AJP - Renal Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 294: F345-F354, 2008. First published November 21, 2007; doi:10.1152/ajprenal.00509.2006
0363-6127/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/2/F345    most recent
00509.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Abdullah, H. I.
Right arrow Articles by Ferreri, N. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Abdullah, H. I.
Right arrow Articles by Ferreri, N. R.

CaR activation increases TNF production by mTAL cells via a Gi-dependent mechanism

Huda Ismail Abdullah, Paulina L. Pedraza, John C. McGiff, and Nicholas R. Ferreri

Department of Pharmacology, New York Medical College, Valhalla, New York 10595

Submitted 20 December 2006 ; accepted in final form 19 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We evaluated the contribution of calcium-sensing receptor (CaR)-mediated Gi-coupled signaling to TNF production in medullary thick ascending limb (mTAL) cells. A selective Gi inhibitor, pertussis toxin (PTX), but not the inactive B-oligomer binding subunit, abolished CaR-mediated increases in TNF production. The inhibitory effect of PTX was partially reversed by using an adenylate cyclase inhibitor. CaR-mediated TNF production also was partially reversed by a cAMP analog, 8-Br-cAMP. IP1 accumulation was CaR dependent and blocked by PI-PLC; partial inhibition also was observed with PTX. CaR increased calcineurin (CaN) activity by approximately threefold, and PTX prevented CaR-mediated increases in CaN activity, an nuclear factor of activated T cells (NFAT)-cis reporter construct, and a TNF promoter construct. The interaction between Gi and PKC was determined, as we previously showed that CaR-mediated TNF production was CaN and NFAT- mediated and Gq dependent. CaR activation increased PKC activity by twofold, an effect abolished by transient transfection with a dominant negative CaR construct, R796W, or pretreatment with PTX. Inhibition with the pan-specific PKC inhibitor GF 109203X (20 nM) abolished CaR-mediated increases in activity of CaN, an NFAT reporter, and a TNF promoter construct. Collectively, the data suggest that Gi-coupled signaling contributes to NFAT-mediated TNF production in a CaN- and PKC-dependent manner and may be part of a CaR mechanism to regulate mTAL function. Moreover, concurrent Gq and Gi signaling is required for CaR-mediated TNF production in mTAL cells via a CaN/NFAT pathway that is PKC dependent. Understanding CaR-mediated signaling pathways that regulate TNF production in the mTAL is crucial to defining novel mechanisms that regulate extracellular fluid volume and salt balance.

calcium-sensing receptor; COX-2; loop of Henle; NFAT; PKC

PREVIOUS STUDIES FROM OUR laboratory identified the effects of tumor necrosis factor (TNF)-{alpha} on ion transport pathways in the medullary thick ascending limb (mTAL) cells, and as a modulator of ANG II-dependent hypertension (18–20, 62). The higher levels of TNF and PGE2 production by mTAL tubules from ANG II hypertensive rats compared with those from normotensive controls were consistent with results of in vitro experiments showing that administration of ANG II increases production of TNF and PGE2 by the mTAL. Thus TNF appears to subserve natriuretic and blood pressure-lowering mechanisms via interactions with PGE2. These effects, in response to low concentrations of TNF produced locally, are distinct from the proinflammatory/hypertensive effects associated with higher concentrations of this cytokine. Accordingly, TNF may exhibit prohypertensive functions under certain circumstances. For instance, while TNF has hypertensive effects in pregnancy it does not increase blood pressure in virgin rats (35). Also, the enhancement of Na+ uptake by distal tubules cells was observed only in diabetic, but not normal rats (16).

The calcium-sensing receptor (CaR) belongs to family C of the G protein-coupled receptor (GPCR) superfamily and initially was characterized as the receptor expressed on parathyroid cells responsible for modulating release of parathyroid hormone in response to changes in plasma ionized Ca2+ (7, 8). However, the CaR is also expressed in kidney, thyroid, brain, bone, gastric epithelial cells, fibroblasts, and endothelial cells (25, 40, 45, 50, 52, 63). The physiological functions of the CaR include regulation of Ca2+ homeostatic mechanisms and modulation of Na+, Cl–, Ca2+, and H2O transport in the kidney (9, 10), where it is expressed in the proximal tubule, cortical and medullary TAL, distal convoluted tubule, inner medullary collecting duct, and macula densa (13, 48, 49, 65). The TAL is responsible for reabsorption of ~25% of filtered Na+, as well as Ca2+, and contributes to the generation of the osmotic gradient that drives vasopressin-dependent water reabsorption by the collecting duct (34, 37). Since most divalent mineral reabsorption in the TAL occurs via a paracellular route and is driven by the lumen-positive transepithelial voltage generated by NaCl reabsorption and K+ recycling, reduced NaCl reabsorption lowers Ca2+ reabsorption. Moreover, since the TAL is water impermeable, the concentration of extracellular Ca2+ will increase at the basolateral side of the TAL as Ca2+ reabsorption occurs. Thus reabsorbed Ca2+ interacts with CaR on the basolateral membrane of TAL cells and attenuates Ca2+/Mg2+ reabsorption (30, 49). Increases in plasma levels of Ca2+ also modulate NaCl transport in the rat TAL (61), as raising serum ionized Ca2+ by 25% in humans increased the urinary excretion of Na+ by 150%; Na+ excretion also increased in an animal model in response to infusion of Ca2+ (15, 17).

The modular design of GPCR provides for diversity that allows for distinct functions in a cell type-dependent manner. CaR activation of Gq rapidly increases levels of intracellular Ca2+, a hallmark of PLC-β activation (56), and activates nuclear factor of activated T cells (NFAT) in a calcineurin (CaN)-dependent fashion, resulting in TNF and PGE2 production, which contributes to a mechanism that regulates ion transport in mTAL cells (2). As the CaR activates both Gq and Gi (59), we determined whether a Gi-dependent mechanism also contributes to CaR-mediated TNF production in mTAL cells. Elucidating the pathways by which CaR increases TNF production is necessary for the study of mechanisms that regulate salt and water balance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Sprague-Dawley rats (Charles River Lab, Wilmington, MA), weighing 100–110 g, were maintained on standard rat chow (Ralston-Purina, Chicago, IL) and given tap water ad libitum. All protocols were in accord with the policies of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and received prior approval by the Institutional Animal Care and Use Committee at New York Medical College.

Reagents. Tissue culture media was obtained from Life Technologies (Grand Island, NY). Reagent-grade chemicals and collagenase (type 1A) were from Sigma (St. Louis, MO). Reagents for preparation of the TNF ELISA were purchased from Pharmingen (San Diego, CA). The luciferase assay kit was from Promega (Madison, WI). The inhibitors U-73122, pertussis toxin (PTX) B-oligomer, MDL-12,330A, 8-bromo-cAMP, and GF109203X were purchased from Biomol; PTX was purchased from Calbiochem. All inhibitors were diluted in H2O, ethanol, or DMSO, when stock solutions were formulated. Before use, they were diluted with RPMI to achieve a vehicle concentration of <0.01%. Vehicle controls were included in all experiments and did not affect any of the parameters measured.

