yun C.H. Donowitz M. J. Biol. Chem. 2002; 277: 23714-23724Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Further support for more specific interactions between NHERF-1, NHERF-2, and PDZK1, and given target proteins emerged from study of NHERF-1 knockout mice and two sets of observations were of particular interest with respect to the present studies (18Shenolikar S. Voltz J.W. Minkoff C.M. Wade J.B. Weinman E.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11470-11475Crossref PubMed Scopus (272) Google Scholar). First, although both NHERF-1 and NHERF-2 supported cAMP regulation of Na+-H+ exchange activity when co-expressed with NHE3 in PS120 fibroblast cells, NHE3 activity assayed in isolated renal brush border membrane vesicles from NHERF-1-/- mice was not inhibited in response to activation of protein kinase A despite the presence of normal amounts of NHERF-2 and PDZK1 (10Yun C. Oh S. Zizak M. Steplock D. Tsao S. Tse C. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (396) Google Scholar, 19Weinman E.J. Steplock D. Shenolikar S. FEBS Lett. 2003; 536: 141-144Crossref PubMed Scopus (70) Google Scholar). Second, whereas in vitro and cell experiments have indicated that Npt2a binds to NHERF-1, NHERF-2, and PDZK1, the NHERF-1-/- mice demonstrate increased urinary excretion of phosphate associated with decreased expression of Npt2a (NaPi IIa), the major regulated renal sodium-dependent phosphate transporter in the apical membrane of renal proximal tubule cells (18Shenolikar S. Voltz J.W. Minkoff C.M. Wade J.B. Weinman E.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11470-11475Crossref PubMed Scopus (272) Google Scholar, 20Gisler S.M. Stagljar I. Traebert M. Bacic D. Biber J. Murer H. J. Biol. Chem. 2001; 276: 9206-9213Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 21Hernando N. Deliot N. Gisler S. Weinman E.J. Lederer E. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11957-11962Crossref PubMed Scopus (155) Google Scholar). Moreover, the NHERF-1-/- mice fail to fully adapt to restriction of the dietary intake of phosphate although the abundance of PDZK1 but not NHERF-2 is increased (22Weinman E.J. Boddeti A. Cunningham R. Akom M. Wang F. Wang Y. Liu J. Steplock D. Shenolikar S. Wade J. Am. J. Physiol. 2003; 285: F1225-F1232Crossref PubMed Scopus (50) Google Scholar). These experiments highlight the fact that conclusions about the specificity of interactions between NHERF-1, NHERF-2, or PDZK1 and specific renal transporters derived from in vitro, yeast, and cell expression studies require complimentary studies in native tissues. Unfortunately, pursuing the mechanisms underlying the abnormal regulation of NHE3 by PTH and of Npt2a by alterations in the dietary intake of phosphate in intact animals is difficult and many of the molecular and pharmacologic probes used to dissect specific pathways cannot be used in whole animals. We reasoned, therefore, that the development of primary cultures of mouse proximal tubules would provide a useful system to detail the role of not only the NHERF proteins but also, potentially, might be applicable to the study of renal tissue from other knockout animals. In this article, we describe methods to isolate and study mice primary renal proximal tubule cells in culture that expresses NHE3 and Npt2a, and retain selected regulatory responses including inhibition of NHE3 activity by PTH and forskolin. NHE3 activity in proximal tubule cells from NHERF-1-/- mice, on the other hand, failed to demonstrate inhibition in response to forskolin or PTH indicating that this form of hormonal control of NHE3 activity requires NHERF-1. This conclusion was confirmed by the observations that the inhibitory response to PTH was restored by transiently expressing NHERF-1 in the NHERF-1-/- cells using adenovirus-mediated gene transfer. In like manner, it has been assumed but not clearly established that the physiologic mechanisms that mediate the response to alterations in the phosphate content of the media bathing cultured cells are equivalent to those operative in modulating renal phosphate transport when the dietary intake of phosphate is altered. By contrast to NHERF-1+/+ cells that increase sodium-dependent phosphate transport and Npt2a expression in response to low phosphate media, the NHERF-1-/- cells fail to adapt to the low phosphate media. This indicates clearly that the phosphate regulatory defect is within the proximal tubules themselves and is not the consequence of systemic changes in hormones, vitamin levels, or other factors. Animals and Preparation of Renal Proximal Tubule Cells—Male NHERF-1 (-/-) mice (B6.129-Slc9a3r1tmSsl/Ss1) breed into a C57BL/6 background for 6 generations and parental wild-type inbred control C57BL/6 mice aged 12 to 16 weeks were used in the current experiments (18Shenolikar S. Voltz J.W. Minkoff C.M. Wade J.B. Weinman E.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11470-11475Crossref PubMed Scopus (272) Google Scholar). To prepare primary renal proximal tubule cell cultures, kidney cortices were dissected and minced, and digested using 1% Worthington collagenase Type II and 0.25% soybean trypsin inhibitor. On completion of digestion, the samples were re-suspended in 35 ml of 45% Percoll® and centrifuged at 26,891 × g for 15 min at 4 °C. Proximal tubule cells sedimented to a layer immediately above the red-cell pellet. The 5-10-ml layer containing the proximal tubule cells was aspirated, centrifuged, washed to remove the remaining Percoll®, and re-suspended in 6 to 10 ml of Dulbecco's modified Eagle's medium/F-12 containing 50 units/ml of penicillin, 50 μg/ml streptomycin, 10 ng/ml epidermal growth factor, 0.5 μm hydrocortisone, 0.87 μm bovine insulin, 50 μm prostaglandin E1, 50 nm sodium selenite, 50 μg/ml human transferrin, and 5 pm 3,3′,5-triiodo-l-thyronine. The proximal tubule cells were plated on Matrigel-coated coverslips or plastic cell culture dishes coated with Matrigel, and maintained in an incubator at 37 °C in 5% CO2. The cultures were left undisturbed for 36 h after which the media was replaced every 2 days until the cells achieved confluence. Transport Assays—Na+-H+ exchange activity was determined using the pH-sensitive fluorescent dye, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) (10Yun C. Oh S. Zizak M. Steplock D. Tsao S. Tse C. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (396) Google Scholar). Cells were loaded with 6 μm BCECF-AM in a sodium assay buffer containing 130 mm NaCl, 5 mm KCl, 1 mm MgSO4, 2 mm CaCl2, 1 mm NaPO4, 25 mm glucose in 20 mm HEPES, pH 7.5, for 20 min at room temperature. They were then pulsed with 40 mm NH4Cl, pH 7.4, for 15-20 min followed by sequential washes with a solution containing 130 mm (tetramethylammonium chloride (TMA-Cl), 5 mm KCl, 1 mm MgSO4, 2 mm CaCl2, 1 mm TMA-PO4, 25 mm glucose in 20 mm HEPES, pH 7.5. BCECF fluorescence was measured at excitation wavelengths of 500 and 440 nm and an emission wavelength of 530 nm. The NH4Cl pulse was targeted to achieve an initial pHi of 6.0 and only cells with initial pHi values between 6.0 and 6.2 were included for analysis. Na+-H+ exchange transport, expressed as ΔpHi/s, was calculated from the slope of the initial 5 to 10 s of sodium-dependent pHi recovery. Over this time period, the relation between pHi and time was essentially linear. In some cases, cells were pretreated with 10-4m forskolin or 10-7m PTH-(1-34) (rat-PTH) during the final 15 min of dye loading and continuously during the perfusion. Control cells were treated with Me2SO, the diluent for forskolin. At the end of each experiment, the cells were equilibrated in pH clamp media containing 20 mm HEPES, 20 mm MES, 110 mm KCl, 14 mm NaCl, 1 mm MgSO4, 1 mm CaCl2, 1 mm TMA, 25 mm glucose, and 10 μm nigericin at pH 6.1 and 7.2. Control and experimental measurements were made on cells from the same proximal tubule cell preparation and were assayed on the same day. Phosphate transport was measured by determination of the sodium-dependent uptake of radiolabeled phosphate (23Lederer E.D. Khundmiri S.J. Weinman E.J. J. Am. Soc. Nephol. 