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J Am Soc Nephrol 13:1750-1756, 2002
© 2002 American Society of Nephrology

Angiotensin II Stimulates Calcineurin Activity in Proximal Tubule Epithelia through AT-1 Receptor-Mediated Tyrosine Phosphorylation of the PLC-{gamma}1 Isoform

Janice P. Lea*, Shao G. Jin*, Brian R. Roberts*, Michael S. Shuler*, Mario B. Marrero{dagger} and James A. Tumlin*

*Renal Division, Emory University, Atlanta, Georgia; and {dagger}Vascular Biology Center Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia.

Correspondence to Dr. Janice P. Lea, Renal Division, WMRB Room 338, Emory University School of Medicine, 1639 Pierce Drive, NE, Atlanta, GA 30322. Phone: 404-727-2525; Fax: 404-727-3425; E-mail: jlea{at}emory.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Angiotensin II (AngII) contributes to the maintenance of extracellular fluid volume by regulating sodium transport in the nephron. In nonepithelial cells, activation of phospholipase C (PLC) by AT-1 receptors stimulates the generation of 1,4,5-trisphosphate (IP3) and the release of intracellular calcium. Calcineurin, a serine-threonine phosphatase, is activated by calcium and calmodulin, and both PLC and calcineurin have been linked to sodium transport in the proximal tubule. An examination of whether AngII activates calcineurin in a model of proximal tubule epithelia (LLC-PK1 cells) was performed; AngII increased calcineurin activity within 30 s. An examination of whether AngII activates PLC in proximal tubule epithelia was also performed after first showing that all three families of PLC isoforms are present in LLC-PK1 cells. Application of AngII increased IP3 generation by 60% within 15 s, which coincided with AngII-induced tyrosine phosphorylation of the PLC-{gamma}1 isoform also observed at 15 s. AngII-induced tyrosine phosphorylation was blocked by the AT-1 receptor antagonist, Losartan. Subsequently, an inhibitor of tyrosine phosphorylation blocked the AngII-induced activation of calcineurin, as did coincubation with an inhibitor of PLC activity and with an antagonist of the AT-1 receptor. It is therefore concluded that AngII stimulates calcineurin phosphatase activity in proximal tubule epithelial cells through a mechanism involving AT-1 receptor–mediated tyrosine phosphorylation of the PLC isoform.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II type 1 (AT-1) receptor signal transduction pathways in both vascular smooth muscle cells (VSMC) and mesangial cells include activation of phospholipase C (PLC) and generation of inositol triphosphate (IP3) (1,2). PLC has been linked in turn to AngII-mediated sodium transport in the proximal tubule (3,4). An increase in intracellular IP3 could act on several pathways, but we were interested in calcium-mediated pathways. For example, an increase in intracellular IP3 will stimulate the release of calcium from the endoplasmic reticulum, resulting in activation of calcineurin and other calcium-dependent enzymes (5). Calcineurin is a heterotrimeric protein phosphatase composed of calmodulin, an {alpha} catalytic subunit, and a {beta} regulatory subunit (5).

The physiologic roles of calcineurin in the kidney are not completely elucidated, but recent studies demonstrate that calcineurin does influence basal and receptor-mediated sodium transport in nephron segments (6–8). In microdissected cortical collecting ducts for example, inhibition of calcineurin activity with FK-506 decreases Na/K-ATPase activity by 85% and reduces Na/K-ATPase activity in medullary thick ascending limbs by 56% (7). Aperia et al. (8) concluded that calcineurin was involved in AngII-stimulated Na/K-ATPase, but no studies have measured AngII’s direct effect on calcineurin phosphatase activity in the kidney. We report for the first time that AngII stimulates calcineurin activity in a proximal tubule cell line (LLC-PK1 cells) and examine the signal transduction pathway by which this occurs.

