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Tissue-specific Regulation of Sodium/Proton Exchanger Isoform 3 Activity in Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) Null Mice

2007; Elsevier BV; Volume: 282; Issue: 34 Linguagem: Inglês

10.1074/jbc.m701910200

ISSN

1083-351X

Autores

Rakhilya Murtazina, Olga Kovbasnjuk, Nicholas C. Zachos, Xuhang Li, Yueping Chen, Ann L. Hubbard, Boris M. Hogema, Deborah Steplock, Ursula Seidler, Kazi Mirajul Hoque, Chung‐Ming Tse, Hugo R. de Jonge, Edward J. Weinman, Mark Donowitz,

Tópico(s)

Ion channel regulation and function

Resumo

The multi-PDZ domain containing protein Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) binds to Na+/H+ exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1-/- mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1-/- ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1-/- mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2′O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1-/- mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC. The multi-PDZ domain containing protein Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) binds to Na+/H+ exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1-/- mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1-/- ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1-/- mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2′O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1-/- mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC. The Na+/H+ exchanger regulatory factor (NHERF) 2The abbreviations used are: NHERF1Na+/H+ exchanger regulatory factor 1SNARF-4Ffor carboxyseminaphthorhodafluors-4FBBbrush borderNHE3Na+/H+ exchanger isoform 3NMDAN-methyl-d-glucamineEPACexchange protein directly activated by cAMP8-pCPT-2′-O-Me-cAMP8-(4-chlorophenylthio)-2′O-methyl-cAMPNHERF1 nullNHERF1-/-DICdifferential interference contrastROIregions of interestPBSphosphate-buffered salineWTwild typePKAprotein kinase A family of multi-PDZ domain proteins consists of four homologous and evolutionarily related proteins (1Weinman E.J. Cunningham R. Wade J.B. Shenolikar S. J. Physiol. (Lond.). 2005; 567: 27-32Crossref Scopus (68) Google Scholar, 2Donowitz M. Cha B. Zachos N.C. Brett C.L. Sharma A. Tse C.M. Li X. J. Physiol. (Lond.). 2005; 567: 3-11Crossref Scopus (183) Google Scholar, 3Thelin W.R. Hodson C.A. Milgram S.L. J. Physiol. (Lond.). 2005; 567: 13-29Crossref Scopus (35) Google Scholar, 4Hernando N. Gisler S.M. Pribanic S. Deliot N. Capuano P. Wagner C.A. Moe O.W. Biber J. Murer H. J. Physiol. (Lond.). 2005; 567: 21-26Crossref Scopus (45) Google Scholar). In renal proximal tubule and small intestinal epithelial cells, all four NHERF proteins are present in the apical domain either in the brush border (BB) or just below it (Refs. 5Wade J.B. Welling P.A. Donowitz M. Shenolikar S. Weinman E.J. Am. J. Physiol. 2001; 280: C192-C198Crossref PubMed Google Scholar and 6Wade J.B. Liu J. Coleman R.A. Cunningham R. Steplock D.A. Lee-Kwon W. Pallone T.L. Shenolikar S. Weinman E.J. Am. J. Physiol. 2003; 285: C1494-C1503Crossref PubMed Scopus (102) Google Scholar; this study). Although named after their role in regulation of NHE3, these proteins affect many classes of proteins. Among them, transport proteins are the most frequently identified, and the most intensely studied function of the NHERF proteins is in regulation of these transport proteins (7Shenolikar S. Voltz J.W. Cunningham R. Weinman E.J. Physiology (Bethesda). 2004; 19: 362-369Crossref PubMed Scopus (136) Google Scholar). The mechanisms of actions involved are multiple in the regulation of transport proteins. These include directing delivery of proteins to the plasma membrane from the synthetic pathway and/or recycling system, affecting plasma membrane retention, and more directly affecting turnover number (7Shenolikar S. Voltz J.W. Cunningham R. Weinman E.J. Physiology (Bethesda). 