2003; 14: 1711-1719Crossref PubMed Scopus (48) Google Scholar). Twenty-four to 72 h prior to study, the cells were incubated in low (0.3 mm) or high (1.9 mm) phosphate media. The cells were washed in serum-free medium, followed by incubation in a transport medium containing 137 mm NaCl or 137 mm TMA-Cl, 5.4 mm KCl, 2.8 mm CaCl2, 1.2 mm MgSO4, and 0.1 mm KH2PO4. Phosphate uptake was initiated by the addition of transport medium containing 32P-radiolabeled orthophosphate. Uptake was continued for 10 min at room temperature, after which the cell were washed with ice-cold medium in which TMA-Cl was substituted for sodium chloride, 32P was omitted, and 0.5 mm sodium arsenate was added. The uptake of 32P from the TMA-Cl solution was used to determine sodium-independent uptake and was subtracted to yield the sodium-dependent uptake of 32P. The cells were solubilized in 1% Triton X-100 for 90 min at 4 °C and an aliquot analyzed by liquid scintillation spectroscopy. Each assay was performed in triplicate and averaged to provide a single data point. Other Methods—The production of intracellular cAMP in cultured cells in response to 10-7m PTH and 10-4m forskolin was measured by non-acetylation EIA (cAMP Biotrak Assay Kit, Amersham) in the presence of 0.4 mm 3-isobutyl-1-methylxanthine. Protein kinase C (PKC) activity was assayed using the SignaTECT™ PKC Assay System form Promega containing a specific PKC substrate and capture membrane. The results of the PKC assays were confirmed by Western immunoblotting using PKCα, phospho-PKC (pan), and phospho-PKC α/βII antibodies. Cells were fixed in paraformaldehyde and prepared for confocal microscopy as previously described (18Shenolikar S. Voltz J.W. Minkoff C.M. Wade J.B. Weinman E.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11470-11475Crossref PubMed Scopus (272) Google Scholar). To obtain membrane preparations from the cultured cells, the cells were washed with sterile ice-cold phosphate-buffered saline, detached by scraping, and centrifuged for 5 min at 800 × g. The supernatant was discarded and the pellet re-suspended in 1.5 ml of buffer containing 50 mm Tris, pH 7.4, 0.1 mm EDTA, 0.1% β-mercaptoethanol, and Complete Protease Inhibitor Mixture (Roche Applied Science). The cells were then disrupted by three 20-s bursts from a probe sonicator followed by a 10-min centrifugation at 1,000 × g to remove large particulates. This supernatant was ultra-centrifuged for 1 h at 100,000 × g. The pellet was then re-suspended in 0.1% SDS and prepared for electrophoresis by the addition of Laemmli buffer. Western immunoblotting was performed using antibodies specific for NHERF-1, NHERF-2, ezrin, PDKZ1, Npt2a, NHE3, and green fluorescent protein (GFP). Infective recombinant adenoviruses were produced using AdEasy (Stratagene), a commercially available system adapted from methods originally developed by He et al. (24He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3216) Google Scholar). Recombinant adenoviruses were produced by inserting the cDNA into a shuttle plasmid (pShuttleCMV) and performing homologous recombination in Escherichia coli with this shuttle vector and a large adenovirus-containing plasmid following electroporation. Recombinants were identified from single colonies and infective adenovirus virions were produced following transfection of the linearized recombinant adenovirus plasmid in HEK293 cells. Virus stocks were amplified in HEK293 cells on 15-cm plates and purified following lysis by CsCl banding using ultracentrifugation. Protein concentrations were determined using the method of Lowry et al. (25Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Reverse transcription-PCR using appropriate primers was used to detect NHE1, NHE2, and NHE3 (9Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Investig. 1995; 95: 2143-2149Crossref PubMed Scopus (301) Google Scholar). Extraction of DNA and Northern blots was performed as previously described (22Weinman E.J. Boddeti A. Cunningham R. Akom M. Wang F. Wang Y. Liu J. Steplock D. Shenolikar S. Wade J. Am. J. Physiol. 2003; 285: F1225-F1232Crossref PubMed Scopus (50) Google Scholar). Statistical comparisons were performed using Peritz analysis of variance (26Harper J.F. Comput. Biol. Med. 1984; 14: 437-445Crossref PubMed Scopus (283) Google Scholar). An enriched suspension of proximal convoluted tubules was obtained using gradient centrifugation following protocols previously described by Mandel and co-workers and modified by our laboratories (27Soltoff S.P. Mandel L.J. J. Gen. Physiol. 