In VSMC, AT-1 receptor activation has been linked to both G protein–coupled stimulation of PLC-{beta} isoforms and to tyrosine phosphorylation of PLC-{gamma} isoforms (9). We recently demonstrated that all three families of PLC isoforms (PLC-{beta}, -{gamma}, and -{delta}) are expressed throughout the rat nephron (10), but the role of PLC in AngII-mediated signaling and sodium transport in the proximal tubule is controversial (11,12,13). Because of the heterogeneity of cell types along the nephron, AngII signaling in specific nephron segments could involve different PLC isoforms. We investigated the role of specific PLC isoforms in AngII- mediated signaling mechanisms in LLC-PK1 cells, a cell culture model of proximal tubule epithelia. We find similar PLC isoforms present in LLC-PK1 cells as we did in microdissected rat proximal tubules (10) and report that AngII stimulates calcineurin activity through a pathway involving tyrosine phosphorylation of the PLC-{gamma}1 isoform.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
LLC-PK1 Cell Culture
LLC-PK1 cells were maintained in Dulbecco modified Eagle medium (DMEM; Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% (vol/vol) fetal bovine serum (FBS, Life Technologies), 100 mg/ml streptomycin, and 100 U/ml penicillin. The cells were grown to confluence on semipermeable supports (0.02 mm; Nunc Inc., Naperville, IL) using 100-mm culture dishes (37°C in a 5% CO2-enriched, humidified atmosphere). To growth-arrest the cells, media was replaced with serum-free DMEM for 24 to 48 h before each experiment.

Calcineurin Substrate
A calcineurin-specific substrate derived from the RII regulatory subunit of protein kinase A (PKA) was synthesized and labeled with 32PO4 using the catalytic subunit of PKA (Sigma, St. Louis, MO) and {gamma} 32PO4-ATP, (10 Ci/mmol; Dupont/New England Nuclear, Boston, MA) (14). Radiolabeled peptide was separated from unincorporated 32PO4 using Sep-Pak C18 Sephadex column chromatography (Millipore Corp., Milford, MA) and 20 ml of 0.1% trifluoroacetic acid (TFA, Sigma). The protein content of the eluted peptide was measured with a BCA protein assay kit (Pierce Immunotechnology, Rockford, IL) and kept frozen at -80°C until used.

Calcineurin Activity in LLC-PK1 Cells
Confluent LLC-PK1 cells on semipermeable supports were removed by scraping and permeabilized with imidazole (100 mM), hypotonic shock, and rapid freeze/thawing (14). Calcineurin activity was measured in 20 mM Tris (pH 8.0) containing 100 mM NaCl, 0.5 mM DTT, 0.1 mM CaCl2, 0.1 mg/ml BSA, 100 nM calmodulin, and 100 nM calyculin, a specific inhibitor of class 1 and 2A phosphatases (L.C. Laboratories, Boston, MA). Distilled H2O was added to a final reaction volume of 10 µl. AngII was incubated with permeabilized LLC-PK1 cells for periods of 30 s to 5 min. The final concentration of radiolabeled calcineurin substrate was 200 p.m. The reaction was stopped with 500 µl of an ice-cold mixture of 10% TCA and 5% activated charcoal. The reaction tubes were centrifuged at 14,000 rpm for 15 min, and the supernatant was filtered through a 450-µm nitrocellulose filter (Millipore). Total radioactivity of hydrolyzed 32PO4 was counted by liquid scintillation, and calcineurin activity is reported as femtomoles of substrate hydrolyzed/mg protein per min. To determine the role of PLC, tyrosine kinases, and AT receptors, LLC-PK1 cells were pretreated with 10 µM U-71322 (a non–isoform-specific PLC antagonist) or with a negative control -10 µM U-73433 (a structural analogue of U-73122 that does not inhibit PLC). Cells were then pretreated with 120 µM genistein (a tyrosine kinase antagonist) as well as with 10-5 M Losartan (AT-1 antagonist) or with 10-6 M PD 123177 (AT-2 antagonist) for 30 min before addition of AngII.