2004; 19: 362-369Crossref PubMed Scopus (136) Google Scholar, 8Cao T.T. Deacon H.W. Reczek D. Bretscher A. von Zastrow M. Nature. 1999; 401: 286-290Crossref PubMed Scopus (571) Google Scholar, 9Gage R.M. Kim K.A. Cao T.T. von Zastrow M. J. Biol. Chem. 2001; 276: 44712-44720Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 10Gage R.M. Matveeva E.A. Whiteheart S.W. von Zastrow M. J. Biol. Chem. 2005; 280: 3305-3313Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Puthenveedu M.A. von Zastrow M. Cell. 2006; 127: 113-124Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 12Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 426-436Crossref PubMed Scopus (350) Google Scholar). In addition, NHERF family members bring a wide variety of signaling molecules into complexes, some of which only bind to one or another of the NHERF family, whereas others bind to all. Na+/H+ exchanger regulatory factor 1 for carboxyseminaphthorhodafluors-4F brush border Na+/H+ exchanger isoform 3 N-methyl-d-glucamine exchange protein directly activated by cAMP 8-(4-chlorophenylthio)-2′O-methyl-cAMP NHERF1-/- differential interference contrast regions of interest phosphate-buffered saline wild type protein kinase A Because of the presence of multiple (if not all) NHERF family members in the proximal tubule and small intestinal apical membrane domains, it has proven difficult to attribute the NHERF regulatory functions to specific members of this gene family in vivo. In contrast, simple cells lacking most or all NHERF family members have been useful in determining the functions of individual NHERF proteins (13Yun C.H. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (403) Google Scholar, 14Kim 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 (114) Google Scholar, 15Mahon M.J. Donowitz M. Yun C.C. Segre G.V. Nature. 2002; 417: 858-861Crossref PubMed Scopus (277) Google Scholar, 16Lee-Kwon W. Kawano K. Choi J.W. Kim J.H. Donowitz M. J. Biol. Chem. 2003; 278: 16494-16501Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 17Lee-Kwon W. Kim J.H. Choi J.W. Kawano K. Cha B. Dartt D.A. Zoukhri D. Donowitz M. Am. J. Physiol. 2003; 285: C1527-C1536Crossref PubMed Scopus (87) Google Scholar, 18Cha B. Kim J.H. Hut H. Hogema B.M. Nadarja J. Zizak M. Cavet M. Lee-Kwon W. Lohmann S.M. Smolenski A. Tse C.M. Yun C. de Jonge H.R. Donowitz M. J. Biol. Chem. 2005; 280: 16642-16650Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This has been done in most detail related to regulation of NHE3 activity. For instance, cAMP inhibition of NHE3 mimics some postprandial aspects of digestion (induced by secretin) and can be reproduced in PS120 fibroblasts by expression of either NHERF1 or NHERF2; whereas in the absence of both proteins, cAMP has a reduced or no effect on NHE3 activity (13Yun C.H. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (403) Google Scholar, 19Emami S. Chastre E. Bodere H. Gespach C. Bataille D. Rosselin G. Peptides (Elmsford). 1986; 7: 121-127Crossref PubMed Scopus (19) Google Scholar). The situation is more complicated in epithelial cell models, such as Caco-2 and OK cells, which are used as intestinal and renal proximal tubule Na+ absorptive cell models, respectively. Both cells express multiple NHERF family members in the apical domain (5Wade J.B. Welling P.A. Donowitz M. Shenolikar S. Weinman E.J. Am. J. Physiol. 2001; 280: C192-C198Crossref PubMed Google Scholar, 20Zachos N.C. Li Hodson C. Chen Y. Milgram S. Donowitz M. Gastroenterology. 2005; 128: 177Google Scholar). Knockdown studies of each NHERF family member have the potential to determine the role of each individual family member, although few studies have been reported using this approach (21Takahashi Y. Morales F.C. Kreimann E.L. Georgescu M.M. EMBO J. 2006; 25: 910-920Crossref PubMed Scopus (168) Google Scholar, 22Yun C.C. Sun H. Wang D. Rusovici R. Castleberry A. Hall R.A. Shim H. Am. J. Physiol. 