Bradykinin B2 Receptors Activate Na+/H+ Exchange in mIMCD-3 Cells via Janus Kinase 2 and Ca2+/Calmodulin*

Yurii V. MukhinDagger , Tamara VlasovaDagger , Ayad A. Jaffa§, Georgiann CollinsworthDagger , John L. BellDagger , Baby G. TholanikunnelDagger , Tobiah Pettus, Wayne FitzgibbonDagger , David W. PlothDagger , John R. RaymondDagger , and Maria N. GarnovskayaDagger ||

From the Medical and Research Services of the Ralph H. Johnson Veterans Affairs Medical Center, and Departments of Medicine (Dagger  Nephrology and § Endocrinology Divisions) and  Pharmacology of the Medical University of South Carolina, Charleston, South Carolina 29425

Received for publication, November 30, 2000, and in revised form, January 25, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We used a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) to examine the regulation of the ubiquitous sodium-proton exchanger, Na+/H+ exchanger isoform 1 (NHE-1), by a prototypical G protein-coupled receptor, the bradykinin B2 receptor. Bradykinin rapidly activates NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry of quiescent cells and by 2'-7'-bis[2-carboxymethyl]-5(6)-carboxyfluorescein fluorescence measuring the accelerated rate of pHi recovery from an imposed acid load. The activation of NHE-1 is blocked by inhibitors of the bradykinin B2 receptor, phospholipase C, Ca2+/calmodulin (CaM), and Janus kinase 2 (Jak2), but not by pertussis toxin or by inhibitors of protein kinase C and phosphatidylinositol 3'-kinase. Immunoprecipitation studies showed that bradykinin stimulates the assembly of a signal transduction complex that includes CaM, Jak2, and NHE-1. CaM appears to be a direct substrate for phosphorylation by Jak2 as measured by an in vitro kinase assay. We propose that Jak2 is a new indirect regulator of NHE-1 activity, which modulates the activity of NHE-1 by increasing the tyrosine phosphorylation of CaM and most likely by increasing the binding of CaM to NHE-1.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ubiquitous isoform of the Na+/H+ exchanger (NHE-1)1 is essential for the regulation of cellular volume, growth, and intracellular pH and may regulate other functions such as bone resorption (1) or tumor invasiveness (2). Despite the potential importance of NHE-1 to many cellular functions, the processes involved in its regulation have not yet been fully elucidated. NHE-1 is nearly quiescent in resting cells but is activated by a variety of hormones, growth factors, and hypertonicity (3, 4). Growth factors activate NHE-1 by mechanisms that are either dependent or independent of phosphorylation of NHE-1 and/or NHE regulatory proteins. The phosphorylation reactions can be carried out by diverse kinases such as protein kinase C (5, 6), extracellular signal-regulated protein kinases family mitogen-activated protein kinases (7-10), phosphatidylinositol 3'-kinase (5), myosin light chain kinase (11), and ribosomal S6 protein kinase (12). The rapid activation of NHE-1 is usually associated with an increase in its phosphorylation (13), but deletion of the phosphorylation sites contained within residues 636-815 of NHE-1 only reduces its response to growth factors by about 50% (14). Those results suggest that mechanisms of regulation of NHE-1 that do not involve its direct phosphorylation are important.

The best characterized nonphosphorylation-dependent mechanism of regulation of NHE-1 involves Ca2+/calmodulin (CaM). NHE-1 has two calmodulin-binding domains. A high affinity CaM domain (residues 637-657) serves as an autoinhibitory domain (15, 16). The functional significance of the low affinity domain (residues 664-684) is uncertain. To this point, clear links between CaM, phosphorylation, and activation of NHE-1 have not been identified.

In this report, we studied the regulation of NHE-1 activity in the murine mIMCD-3 cell culture model of the kidney inner medullary collecting duct (17). Because cells in the inner medulla must be able to tolerate extremely high osmolarities, we reasoned that these cells would have highly responsive cell volume defense pathways including sodium-proton exchangers (18). In that regard, native cells from the inner medullary collecting duct have been shown to express three subtypes of sodium-proton exchangers (NHE-1, NHE-2, and NHE-4) on their basolateral surfaces (19), and mIMCD-3 cells contain mRNA for both NHE-1 and NHE-2 (20).

Previously, we provided physiological data suggesting that functional B2 receptors were present in the inner medullary collecting ducts of rats (21). Because B2 receptors have been shown to activate CaM (22, 23) and sodium-proton exchange (24), we felt that B2 receptors were excellent candidates for regulating NHE-1 in mIMCD-3 cells. Thus, we evaluated mIMCD-3 cells for the expression of functional B2 receptors and determined whether they were coupled to NHE activation in those cells. Further, we explored the interactions between CaM and phosphorylation reactions in the process of activation of sodium-proton exchange after stimulation of the B2 receptors. The data presented herein support a link between Jak2-dependent phosphorylation of CaM and activation of NHE-1 by bradykinin.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Fluo-3 and BCECF were purchased from Molecular Probes, Inc. (Eugene, OR). 5-(N-ethyl-N-isopropyl)-amiloride was purchased from RBI (Natick, MA). ET-18-OCH3, AG490, and D609 were from Biomol (Plymouth Meeting, PA). Tetramethylammonium (TMA) chloride, probenecid, calmidazolium, fluphenazine, W-7, phorbol 12-myristate 13-acetate, GF109203X, and various salts were from Sigma. PD98059 and anti-phosphotyrosine monoclonal antibody were from Calbiochem. HOE-140 (icatibant) and [des-Arg10]HOE-140 were from Hoechst AG (Frankfurt, Germany). Purified CaM, anti-CaM monoclonal antibody, anti-Jak2 agarose-conjugated antibody, and anti-phosphotyrosine polyclonal antibody were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-phosphospecific Jak2 antibody was from BIOSOURCE International (Camarillo, CA). Anti-NHE-1 and anti-NHE-2 polyclonal antibodies were from Chemicon International (Temecula, CA). All cell culture media and supplements were from Life Technologies, Inc. mIMCD-3 cells were purchased from the American Type Culture Collection (Manassas, VA). Polycarbonate cell culture inserts for microphysiometry and black 96-well microtiter plates needed for the FLIPR were from Corning Costar (Cambridge, MA). Black pipette tips were from Molecular Devices Corp. (Sunnyvale, CA).