Isolation of mTAL cells. The isolation and characterization of mTAL cells (~95% purity) were performed as previously described (11, 18). Briefly, male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (0.65 mg/100 g body wt). The kidneys were perfused with sterile 0.9% saline, via retrograde perfusion of the aorta, and cut along the corticopapillary axis. The inner stripe of the outer medulla was excised, minced with a sterile blade, and incubated for 10 min at 37°C in a 0.1% collagenase solution gassed with 95% oxygen. The suspension was sedimented on ice, mixed with Hanks' balanced salt solution (HBSS) containing 2% BSA, and the supernatant containing the crude suspension of tubules was collected. The collagenase digestion was repeated three times with the remaining undigested tissue. The combined supernatants were centrifuged for 10 min, resuspended in HBSS, and then filtered through a 52-µm nylon mesh membrane (Fisher Scientific, Springfield, NJ). The filtrated solution was discarded, and the tubules retained on the mesh were resuspended in HBSS. Then, the solution was centrifuged at 500 rpm for 10 min, the supernatant was aspirated, and the cells were cultured in renal epithelial cell basal medium (REBM, Cambrex), containing renal epithelial cell growth medium (REGM, Cambrex), consisting of rhEGF, insulin, hydrocortisone, GA-1000 (gentamycin sulfate and amphotericin B), FBS, epinephrine, T3 (triiodothronine), and transferrin. After 3–4 days, monolayers of cells were 80–90% confluent. The cells were quiesced for 24 h in RPMI containing 0.42 mM CaCl2 and 0.5% FBS, L-glutamine (2 mM), 100 U/ml streptomycin-penicillin (GIBCO), MEM nonessential amino acids (GIBCO), MEM sodium pyruvate, and β-mercaptoethanol before their use. In all experiments, "control conditions" (i.e., no addition of CaCl2) reflects that cells were incubated in media containing 0.42 mM Ca2+. This amount of Ca2+ should be added to the amounts used to challenge the cells to obtain total extracellular Ca2+ present. These control conditions were selected based on previous work showing that the CaR is functionally insensitive when extracellular Ca2+ concentrations are <1 mM (58).

Measurement of TNF. CaR-mediated TNF production was assessed by ELISA as previously described (57). Briefly, primary cultured rat mTAL cells were quiesced overnight and then challenged with CaCl2 or various inhibitors for 6 h at 37°C/5% CO2. TNF levels in cell-free supernatants were determined by ELISA (Pharmingen), according to the protocol provided by the manufacturer (57).

Gene transfection. mTAL cells were cultured to 70–80% confluence and transfected as previously described (55). Briefly, the medium was removed and cells were placed in 1 ml of serum-free OPTI-MEM medium containing 10 µl Lipofectamine 2000 reagent (Invitrogen) and plasmid DNA containing a pGV-B2-TNFprom-driven firefly luciferase promoter construct (0.05 µg/well), an NFAT binding site, TATA-driven firefly luciferase promoter (2 µg/well; Stratagene), R796W (7 µg/well), or empty plasmid vectors (pCIS-CK and pGV-B2). The pGV-B2-TNFprom promoter construct was a generous gift from Dr. Akio Nakamura (Teikyo University School of Medicine). Each of the constructs, except R796W, was dually transfected with a control pSV40-driven Renilla luciferase promoter (0.025 µg/well) to normalize the data for 4 h at 37°C/5% CO2. After the transfection period, 1 ml of REGM medium was added and the cells were incubated overnight at 37°C/5% CO2. The medium was then removed, and cells were cultured for an additional 12 h in REGM. The cells were quiesced overnight in RPMI medium containing 0.5% FBS, then treated with the appropriate reagents for the indicated times; washed once with PBS, and collected with appropriate lysis buffer. Each sample set was quantitated with a Stop and Glo assay according to the manufacturer's directions (Promega). The transfection efficiency, for R796W, was evaluated by staining for β-galactosidase activity in cells transiently transfected with the pSV-β-galactosidase vector. Expression of β-galactosidase was visualized by microscopy, after incubation with the X-Gal substrate. β-Galactosidase expression was ~35–40 and 65–70%, after addition of 3 or 7 µg/well of the pSV-β-galactosidase vector, respectively.

Calcineurin assay. CaN activity was determined using a Biomol Green Cellular Calcineurin Assay Kit Plus. The protocol was performed according to the manufacturer's protocol with minor modifications, as previously described (2). Briefly, cells were washed three times with TBS and collected with the provided lysis buffer. Background phosphate released was subtracted from each sample, and the CaN activity was determined by the following equation: PP2B = total phosphatase activity (PP1, PP2A, PP2B, and PP2C) – EGTA buffer (PP1 and PP2A).

PKC activity assay. CaR-mediated PKC activity was analyzed by a nonradioactive ELISA purchased from Stressgen Bioreagents. The assay utilizes a specific synthetic peptide that is a substrate for PKC and detected with a phospho-specific antibody that recognizes the phosphorylated form of the substrate; color development is in proportion to PKC phosphotransferase activity. mTAL cells were grown to ~80–90% confluence and challenged with extracellular Ca2+ for 15 min, following a 15-min to 1-h preincubation with vehicle or inhibitors. Subsequently, cells were washed with ice-cold TBS, then incubated for 10 min on ice with 200 µl/well of 1x RIPA lysis buffer (Santa Cruz Biotechnology) with minor modifications (1 mM PMSF, 1 mM sodium vanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1% NP-40); added before collection. Cells were collected in microcentrifuge tubes and subjected to a burst of sonication. Tubes were centrifuged at 13,000 rpm for 15 min, and 30 µl of clear supernatants (cytosolic fraction) was assayed for PKC activation. All additional reagents used were provided in the kit including the PKC substrate, 96-well microtiter plate, ATP, phosphospecific substrate antibody, anti-rabbit IgG:horseradish peroxidase conjugate, wash buffer, and stop solution. Wells were developed with the provided TMB substrate and read at 450 nm. In addition, sample lysate was used to determine protein concentration, according to the BCA method, for each individual sample to normalize relative PKC activity.

D-myo-Inositol 1 phosphate assay. D-myo-Inositol 1,4,5 (IP3) is typically produced within seconds of phosphoinositol (PI)-PLC activation and then degraded to D-myo-inositol 2 phosphate (IP2) and D-myo-inositol 1 phosphate (IP1), and, finally, into myo-inositol; incubation of cells with LiCl prevents the degradation of IP1 into myo-inositol. IP3 levels were assayed by indirectly measuring the accumulation of IP1, a metabolite of IP3, with an IP1 Assay Kit purchased from Cisbio, with a few modifications made to the manufacturer's protocol. Primary mTAL cells were grown in 24-well plates until 80–90% confluent and then quiesced overnight. Then, cells were incubated in stimulation buffer (100 µl), less drugs and Ca2+ (1.2 mM), for 2 h at 37°C/5% CO2 in an incubator. We modified the stimulation buffer, since the manufacturer's stimulation buffer contained 1.0 mM CaCl2. The modified stimulation buffer (pH 7.4), without phosphate salts, contains 10 mM HEPES, 0.42 mM CaCl2, 0.5 mM MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, and 50 mM LiCl. Next, we either pretreated cells with various inhibitors or their negative controls for 1 h, before Ca2+ stimulation for 5 min. Subsequently, we added 100 µl of 2.5% lysis buffer (kit component) and incubated the cells for 30 min in a 37°C/5% CO2 incubator. Cell supernatants were assayed in 96-well ELISA plate format and for protein normalization. Samples and IP1 standard were incubated with IP1-horseradish peroxidase conjugate and anti-monoclonal antibody for 3 h at room temperature. Subsequently, cells were washed six times and incubated with 100 µl TMB for ~30 min in the dark at room temperature, followed by the addition of 100 µl of the provided stop solution and read at 450 nm with corrections at 620 nm. Each sample was normalized for protein concentration, using a spectrophotometer that estimates protein concentration by absorbing light at a wavelength of 280 nm and then corrected at a wavelength of 230 nm for possible nucleic acid contamination. This was a necessary measure, since the lysis buffer contains BSA, which interferes with the various protein assay methods. The sample protein concentration is a function of the calculated protein concentration minus background (protein concentration of lysis buffer with stimulation buffer alone).