Measurement of 1,4,5-IP3 in LLC-PK1 Cells
After exposure of LLC-PK1 cells to Ang-II (10-7 M) for 15 s to 6 min, cells were lysed and proteins were precipitated using 1 ml of ice-cold TCA 10% (TCA). The lysate was centrifuged for 10 min at 1000 x g, and 1,4,5-IP3 was measured by radioimmunoassay (RIA, New England Nuclear). IP3 generation is reported as picomoles per 106 cells.

Western Blot Analyses of PLC Isoforms
After removal of media, LLC-PK1 cells were treated with sodium dodecyl sulfate (SDS) buffer containing 0.5 M Tris, glycerol, 10% SDS, 0.05% bromophenol blue, and 10% 2{beta}-mercaptoethanol. The plates were scraped, and cells were disrupted by repeated passages through an insulin syringe needle and stored at 4°C. Isolated glomeruli were suspended in 6% SDS buffer containing 0.125 M Tris HCl (pH 7.5), 20% glycerol (by vol), 10 mg/ml leupeptin, 5 mg/ml chymostatin, 10 mg/ml aprotinin, 50 mM benzamidine, 10 mg/ml PMSF, 10 mg/ml pepstatin, and 5 mg/ml sodium trypsin inhibitor. In other experiments, cortical and medullary slices of normal rat kidneys were placed in lysis buffer (see above); proteins were collected and size-separated by SDS-PAGE (7.5%). The cells and tissues were transferred to nitrocellulose membranes and probed with isoform-specific antibodies to the {beta}1 and 2, {gamma}1 and 2, and {delta}1 and 2 isoforms of PLC (Santa Cruz Biotechnology, Santa Cruz, CA, or Upstate Biotechnology Incorporated, Lake Placid, NY). Proteins from rat brain and liver were used as positive controls (10).

Immunoprecipitation of Tyrosine Phosphorylated Proteins
LLC-PK1 cells were incubated with 10-7 M AngII from 30 s to 6 min. The plates were scraped, and cell lysates were obtained as described before centrifugation at 6000 x g for 20 min. Cell lysates were incubated with monoclonal phosphotyrosine antibodies (Upstate Biotechnology, Inc.) for 24 h and then with protein G agarose beads for 2 h at 4°C. The immunoprecipitates were size-separated by SDS-PAGE, transferred to nitrocellulose, and probed with monoclonal antibodies to the PLC-{gamma}1, -{beta}1, and -{delta}1 isoforms. To determine if the AngII-induced tyrosine phosphorylation was mediated by the AT-1 or AT-2 receptor, cells were preincubated with 10-5 M Losartan (AT-1 antagonist) or with 10-6 M PD 123177 (AT-2 antagonist) for 30 min before addition of AngII.

Statistical Analyses
Statistical significance was calculated by a t test when two groups were compared on ANOVA to compare results from three or more groups, followed by a multiple comparisons. A Tukey’s protected t test was performed to determine statistically significant values. Data are presented as mean ± SEM, and values of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AngII Stimulates Calcineurin Activity in LLC-PK1 Cells
AngII (10-7M) was added to the apical surface of LLC-PK1 cells grown on semipermeable supports for periods of 30 s to 4 min. Apical application of AngII increased calcineurin activity at 30 s, with a 79% increase at 1 min (Figure 1). Calcineurin buffer served as a negative control. Longer incubations (up to 30 min) did not increase calcineurin activation further (data shown only up to 4 min). Lower concentrations of AngII (10-11 M) did not activate calcineurin activity (data not shown).



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  		Figure 1. Apical angiotensin II (AngII) increases calcineurin activity in LLC-PK1 Cells. LLC-PK1 cells were grown to confluence on semipermeable supports in six-well plates at 37°C. AngII (10-7 M) was added to the apical surface for 30 s to 4 min. Reactions were stopped by and lysates incubated with calcineurin substrate for 15 min. AngII (solid line) significantly increased calcineurin activity at 30 s. Calcineurin buffer was used as a vehicle control (dashed line). * P < 0.01 versus control value.