Cell Culture-- The mIMCD-3 cell line is derived from the inner medullary collecting duct of an SV-40 transgenic mouse (17). This line retains many differentiated properties of terminal IMCD cells including the expression of an amiloride-inhibitable sodium channel, inhibition of apical to basal Na+ fluxes by atrial natriuretic peptide and amiloride, tolerance to high osmotic growth conditions, and accumulation of intracellular osmolytes in response to hypertonic stress. In addition, these cells form tight monolayers with high transepithelial resistance (~1350 ohms × cm2). This high electrical resistance feature was recently shown also to be exhibited in primary cultures of rat IMCD cells, where resistance exceeded 1000 ohms × cm2 (25). mIMCD-3 cells were grown in a mixture of Dulbecco's modified Eagle's medium and Ham's F-12 (equal parts) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml of streptomycin at 37 °C in 95% air and 5% CO2.

Microphysiometry-- The microphysiometer uses a light-addressable silicon sensor to detect extracellular protons (26). Each of eight channels has two inlet ports for buffers, one of which usually contains a vehicle control, and the other of which carries the test substance. The cells are perfused with buffer, and valve switches and stop-start cycles are totally controlled by a programmable computer. Acidification rate data are transformed by a personal computer running CytosoftTM version 2.0 and are presented as the extracellular acidification rate in µV/s, which roughly correspond to milli-pH units/min (Nernst equation). In order to facilitate comparison of data between two channels, values are expressed as a percentage of a base line determined by computerized analysis of the five data points prior to exposure of the cell monolayers to a test substance.

For all of the experiments, mIMCD-3 cells were plated onto polycarbonate membranes (3-µm pore size, 12-mm size) at a density of 300,000 cells/insert the night prior to experimentation. After cells were attached to the membranes, they were growth-arrested in serum-free culture medium for 20 h before the experiment. The day of the study, cells were washed with serum-free, bicarbonate-free Ham's F-12 medium, placed into the microphysiometer chambers, and perfused at 37 °C with the same medium or balanced salt solutions. For most studies, the pump cycle was set to perfuse cells for 60 s, followed by a 30-s "pump-off" phase, during which proton efflux was measured from the 8th through the 28th second. Cells were exposed to the test agent for three or four cycles (270-360 s). Valve switches (to add or remove test agents) were performed at the middle of the pump cycle. Data points were then acquired every 90 s. The peak effect during stimulation was expressed as the percentage increase from base line.

Isolation of Cellular RNA-- Total cellular RNA was isolated by a single-step guanidine isothiocyanate/phenol-chloroform extraction method (27) from cultured mIMCD-3 cells using the RNA STAT-60 reagent (TEL-TEST B, Inc., Tyler, TX). Cell monolayers were lysed directly in the culture dish by adding RNA STAT-60 and passing the cell lysate several times through a Pasteur pipette. The crude homogenates were allowed to stand at room temperature for 5 min. Chloroform was added to the homogenates (0.2 ml of chloroform/ml of RNA STAT-60). Samples were vortexed for 15 s and then allowed to stand at room temperature for 2-3 min. Samples were then centrifuged at 12,000 × g for 15 min at 4 °C, and the aqueous phase was removed into fresh tubes and extracted again with 1 ml of chloroform/isoamyl alcohol (24:1). Samples were recentrifuged as in the previous step, the aqueous phase was removed, and the RNA was precipitated by the addition of isopropyl alcohol (0.5 ml of isopropyl alcohol/ml of the RNA STAT-60 used for sample homogenization). Samples were stored at room temperature for 5-10 min and then centrifuged at 12,000 × g for 10 min. The well-formed white pellet was washed using 75% ethanol by vortexing the mixture vigorously collected by subsequent centrifugation at 7,500 × g for 10 min. The pellet was air-dried for 20-30 min and then was dissolved in RNase-free water. The integrity of the RNA was tested directly by agarose gel electrophoresis.

Ribonuclease Protection Assay-- Ribonulease protection assay was carried out following the manufacturer's instructions provided with the Ambion RPA II kit. In brief, 20 µg of sample RNA was hybridized with 50 × 104 cpm of antisense riboprobe in hybridization buffer (80% deionized formamide/100 mM sodium citrate (pH 6.4), 300 mM sodium acetate (pH 6.4), and 1 mM ethylene diamine tetraacetic acid) for 16-20 h at 48 °C. After hybridization, excess single-strand riboprobe and the unhybridized portion of sample RNA was digested away with ribonucleases A and T1 mixture (Solution Bx, Ambion) supplemented with 1% blue coprecipitant solution (Ambion) to visualize the pellet. The reaction mixture was incubated at 34 °C for 30 min. The ribonucleases were inactivated, and protected double-stranded RNA was precipitated in a single step by adding Solution Dx (Ambion) and incubating at -20 °C for 20 min. Samples were then centrifuged for 15 min, and the pellets were air-dried and reconstituted in 8 µl of gel loading buffer (8% sucrose, 0.025% bromphenol blue, 0.025% xylene cyanol). The protected segments were isolated on a 5% native polyacrylamide gel. Gels were dried and exposed to Kodak X-Omat AR films for 4-16 h. beta -Actin or glyceraldehyde-3-phosphate dehydrogenase was used to normalize the mRNA loading on the gel. Dried gels were also exposed to a PhosphorImager intensifying screen for the same period for further analysis by the Storm 860 imaging system. (Molecular Dynamics, Inc., Sunnyvale, CA). Densitometric analysis was performed using ImageQuantTM supplied by the same manufacturer.

