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Intracellular ANG II induces cytosolic Ca²⁺ mobilization by stimulating intracellular AT₁ receptors in proximal tubule cells

¹Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan; and ²Department of Physiology, Tulane University Health Sciences Center, New Orleans, Louisiana

Submitted 1 July 2005 ; accepted in final form 19 December 2005

	ABSTRACT

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Intracellular ANG II induces biological effects in nonrenal cells, but it is not known whether it plays a physiological role in renal proximal tubule cells (PTCs). PTCs express angiotensinogen, renin, and angiotensin-converting enzyme mRNAs, suggesting the presence of high levels of intracellular ANG II. We determined if microinjection of ANG II directly in single PTCs increases intracellular calcium concentration ([Ca²⁺]_i) and, if so, elucidated the cellular mechanisms involved. Changes in [Ca²⁺]_i responses were studied by fluorescence imaging using the Ca²⁺ indicator fluo 3. ANG II (1 nM) was microinjected directly in the cells, whereas cell-surface angiotensin type 1 (AT₁) receptors were blocked by losartan (10 µM). When ANG II (1 nM) was added to the perfusate, there was a marked increase in [Ca²⁺]_i that was blocked by extracellular losartan. With losartan in the perfusate, intracellular microinjection of ANG II elicited a robust increase in cytoplasmic [Ca²⁺]_i that peaked at 30 s (basal: 2.2 ± 0.3 vs. ANG II: 14.9 ± 0.4 relative fluorescence units; P < 0.01). Chelation of extracellular Ca²⁺ with EGTA (2 mM) did not alter microinjected ANG II-induced [Ca²⁺]_i responses (Ca²⁺ free + ANG II: 12.3 ± 2.6 relative fluorescence units, not significant vs. ANG II); however, pretreatment with thapsigargin to deplete intracellular Ca²⁺ stores or with U-73122 to inhibit phospholipase C (1 µM each) markedly attenuated microinjected ANG II-induced [Ca²⁺]_i responses. Combined microinjection of ANG II and losartan abolished [Ca²⁺]_i responses, whereas a combination of ANG II and PD-123319 had no effect. These data demonstrate for the first time that direct microinjection of ANG II in single PTCs increases [Ca²⁺]_i by stimulating intracellular AT₁ receptors and releases Ca²⁺ from intracellular stores, suggesting that intracellular ANG II may play a physiological role in PTC function.

intracellular calcium; kidney; microinjection; proximal tubules; receptor-mediated endocytosis

Although it is widely accepted that ANG II exerts diverse effects by activating cell-surface AT₁ receptors in targeted cells (6, 39), recent studies suggest that intracellular ANG II, either accumulated through internalization or synthesized within the cells, is also capable of inducing intracellular responses (5, 7–10). Haller et al. (10) demonstrated that microinjection of ANG II in VSMCs increased intracellular [Ca²⁺]_i, and this response was blocked by intracellular administration of candesartan, an AT₁ receptor antagonist (10). Similarly, dialysis of ANG II in VSMCs or cardiac myocytes has been shown to stimulate voltage-operated Ca²⁺ channels (8) or reduce inward calcium currents (7). Cook et al. (5) recently reported that ANG II generated within hepatocytes appeared to exert mitogenic effects. Although PTCs are known to produce and accumulate high levels of ANG II, it remains unclear whether intracellular ANG II exerts any role in regulating proximal tubular cell function. PTCs are structurally and functionally different from VSMCs, hepatocytes, or cardiac myocytes, and they may have completely different responses to extracellular and intracellular ANG II. Studies by Schelling and Linas (25) suggested that endocytosis of the ANG II-AT₁ receptor complex may be required for full expression of biological actions of ANG II in PTCs. Moreover, Thekkumkara et al. (31, 32) demonstrated that internalization of an AT_1A receptor-ANG II complex increases transcellular sodium transport in opossum kidney cells. Although these results suggest possible roles for intracellular ANG II, the influence of intracellular ANG II in PTC function remains poorly understood. In the present study, we hypothesized that intracellular ANG II activates cytoplasmic AT₁ receptors to increase intracellular calcium concentration ([Ca²⁺]_i) by mobilizing Ca²⁺ from intracellular stores in PTCs. We found that microinjection of ANG II directly into single PTCs induced intracellular Ca²⁺ responses that were mediated by intracellular AT₁ receptors and mobilization of intracellular Ca²⁺ stores but not dependent on influx of extracellular Ca²⁺.