 
To investigate if PLC is involved in the stimulation of calcineurin activity by AngII, we first determined which PLC isoforms were present in LLC-PK1 cells. Protein extracts from LLC-PK1 cells were size-separated by SDS-PAGE and probed with monoclonal antibodies recognizing the {beta}1 and {beta}2, {gamma}1 and {gamma}2, or {delta}1 and {delta}2 isoforms of PLC. Figure 2 is a representative Western blot that demonstrates the presence of 150-, 145-, and 85-kD bands consistent with the {beta}, {gamma}, and {delta} isoforms, respectively. These results are similar to our findings in freshly microdissected proximal tubules (10). We then examined changes in the product of PLC activity, inositol trisphosphate, or IP3 by measuring the time course of AngII-induced IP3 generation. AngII (10-7 M) was added to the apical surface of LLC-PK1 cells, resulting in a 60% increase in IP3 generation after 15 s (Figure 3). Note that this response precedes AngII’s activation of calcineurin that occurred at 30 s (Figure 1). We then pretreated the apical surface of LLC-PK1 cells with 10 µM U-73122 (a non–isoform-specific PLC antagonist) for 10 min before adding AngII at various timepoints. Figure 4 shows that U-73122 completely blocked AngII-stimulated calcineurin activity. We then incubated cells with a structural analogue of U-73122, U-73433 (10 µM), which has negligible inhibitory activity and is a useful control compound (15), for 10 min before adding AngII. Inhibition of PLC activity induced by calcineurin occurred with U-73122 but not with U-73433 (Figure 5).



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  		Figure 2. Phospholipase C (PLC) isoform expression in LLC-PK1 cells. Proteins from LLC-PK1 cells grown on plastic were harvested and size-separated by SDS-PAGE. Western blots were probed with monoclonal antibodies recognizing the {beta}1, {beta}2, {gamma}1, {gamma}2, {delta}1, and {delta}2 isoforms of PLC. Bands of the appropriate size for each of these six PLC isoforms were detected.

 


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  		Figure 3. AngII stimulates 1,4,5-trisphosphate (IP3) generation in LLC-PK1 cells. LLC-PK1 cells were grown to confluence in six-well plates at 37°C. AngII (10-7 M) was added to the cells for 15 s to 6 min. Reactions were stopped with ice-cold TCA and cell scraping. AngII significantly increased IP3 levels at 15 s and 3 min. IP3 levels were reported as pmoles of IP3 per 107 cells. * P < 0.05.

 


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  		Figure 4. AngII stimulates calcineurin activity through a PLC-dependent pathway. LLC-PK1 cells were grown to confluence on semipermeable supports and incubated with the PLC inhibitor, U-73122 (10 µM; dashed line) for 10 min. AngII (10-7 M) was added to the apical surface of U-73122– or vehicle-treated cells for 30 s to 4.0 min. U-73122 blocked calcineurin activation by AngII. * P < 0.05 versus control.

 


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  		Figure 5. AngII-stimulated calcineurin activity blocked by U73122 but not by U73433. LLC-PK1 cells were grown to confluence on semipermeable supports and incubated with the structural analogue of U-73122, U-73433 (10 µM) for 10 min before the application of AngII (10-7 M) for 4 min. U-73122 blocked calcineurin activation by AngII but not U-73433. * P < 0.05.