Measurement of Intracellular Ca2+-- We used a FLIPRTM fluorescence laser imaging plate reader (28) to measure intracellular Ca2+ ([Ca2+]i) in mIMCD-3 cells plated into 96-well microtiter plates. Cells were seeded (~50,000 cells/well) in 96-well clear bottom black microplates (Corning Costar) and left overnight in CO2 incubator at 37 °C. On the day of assay, cells were loaded with 4 µM Fluo-3 for 1 h in Hanks' balanced salt solution, pH 7.4, containing 20 mM HEPES and 2.5 mM probenecid. After loading, cells were washed four times with Hanks' balanced salt solution on an automated plate washer (Labsystems, Helsinki, Finland). The plates were then placed into the FLIPR. The 96 wells were simultaneously illuminated for 0.4 s by an argon laser (488 nm) set at ~0.3 watts (Coherent Inc., Santa Clara, CA). Fluorescence emission readings were obtained using a 540-nm bandpass filter at 1-s intervals until a base line was obtained (about 10 readings). Then cells were simultaneously exposed to a range of BK concentrations (10-5 to 3 × 10-10 M) in the presence or absence of B1 ([des-Arg10]HOE-140) or B2 (HOE-140) antagonists (29). Fluorescence readings were then obtained from each well every second for a total of 2 min and then every 6 s for 2 min. Replicate readings were averaged. Each tracing was normalized using background fluorescence from wells treated with buffer controls. Tracings were acquired, averaged, and normalized using the FLIPRTM Control software program (Molecular Devices). Peak values from each tracing were then used to construct concentration-response plots for the various conditions.

Measurement of Intracellular pH-- The FLIPRTM was also used to measure pHi in mIMCD-3 cells plated into 96-well microtiter plates. Cells were treated as described above and then were loaded with a dye (5 µM BCECF) in a loading buffer (Hanks' balanced salt solution, pH 7.4, containing 20 mM HEPES, 2.5 mM probenecid, and 1% fetal bovine serum) for 1 h at 37 °C. 20 min before the end of the loading phase, 20 mM NH4Cl was added to each well. Cells were then washed four times with the loading buffer containing 20 mM NH4Cl. In the FLIPR, cells were acid-loaded by an ammonium chloride prepulse protocol. In this method, the extracellular buffer contains NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and NH3 in an equilibrium that is essentially recapitulated in the cell interior. When the extracellular medium is changed to a buffer lacking NH4Cl, intracellular NH3 diffuses rapidly out of the cell, causing the cells to become acutely "loaded" with protons donated from NH4 (30). Some cells were pretreated with 100 nM HOE-140 (B2 receptor antagonist) or with 100 nM [des-Arg10]HOE-140 (B1 receptor antagonist) for 10 min. Cells were allowed to recover from the acid load in the presence and absence of sodium and various test substances. Fluorescence excitation was obtained using an argon laser (488-nm wavelength at 300 milliwatts), and emission (>540 nm) was monitored kinetically. Calibration of the BCECF fluorescence to the intracellular pH was performed on similar 96-well plates using the electroneutral H+/K+ ionophore, nigericin. For calibration, cells were equilibrated with buffers of varied pH and 130 mM of KCl (the estimated intracellular concentration of K+). Calibration plots were then generated by equalizing intracellular and extracellular pH by adding 20 µM nigericin to cells in various pH buffers (31).

Immunoprecipitation-- Quiescent mIMCD-3 cell monolayers were treated with 100 nM bradykinin or vehicle for 10 min, lysed in 1 ml/100-mm dish of radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% Nonidet P-40, 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and pepstatin at 1 µg/ml each). Cell lysates were precleared by incubating with a protein A-agarose bead slurry for 30 min at 4 °C. Precleared lysates (1 µg/µl total cell protein) were incubated with anti-Jak2/protein A-agarose, with anti-NHE-1 or anti-NHE-2 antibody or with polyclonal anti-phosphotyrosine antibody overnight at 4 °C. Phosphotyrosine, NHE-1, and NHE-2 immunoprecipitates were captured by the addition of protein A-agarose. The agarose beads were collected by centrifugation, washed three times with radioimmune precipitation buffer, resuspended in 2× Laemmli sample buffer, boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis and subsequent immunoblot analysis with monoclonal anti-CaM or anti-phosphotyrosine IgG. The same Western blots were reprobed with the antibody used for immunoprecipitation to assure that equal amounts of protein were loaded in each lane.

Jak2 Phosphorylation Assay-- Phosphorylation of Jak2 in response to BK was assessed using a Jak2 dual phosphospecific antibody. Quiescent cells were treated with 100 nM BK for 10 min and lysed in radioimmune precipitation buffer. The lysates were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions with 4-20% precast gels (Novex, San Diego, CA). After semidry transfer to polyvinylidine difluoride membranes, membranes were blocked with a Blotto buffer and incubated with the phospho-Jak2 antibody (0.5 µg/ml). After incubation with alkaline phosphatase-linked secondary antibodies, immunoreactive bands were visualized by a chemiluminescent method (CDP StarTM, New England Biolabs, Beverly, MA) using preflashed Eastman Kodak Co. X-AR film and quantified using a GS-670 densitometer and Molecular Analyst software (Bio-Rad). To make sure that equal amounts of protein were loaded in each lane, blots were stripped and reprobed with control Jak2 antibody, which recognizes equally well phosphorylated and nonphosphorylated Jak2.

Jak2 in Vitro Kinase Assay-- 10 µl of Jak2 enzyme-agarose complex was incubated in kinase assay buffer (50 mM NaCl, 10 mM HEPES, pH 7.4, 5 mM MgCl2, 5 mM MnCl2, 0.1 mM sodium orthovanadate), containing 1 µCi/µl [gamma -32P]ATP and 4 µg of purified CaM for different periods of time (5, 10, 15, and 30 min) at 30 °C. The reactions were stopped by adding 50 µl of 2× Laemmli sample buffer and boiling for 5 min. Samples were resolved by SDS-polyacrylamide gel electrophoresis, and radiolabeled bands were visualized by autoradiography. Data analysis was performed with the Storm 860 Imagine system (Molecular Dynamics) using ImageQuantTM software.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

mIMCD-3 Cells Express Bradykinin B2 Receptors-- In order to demonstrate the presence of BK receptor mRNA in mIMCD-3 cells, we used an RNase protection assay. Using an antisense riboprobe of ~400 nucleotides and total RNA isolated from mIMCD-3 cells, we obtained a single protected band on the autoradiogram (Fig. 1A). To confirm that the protected band corresponds to B2 receptor mRNA, we hybridized increasing quantities of in vitro transcribed sense strand B2 receptor mRNA with this riboprobe. The intensity and the size of the protected band confirmed the presence of B2 receptor mRNA in mIMCD-3 cells and suggested that receptor protein might also be expressed in those cells.