Statistical analysis. Data were compared by a one-way ANOVA followed by the Newman-Kuels test, using the GraphPad Prism 2.01 statistical program, when multiple comparisons were made. Data columns were statistically analyzed to derive exact means, SE, and SD for graphic analysis. Data are presented as means ± SE; P ≤ 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CaR-mediated activation of Gi increases TNF production and promoter activity in mTAL cells. Stimulation of the CaR in several cell types has been linked to activation of both Gq- and Gi-dependent signaling mechanisms (43). The extent to which Gi-dependent signaling contributes to CaR-mediated TNF production was assessed in mTAL cells. The selective Gi inhibitor PTX holotoxin is a heterohexameric protein made up of an A-protomer and B-oligomer (33). The A-protomer containing a single polypeptide (S1 subunit) disrupts transmembrane signaling by ADP-ribosylation of the fourth amino acid (cysteine) from the COOH terminus of the G protein {alpha}-subunit; while the B-oligomer, containing five polypeptides, binds to cell receptors (most likely containing carbohydrate) and delivers the S1 subunit (33). Treatment with PTX (100 ng/ml) abolished Ca2+-mediated increases in TNF production at 6 h, but did not alter basal TNF production (Fig. 1A) or affect mTAL cell viability (not shown). These data suggest that Gi{alpha}-coupled signaling is part of a mechanism by which activation of the CaR stimulates TNF production in mTAL cells. Treatment with the PTX B-oligomer (binding subunit), at the same concentration as PTX, did not inhibit the Ca2+-mediated increase in TNF production, suggesting that the PTX holotoxin, not the binding subunit alone, is required for the Gi inhibitory effect and that the B-oligomer, which does not possess any intrinsic ribosyltransferase activity, does not inhibit signal transduction by the CaR (Fig. 1A).


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 1. Pertussis toxin (PTX) decreases calcium-sensing receptor (CaR)-mediated TNF production and promoter activity/role of cAMP. A: medullary thick ascending limb (mTAL) cells were pretreated with PTX (100 ng/ml) or the PTX B-oligomer (inactive binding subunit; 100 ng/ml) for 1 h at 37°C/5% CO2, then challenged with 1.2 mM Ca2+ for 6 h with or without MDL-12,330A (500 µM). TNF in cell-free supernatants was determined by ELISA; n = 6. B: cells were pretreated for 15 min with 8-Br-cAMP before Ca2+ treatment for 6 h; n = 6. C: cells were cotransfected with a pGV-B2-TNFprom construct (0.05 µg/well) and SV40 Renilla luciferase promoter construct (0.025 µg/well), which was used to normalize the data in a dual transfection assay. Cells were pretreated with PTX for 1 h, then challenged with 1.2 mM Ca2+ for 6 h; n = 3; representative experiment from 5 similar experiments.

 
A possible role for cAMP was addressed by attempting to reverse the effects of PTX with a cell-permeable and irreversible inhibitor adenylate cyclase (AC), MDL-12,330A (500 µM). Cells were pretreated with PTX for 1 h and then incubated with Ca2+ for 30 min to allow early events of signal transduction to be initiated; MDL-12,330A was then added to the cells for an additional 5.5 h. Neither PTX nor MDL-12,330A affected basal TNF levels, suggesting that changes in cAMP levels per se are not sufficient to influence TNF production in the absence of CaR activation. However, MDL-12,330A partially reversed the inhibitory effects of PTX, suggesting that cAMP contributes, at least in part, to TNF production in mTAL cells (Fig. 1A). Moreover, 8-Br-cAMP (1 mM), a cell-permeable analog of cAMP, which is relatively resistant to phosphodiesterase and preferentially activates cAMP-dependent protein kinase, attenuated Ca2+-stimulated increases in TNF production (Fig. 1B).

The effect of Gi inhibition on TNF promoter activity was determined since an increase in TNF gene transcription contributes to TNF production in mTAL cells (57). mTAL cells were transiently cotransfected with TNF promoter firefly luciferase (pGV-B2-TNFprom) and pSV40 Renilla luciferase constructs. Activity of the TNF promoter construct increased approximately threefold after cells were challenged with 1.2 mM Ca2+ (Fig. 1C). In contrast, the CaR-mediated increase in TNF promoter activity was inhibited when cells were pretreated with PTX, suggesting that Gi-coupled signaling contributes to increases in TNF production by increasing TNF gene transcription.

IP1 accumulation in mTAL cells is partially Gi dependent. We previously showed that CaR-mediated TNF production was blocked by the PI-PLC inhibitor U73122 [GenBank] (2). To investigate whether there is early Gi and Gq cross talk, we assessed the role of Gi-coupled signaling on IP1 accumulation. Typically, IP3 has a very short half-life (30 s) before degradation in mammalian cells to IP2, IP1, and, finally, myo-inositol. Since IP3 is released within seconds, it is difficult to measure accurately, while IP1 accumulation can be measured more precisely since LiCl can prevent its degradation. Cells were incubated for 2 h in a media containing LiCl (50 mM) to prevent the degradation of IP1 into myo-inositol and to allow IP1 to accumulate intracellularly. Next, cells were stimulated with Ca2+ (1.2 mM) for 5 min, based on a time course study indicating that this time period was optimal for IP1 accumulation in mTAL cells (not shown). Ca2+ stimulation increased CaR-mediated IP1 accumulation by approximately twofold (Fig. 2). Moreover, cells pretreated with the selective PI-PLC inhibitor (U73122 [GenBank] ; 4 µM) for 1 h decreased Ca2+-mediated increases in IP1 accumulation to basal levels; while pretreatment with the inactive analog (U73343 [GenBank] ; 4 µM) did not have any effect (Fig. 2). These data support the notion that CaR is coupled to Gq in mTAL cells and verifies that PI-PLC mediates IP1 accumulation in these cells. Notably, PTX pretreatment decreased Ca2+-mediated increases in IP1 accumulation by approximately one-third, while pretreatment with the PTX B-oligomer (100 ng/ml) did not alter basal levels or Ca2+-mediated IP1 accumulation (Fig. 2). These data suggest that upon CaR activation, Gq-coupled signaling is immediately initiated and partially regulated by Gi-coupled signaling.