 
Tyrosine Kinase Activation by AngII
To determine if AngII-induced phosphorylation of PLC is required for activating calcineurin, the apical surface of LLC-PK1 cells was incubated with 120 µM genistein (a tyrosine kinase antagonist) for 60 min. Genistein completely blocked the activation of calcineurin by AngII, suggesting that tyrosine phosphorylation mediates AngII signaling in LLC-PK1 cells (Figure 6). The PLC-{gamma}1 isoform in VSMC has been linked to tyrosine phosphorylation events; we therefore examined if the PLC-{gamma}1 isoform is tyrosine phosphorylated in response to AngII. Proteins from LLC-PK1 cells were treated with AngII (10-7 M) for periods of 15 s, 30 s, and 3 min. Proteins were immunoprecipitated using phosphotyrosine antibodies and size-separated by SDS-PAGE before probing with PLC-{gamma}1 monoclonal antibodies. As shown in Figure 7, AngII stimulated tyrosine phosphorylation of the PLC-{gamma}1 isoform, with a peak in phosphorylation at 15 s. We also performed similar immunoprecipitation experiments probing with PLC-{beta}1 and {delta}1 antibodies and found no change in tyrosine phosphorylation (data not shown). Thus, only the PLC-{gamma}1 isoform appears to be tyrosine phosphorylated in response to AngII.



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  		Figure 6. AngII stimulates calcineurin activity through activation of tyrosine kinases. LLC-PK1 cells were grown to confluence on semipermeable supports and incubated with genistein (120 µM; dashed line) or vehicle (solid line) for 60 min. AngII (10-7 M) was added to the apical surface of genistein- and vehicle-treated cells for 30 s to 4.0 min. Genistein blocked calcineurin activation by AngII. * P < 0.01.

 


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  		Figure 7. AngII stimulates tyrosine phosphorylation of PLC {gamma}-1 isoforms in LLC-PK1 cells. LLC-PK1 cells were incubated with AngII (10-7 M) for 15 s, 30 s, or 3 min. After stopping the reactions, cell lysates were incubated with phosphotyrosine antibodies for 24 h. Tyrosine phosphorylated proteins were separated with protein G agarose beads and size-separated by SDS-PAGE. Western blots were probed with monoclonal PLC-{gamma}1 antibodies (top panel). Laser densitometry analysis demonstrates that AngII significantly increased tyrosine phosphorylation of PLC-{gamma}1 isoforms at 15 s. * P < 0.05; n = 5.

 
AngII’s Effects on Tyrosine Phosphorylation and Calcineurin Are Mediated by the AT-1 Receptor
We preincubated LLC-PK1 cells with either the AT-1 antagonist Losartan (10-5 M) or with the AT-2 antagonist PD 123177 (10-6 M) for 30 min before AngII application. Figure 8 shows that the AngII-induced tyrosine phosphorylation that occurred at 15 s is blocked by the AT-1 antagonist but not by the AT-2 antagonist. Finally, we again preincubated LLC-PK1 cells with either the AT-1 antagonist Losartan (10-5 M) or with the AT-2 antagonist PD 123177 (10-6 M) for 30 min before AngII application and measured calcineurin activity. As in Figure 9, Losartan completely blocked AngII-induced calcineurin activity and PD 12377 did not.



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  		Figure 8. AngII-induced tyrosine phosphorylation of PLC-{gamma}1 is mediated by the AT-1 receptor. LLC-PK1 cells were pretreated with Losartan (10-5 M) or PD 123177 (10-6 M) for 30 min before incubation with AngII (10-7 M) for 15 s, 1.5 min, or 3 min. Immunoprecipitation followed by Western blot analysis was performed as in Figure 7 (panel A, showing increased tyrosine phosphorylation at 15 s). The AT-1 antagonist Losartan completely blocked AngII-induced tyrosine phosphorylation at 15 s (B), but no decrease was observed with the AT-2 blocker PD 123177 (C); compare all panels at 15 s.