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Fig. 1.   Ribonuclease protection assay demonstrating the presence of B2 receptor mRNA in mIMCD-3 cells. A, autoradiogram obtained from RNase protection assay using increasing quantities of RNA isolated from mIMCD-3 cells. Antisense RNA corresponding to ~400 nucleotides from the coding region of B2 receptor was employed as a riboprobe. B, autoradiogram obtained from RNase protection assay using increasing quantities of in vitro transcribed sense B2 receptor mRNA shows a linear relationship between the amount of B2 receptor mRNA and the intensity of the protected band.

mIMCD-3 Cells Express a Sodium-dependent Proton Efflux Pathway-- We used a CytosensorTM microphysiometer (Molecular Devices) to measure proton efflux from intact monolayers of mIMCD-3 cells plated onto polycarbonate membranes. The microphysiometer was designed to measure the rate of production of extracellular protons in the monolayers during stop-flow conditions by assessing in real time the rate of decrease in extracellular pH in intact cells (26). We have previously used the microphysiometer to specifically study Na+/H+ exchange in fibroblasts (34, 35) and enterocytes (36). Fig. 2A shows that mIMCD-3 cells exposed to a sodium-free balanced salt solution (in which TMA is substituted for sodium) establish a relatively stable basal rate of proton efflux (extracellular acidification rate (ECAR)). When the perfusate is switched to a sodium-replete solution (in which sodium is substituted mM per mM for TMA), the mIMCD-3 cells produce a transient but vigorous burst of proton efflux, which peaks at 500% of basal values. This burst of sodium-dependent proton efflux could be blocked by preincubation with 1 µM EIPA (ethylisopropylamiloride), which is an inhibitor of sodium-proton exchangers types 1 and 2 (NHE-1 and NHE-2).


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Fig. 2.   mIMCD-3 cells express a sodium-dependent proton efflux pathway as determined by proton microphysiometry (A) and BCECF fluorescence measurements (B). Microphysiometry was performed on quiescent mIMCD-3 cell monolayers as described under "Experimental Procedures." A, cells exposed to a Na+-free balanced salt solution in which TMA is substituted for Na+ established a relatively stable basal rate of H+ efflux (ECAR). When the perfusate is switched to a Na+-replete solution (in which Na+ is substituted mM per mM for TMA), the cells produced a transient but vigorous burst of H+ efflux, which peaked at ~500% of basal values. This burst of Na+-dependent H+ efflux could be blocked by preincubation with 1 µM EIPA (ethylisopropylamiloride). B, cells were acid-loaded by the ammonium chloride prepulse method as described under "Experimental Procedures" and then were allowed to recover in the presence and absence of sodium or in the presence of 1 µM EIPA. Plots presented in A and B are representative of at least four independent experiments, each of which had similar results.

Fig. 2B shows results of a protocol in which an acid load was imposed upon mIMCD-3 cells by the ammonium chloride prepulse method. After removal of the ammonium chloride from the salt solution, intracellular pH dropped from a resting pHi of ~7.24 to 6.67. In the presence of sodium, pHi recovered close to base line (>7.1) in ~8 min. Most of the recovery of pHi could be blocked by removing sodium from the balanced salt solution or by including 1 µM EIPA in sodium-replete recovery solution. Although a small sodium-independent and EIPA-resistant recovery of pHi did occur (to pHi of ~6.75 after 1 min of recovery and 6.8 after 8 min of recovery), >80% of the recovery appeared to be mediated by NHE-1 or NHE-2 as evidenced by its dependence on sodium and sensitivity to EIPA.

BK Stimulates a Sodium-dependent, EIPA-inhibitable Proton Efflux Pathway in mIMCD-3 Cells through a B2 Receptor-- Fig. 3A shows that cells treated with 100 nM BK (open circles) had a rapid increase in extracellular acidification rate that did not occur when cells were exposed to vehicle (black circles) during the valve switch. The magnitude of the response depended upon the concentration of BK, as shown in Fig. 3B, with half-maximal values occurring at 30-100 nM BK. Fig. 3C shows that the stimulatory effect of BK occurred in sodium-containing Ham's F-12 medium or in sodium-containing balanced salt solution but not in a solution in which TMA was substituted for sodium. The effect could also be blocked by EIPA. Fig. 3D shows studies in which two specific BK receptor antagonists were examined for inhibition of BK-stimulated ECAR in mIMCD-3 cells. These studies showed that the receptor is a B2 receptor because proton efflux was blocked by the B2 receptor antagonist (HOE-140, icatibant) but not by the B1 receptor antagonist [des-Arg10]HOE-140. Thus, Fig. 3 presents evidence that the B2 receptor in mIMCD-3 cells activates proton efflux through stimulation of a NHE (probably NHE-1 or NHE-2).


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Fig. 3.   ECAR measurements from mIMCD-3 cell monolayers were obtained as described under "Experimental Procedures." A, bradykinin (white circles) stimulates ECAR, whereas vehicle (dark circles) does not. Cells were exposed to perfusate containing bradykinin (BK) or vehicle during the time span encompassed by the two-headed arrow and gray box at the top. B, bradykinin stimulates ECAR in a concentration-dependent manner. Each white circle represents the peak reading within the first 6 min of bradykinin exposure. C, ECAR stimulated by 100 nM in various buffers, including Ham's F-12 medium without and with 1 µM EIPA, a balanced salt solution containing NaCl or TMA substituted mM per mM for sodium. D, effects of B1 (HOE-140, icatibant) and B2 ([des-Arg10]HOE-140) receptor antagonists on BK-stimulated ECAR. Antagonists were added 30 min prior to the addition of BK. All experiments were performed at least four times. Error bars in C and D represent the S.E.