2005; 289: C2-C11Crossref PubMed Scopus (100) Google Scholar, 23Khundmiri S.J. Weinman E.J. Steplock D. Cole J. Ahmad A. Baumann P.D. Barati M. Rane M.J. Lederer E. J. Am. Soc. Nephrol. 2005; 16: 2598-25607Crossref PubMed Scopus (30) Google Scholar). Nonetheless, these models do not reproduce the complexity of intact tissue, in which NHERF family proteins may be present in adjacent cells of different types that interact. Knocking out individual and multiple NHERF family members in mouse intestine and proximal tubule is a promising approach, and NHERF1 and NHERF3/PDZK1 knock-out mice have been partially characterized (1Weinman E.J. Cunningham R. Wade J.B. Shenolikar S. J. Physiol. (Lond.). 2005; 567: 27-32Crossref Scopus (68) Google Scholar, 24Kocher O. Yesilaltay A. Cirovic C. Pal R. Rigotti A. Krieger M. J. Biol. Chem. 2003; 278: 52820-52825Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 25Capuano P. Bacic D. Stange G. Hernando N. Kaissling B. Pal R. Kocher O. Biber J. Wagner C.A. Murer H. Pfluegers Arch. Eur. J. Physiol. 2005; 449: 392-402Crossref PubMed Scopus (69) Google Scholar, 26Shenolikar 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 (285) Google Scholar, 27Cunningham R. Steplock D. Wang F. Huang H.E.X. Shenolikar S. Weinman E.J. J. Biol. Chem. 2004; 279: 37815-37821Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Renal proximal tubule and small intestine both use BB NHE3 to absorb the majority of luminal Na+. It has been assumed that regulation of NHE3 by common agonists acts by similar mechanisms in these two epithelia. However, suggestions have also been made that regulation of NHE3 by trafficking is very different between these two tissues, with the possibility that NHE3 trafficking only occurs between the microvilli and intervillus clefts in proximal tubule, whereas NHE3 traffics in the intestine by the more conventional plasma membrane → intervillus clefts (clathrin-coated pits and lipid rafts) → common and recycling endosomes → return to surface and some entry to degradative pathway of late endosomes → microvesicular bodies → lysosomes (28Li X. Zhang H. Cheong A. Leu S. Chen Y. Elowsky C.G. Donowitz M. J. Physiol. (Lond.). 2004; 556: 791-804Crossref Scopus (62) Google Scholar, 29McDonough A.A. Biemesderfer D. Curr. Opin. Nephrol. Hypertens. 2003; 12: 533-541Crossref PubMed Scopus (41) Google Scholar). Comparisons of regulation of NHE3 between small intestine and proximal tubule by the same agonist has the potential to provide further insights into how NHE3 is regulated differently in these two epithelia. A role for NHERF1 in cAMP inhibition in the mouse proximal tubule has been suggested based on comparison of wild type and NHERF1-/- mouse NHE3 activity in BB membrane vesicle preparations and in primary cultures of these cells (26Shenolikar 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 (285) Google Scholar, 30Weinman E.J. Steplock D. Shenolikar S. FEBS Lett. 2003; 536: 141-144Crossref PubMed Scopus (76) Google Scholar). However, studies of the effect of cAMP on NHE3 activity in intact renal proximal tubule or ileal Na+ absorptive cells from wild type compared with NHERF-/- mice have not been reported. This study describes development of a method using multi-photon microscopy to quantitate apical membrane NHE3 activity in intact mouse small intestine and proximal tubule and demonstrates important differences between cAMP inhibition of NHE3 in these two epithelial tissues and in the dependence of NHE3 regulation on NHERF1. Materials—SNARF-4F acetoxymethyl ester was from Invitrogen; HOE694 was from Sonafi-Aventis; 8-pCPT-2′-O-Me-cAMP was from BioLog; salts and other chemicals were from Sigma or Fisher at the highest grade available. Rabbit polyclonal antibody to NHE3 (Ab1381 and Univ. of Arizona), NHERF1 (Ab5199), and NHERF2 (Ab2570) were previously characterized (31Hoogerwerf W.A. Tsao S.C. Devuyst O. Levine S.A. Yun C.H. Yip J.W. Cohen M.E. Wilson P.D. Lazenby A.J. Tse C.M. Donowitz M. Am. J. Physiol. 