 


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  		Figure 9. AngII-induced calcineurin activity is mediated by the AT-1 receptor. LLC-PK1 cells were pretreated with Losartan (10-5 M) or PD 12377 (10-6 M) for 30 min before incubation with AngII (10-7 M) for 4 min. Losartan completely blocked AngII-induced calcineurin activation, but PD 12377 did not. PD12 indicates PD 12377. * P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results demonstrate that apical administration of AngII stimulates calcineurin phosphatase activity in a model of proximal tubule epithelia, LLC-PK1 cells. The activation of calcineurin in response to apical AngII application is important because apical AngII stimulates proximal tubule sodium transport (13). All three PLC isoform families are expressed in LLC-PK1 cells; which suggests that LLC-PK1 cells may be a good cell culture model system for studying PLC signal transduction events in the proximal tubule in vivo, because the same pattern is found in fresh microdissected rat tubules (10). We found evidence of a link between PLC and calcineurin on the basis of the following observations. First, before the activation of calcineurin at 30 s, AngII activates the AT-1 receptor and induces tyrosine phosphorylation of the PLC- {gamma}1 isoform in LLC-PK1 cells, leading to a 60% increase in IP3 levels at 15 s. Second, coincubation of cells on the apical surface with an inhibitor of phospholipase C, tyrosine kinases, or an AT-1 receptor antagonist blocks activation of calcineurin by AngII.

AngII contributes to the maintenance of extracellular fluid volume by regulating sodium transport in the proximal and distal nephron (3,16,17). Aperia et al. (8) found that AngII increases Na/K-ATPase in microdissected proximal tubules. Pretreatment with FK-506 (an inhibitor of calcineurin phosphatase activity) reversed AngII-mediated stimulation of Na/K-ATPase activity. They concluded that calcineurin must be involved in the pathway between AngII-mediated activation of the AT-1 receptor and the sodium pump. Indeed, in vitro and in vivo studies have shown that phosphorylation of the {alpha} catalytic subunit of the Na/K-ATPase can reduce activity of this enzyme (18,19). We have extended these studies by showing that calcineurin can regulate Na/K-ATPase activity in the proximal and distal nephron (20,7). Regarding the signaling pathway between AngII and calcineurin, Schelling et al. (3) isolated proximal tubules from rat kidney and demonstrated that apical application of AngII (10-7 M) stimulated 22Na+ transport within 10 min. When the tubules were pretreated with U-73122 (a non–isoform-specific inhibitor of PLC) (21), stimulation of sodium transport by AngII was completely blocked (3).

We decided to examine this pathway in more detail and applied 10-7 M AngII to the apical surface of LLC-PK1 cells and measured calcineurin activity. Calcineurin activity increased at 30 s with a 79% increase at 1 min. To confirm our hypothesis that PLC could be involved in AngII-stimulated calcineurin activity in the proximal tubule, we pretreated LLC-PK1 cells with a PLC-inhibitor, U-73122, before applying AngII and found no calcineurin activation (Figure 4). To further confirm the involvement of PLC, we measured IP3 and found that AngII stimulated the generation of IP3 at 15 s (Figure 3). This increase precedes the activation of calcineurin and agrees with the time course of AngII-induced IP3 generation noted by Poggioli et al. (22), which occurred at 10 s and was followed by a peak in intracellular calcium at 15 s. Thus, AngII could be stimulating calcineurin phosphatase activity through IP3-mediated increases in intracellular calcium.

Alternative PLC-independent pathways that increase intracellular calcium have been described in the proximal tubule. Douglas et al. (12) found that 10-8 M AngII stimulated PLA2 activity in proximal tubule cells, resulting in the release of arachidonic acid and lysophosphatidylcholine. In subsequent studies, Madhun et al. (23) found that metabolism of arachidonic acid by the cytochrome P-450 pathway resulted in the synthesis of 5,6-epoxyeicosatrienoic acid, a metabolite that increases extracellular calcium (Ca++) flux through voltage-sensitive Ca++ channels. Thus, signaling through the apical membrane could occur through PLC-independent pathways. Our results demonstrate that PLC is involved in AngII-induced calcineurin activity in LLC-PK1 cells; incubation of cells with U-73122 blocked calcineurin activation by AngII, and the structural analogue of U-73122, U-73433, which has negligible inhibitory activity (15) did not block calcineurin (Figures 4 and 5). U-73122 has been characterized in other systems as a specific PLC inhibitor; it has no effect on adenylyl cyclase or phospholipase A2 (21).