BK Stimulates a Sodium-dependent, EIPA-inhibitable pHi Recovery Pathway in mIMCD-3 Cells through a B2 Receptor-- Fig. 4A shows that 100 nM BK accelerated the rate of recovery from an acid load and that this effect was inhibitable by EIPA. In the presence of BK, pHi rose to ~7.0 in the first minute and to >7.25 after 8 min, whereas in the absence of BK it rose to ~6.8 in the first minute and to ~7.15 after 8 min. Fig. 4B shows that the B2 receptor antagonist, icatibant, but not the B1 receptor antagonist, [des-Arg10]HOE-140, blocked the increased rate of recovery of pHi induced by BK. Thus, Fig. 4 presents evidence that the B2 receptor in mIMCD-3 cells activates a sodium-dependent, EIPA-inhibitable pHi recovery pathway probably through stimulation of NHE-1 or NHE-2.


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Fig. 4.   BK accelerates the rate of recovery of pHi from an acid load. Cells were acid-loaded by the ammonium chloride prepulse method as described under "Experimental Procedures" and then were allowed to recover in the presence and absence of 100 nM BK with or without 1 µM EIPA (A) or a 100 nM concentration of of specific inhibitors of B1 ([des-Arg10]HOE-140) or B2 (HOE-140) receptors (B). The inhibitors were added at the same time as BK. The plots are representative of at least six tracings for each condition, all of which showed qualitatively similar results.

Inhibitor Studies Suggest the Involvement of Phospholipase C in BK-induced Increases in ECAR-- Fig. 5 shows studies in which various inhibitors were examined for effects on BK-stimulated ECAR in mIMCD-3 cells. These studies showed that the response is not sensitive to pertussis toxin. Two phospholipase C (PLC) inhibitors (D609 and ET-18-OCH3) blocked proton efflux, suggesting that the B2 receptor couples to PLC in mIMCD-3 cells (Fig. 5A). Similar results were obtained with another PLC inhibitor, U73122 (not shown). In contrast, PI-3'-kinase inhibitors (20 nM wortmannin and 50 µM LY294002) had no effect (not shown). Because PLC activation can lead to stimulation of PKC through the intermediate actions of diacylglycerol, we tested the effects of a PKC inhibitor (GF109203X) on BK-induced proton efflux. GF109203X (1 µM) did not attenuate BK-induced proton efflux but was able to inhibit PMA (1 µM)-elicited proton efflux. Those studies showed that a PKC inhibitor in a concentration that was adequate to block ECAR induced by direct activation of PKC by PMA did not block B2 receptor-elicited ECAR. That result is consistent with the noninvolvement of PKC in B2 receptor-elicited ECAR in mIMCD-3 cells. PLC activation also leads to increases in intracellular Ca2+, which in turn can stimulate a number of cellular signaling targets. Thus, we next established that B2 receptors increase intracellular Ca2+ in mIMCD-3 cells.


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Fig. 5.   Effects of various inhibitors on the activation of ECAR. ECAR was measured by microphysiometry as described under "Experimental Procedures." Cells were preincubated with pertussis toxin (PTX; 200 ng/ml) overnight and with other inhibitors for 30 min prior to stimulation with 100 nM BK (A) or 1 µM PMA (B). D609 and ET-18-OCH3 (ET-18) are inhibitors of phospholipase C, and GF109203X (GF) is an inhibitor of PKC. All experiments were performed at least four times. Error bars represent S.E.

Confirmation that BK Increases [Ca2+]i through a B2 Receptor in mIMCD-3 Cells-- We used a FLIPRTM plate reader to simultaneously measure [Ca2+]i in mIMCD-3 cells plated into 96-well microtiter plates. Those experiments (Fig. 6A) show that BK induced a rapid and sustained elevation of intracellular Ca2+ in mIMCD-3 cells preloaded with 10 µM Fluo-3, a Ca2+-sensitive fluorescent dye. The BK signal was dependent on concentration, with a half-maximal effect (EC50) at ~10 nM. Co-incubation with the B1 receptor antagonist, [des-Arg10]HOE-140 (100 nM) had no effect on either the Vmax or EC50. In contrast, inclusion of 10 nM icatibant nearly eliminated the Ca2+ transient initiated by 100 nM BK and shifted the EC50 of BK to the right without affecting the Vmax. Graded concentrations of icatibant shifted the EC50 further rightward and also induced graded decrements in Vmax. This configuration of plots is classical for competitive antagonism and provides very strong pharmacological evidence that the effect is mediated by B2 (and not B1) receptors.


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Fig. 6.   Involvement of Ca2+/calmodulin in B2 receptor signaling in mIMCD-3 cells. A, increases in intracellular calcium correspond to the B2 receptor subtype. Calcium fluxes were measured using the FLIPR to detect changes in Fluo-3 fluorescence as described under "Experimental Procedures." Individual values expressed in Delta  fluorescence units were normalized to the maximal increase in calcium detected in the first 2 min of exposure to 10 µM BK. [des-Arg10]HOE-140 is a B1 receptor antagonist, and HOE-140 (icatibant) is a B2 receptor antagonist. Cells were preincubated with antagonists for 15 min prior to the addition of BK. The experiment shown in A is representative of four experiments performed in eight replicates in which very similar results were obtained. B shows the effects of various CaM inhibitors on the activation of ECAR by 100 nM BK. ECAR was measured by microphysiometry as described under "Experimental Procedures." Cells were preincubated with CaM inhibitors for 30 min prior to the addition of BK. CMDZ, calmidazolium; Fluphen, fluphenazine. Experiments in this panel were performed at least three times in duplicate or triplicate. Error bars represent S.E.

Inhibitor Studies Suggest the Involvement of CaM in BK-induced Increases in ECAR-- One target of increased intracellular calcium is CaM, which regulates a variety of kinases, phosphodiesterases, and other effectors. CaM also has been shown to regulate the activity of sodium proton exchangers (37), so we tested the effects of three structurally distinct CaM inhibitors on BK-activated proton efflux. Those experiments (Fig. 6B) showed that W-7 (50 µM), calmidazolium (5 µM), and fluphenazine (10 µM) each blocked the stimulation of proton efflux elicited by 100 nM BK by ~80%. Thus, these studies support a role for CaM in the stimulatory effect of BK on NHE in mIMCD-3 cells.