1996; 270: G29-G41PubMed Google Scholar, 32Collins J.F. Xu H. Kiela P.R. Zeng J. Ghishan F.K. Am. J. Physiol. 1997; 273: C1937-C1946Crossref PubMed Google Scholar, 13Yun C.H. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (403) Google Scholar). Anti-ezrin and anti-actin antibodies were from Sigma; anti-EPAC1 antibodies were from Santa Cruz. Animals—Male NHERF1-/- mice bred into a C57Bl/6 background (Charles River) for at least six generations were produced from heterozygotes as initially reported (26Shenolikar 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 (285) Google Scholar). NHERF1-/- mice and wild type male C57Bl/6 mice were studied between 10 and 14 weeks of age. The mice were maintained under standard light and climate conditions in the animal facility of the Johns Hopkins University School of Medicine with ad libitum access to water and chow. Experiments with animals were carried out using protocols approved by the Animal Use Committee of the Johns Hopkins University. Isolation of Jejunum, Ileum, and Kidney Cortex for Na+/H+ Exchange Activity Assays—Mice were briefly anesthetized with ether and then sacrificed by cervical dislocation. The abdomen was immediately opened by midline incision and proximal jejunum (∼2 cm in length starting ∼1 cm distal to ligament of Treitz), and distal ileum (∼3 cm in length ending 1 cm proximal to the ileo-cecal junction) were excised and placed immediately in cold "Na+ buffer" (138 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgSO4, 1 mm NaH2PO4, 25 mm glucose, and 20 mm HEPES, 1 mm probenecid, pH 7.4) and opened along the antimesenteric border. 6–8-mm pieces were mounted with Krazy Glue (Elmer's Products Inc., Columbus, OH) onto a glass coverslip with the mucosal surface facing up. The kidney was cut in half with a razor blade, the capsule was removed, and the cortex was excised using a razor blade. 1–1.5-mm-thick slices were glued onto coverslips. All preparations were performed on ice. The glue used for mounting had no autofluorescent signal and did not affect viability of cells since the percent of cells taking up propidium iodide at the end of the experiment was similar to non-glued tissue maintained in Na+ buffer (data not shown). cAMP Treatment before Study for Electron Microscopy or Immunochemistry—Ileum or kidney segments were preincubated for 10 min at 37 °C in Ringer-HCO3, 10 mm glucose, gassed at 95% O2, 5% CO2, and then exposed at 37 °C to 100 μm 8-Br-cAMP or an equal volume of H2O (as vehicle control) for 30 min and then examined for electron microscopy or immunohistochemistry. Measurement of Mouse Jejunum, Ileal, and Renal Cortical NHE3 Activity by Multi-photon Microscopy—The wavelength for excitation of SNARF-4F for conventional confocal microscopy is between 488 and 530 nm with emission 580 and 640 nm. The emission wavelengths are the same for the two-photon microscope, whereas the wavelength for dye excitation was determined empirically. Wavelengths between 740 and 800 nm produced the largest emission fluorescence without visible tissue damage with average power from the Ti: Sa laser ∼800–860 milliwatts and pulse bandwidth ∼16–18 nm, corresponding to <50 fs pulse duration at 80 MHz repetition rate. This excitation was attenuated by the optical system and by a combination of neutral density filters such that the average intensity at the focal plane was <10 milliwatts. SNARF-4F Loading and Imaging—The protocol for imaging intracellular pH of intact mouse jejunum, ileum, or kidney cortex using multi-photon microscopy is described under "Results." By using a 60×/1.00 water immersion objective (Nikon), the images of the jejunal and ileal villus or renal cortical proximal tubule cells loaded with the "dual