We demonstrated the expression of PLC-{beta}1 and 2, {gamma}1 and 2, and {delta}1 and 2 isoforms in LLC-PK1 cells (Figure 2). Previous studies in VSMC demonstrated that AT-1 receptor activation resulted in the tyrosine phosphorylation of the PLC-{gamma}1 isoform (1). To determine if the PLC-{gamma}1 isoform undergoes tyrosine phosphorylation in LLC-PK1 cells, we immunoprecipitated proteins from AngII-treated cells using phosphotyrosine antibodies. We observed a peak phosphorylation of the {gamma}1 isoform at 15 s after AngII addition (Figure 7). Moreover, inhibition of tyrosine kinases by genistein blocked the activation of calcineurin by AngII (Figure 6). Thus, our data show that tyrosine phosphorylation of the PLC-{gamma}1 isoform precedes the time course for AngII-induced activation of calcineurin. As has been reported in VSMC, we found that AT-1 receptor activation is responsible for the induction of PLC-{gamma}1 tyrosine phosphorylation in LLC-PK1 cells (Figure 8). In addition, we report direct evidence that the AT-1 receptor mediates calcineurin activation by AngII because calcineurin activation was blocked by an AT-1 antagonist, Losartan, but not by an AT-2 antagonist, PD12377 (Figure 9).

Few studies have investigated the link between AngII receptor signaling and calcineurin activity. In a study of cultured bovine adrenal glomerulosa cells, calcineurin was required for AngII to augment ACTH-induced stimulation of adenylyl cyclase (24). Others reported that calcineurin is activated in response to AngII stimulation in VSMC and is translocated into the nucleus (25). We report that the AT-1 receptor mediates changes in calcineurin activity in proximal tubule epithelia. Lastly, AngII-stimulated cardiac hypertrophy involves calcineurin and subsequent dephosphorylation of the transcription factor NF-AT3 (nuclear factor of activated T cells) (26). Additional studies are needed to determine whether calcineurin is important to similar hypertrophic processes in the kidney.

On the basis of our results, we suggest the following scheme for AngII-mediated signal transduction events in the proximal tubule. AngII activates the AT-1 receptor leading to increased tyrosine phosphorylation of the PLC-{gamma}1 isoform, resulting in IP3 release within 15 s. Subsequent activation of intracellular calcium could stimulate calcineurin phosphatase activity within 30 s. Our data do not exclude additional mechanisms of AngII-dependent calcineurin activation, including other non–IP3-dependent mediators of intracellular calcium, nor do they exclude a role for other PLC isoforms. Only the PLC-{gamma}1 isoform was tyrosine phosphorylated, and inhibition of PLC and of tyrosine kinases completely blocked calcineurin activation by AngII; it would therefore appear that the PLC-{gamma}1 signaling system transduces the majority of AngII-mediated calcineurin phosphatase activity in cultured proximal tubule cells. The physiologic significance of this finding may be that calcineurin phosphatase activity is closely linked to changes in intracellular calcium related to changes in IP3. Via this mechanism, activation of the AT-1 receptor could increase proximal tubular Na/K-ATPase activity through a calcineurin-mediated reduction in {alpha} subunit phosphorylation.


   Acknowledgments
 
The authors thank Drs. William E. Mitch and Jeff M. Sands for their critical review of the manuscript. This work was supported by a Robert Wood Johnson Foundation Faculty Development Award and an NIH minority supplement award (3 P01 DK50268–02S1) to J. P. Lea, an NIH grant (P01-DK50268) to M. B. Marrero, and NIH grants (P01-DK50268 and RO1 HL59978) and the Marguerite Mason Transplantation trust to J. A. Tumlin.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication March 20, 2002. Accepted for publication April 3, 2002.




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