Role for Janus Kinase 2 in BK-induced Increases in ECAR-- The involvement of Jak2 in regulating ECAR in mIMCD-3 cells was supported by studies with tyrosine kinase inhibitors (Fig. 7). A broad spectrum tyrosine kinase inhibitor, genistein (50 µM), markedly (75%) attenuated the ability of 100 nM BK to induce ECAR, while an inactive analog of genistein (daidzein) was without effect. AG490 (50 µM), a specific inhibitor of nonreceptor tyrosine kinase Jak2 (38) blocked the BK-induced ECAR by 80%. AG1478, an EGF receptor tyrosine kinase inhibitor, did not prevent the stimulation of ECAR by BK but completely blocked EGF-induced ECAR. We also determined that the Ras-extracellular signal-regulated protein kinase pathway inhibitor (20 µM apigenin) and the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 (50 µM) were without effect on BK-stimulated ECAR (not shown). Thus, this result suggests the involvement of the nonreceptor tyrosine kinase Jak2 in the regulation of NHE in mIMCD-3 cells. In order to confirm the involvement of Jak2 in BK-mediated signaling, we measured the activation of Jak2 by BK using Western blotting with a phosphospecific anti-Jak2 antibody. Reprobing of the same blots with a control Jak2 antibody, which recognizes Jak2 independent of its phosphorylation state showed equal amounts of Jak2 in the samples (data not shown). BK treatment (100 nM for 10 min) induced a ~100% increase in the level of phosphorylation of Jak2 (Fig. 8A). Maximum phosphorylation of Jak2 occurred after 5-10 min of incubation with BK and persisted for at least 20 min. Increased phosphorylation of Jak2 was detectable well after the BK-induced elevation of intracellular Ca2+ (Fig. 8B). This is consistent with a role for elevation of intracellular Ca2+ leading to Jak2 activation. We tested this possibility by exposing cells to a calcium ionophore, A23187 (1 µM), which leads to elevations of intracellular Ca2+ by non-receptor-dependent mechanisms. Fig. 8C shows that exposure of mIMCD-3 cells to A23187 for 5 min induces an increase of ~75% in the phosphorylation of Jak2.


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Fig. 7.   Involvement of tyrosine kinases in B2 receptor signaling in mIMCD-3 cells. A, ECAR was measured by microphysiometry as described under "Experimental Procedures." Tyrosine kinase inhibitors were added 30 min prior to stimulation with 100 nM BK (A) or with 10 ng/ml epidermal growth factor (EGF; B). Daidz, daidzein; Genist, genistein. Experiments in this panel were performed at least three times in duplicate or triplicate. Error bars represent S.E.


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Fig. 8.   BK and increased intracellular Ca2+ induce tyrosine phosphorylation of Jak2. The phosphorylation state of Jak2 was determined in whole cell lysates from mIMCD-3 cells using a phosphorylation state-specific antibody for Jak2 in an immunoblot as described under "Experimental Procedures." A, cells were treated with 100 nM BK or vehicle, lysed, and subjected to immunoblot. Inset, a representative immunoblot. B, the phosphorylation of Jak2 induced by BK is dependent upon the time of exposure to BK. The inset shows that the BK-induced Ca2+ transients occur prior to detectable phosphorylation of Jak2 by BK. C, cells incubated with the calcium ionophore A23187 (1 µM) for 5 min show induction of Jak2 phosphorylation similar in magnitude to that induced by BK. All experiments were repeated at least three times. Error bars represent S.E.

Convergence of the CaM and Jak2 Signals-- The next series of experiments was designed to probe possible interactions between CaM and Jak2 in the pathway of activation of NHE by expanding on the results shown in Fig. 8C. We explored the possibility of a BK-induced physical interaction between Jak2 and CaM by using immunoprecipitation of lysates from cells treated with vehicle or BK with anti-Jak2/protein A-agarose followed by Western blotting with a CaM antibody. Fig. 9A shows that CaM and Jak2 are co-precipitated and that this can be increased by treatment of mIMCD-3 cells with BK. Pretreatment of the cells with AG490 (50 µM for 30 min) significantly decreased the amount of CaM in Jak2 immunoprecipitates (Fig. 9B), suggesting that BK-induced Jak2 activity is necessary for the formation of the complex that includes CaM and Jak2.


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Fig. 9.   Relationship between Jak2 and CaM in BK-induced signaling complex. Co-immunoprecipitations were performed as described under "Experimental Procedures." A, co-immunoprecipitation experiments show that 100 nM BK induces a complex that includes CaM and Jak2. Formation of this complex is prevented by preincubation with AG490 (50 µM) for 30 min (B). C shows that BK increases the CaM present in phosphotyrosine (PY) immunoprecipatates. The left inset shows a band of the same relative mobility as CaM on a phosphotyrosine immunoblot from phosphotyrosine immunoprecipitates. Immunoprecipitation was performed with polyclonal phosphotyrosine antibody, whereas monoclonal phosphotyrosine antibody was used for immunoblotting. The blot was stripped and reprobed with a CaM antibody (right inset) to confirm that CaM is tyrosine-phosphorylated in response to BK. D shows that purified Jak2 phosphorylates purified CaM in a time-dependent manner in an in vitro phosphorylation assay. IP, immunoprecipitation; IB, immunoblot.

We also tested the hypothesis that BK stimulates tyrosine phosphorylation of CaM, because Jak2 is known to activate downstream signals by phosphorylating substrates such as Stat1 and Stat3. Cells were treated with 100 nM BK or vehicle for 10 min, lysed, and immunoprecipitated with either a polyclonal phosphotyrosine antibody or with anti-Jak2/protein A-agarose as described under "Experimental Procedures." Subsequent immunoblot analyses were performed with monoclonal anti-CaM and anti-phosphotyrosine immunoglobulins. The data presented in Fig. 9C confirm that CaM becomes tyrosine-phosphorylated in response to BK treatment. Pretreatment of the cells with AG490 (50 µM for 30 min) blocked tyrosine phosphorylation of CaM in response to BK treatment (not shown), suggesting that this phosphorylation is caused by Jak2, although those experiments do not prove that CaM is a substrate for Jak2. Fig. 9D shows the results of experiments in which purified CaM was used as a substrate for purified Jak2 in an in vitro phosphorylation assay. Those experiments showed that Jak2 can phosphorylate CaM in a time-dependent manner. Thus, CaM can be a direct substrate for phosphorylation by Jak2.