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 2. Effects of Gq and Gi activation on D-myo-inositol 1 (IP1) accumulation. mTAL cells were incubated for 2 h in modified isosmotic media containing LiCl (50 mM) and CaCl2 (0.42 mM). Cells that were stimulated with Ca2+ (1.2 mM) for 5 min increased IP1 accumulation by ~2-fold; n = 16. Pretreatment with the phosphoinositol (PI)-PLC inhibitor (U73122; 4 µM) or a Gi inhibitor (PTX; 100 ng/ml) for 1 h, before stimulation, abolished or decreased by approximately one-third the Ca2+-mediated increases in IP1 accumulation, respectively; n = 8. Moreover, the negative controls, U73343 and PTX B-oligomer, at the same concentrations, did not alter either basal level or Ca2+-mediated increases in IP1 levels; n = 8.

 
Gi-mediated TNF production is NFAT dependent. To test whether Gi-coupled signaling contributes to TNF production via NFAT, mTAL cells were cotransfected cells with a TATA-driven NFAT binding site firefly luciferase construct and SV40-driven Renilla luciferase construct. Pretreatment for 1 h with PTX did not alter basal levels of NFAT luciferase activity but markedly attenuated the Ca2+-stimulated increase in NFAT binding site luciferase activity (Fig. 3); suggesting that Gi-coupled signaling is involved in NFAT activation.


Figure 3
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 3. Inhibition of Gi-dependent signaling abolishes CaR-mediated NFAT luciferase activity. mTAL cells were transiently cotransfected with a NFAT cis-reporter construct (2 µg/well) and SV40 Renilla luciferase promoter construct (0.025 µg/well), which was used to normalize the data. Treatment with 1.2 mM Ca2+ increased NFAT binding site luciferase activity; n = 5.

 
Transfection with a dominant negative CaR construct inhibits Ca2+-mediated CaN activity in mTAL cells. The dominant negative CaR construct R796W was used to demonstrate that the increase in CaN activity in response to challenge with extracellular Ca2+ was dependent on CaR activation. Primary cultures of mTAL cells were grown in six-well plates, quiesced overnight in RPMI 1640 containing 0.5% FBS and 0.4 mM CaCl2, washed in RPMI 1640, and challenged at 37°C/5% CO2 for 25 min with Ca2+ (1.2 mM). CaN activity increased in cells challenged with extracellular Ca2+ (Fig. 4A). However, challenge with extracellular Ca2+ failed to increase CaN activity in cells transiently transfected with R796W, a construct that permits binding of Ca2+ to the CaR but prevents signaling (40); transient transfection with R796W alone did not alter cell viability (not shown). Moreover, transient transfection with the empty vector did not alter basal or Ca2+-induced levels of TNF production (control: 35.5 ± 2.4; Ca2+: 765.0 ± 80.3; pcDNA3.1: 38.8 ± 12.8; Ca2+ and pcDNA3.1: 832.0 ± 73.0 pg/ng protein; n = 4). These data indicate that the increase in CaN activity in response to extracellular Ca2+ requires activation of a functional CaR.


Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 4. Calcineurin (CaN) activity is inhibited by the dominant negative CaR construct R796W and the Gi inhibitor PTX. A: mTAL cells were transiently transfected with R796W (7 µg/well) and calcineurin activity determined after challenge with 1.2 mM Ca2+ for 25 min. The free phosphate released was divided by the amount of protein in each sample to normalize for differences in protein levels per sample; n = 4. B: pretreatment with PTX inhibited CaR-mediated increases in CaN activity, as indicated by RII phospho-substrate cleavage, but did not alter basal CaN activity; n = 4.

 
Gi-mediated TNF production is CaN dependent. The mechanisms by which NFAT activity was increased following CaR activation have not yet been explored. Accordingly, a CaN cellular activity assay was performed to test whether Gi-coupled signaling impacts NFAT-mediated TNF production in a CaN-dependent manner. Pretreatment of mTAL cells with PTX inhibited Ca2+-mediated increases in CaN activity, suggesting that Gi-coupled signaling is essential to the mechanism by which activation of the CaR increases CaN activity (Fig. 4B). Collectively, the data suggest that the CaR increases TNF production via a mechanism involving TNF transcription that requires NFAT activation as an obligate step, following Gi-dependent activation of CaN.

Contribution of PKC to CaN- and NFAT-mediated increases in TNF promoter activity. Since Gq- and Gi-coupled signaling contribute to TNF synthesis in a CaN- and NFAT-dependent manner, interactions downstream of G protein activation were evaluated to determine whether these pathways interact with each other or function in a parallel manner. We previously showed that the CaR induces PKC-mediated TNF production (57). Accordingly, the contribution of PKC to the CaN- and NFAT-dependent mechanism that regulates TNF promoter activity was determined. mTAL cells were transiently cotransfected with pGV-B2-TNFprom promoter (0.05 µg/well) and SV40 Renilla luciferase constructs and treated with 1.2 mM Ca2+ for 6 h. Pretreatment with the pan-specific PKC inhibitor GF 109203X (20 nM) attenuated CaR-mediated increases in TNF promoter activity but did not alter basal activity, suggesting that activation of PKC is part of the mechanism whereby CaR activation increases TNF gene transcription (Fig. 5). Pretreatment with GF 109203X did not alter mTAL cell viability (not shown). To assess whether NFAT was involved in PKC-mediated increases in TNF promoter activity, mTAL cells were transiently cotransfected with the NFAT cis-reporter- and SV40-Renilla luciferase promoter constructs. Increased NFAT binding site luciferase activity was observed in cells treated with 1.2 mM Ca2+; pretreatment with GF 109203X decreased CaR-mediated increases in luciferase activity but did not alter basal luciferase activity (Fig. 6A). The contribution of PKC to CaN activation was also determined, since CaR-mediated NFAT activation is PKC dependent. Pretreatment of mTAL cells with GF 109203X, prevented dephosphorylation of the RII phospho-substrate for CaN, suggesting that CaR-mediated activation of CaN also is PKC dependent (Fig. 6B). Collectively, these data suggest that PKC-dependent activation of the CaN-NFAT pathway contributes to TNF gene transcription induced by CaR activation in mTAL cells.


Figure 5
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 5. PKC inhibitor, GF 109203X, attenuates CaR-mediated activation of a TNF promoter construct. mTAL cells were transiently transfected with pGV-B2-TNFprom (0.05 µg/well) and treated with 1.2 mM Ca2+ for 6 h. Pretreatment with GF 109203X (20 nM) inhibited CaR-activated increases in luciferase activity but did not alter basal luciferase activity; normalization was accomplished with a dual transfection using a SV40 Renilla luciferase promoter construct (0.025 µg/well); n = 3.

 

Figure 6
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 6. PKC inhibition attenuates CaR-mediated activation of an NFAT reporter construct and inhibits calcineurin activation. A: mTAL cells were transiently transfected with an NFAT cis-reporter construct (2 µg/well). Treatment with 1.2 mM Ca2+ increased NFAT binding site luciferase activity that was attenuated by pretreatment with GF 109203X; normalization was accomplished by cotransfection with an SV40 Renilla luciferase promoter construct (0.025 µg/well); n = 3. B: pretreatment of mTAL cells with GF 109203X inhibited Ca2+-mediated increases in CaN activity, as indicated by RII phospho-substrate cleavage but does not alter basal level CaN activity; n = 4.