emission" pH-sensitive dye, SNARF-4F in Na+ buffer, pH 7.4, were visualized using a multi-photon laser scanning microscope (MRC-1024MP, Bio-Rad) powered by a wide band, infrared (780 nm) combined photo-diode pump laser and mode-locked titaniumsapphire laser (Tsunami Ti: Sa laser, Spectra-Physics, Mountain View, CA). The 8-bit images were recorded and stored, after which fluorescence intensity was calculated off-line using MetaMorph 5.0 rl software (Molecular Devices Corp.) as described below. Jejunum, ileum, and renal cortical slices were loaded with 20 μm SNARF-4F in Na+ buffer at 37 °C for 35 min with 95% O2, 5% CO2 gassing. The coverslip with the glued tissue was then placed in a perfusion chamber (RC-21BDW, Warner Instrument), mounted onto a heated microscope stage at 25 °C (PH series, Warner Instrument), and perfused using a peristaltic pump (Imatec; Reglo, Switzerland) at 1 ml/min with Na+ buffer for 15 min at room temperature. Tissue was then acidified using a prepulse, which consisted of perfusing with 60 mm NH4Cl for 30 min followed by N-methyl-d-glucamine ("NMDA") buffer (same as Na+ buffer with NMDA replacing Na+) for 20–25 min. To monitor Na+/H+ exchange activity as the initial rate of pH recovery, the NMDA buffer was switched to Na+ buffer. Both buffers contained 50 μm HOE694 to eliminate the contributions of NHE1 and NHE2. As described under Results, reagents of interest (100 μm 8-Br-cAMP, 100 μm 8-pCPT-2′-O-Me-cAMP, 50 or 1 μm H-89) were added to all perfusion buffers. 1 mm probenecid was in all perfusates to prevent SNARF-4F leakage (33Steinberg T.H. Newman A.S. Swanson J.A. Silverstein S.C. J. Cell Biol. 1987; 105: 2695-2702Crossref PubMed Scopus (128) Google Scholar, 34Di Virgilio F. Steinberg T.H. Swanson J.A. Silverstein S.C. J. Immunol. 1988; 140: 915-920PubMed Google Scholar, 35Chu S. Montrose M.H. J. Physiol. (Lond.). 1996; 494: 783-793Crossref Scopus (37) Google Scholar). The leakage of dye was greater in small intestine than in kidney. In all tissues the leakage of dye was increased when specimens were perfused with Na+-free buffer (NMDA buffer). This suggested that extracellular Na+ retained the SNARF-4F inside the cells. Because SNARF-4F leakage in kidney was much less than in small intestine, one slice of kidney cortex was used for both control and treated conditions sequentially (time control studied in parallel), whereas in experiments in small intestine, two separate pieces of tissues obtained from two animals were used for the control/treated conditions. SNARF-4F Emission—For images for each optical section (small intestine and kidney cortex), 0–50 μm from villus tip and cut surface, both at 10 μm steps (Fig. 1, A and B), were taken at 580 and 640 nm and stored. These conditions allowed quantifiable signals to be studied at depths up to 40–50 μm from the villus tip or cortical surface. Below that, the signal became too dim to obtain quantitative ratiometric data. Analysis of Collected Images—Optical images for analysis were taken typically starting at 20 and 30 μm from the tip of villus or 10 μm below the cut surface of the kidney cortex to avoid villus cells potentially close to shedding into the small intestinal lumen and damaged cortical cells from the slice preparation. Regions of interest (ROI) including regions for measurement of background were randomly chosen in 2–3 individual villi (Fig. 1C) or proximal tubules. Fluorescence intensity in gray levels that correspond to relative amounts of SNARF-4F for each ROI (16–21 ROIs per time point) for both 640- and 580-nm emissions was calculated using MetaMorph. The intensity of background was subtracted from each chosen ROI. The 640/580 ratio for each ROI was calculated, average value of ROIs were determined for each time point, and 640/580 ratios over time were determined followed by conversion to pH values with internal pH standards using Microsoft Excel. The