Presence of NHE-1 in CaM Immunoprecipitates-- In order to establish whether NHE-1, NHE-2, or both were present in the BK-induced signaling complex, we performed co-immunoprecipitation experiments in which NHE-1 and NHE-2 were immunoprecipitated from lysates of mIMCD-3 cells treated or not treated with BK. Immunoprecipitates were next probed with CaM antibodies.

Fig. 10 shows that immunoblots document the presence of CaM in NHE-1 immunoprecipitates and further shows that the amount of CaM complexed with NHE-1 increases after treatment of mIMCD-3 cells with 100 nM BK (Fig. 10A). NHE-2 immunoprecipitates also show the presence of CaM, which is not surprising because many of the amino acids in the high affinity CaM binding site of NHE-1 are conserved in NHE-2 (4). However, there was no change in the amount of CaM in NHE-2 immunoprecipitates after BK treatment (Fig. 10B). When the same blots were stripped and reprobed with NHE-1 or NHE-2 antibodies, equal amounts of NHE-1 and NHE-2 appeared in the samples from cells treated or not treated with BK (data not shown).


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Fig. 10.   BK induces a complex of CaM and NHE-1 but not NHE-2. Co-immunoprecipitations were performed as described under "Experimental Procedures." A shows that treatment with 100 nM BK for 10 min increases the amount of CaM in NHE-1 immunoprecipitates. The same experiment with NHE-2 does not show any significant change in CaM contents after BK treatment (B). IP, immunoprecipitation; IB, immunoblot.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The current work shows that B2 receptors endogenous to mIMCD-3 cells activate NHE-1 activity through a pathway that involves PLC, elevated intracellular Ca2+, CaM, and Jak2 (Fig. 11). What is new about this work is that 1) BK stimulates the assembly of a signal transduction complex that includes CaM, Jak2, and NHE-1; 2) Jak2 is involved in the activation of NHE-1; 3) the regulation of NHE-1 by Jak2 involves tyrosine phosphorylation of CaM; and 4) CaM appears to be a direct substrate for phosphorylation by Jak2. Our studies identify a new regulator of NHE-1 activity (Jak2) and point to a regulatory relationship with CaM, which has been shown previously to be a key regulator of NHE-1 activity through binding interactions with two CaM domains of NHE-1 (15, 16).


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Fig. 11.   Scheme of putative signal transduction pathway used by BK to activate NHE-1 in mIMCD-3 cells. B2R, bradykinin type 2 receptor; P, phosphorylation.

The kidney inner medullary collecting duct plays major roles in volume and acid-base homeostasis. We have previously presented evidence that B2 receptors regulate chloride and water transport in medullary collecting ducts of rats (21). The current studies present evidence for functional B2 receptors in immortalized cultures of murine inner medullary collecting duct cells (mIMCD-3). They also support a potential role for B2 receptors in the regulation of sodium-proton exchange in these cells. This latter finding is not surprising in that bradykinin (BK) has been shown to induce alkalization of rat IMCD cells (39). BK was also previously shown to stimulate NHE-1 activity in renal tubular epithelial cells (24).

In our study, mIMCD-3 cells treated with 100 nM BK had a rapid increase in ECAR as measured by microphysiometry. The evidence that the B2 receptor is involved in this effect is based on the presence of mRNA of the B2 receptor in cultured mIMCD-3 cells and on pharmacological studies with specific antagonists. Moreover, BK activates sodium proton exchange in these cells based on the existence of BK-stimulated, EIPA-inhibited, and sodium-dependent proton efflux and pHi recovery pathways. The burst of Na+-dependent H+ efflux in response to BK could be blocked by preincubation with 1 µM EIPA, which is an inhibitor of sodium-proton exchangers types 1 and 2 (NHE-1 and NHE-2). At this concentration, it would be expected to block the activity of NHE-1 completely and NHE-2 partially (37), suggesting that B2 receptor most likely activates NHE-1. The data presented in Fig. 10 also support the involvement of NHE-1 rather than NHE-2 in this process, in that BK increases complexation of CaM with NHE-1 (but not with NHE-2).

We also attempted to delineate other components of the B2 receptor-activated signaling pathway that leads to the activation of NHE-1 in mIMCD-3 cells. The response was not sensitive to pertussis toxin, showing the lack of involvement of pertussis toxin-sensitive G proteins. Most likely, the B2 receptor acts through Galpha q, which has been shown to activate amiloride-sensitive Na+/H+ exchanger (40). We found that NHE-1 activation was suppressed by two different PLC inhibitors (D609 and ET-18-OCH3), suggesting that the B2 receptor couples to PLC in mIMCD-3 cells. Because PLC activation could lead to stimulation of PKC through the intermediate actions of diacylglycerol, we tested the effects of a PKC inhibitor (GF109203X) on BK-induced proton efflux. This inhibitor did not block B2 receptor-elicited ECAR but was able to inhibit PMA-induced activity of NHE, suggesting the noninvolvement of PKC in BK-induced NHE activation. PLC activation also leads to increase in intracellular Ca2+, which in turn can stimulate different signaling targets inside the cell. One such target is CaM, so we used several structurally different CaM inhibitors to study its possible role in the BK-induced activation of NHE. Structurally distinct CaM inhibitors blocked the stimulation of ECAR elicited by 100 nM BK by ~80%. A potential role for CaM is not surprising, because NHE-1 possesses CaM-binding sites that are critical for growth factor-stimulated activity of the exchanger (15).

We used several tyrosine kinase inhibitors to study the possible role of tyrosine kinases in the BK-induced activation of NHE. A broad spectrum tyrosine kinase inhibitor, genistein, but not its inactive analog (daidzein) blocked BK-stimulated ECAR. AG1478, an EGF receptor tyrosine kinase inhibitor, had no effect on the BK-induced activation of NHE but could block EGF-induced ECAR in mIMCD-3 cells. The specific Jak2 inhibitor (AG490) effectively blocked BK-stimulated ECAR, supporting a surprising role for Jak2 in the regulation of NHE in mIMCD-3 cells. The Jak family of nonreceptor tyrosine kinases induce gene regulation through the signal transducers and activators of transcription (41), although Jak2 may regulate other signal transduction functions (38). Our current work suggests that Jak2 regulates NHE-1 by phosphorylating CaM and modulating the interactions of CaM with NHE-1.