 
CaR activation increases PKC activity in mTAL cells. To verify that Ca2+ increased PKC activity in a CaR-dependent manner, cells were transiently transfected with R796W, a dominant negative CaR construct. Overexpression of R796W abolished Ca2+-mediated increases in PKC substrate phosphorylation, an in vitro correlate of PKC activation, but did not alter basal PKC activity (Fig. 7A). Pretreatment with a PI-PLC inhibitor, U73122 [GenBank] , also blocked CaR-mediated increases in PKC activity but did not alter basal activity (Fig. 7A) or cell viability (not shown). GF 109203X, the pan-specific PKC inhibitor was used as a positive control and, as expected, blocked CaR-mediated PKC activation (Fig. 7A). Increases in CaR-mediated PKC activity also were blocked when cells were preincubated with the Gi inhibitor PTX; basal activity was not affected (Fig. 7B). Collectively, these data suggest that activation of CaR increases PKC activity in mTAL cells via Gq- and Gi-dependent mechanisms.


Figure 7
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 7. Activation of CaR stimulates PKC activity via Gq and Gi. A: PKC activity was determined in mTAL cells transiently transfected with dominant negative CaR construct R796W for 24 h before stimulation with Ca2+ in the absence or presence of a PI-PLC inhibitor, U73122, or the pan-specific PKC inhibitor GF 109203X. B: PKC activity in response to CaR activation was determined in the absence or presence of PTX (1 h preincubation). The relative amount of PKC activity in cellular lysates was normalized according to the amount of protein in each sample; n = 4.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We demonstrated that inhibition of a Gi-coupled mechanism prevented CaR-mediated increases in CaN activity, NFAT activity, TNF promoter construct activity, and TNF production by mTAL cells. Stimulation of CaN and NFAT activity via CaR-mediated activation of a Gq-coupled mechanism increases TNF production, and activation of PKC contributes to production of this cytokine by mTAL cells (2, 57). In the present study, preincubation of mTAL cells with either U73122 [GenBank] or PTX completely inhibited CaR-mediated increases in PKC activity, suggesting that concurrent coupling of CaR to functional Gq- and Gi-dependent pathways is required for stimulation of PKC activity and TNF production. Moreover, cross talk between these two pathways at the level of PLC activation may contribute to CaR-mediated TNF production in these cells. Since the CaR exists as a homodimer on the plasma membrane, it may bind and activate two G proteins at the same time in a configuration that is cell type dependent (4, 61). The spatial proximity of the two sets of G proteins may impact signal transduction via the CaR. Collectively, these findings suggest that signaling by means of synchronized CaR coupling to Gq and Gi is required for activation of a CaN- and NFAT-dependent mechanism that regulates TNF production by mTAL cells via a pathway involving PKC. A model to illustrate the possible influence exerted by one CaR monomer on the functional CaR dimer is presented in Fig. 8.


Figure 8
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 8. Putative interactions that contribute to CaR-mediated signaling in mTAL cells. The CaR is a constitutively expressed receptor and present as a homodimer or potentially as a heterodimer with other mTAL-expressed G protein-coupled receptors (GPCR) in the plasma membrane, on the basolateral side of mTAL epithelial cells. Upon Ca2+ sensing, cooperative binding to the extracellular binding domain is triggered in accordance to Hill's coefficient. Activation by multiple Ca2+ ions elicits a conformational change in the homodimer or potential GPCR heterodimer, enabling G protein coupling most possibly by the i2 and i3 intracellular domains. Both heterotrimeric Gi- and Gq-coupled signaling appear to be initiated in a cooperative manner, both being obligate signaling entities. Interruption of PTX-sensitive or -insensitive signaling pathways suggests a necessary upstream signaling cross talk, as inhibiting either side of the pathway completely abolishes PKC, CaN, and NFAT activation, as well as TNF production. The all-or-none response may be a composite of regulatory signaling mechanisms by various G protein subunits or PLC, as shown in other biological systems. Dashed arrow, partial regulation; solid arrow, full regulation; AC, adenylate cyclase; COX-2, cyclooxygenase-2; DAG, diacylglycerol; IP3, inositol triphosphate; NFAT, nuclear factor of activated T cells; GPCR*, potential heterodimeric partner for the CaR in the mTAL.

 
The precise mechanism by which CaR-mediated activation of Gq and Gi interact to affect PKC activity and subsequent downstream signaling events in mTAL cells remains to be determined. CaR form disulfide-linked dimers via their extracellular domains, and intermolecular interactions within the dimeric receptor contribute to receptor function (5). These covalently bound dimers, the expression of which is independent of the activation status of the CaR, have been observed in HEK cells transfected with various CaR constructs and in rat kidney, where the receptor is expressed endogenously (4, 60). Although the molecular basis has yet to be defined, the CaR exhibits a large Hill coefficient, suggesting that binding of ligand and/or G protein by the receptor is subject to positive cooperativity (21, 41). However, cross talk between the subunits and the close spatial relationship between two sets of heterotrimeric G proteins also may affect function in a cooperative manner, as CaR homodimers expressed at the plasma membrane represent the predominant form of this receptor; higher oligomers also may be expressed while monomers are rare (4, 59, 60). The possibility of CaR heterodimerization with other members of its own family or even other GPCR families cannot be ruled out. Sequence homology and cloning across species suggest that the CaR exists in nature as one form; hence one CaR monomer can only homodimerize with another CaR monomer. Nonetheless, CaR may be able to heterodimerize with the metabotropic glutamate (mGlu) receptors, GABAB receptors, or various GPCR that can heterodimerize with other receptors, exemplified by AT1 receptors-angiotensin II with B2 receptors-bradykinin and D1 receptors-dopamine with A1 receptors-adenosine (1, 22, 24).

Previous studies have shown that point mutations in CaR intracellular loops known to interact with G proteins exert dominant negative effects on the function of wild-type receptors (4). We demonstrated that overexpression of a CaR with a point mutation in the third intracellular loop prevented CaR-mediated increases in PKC and CaN activity. This mutated receptor exerts a dominant negative function (40) but does not affect surface expression of the CaR (5). Moreover, experiments in which various mutant CaR were coexpressed in HEK cells demonstrated that an inactivating mutation in the intracellular loop of a CaR monomer resulted in greatly reduced signal transduction (5). This model may be similar to the mechanism that accounts for the dysfunction of inactivating mutations of the CaR. For example, patients with familial hypocalciuric hypercalcemia likely express a normal CaR on one allele and a mutant CaR on the other allele. Some of these patients exhibit dominant negative phenotypes that may result from formation of CaR heterodimers consisting of wild-type and mutant-type receptors, the latter type negatively affecting function of the former (4). The structural topology of the CaR may dictate that interaction of Ca2+ with the extracellular domain and intracellular loop 3 of one CaR monomer may be influenced by its dimeric partner (5). Indeed, GABAB receptors, as well as CaR, which are GPCR family C members, form functional homodimers that interact with G proteins via intracellular loop 3. Within the class III GPCRs, intracellular loop 2 also plays an important role in selectively coupling the receptor to G proteins (26, 46, 54). We postulate that inhibition of either Gq or Gi blocks CaR-mediated increases in PKC activity via generation of a functionally inactive monomer that exerts a dominant negative effect and is able to block activity of the homodimerized receptor.