Na+/H+ exchange activity of NHE3 was determined as the initial rate in pHi change by calculating the initial steep pHi slope after the addition of Na+ buffer using linear curve fit analysis (Origin 6.0) and presented as ΔpH/min. Immunofluorescence Staining of Ileum for NHE3, NHERF1, and NHERF2—Tissue samples exposed to cAMP or control solutions were obtained as described above and fixed in 3.5% paraformaldehyde in PBS at 4 °C and paraffin-embedded. Histological sections (4 μm thick) were mounted onto Superfrost microscope slides (Fisher) and heat-fixed. Slides were microwaved for antigen recovery in 10 mm sodium citrate buffer, pH 6 (Sigma) at power level setting 9 (Panasonic model NN-C980B Conventional Microwave Oven, Secaucus, NJ) for 2–5 min. After cooling for 30 min, sections were washed in PBS and preblocked with 5% normal goat serum in PBS for 30 min at room temperature. Sections were incubated for 1 h at room temperature and then for 48 h (4 °C) with polyclonal NHERF1, NHERF2, or NHE3 antibodies, diluted 1:500 in 5% normal goat serum-PBS. In parallel studies using the same animals, kidney cortex slices also were immediately fixed in fresh PLP (2% paraformaldehyde, 0.875 m l-lysine-HCl, 0.1 m sodium-metaperiodate) for 2 h at room temperature. Tissues were rinsed twice in 0.5 m sucrose, 0.1 m sodium phosphate for 10 min before leaving tissues in rinsing solution overnight at 4 °C. Cortical kidney slices were placed in plastic embedding molds and filled with OCT embedding medium (Tissue-Tek). Molds were then immediately submerged in isopentane and placed on dry ice. After excess solvent had evaporated, molds were stored at −80 °C until sectioned. 8-μm kidney sections were fixed in ice-cold 95% methanol for 10 min. Sections were rinsed three times in 1× PBS and blocked in 5% normal goat serum in PBS containing 0.2% Triton X-100 and 0.02% sodium azide for 30 min at room temperature. Kidney sections were incubated with a polyclonal primary antibody to NHE3 (Ab1381) diluted 1:100 in blocking solution for 1 h at room temperature With both methods of fixation, ileal and kidney sections were then washed twice in PBS for 10 min and incubated with anti-rabbit Alexa-fluor secondary antibodies, each diluted 1:100 for 1 h at room temperature. Sections were washed twice with PBS, autofluorescence-quenched with 1% Sudan Black (Sigma) in 70% methanol for 10 min at room temperature, counterstained with Hoechst 33342 (Invitrogen), and mounted with Gel Mount (Sigma). Ileal and kidney sections were imaged using a Zeiss LSM510 confocal fluorescence microscope (63× objective, oil immersion) for fluorescence and Nomarski differential interference contrast (DIC). Electron Microscopy of Ileum from Wild Type and NHERF1-/- Mice—Tissue samples exposed to cAMP or control solutions in vitro were obtained as described above and fixed by immersion in ice-cold 1.6–2% glutaraldehyde, 0.1 m sodium cacodylate, pH 7.4, for 60 min. During that time tissue was cut into smaller segments (∼3 mm on a side). After rinsing in 0.1 m sodium cacodylate, 3.5% sucrose, the tissue was incubated in reduced osmium (1.5% potassium ferrocyanide, 1% OsO4 in the same buffer) for 60 min on ice, rinsed several times in water, dehydrated through a series of graded EtOH, and embedded in EPON 812. Ultrathin sections were prepared, stained with uranyl acetate and then lead citrate, and viewed with a Hitachi 7600 microscope. Microvillar lengths were determined at a magnification of ×25000 at the microscope using the "measurement" tool. Three independent experiments were performed, and the microvilli of 5–13 cells were measured for each treatment. Ileal and Proximal Tubule Total Membrane Preparation/Immunoblot from Wild Type and NHERF1-/- Mice—Ileum (distal half of small intestine) and kidney cortex were excised from animals. Ilea were rinsed with ice-cold 0.9% saline and opened along the