	MATERIALS AND METHODS

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PTC culture. Cultured PTCs were obtained from American Type Culture Collection and subcultured as described previously (16, 24, 28). These cells were initially derived from the S₁ segment of rabbit kidney proximal tubules and have been shown to express electrolyte transporters and major components of the RAS, including angiotensinogen, renin, ACE, and ANG II receptors (24). In the present study, unless specified otherwise, PTCs were subcultured on glass coverslips for measurements of [Ca²⁺]_i responses in complete DMEM-F-12 growth medium supplemented with 50 nM hydrocortisone, 5% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained at 37°C and 95% O₂-5% CO₂ and fed every 2–3 days. Serum was removed from the medium for 24 h before the experiments began (16, 28).

Expression of AT₁ receptor protein in PTCs. To confirm that PTCs we used express AT₁ receptors, the cells were split into six-well plates and grown to 80% confluence as described above. After being serum starved for 24 h, cells were washed two times with ice-cold PBS and lysed with a modified RIPA buffer [50 mM Tris·HCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM Na₃VO4, and 1 mM NaF, pH 7.4]. Samples were extracted as described previously (12), and protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce). Proximal tubule protein samples (10 µg each) were electrophoretically separated on 8–16% Tris-glycine gels at 120 volts for 1.5–2 h. After SDS separation, proteins were transferred to Millipore Immobilon-P membranes using a Bio-Rad Trans-Blot Semi-Dry system powered by a Bio-Rad Power-Pac HC (25 volts, 0.12 ampere, 1.5 h). The membranes were blotted overnight at 4°C with 5% nonfat dry milk and incubated for 3 h at room temperature with a primary rabbit anti-AT₁ receptor polyclonal antibody raised against the NH₂-terminal extracellular domain of AT₁ receptors (1:200, SC-1173; Santa Cruz; see Ref. 12). To determine the specificity of the antibody, an AT₁ receptor-selective blocking peptide was used to treat the samples before running the Western blots (SC-1173BP; Santa Cruz). To ensure equal protein loading, the same membranes were treated with a stripping buffer (Pierce) for 20 min, blotted with 5% nonfat dry milk, and reprobed with a mouse anti--actin monoclonal antibody at 1:2,000 (Sigma-Aldrich). Western blot signals were detected using enhanced chemiluminescence (Amersham) and analyzed using a microcomputer imaging device with a digital camera (MCID; Imaging Research).

Measurement of intracellular ANG II in PTCs. To confirm that PTCs we used produce endogenous ANG II and exposure of these cells to extracellular ANG II increases intracellular ANG II levels via AT₁ receptor-mediated endocytosis, cells were treated with vehicle (serum-free medium), ANG II (1 nM), or ANG II plus losartan (10 µM) to block receptor-mediated endocytosis for 60 min at 37°C. After treatment, the medium was removed, and the cells were washed two times with ice-cold PBS and then washed one time with ice-cold acid buffer (5 mM acetic acid, 150 mM NaCl, pH 2.5) to remove any remaining cell membrane-bound ANG II as described previously (1, 9, 13, 32). ANG II was extracted from PTCs in a buffer containing 20 mM Tris·HCl, 10 mM EDTA, 5 mM EGTA, 5 mM mercaptoethanol, 50 g/ml PMSF, 1 µg/ml aprotinin, and 1 µg/ml pepstatin and measured using a sensitive and specific ANG II enzyme immunoassay kit (Biochem/Peninsula). Protein concentration in each sample was used to calculate final ANG II levels expressed as picograms per milligram protein.

Monitoring [Ca²⁺]_i responses in single PTCs. Subconfluent PTCs were sparsely plated on glass coverslips and loaded with the calcium-sensitive fluorescent dye fluo 3-AM (2 µM; Molecular Probes), which was prepared in PBS for 30 min at 37°C (10, 18). After incubation, coverslips with attached cells were rinsed two times with PBS to remove extracellular dye and extracellular agonist(s) and placed inside a perfusion chamber mounted on a Nikon Diaphot 200 inverted microscope with a TE-FM epifluorescence attachment and a fluorescence filter suitable for fluo 3 (excitation: 488-nm wavelength; emission: 526; see Ref. 10). The cells were continuously maintained in the perfusate (10 mM HEPES, 1.5 mM CaCl₂, 145 mM NaCl, 5 mM KCl, 0.5 Na₂HPO₄, 6 mM glucose, and 0.5 mM MgSO₄, pH 7.4) at 37°C. The [Ca²⁺]_i responses to extracellular and intracellular ANG II were recorded with a digital camera and microcomputer imaging device (Imaging Research), first at basal levels and then at 5-s intervals after microinjection of ANG II until responses returned to baseline.