In the present study, inhibition of either Gq or Gi blocked CaR-mediated PKC activity, which was required for activation of CaN and NFAT. Efficient activation of PLC-β in response to CaR activation is mediated by the second and third intracellular loops of the receptor; primary as well as secondary structure is critical for CaR signaling (12). PI-PLC cleaves phosphatidylinositol-4,5-bisphosphate into IP3 and diacylglycerol (DAG), which increases intracellular Ca2+ mobilization and PKC activity, respectively. Transient transfection of mTAL cells with a dominant negative CaR construct revealed that increases in PKC activity in response to extracellular Ca2+ were mediated by activation of endogenous CaR. Moreover, CaR-mediated increases in PKC activity were prevented when signaling via either Gq or Gi was blocked. Stimulation of PLC-β by several types of GPCR is mediated by pathways involving Gq, as well as Gi; this may be one mechanism by which coupling to Gi, following CaR activation, can also activate PKC (32, 47, 53). Indeed, accumulation of IP1 in mTAL cells was partially dependent on activation of Gi. Alternatively, GPCR including CaR can stimulate PKC in a PLC-independent manner via coupling to Gi (23). The rat mTAL expresses several of the 11 known isoforms of PKC including Ca2+-dependent ({alpha},β II) and Ca2+-independent ({delta}, {varepsilon}, {zeta}) PKC isoforms, and several studies have illustrated the functional importance of PKC activation in the mTAL (3). For instance, PKC-{alpha} is required for superoxide-mediated NaCl absorption in the TAL, while PKC increases HCO3– reabsorption by inhibiting AVP-stimulated cAMP production via a Gi-dependent mechanism (28, 44). The contribution of CaR-dependent PKC activation to a TNF-dependent mechanism that regulates mTAL function is currently being studied.

Many downstream events subsequent to CaR activation are PKC dependent (38, 39). In the present study, we demonstrated that CaR-mediated CaN activation is PKC dependent, a mechanism that also has been described in airway remodeling, where an inhibitor of PKC prevented urotensin-mediated increases in CaN activity (14). Inhibition of PKC activity also abolished CaR-mediated NFAT activation and TNF gene transcription, indicating that PKC is an obligate step in the cascade culminating in TNF production. CaR signaling via Gq- or Gi-coupled mechanisms occurs in a cell type-dependent manner (59). The demonstration that a CaN- and NFAT-dependent, Gq-coupled mechanism increases TNF production in mTAL cells prompted us to determine whether activation of this pathway is restricted to Gq-coupling. Pretreatment of mTAL cells with PTX inhibited CaR-mediated CaN and NFAT activity, suggesting that a Gi-coupled mechanism was involved. While a role for Gq-coupled pathways that activate NFAT has been described (31), the link between signaling via Gi and activation of NFAT is less clear. However, inhibition of insulin secretion from pancreatic islets in response to activation of GABAB receptors was prevented by Gi-coupled activation of CaN and NFAT activity increased via a Gi-dependent mechanism, after CXC chemokine receptor activation (6, 51). These studies demonstrate that Gi-coupled signaling through varied receptors regulates CaN and NFAT activation.

Interestingly, immune suppression, as well as renal dysfunction or nephrotoxicity, is a feature of CaN-{alpha} knockout mice. Recent studies show that CaN inhibitors given to kidney transplant patients evoked edema of the lower extremities due to increased venous pooling (27). It is well established that nonsteroidal anti-inflammatory drug therapy should be avoided in renal transplant patients, or salt-depleted patients, as well as in cases of renal insufficiency because of the possible deterioration of renal function, a COX-2-mediated effect (64). It is intriguing to speculate that inhibition of a CaN-/NFAT-dependent mechanism that regulates TNF production and renal tubular function via a COX-2-dependent mechanism contributes to these events.