1984; 84: 643-662Crossref PubMed Scopus (66) Google Scholar, 28Dolson G.M. Hise M.K. Weinman E.J. Am. J. Physiol. 1985; 249: F409-F416Crossref PubMed Google Scholar). We plated the suspended tubules on culture dishes for biochemical and phosphate transport experiments or on glass coverslips for NHE3 assays. Plating of cells on basement membrane matrix (Matrigel) increased the viability of the cells and was used in most experiments. The cells remained relatively quiescent for several days immediately following plating and any attempts to change media or otherwise manipulate the cells resulted in decreased viability. Accordingly, the cells were left undisturbed for 3 to 4 days before changing the media after which the media was changed every other day till the cells were ready for study. Attempts to pass cells that had achieved confluence were unsuccessful. Confluent or near confluent cultures contained a uniform cell type although in every culture, there were clusters of cells with a different morphology. We estimated that these other cells constituted 5% or less of the total population of cells. As will be discussed, we did not attempt to identify this minority population of cells because it would be unlikely that they would express the two transporters of interest in this study, namely NHE3 and Npt2a. Assays for cAMP and protein kinase C activity were used to characterize the response to PTH. In wild-type cells, PTH (10-7m) stimulated cAMP generation 33.3 ± 8.4-fold and protein kinase C activity by 34.4 ± 6.2%. In NHERF-1 null proximal tubule cells, PTH (10-7m) stimulated cAMP generation 33.8 ± 7.2-fold and protein kinase C activity by 37.6 ± 6.1%. There were no differences between wild-type and knockout cells (Table I). In many of the above experiments, cAMP generation in response to vasopressin (10-7m) was also examined. The effect of vasopressin was variable but always severalfold lower than the response to PTH (data not shown).Table IThe effect of PTH (10−7m) on cAMP generation and protein kinase C activity in cultured proximal tubule cells from wild-type and NHERF-1−/− cellscAMP generationProtein kinase C activityWild-type cells33.3 ± 8.4aIndicates p < 0.05. n = number of preparations studied. (n = 5)34.4 ± 6.2aIndicates p < 0.05. n = number of preparations studied. (n = 5)NHERF-1−/− cells33.8 ± 7.2aIndicates p < 0.05. n = number of preparations studied. (n = 7)37.6 ± 6.1aIndicates p < 0.05. n = number of preparations studied. (n = 5)a Indicates p < 0.05. n = number of preparations studied. Open table in a new tab Reverse transcriptase-PCR and immunoblotting indicated that the cultured cells expressed NHE1 and NHE2 in addition to NHE3. Accordingly, we next determined the Na+-H+ exchange activity in wild-type and NHERF-1 null proximal tubule cells, and the response to 50 nm ethyl isopropylamiloride (EIPA), a concentration that would inhibit NHE1 and NHE2 activity but not affect NHE3 activity (29Park K. Olschowka J.A. Richardson L.A. Bookstein C. Chang E.B. Melvin J.E. Am. J. Physiol. 1999; 276: G470-G478PubMed Google Scholar). Na+-H+ exchange averaged 0.044 ± 0.002 ΔpHi/s and 0.042 ± 0.02 in the absence and presence of 50 nm EIPA in wild-type cells and 0.042 ± 0.004 ΔpHi/s and 0.039 ± 0.004 in the absence or presence of 50 nm EIPA in null cells. In both cell types, 50 μm EIPA abolished sodium-dependent pHi recovery. In all remaining experiments, 50 nm EIPA was included in the perfusion solutions to permit specific study of NHE3 activity. NHERF-1 facilitates the formation of a multiprotein signal complex that phosphorylates NHE3 and down-regulates its activity (30Zizak M. Lamprecht G. Steplock D. Tariq N. Shenolikar S. Donowitz M. Yun C. Weinman E.J. J. Biol. Chem. 1999; 274: 24753-24758Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 31Weinman E.J. Steplock D. Donowitz M. Shenolikar S. Biochemistry. 