The most novel finding presented in this report is that Jak2 regulates the function of NHE-1 through tyrosine phosphorylation of CaM. It should be noted that CaM has not previously been shown to be a substrate for Jak2. Further, the information in Fig. 11 suggests that CaM is a direct substrate for tyrosine phosphorylation by Jak2. CaM has previously been shown to be as substrate for phosphorylation by both tyrosine kinases and serine-threonine kinases (42-44). Activation of the insulin receptor (a receptor tyrosine kinase) has been shown to result in phosphorylation of Tyr-99 and Tyr-138 of CaM in CHO-IR cells (45, 46). The EGF receptor (another receptor tyrosine kinase) phosphorylates Tyr-99 of bovine brain CaM (47, 48) with a stoichiometry of 1:1 (49). In contrast, casein kinase II, an insulin-sensitive nonreceptor kinase, phosphorylates CaM in vitro on serine and threonine residues (Thr-79, Ser-81, Ser-101, and Thr-117) (42). Our current study presents new information that documents that activation of a G protein-coupled receptor (B2 receptor for BK) also can result in phosphorylation of CaM. Previously Fukami et al. (50) proposed, using in vitro experiments, that Ca2+ may down-regulate the level of tyrosine phosphorylation of CaM in Rous sarcoma virus-transformed cells. In contrast, our results have led us to suggest that elevated intracellular Ca2+ contributes to the activation of Jak2 and the subsequent phosphorylation of CaM.

There is no consensus on the effects of phosphorylation of CaM on its ability to interact with and activate its downstream targets. Our work suggests that tyrosine phosphorylation of CaM results in increased binding to and activation of NHE-1. Unlike the current study, Fukami et al. (50) previously suggested that Ca2+-dependent phosphorylation of CaM may attenuate its function in vivo. Phosphorylation of CaM on Tyr-99 was shown to selectively attenuate the action of CaM antagonists on type I cyclic nucleotide phosphodiesterase activity (51). In contrast, phosphorylation of Tyr-99 increases the affinity of CaM for Ca-ATPase (52). In order to address that discrepancy, Corti et al. (44) studied the effects of CaM phosphorylated on Tyr-99 on the binding affinities and activation of six different CaM target enzymes (myosin light chain kinase MLCK, 3'-5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric-oxide synthase, and type II Ca2+/CaM-dependent protein kinase). They concluded that tyrosine phosphorylation of CaM Tyr-99 generally led to an increase in the ability of CaM to activate its targets. For three of the enzymes (3'-5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, and type II Ca2+/CaM-dependent protein kinase), the primary effect was a decrease in the concentration at which half-maximal activation was attained. In contrast, for calcineurin and neuronal nitric-oxide synthase, phosphorylation of CaM significantly increased the Vmax. For MLCK, however, tyrosine phosphorylation of CaM had no effect (44). Thus, the idea that tyrosine phosphorylation of CaM results in increased binding to and activation of NHE-1 is supported by precedent with other CaM-activated enzymes.

We acknowledge that our results do not rule out the possibility that NHE-1 is also phosphorylated in response to BK. Although tyrosine phosphorylation of the exchanger has not been reported yet, there are two tyrosine residues in the CaM-binding domain of NHE-1 (Tyr-682 and Tyr-648) that could be potential substrates for phosphorylation by Jak2. Further work will be required to show if BK-stimulated Jak2 is efficient in phosphorylating NHE-1 and whether this phosphorylation contributes to BK-mediated activation of the exchanger.

In summary, we used a cultured murine cell model (mIMCD-3) of the inner medullary collecting duct to examine the regulation of NHE-1 activity by the bradykinin B2 receptor, a prototypical G protein-coupled receptor. The current work shows that B2 receptors endogenous to mIMCD-3 cells activate NHE-1 activity through a pathway that involves PLC, elevated intracellular Ca2+, CaM, and Jak2 (Fig. 11). BK rapidly stimulates the assembly of a signal transduction complex that includes CaM, Jak2, and NHE-1. We suggest that Jak2 is involved in the activation of NHE-1 by increasing the tyrosine phosphorylation of CaM, which appears to be a direct substrate for phosphorylation by Jak2. Thus, our studies identify a new indirect regulator of NHE-1 activity (Jak2), which regulates the activity of NHE-1 by modulating the binding of CaM and NHE-1.

    FOOTNOTES

* This work was supported by grants from the Department of Veterans Affairs (Merit Awards to M. N. G., D. W. P., and J. R. R. and a REAP Award to Y. V. M., A. A. J., J. R. R., and M. N. G.), National Institutes of Health Grants DK52448 (to J. R. R.) and K01-DK02694 (to Y. V. M.), laboratory endowments jointly supported by the Medical University of South Carolina Division of Nephrology and Dialysis Clinics, Incorporated (to D. W. P. and J. R. R.), an American Heart Association Fellowship Award (to Y. V. M.), and a Medical University of South Carolina University Research Foundation award (to M. N. G.). The FLIPRTM is a shared Medical University of South Carolina resource obtained with Public Health Service Grant S10 RR13005. The microphysiometer is a shared Veterans Affairs resource obtained with a Veterans Affairs large equipment grant.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Rm. 829 CSB, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425-2227. Tel.: 843-876-5128; Fax: 843-792-8399; E-mail: garnovsk@musc.edu.

Published, JBC Papers in Press, February 16, 2001, DOI 10.1074/jbc.M010834200

    ABBREVIATIONS

The abbreviations used are: NHE-1 and -2, Na+/H+ exchanger isoform 1 and 2, respectively; NHE, Na+/H+ exchange; CaM, calmodulin; Jak2, Janus kinase 2; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; TMA, tetramethylammonium; FLIPR, fluorescent imaging plate reader; BK, bradykinin; ECAR, extracellular acidification rate; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; BCECF, 2'-7'-bis[2-carboxymethyl]-5(6)-carboxyfluorescein; EGF, epidermal growth factor.

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ABSTRACT
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RESULTS
DISCUSSION
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