In summary, signaling through the CaR coupled to a Gi-dependent pathway plays an essential role in activation of CaN and NFAT, which increases TNF gene transcription in a PKC-dependent manner. Moreover, it is necessary to have concurrent activation of Gq and Gi in response to CaR stimulation in mTAL cells to elicit these responses. Several regions along the COOH-terminal structure of G{alpha} subunits are critical for interactions with GPCRs (29, 36, 42). For instance, residue -4 in the Gi family is the cysteine residue that is ADP-ribosylated by PTX, a covalent modification that prevents the G protein from interacting with the receptor; conformational changes in the COOH terminus may affect the activation status of the receptor. Perhaps manipulation of these signaling cascades via highly selective interventions may prove to be important in treating various metabolic and hypertensive disorders.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-56423 and PPG-HL-34300.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. R. Ferreri, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (e-mail: nick_ferreri{at}nymc.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. AbdAlla S, Lother H, Quitterer U. AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration. Nature 407: 94–98, 2000.[CrossRef][Medline]
  2. Abdullah HI, Pedraza PL, Hao S, Rodland KD, McGiff JC, Ferreri NR. NFAT regulates calcium-sensing receptor-mediated TNF production. Am J Physiol Renal Physiol 290: F1110–F1117, 2006.[Abstract/Free Full Text]
  3. Aristimuno PC, Good DW. PKC isoforms in rat medullary thick ascending limb: selective activation of the delta-isoform by PGE2. Am J Physiol Renal Physiol 272: F624–F631, 1997.[Abstract/Free Full Text]
  4. Bai M, Trivedi S, Brown EM. Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. J Biol Chem 273: 23605–23610, 1998.[Abstract/Free Full Text]
  5. Bai M, Trivedi S, Kifor O, Quinn SJ, Brown EM. Intermolecular interactions between dimeric calcium-sensing receptor monomers are important for its normal function. Proc Natl Acad Sci USA 96: 2834–2839, 1999.[Abstract/Free Full Text]
  6. Braun M, Wendt A, Buschard K, Salehi A, Sewing S, Gromada J, Rorsman P. GABAB receptor activation inhibits exocytosis in rat pancreatic beta-cells by G-protein-dependent activation of calcineurin. J Physiol 559: 397–409, 2004.[Abstract/Free Full Text]
  7. Brown EM, Chattopadhyay N, Vassilev PM, Hebert SC. The calcium-sensing receptor (CaR) permits Ca2+ to function as a versatile extracellular first messenger. Recent Prog Horm Res 53: 257–280, 1998.[Medline]
  8. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 366: 575–580, 1993.[CrossRef][Medline]
  9. Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 81: 239–297, 2001.[Abstract/Free Full Text]
  10. Brown EM, Vassilev PM, Hebert SC. Calcium ions as extracellular messengers. Cell 83: 679–682, 1995.[CrossRef][Web of Science][Medline]
  11. Carroll MA, McGiff JC, Ferreri NR. Products of arachidonic acid metabolism. Methods Mol Med 86: 385–397, 2003.[Medline]
  12. Chang W, Chen TH, Pratt S, Shoback D. Amino acids in the second and third intracellular loops of the parathyroid Ca2+-sensing receptor mediate efficient coupling to phospholipase C. J Biol Chem 275: 19955–19963, 2000.[Abstract/Free Full Text]
  13. Chattopadhyay N, Baum M, Bai M, Riccardi D, Hebert SC, Harris HW, Brown EM. Ontogeny of the extracellular calcium-sensing receptor in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 271: F736–F743, 1996.[Abstract/Free Full Text]
  14. Chen YH, Yao WZ, Zhao MW, Pang YZ, Tang CS. Cross-talk between calcineurin and protein kinases in airway remodeling in asthma. Beijing Da Xue Xue Bao 37: 599–602, 2005.[Medline]
  15. Chomdej B, Bell PD, Navar LG. Renal hemodynamic and autoregulatory responses to acute hypercalcemia. Am J Physiol Renal Fluid Electrolyte Physiol 232: F490–F496, 1977.[Free Full Text]
  16. DiPetrillo K, Coutermarsh B, Gesek FA. Urinary tumor necrosis factor contributes to sodium retention and renal hypertrophy during diabetes. Am J Physiol Renal Physiol 284: F113–F121, 2003.[Abstract/Free Full Text]
  17. el-Hajj Fuleihan G, Seifter J, Scott J, Brown EM. Calcium-regulated renal calcium handling in healthy men: relationship to sodium handling. J Clin Endocrinol Metab 83: 2366–2372, 1998.[Abstract/Free Full Text]
  18. Escalante BA, Ferreri NR, Dunn CE, McGiff JC. Cytokines affect ion transport in primary cultured thick ascending limb of Henle's loop cells. Am J Physiol Cell Physiol 266: C1568–C1576, 1994.[Abstract/Free Full Text]
  19. Ferreri NR, Escalante BA, Zhao Y, An SJ, McGiff JC. Angiotensin II induces TNF production by the thick ascending limb: functional implications. Am J Physiol Renal Physiol 274: F148–F155, 1998.[Abstract/Free Full Text]
  20. Ferreri NR, Zhao Y, Takizawa H, McGiff JC. Tumor necrosis factor-alpha/angiotensin interactions and regulation of blood pressure. J Hypertens 15: 1481–1484, 1997.[CrossRef][Web of Science][Medline]
  21. Gama L, Breitwieser GE. A carboxyl-terminal domain controls the cooperativity for extracellular Ca2+ activation of the human calcium sensing receptor. A study with receptor-green fluorescent protein fusions. J Biol Chem 273: 29712–29718, 1998.[Abstract/Free Full Text]
  22. Gama L, Wilt SG, Breitwieser GE. Heterodimerization of calcium sensing receptors with metabotropic glutamate receptors in neurons. J Biol Chem 276: 39053–39059, 2001.[Abstract/Free Full Text]
  23. Gao Y, Tang S, Zhou S, Ware JA. The thromboxane A2 receptor activates mitogen-activated protein kinase via protein kinase C-dependent Gi coupling and Src-dependent phosphorylation of the epidermal growth factor receptor. J Pharmacol Exp Ther 296: 426–433, 2001.[Abstract/Free Full Text]
  24. Gines S, Hillion J, Torvinen M, Le Crom S, Casado V, Canela EI, Rondin S, Lew JY, Watson S, Zoli M, Agnati LF, Verniera P, Lluis C, Ferre S, Fuxe K, Franco R. Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proc Natl Acad Sci USA 97: 8606–8611, 2000.[Abstract/Free Full Text]
  25. Godwin SL, Soltoff SP. Extracellular calcium and platelet-derived growth factor promote receptor-mediated chemotaxis in osteoblasts through different signaling pathways. J Biol Chem 272: 11307–11312, 1997.[Abstract/Free Full Text]
  26. Gomeza J, Joly C, Kuhn R, Knopfel T, Bockaert J, Pin JP. The second intracellular loop of metabotropic glutamate receptor 1 cooperates with the other intracellular domains to control coupling to G-proteins. J Biol Chem 271: 2199–2205, 1996.[Abstract/Free Full Text]
  27. Gooch JL. An emerging role for calcineurin A{alpha} in the development and function of the kidney. Am J Physiol Renal Physiol 290: F769–F776, 2006.[Abstract/Free Full Text]
  28. Good DW. PGE2 reverses AVP inhibition of HCO3– absorption in rat MTAL by activation of protein kinase C. Am J Physiol Renal Fluid Electrolyte Physiol 270: F978–F985, 1996.[Abstract/Free Full Text]
  29. Hamm HE. The many faces of G protein signaling. J Biol Chem 273: 669–672, 1998.[Free Full Text]
  30. Hebert SC. Extracellular calcium-sensing receptor: implications for calcium and magnesium handling in the kidney. Kidney Int 50: 2129–2139, 1996.[Web of Science][Medline]
  31. Hill SJ, Baker JG, Rees S. Reporter-gene systems for the study of G-protein-coupled receptors. Curr Opin Pharmacol 1: 526–532, 2001.[CrossRef][Medline]
  32. Husain S, Shearer TW, Crosson CE. Mechanisms linking adenosine A1 receptors and ERK1/2 activation in human trabecular meshwork cells. J Pharmacol Exp Ther. : 2006.
  33. Kaslow HR, Burns DL. Pertussis toxin and target eukaryotic cells: binding, entry, and activation. FASEB J 6: 2684–2690, 1992.[Abstract]
  34. Kurokawa K. The kidney and calcium homeostasis. Kidney Int Suppl 44: 97–105, 1994.