2000; 39: 6123-6129Crossref PubMed Scopus (125) Google Scholar). To determine whether cultured proximal tubules retain this regulatory pathway, wild-type proximal tubule cells were treated with forskolin and sodium-dependent pHi recovery was measured in the presence of 50 nm EIPA. Forskolin inhibited NHE3 activity by 34.7 ± 3.2% (p < 0.05, n = 7) in wild-type cells but failed to inhibit NHE3 activity (-4.2 ± 3.5%, n = 7) in cultured proximal tubule cells from NHERF-1-/- mice although basal NHE3 activity was similar in both cell types (0.041 ± 0.004 ΔpHi/s in wild-type cells and 0.045 ± 0.003 in NHERF-1 null cells). Recombinant adenovirus-NHERF-1 containing an enhanced GFP tag at the N terminus was used to determine whether we could rescue the null cells. Preliminary studies in PS120 cells indicated that expressed N-terminal GFP-tagged NHERF-1 was fully functional in mediating cAMP inhibition of co-expressed NHE3. We initially infected wild-type cells with various concentrations of adenovirus-GFP or adenovirus-GFPNHERF-1. For both viruses, 109 plaque-forming units were found to result in preserved cell morphology. Comparison of fluorescent labeled cells with bright field images indicated the efficiency of expression of NHERF-1 was 99% or greater at these concentrations of virus particles. The efficiency of infection can also be appreciated in Fig. 1, which shows representative confocal images of NHERF-1-/- cells stained for NHERF-2. The panels on the left (A and C) are non-infected cells showing the presence of native NHERF-2 (panel C) but no green fluorescence (panel A). Panels B and D show cells infected with adenovirus-GFP-NHERF-1. There is clear expression of NHERF-1 as seen in panel B with no change in expression of NHERF-2 (panel D). Moreover, comparison of panels D and B indicate that virtually all NHERF-2 positive cells in this field are expressing NHERF-1. We next wanted to determine whether the expressed NHERF-1 was present in the plasma membrane. Fig. 2 (panel A) shows a Western immunoblot of a membrane preparation from NHERF-1-/- cultured renal proximal tubule cells infected with adenovirus-GFP or adenovirus-GFP-NHERF-1. A band of ∼85 kDa representing the GFPNHERF-1 fusion protein is displayed using an anti-GFP antibody in the plasma membrane of adenovirus-GFP-NHERF-1-infected cells but not in cells infected with adenovirus-GFP. This band was also recognized using an anti-NHERF-1 antibody (data not shown). Fig. 2, panel B, shows immunoblots for ezrin, NHE3, NHERF-2, and PDZK1 indicating that the membrane expression of these proteins was not affected by expression of NHERF-1 in the NHERF-1-/- proximal tubule cells. Adenovirus-GFP infection of wild-type cells did not affect basal, or forskolin- or PTH-mediated inhibition of NHE3 activity (Table II). Adenovirus-GFP infection of NHERF-1 null proximal tubule cells also did not affect basal NHE3 activity and the cells remained unresponsive to forskolin or PTH. Infection of the NHERF-1 null cells with adenovirus-GFP-NHERF-1, however, restored the inhibitory effect of forskolin and PTH to cells derived from null animals. NHE3 activity was inhibited by 28.2 ± 3.0 and 33.2 ± 3.5% in response to forskolin and PTH, respectively.Fig. 2Panel A, Western immunoblot using anti-GFP antibody of membranes obtained from proximal convoluted tubule cells infected with adenovirus-GFP (GFP) or adenovirus-GFP-NHERF-1 (GFP+N-1). A band of ∼85 kDa representing the GFP-NHERF-1 fusion protein is detected in the cells infected with adenovirus-GFP-NHERF-1 (right lane). Panel B shows Western immunoblots for ezrin, NHE3, NHERF-2, and PDZK1 from the same membrane preparations.View Large Image Figure ViewerDownload (PPT)Table IINHE3 activity in transfected cultured proximal tubule cells from wild-type and NHERF-1−/− mice treated with forskolin or PTHControlExperimentalChange%Wild-type cells (GFP)Forskolin (n = 6)0.041 ± 0.0040.026 ± 0.00134.8 ± 6.2aIndicates p < 0.05. n = number of preparations studied.PTH (n = 7)0.045 ± 0.0030.031 ± 0.00229.7 ± 1.8aIndic" @default.
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• W2016516475 title "Defective Parathyroid Hormone Regulation of NHE3 Activity and Phosphate Adaptation in Cultured NHERF-1-/- Renal Proximal Tubule Cells" @default.
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