anti-mesenteric borders. Kidney cortices were rinsed in 0.9% saline and then transferred into homogenization buffer A (20 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm Na3VO4, 50 mm NaF). Ileal villus cells were scraped with a glass slide and placed into homogenization buffer B (60 mm mannitol, 2.4 mm Tris, pH 7.1, 1 mm EGTA, 2 mm Na3VO4, 1 mm β-glycerol phosphate, 1 mm phenylalanine). Protease inhibitor mixture (Sigma) 1:100 was added to buffers A and B, and phosphoramidon (1:1000) was added only to buffer B. Scraped ileal villus cells in buffer B and kidney cortex in buffer A were homogenized at 4 °C with a Polytron (10 times for 10 s at speed 5 with a 20 s interval between each burst) followed by homogenization of samples in a glass-Teflon homogenizer. The homogenates were centrifuged at 4000 rpm for 10 min at 4 °C to remove cell debris and nuclei. Supernatants were then centrifuged at 40000 rpm for 60 min, and total membrane pellets were collected. The resulting total membranes were resuspended in buffer A for kidney preparations and in buffer B for ileal mucosa. The protein concentrations in total membrane were measured with BCA (Sigma). Samples were analyzed with SDS-PAGE Western blotting using primary antibodies for EPAC1, ezrin, actin, or NHERF1 with fluorescently labeled secondary goat anti-mouse IRDye TM800 antibodies (Rockland). The fluorescence intensity of detected protein bands was quantified by the Odyssey system (LI-COR). Statistics—Values are presented as the mean ± S.E. Statistical significance was determined using Student's unpaired and paired t-tests. p values <0.05 were considered significant. Calibration of the Fluorescence Response of SNARF-4F in Kidney Cortex and Small Intestine at Different pH Values—Multi-photon technology rather than conventional confocal microscopy was selected to measure intracellular pH because it caused minimal bleaching in the bulk of the sample and induced less phototoxicity, which made longer study periods possible. These studies typically lasted on single pieces of tissue for a total of 2.5 h in jejunum and ileum and 3.5 h in the kidney cortex. The pKa for small intestine and kidney cortex for SNARF-4F acetoxymethyl ester was experimentally determined. Because the fluorescence response of SNARF-4F in solution and intracellularly is often different (pKa of free SNARF-4F is 6.4 (Invitrogen "Molecular Probes" manual), the SNARF-4F response in kidney cortex and in ileum was calculated by the K+ ionophore/nigericin (10 μm) method (36Levine S.A. Nath S.K. Yun C.H. Yip J.W. Montrose M. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) in the presence of 150 mm K+ extracellularly to equilibrate the intracellular pH with the pH-controlled extracellular media. Fig. 2 shows the calibration of the pH response as the ratio of fluorescence intensities of SNARF-4F measured at two different emission wavelengths 640 and 580 nm for kidney cortex (panels A) and ileum (panels C). The calculation of the fluorescence response of the dye to different pH values for kidney cortex (panel B) and ileum (panel D) yields apparent pKa values ∼6.8 and ∼7.4, respectively. Basal NHE3 Activity—The experimental protocol is shown in Fig. 3, A and D. Typically, before acidification, fluorescence was analyzed at 3 time points 1 min apart, and similar numbers of time points and timing were analyzed after prepulse with NH4Cl and during incubation in NMDA buffer to determine basal Na+/H+ exchange activity under acidified conditions. Then 12–15 readings were obtained at 1-min intervals during pH recovery in Na+ buffer. Calibration of 640/580 ratio was performed using the K+/nigericin method for external pH using pH standards of 6.1–6.3, 6.7–6.8, and 7.3–7.4 (36Levine S.A. Nath S.K. Yun C.H. Yip J.W. Montrose M. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Abstrac

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