  35. LaMarca BB, Cockrell K, Sullivan E, Bennett W, Granger JP. Role of endothelin in mediating tumor necrosis factor-induced hypertension in pregnant rats. Hypertension 46: 82–86, 2005.[Abstract/Free Full Text]
  36. Lichtarge O, Bourne HR, Cohen FE. Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex. Proc Natl Acad Sci USA 93: 7507–7511, 1996.[Abstract/Free Full Text]
  37. Liu J, Farmer JD Jr, Lane WS, Friedman J, Weissman I, Schreiber SL. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66: 807–815, 1991.[CrossRef][Web of Science][Medline]
  38. Liu K, Hsiung S, Adlersberg M, Sacktor T, Gershon MD, Tamir H. Ca2+-evoked serotonin secretion by parafollicular cells: roles in signal transduction of phosphatidylinositol 3'-kinase, and the gamma and zeta isoforms of protein kinase C. J Neurosci 20: 1365–1373, 2000.[Abstract/Free Full Text]
  39. Liu KP, Russo AF, Hsiung SC, Adlersberg M, Franke TF, Gershon MD, Tamir H. Calcium receptor-induced serotonin secretion by parafollicular cells: role of phosphatidylinositol 3-kinase-dependent signal transduction pathways. J Neurosci 23: 2049–2057, 2003.[Abstract/Free Full Text]
  40. McNeil SE, Hobson SA, Nipper V, Rodland KD. Functional calcium-sensing receptors in rat fibroblasts are required for activation of SRC kinase and mitogen-activated protein kinase in response to extracellular calcium. J Biol Chem 273: 1114–1120, 1998.[Abstract/Free Full Text]
  41. Miedlich S, Gama L, Breitwieser GE. Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations. J Biol Chem 277: 49691–49699, 2002.[Abstract/Free Full Text]
  42. Onrust R, Herzmark P, Chi P, Garcia PD, Lichtarge O, Kingsley C, Bourne HR. Receptor and betagamma binding sites in the alpha subunit of the retinal G protein transducin. Science 275: 381–384, 1997.[Abstract/Free Full Text]
  43. Ortiz PA, Garvin JL. Role of nitric oxide in the regulation of nephron transport. Am J Physiol Renal Physiol 282: F777–F784, 2002.[Abstract/Free Full Text]
  44. Ortiz-Capisano MC, Ortiz PA, Harding P, Garvin JL, Beierwaltes WH. Decreased intracellular calcium stimulates renin release via calcium-inhibitable adenylyl cyclase. Hypertension 49: 162–169, 2006.[CrossRef][Web of Science][Medline]
  45. Pazianas M, Adebanjo OA, Shankar VS, James SY, Colston KW, Maxwell JD, Zaidi M. Extracellular cation sensing by the enterocyte: prediction of a novel divalent cation "receptor". Biochem Biophys Res Commun 210: 948–953, 1995.[CrossRef][Web of Science][Medline]
  46. Pin JP, Joly C, Heinemann SF, Bockaert J. Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors. EMBO J 13: 342–348, 1994.[Web of Science][Medline]
  47. Popova JS, Garrison JC, Rhee SG, Rasenick MM. Tubulin, Gq, and phosphatidylinositol 4,5-bisphosphate interact to regulate phospholipase Cbeta1 signaling. J Biol Chem 272: 6760–6765, 1997.[Abstract/Free Full Text]
  48. Riccardi D, Hall AE, Chattopadhyay N, Xu JZ, Brown EM, Hebert SC. Localization of the extracellular Ca2+/polyvalent cation-sensing protein in rat kidney. Am J Physiol Renal Physiol 274: F611–F622, 1998.[Abstract/Free Full Text]
  49. Riccardi D, Lee WS, Lee K, Segre GV, Brown EM, Hebert SC. Localization of the extracellular Ca2+-sensing receptor and PTH/PTHrP receptor in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 271: F951–F956, 1996.[Abstract/Free Full Text]
  50. Riccardi D, Park J, Lee W, Gamba G, Brown EM, Hebert SC. Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation/sensing receptor. Proc Natl Acad Sci USA 92: 131–135, 1995.[Abstract/Free Full Text]
  51. Rosenkilde MM, McLean KA, Holst PJ, Schwartz TW. The CXC chemokine receptor encoded by herpes virus saimiri, ECRF3, shows ligand-regulated signaling through Gi, Gq, and G12/13 proteins but constitutive signaling only through Gi and G12/13 proteins. J Biol Chem 279: 32524–32533, 2004.[Abstract/Free Full Text]
  52. Ruat M, Molliver ME, Snowman AM, Snyder SH. Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals. Proc Natl Acad Sci USA 92: 3161–3165, 1995.[Abstract/Free Full Text]
  53. Schmidt M, Lohmann B, Hammer K, Haupenthal S, Nehls MV, Jakobs KH. Gi- and protein kinase C-mediated heterologous potentiation of phospholipase C signaling by G protein-coupled receptors. Mol Pharmacol 53: 1139–1148, 1998.[Abstract/Free Full Text]
  54. Terakita A, Yamashita T, Nimbari N, Kojima D, Shichida Y. Functional interaction between bovine rhodopsin and G protein transducin. J Biol Chem 277: 40–46, 2002.[Abstract/Free Full Text]
  55. Wang D, An SJ, Wang WH, McGiff JC, Ferreri NR. CaR-mediated COX-2 expression in primary cultured mTAL cells. Am J Physiol Renal Physiol 281: F658–F664, 2001.[Abstract/Free Full Text]
  56. Wang D, McGiff JC, Ferreri NR. Regulation of cyclooxygenase isoforms in the renal thick ascending limb: effects of extracellular calcium. J Physiol Pharmacol 51: 587–595, 2000.[Web of Science][Medline]
  57. Wang D, Pedraza PL, Abdullah HI, McGiff JC, Ferreri NR. Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells. Am J Physiol Renal Physiol 283: F963–F970, 2002.[Abstract/Free Full Text]
  58. Wang W, Lu M, Balazy M, Hebert SC. Phospholipase A2 is involved in mediating the effect of extracellular Ca2+ on apical K+ channels in rat TAL. Am J Physiol Renal Physiol 273: F421–F429, 1997.[Abstract/Free Full Text]
  59. Ward DT. Calcium receptor-mediated intracellular signalling. Cell Calcium 35: 217–228, 2004.[CrossRef][Web of Science][Medline]
  60. Ward DT, Brown EM, Harris HW. Disulfide bonds in the extracellular calcium-polyvalent cation-sensing receptor correlate with dimer formation and its response to divalent cations in vitro. J Biol Chem 273: 14476–14483, 1998.[Abstract/Free Full Text]
  61. Ward DT, Riccardi D. Renal physiology of the extracellular calcium-sensing receptor. Pflügers Arch 445: 169–176, 2002.[CrossRef][Web of Science][Medline]
  62. Wei Y, Babilonia E, Pedraza PL, Ferreri NR, Wang WH. Acute application of TNF stimulates apical 70-pS K+ channels in the thick ascending limb of rat kidney. Am J Physiol Renal Physiol 285: F491–F497, 2003.[Abstract/Free Full Text]
  63. Weston AH, Absi M, Ward DT, Ohanian J, Dodd RH, Dauban P, Petrel C, Ruat M, Edwards G. Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231. Circ Res 97: 391–398, 2005.[Abstract/Free Full Text]
  64. Woywodt A, Schwarz A, Mengel M, Haller H, Zeidler H, Kohler L. Nephrotoxicity of selective COX-2 inhibitors. J Rheumatol 28: 2133–2135, 2001.[Abstract/Free Full Text]
  65. Yang T, Hassan S, Huang YG, Smart AM, Briggs JP, Schnermann JB. Expression of PTHrP, PTH/PTHrP receptor, and Ca2+-sensing receptor mRNAs along the rat nephron. Am J Physiol Renal Physiol 272: F751–F758, 1997.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
S. Hao, H. Zhao, Z. Darzynkiewicz, S. Battula, and N. R. Ferreri
Expression and function of NFAT5 in medullary thick ascending limb (mTAL) cells
Am J Physiol Renal Physiol, June 1, 2009; 296(6): F1494 - F1503.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
H. I. Abdullah, P. L. Pedraza, J. C. McGiff, and N. R. Ferreri
Calcium-sensing receptor signaling pathways in medullary thick ascending limb cells mediate COX-2-derived PGE2 production: functional significance
Am J Physiol Renal Physiol, October 1, 2008; 295(4): F1082 - F1089.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
V. L. M. Herrera, P. Bagamasbad, T. Didishvili, J. L. Decano, and N. Ruiz-Opazo
Overlapping genes in Nalp6/PYPAF5 locus encode two V2-type vasopressin isoreceptors: angiotensin-vasopressin receptor (AVR) and non-AVR
Physiol Genomics, June 1, 2008; 34(1): 65 - 77.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/2/F345    most recent
00509.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Abdullah, H. I.
Right arrow Articles by Ferreri, N. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Abdullah, H. I.
Right arrow Articles by Ferreri, N. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.