Effect of extracellular ANG II and activation of cell-surface AT₁ receptors on [Ca²⁺]_i in PTCs. Experiments were first carried out to confirm that extracellular ANG II increases [Ca²⁺]_i in PTCs and that these effects are blocked when losartan is added to the perfusate. ANG II was added to the perfusate at 1 nM and losartan at 10 µM. These concentrations were based on the observations that proximal tubule fluid and interstitial fluid contain nanomolar levels of ANG II (2, 22, 27) and that at 10 µM losartan completely displaced ANG II receptor binding in rat proximal tubules (36–38).

Effect of intracellular microinjection of ANG II on [Ca²⁺]_i in single PTCs. To determine whether intracellular ANG II increases [Ca²⁺]_i, ANG II (1 nM; Bachem) was microinjected directly into single PTCs while blocking cell-surface AT₁ receptors with losartan in the perfusate (10 µM; Fig. 1). Intracellular microinjection of ANG II was carried out using a Narishige micromanipulator and microinjector at a volume of 70–100 fl/cell with a glass micropipette (10). Success of intracellular microinjection of ANG II was confirmed using FITC-labeled ANG II (Molecular Probes) to follow intracellular trafficking of microinjected FITC-ANG II and to exclude possible leaking of microinjected ANG II in the extracellular space (Fig. 1).

View larger version (48K): [in this window] [in a new window]   Fig. 1. Intracellular microinjection of unlabeled or FITC-ANG II in single proximal tubule cells (PTC). A: phase contrast micrograph taken before insertion of a glass micropipette. B: micrograph taken after insertion of a glass micropipette. C: fluorescence micrograph before microinjection of FITC-ANG II. D: fluorescence micrograph 30 s after microinjection of FITC-ANG II. Note that FITC-ANG II was distributed throughout the cytoplasm. N, nucleus. Magnification: x40.

Role of extracellular Ca²⁺ in [Ca²⁺]_i responses to microinjected ANG II in single PTCs. To determine whether the effects of microinjected ANG II on [Ca²⁺]_i are dependent on influx of extracellular Ca²⁺, cells were perfused in a medium containing zero Ca²⁺ with 2 mM EGTA added while ANG II was injected into the cells (10). [Ca²⁺]_i responses in a single cell on a coverslip were recorded for up to 10 min after ANG II microinjection.

Role of intracellular calcium stores in [Ca²⁺]_i responses to microinjected ANG II in single PTCs. Cells were first pretreated with 1 µM thapsigargin for 30 min to deplete intracellular calcium stores before microinjecting ANG II in the cells (10, 18). [Ca²⁺]_i responses in a single cell on a cover slip were recorded for up to 10 min after microinjection.

Role of phospholipase C/protein kinase C signaling pathway(s) in [Ca²⁺]_i responses to microinjected ANG II in single PTCs. Cells were pretreated with U-73122 (1 µM), a selective phospholipase C (PLC) inhibitor, for 30 min before microinjecting ANG II in the cells. [Ca²⁺]_i responses in a single cell on a cover slip were recorded for up to 10 min after microinjection.

Statistical analysis. Data are presented as means ± SE. [Ca²⁺]_i responses as determined by fluo 3 fluorescence imaging, represented as relative fluorescence units. Differences in intracellular ANG II levels or [Ca²⁺]_i responses between different groups of cells were analyzed using one-way ANOVA followed by Dunnett’s comparison between group means, taking P < 0.05 as significant.

	RESULTS

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Western blot of AT₁ receptor protein in PTCs. Western blot showed a single protein band of 42 kDa (Fig. 2A, top) in PTCs, which was markedly inhibited by an AT₁ receptor-selective blocking peptide (Fig. 2, A and B). The AT₁ receptor protein detected is consistent with the AT₁ receptor reportedly expressed in the rat kidney (12). When the same membranes were stripped and reprobed with an anti--actin antibody, equal protein loading was confirmed (Fig. 2A, bottom). Thus these results confirm that PTCs used in the present study express specific AT₁ receptor protein.

View larger version (16K): [in this window] [in a new window]   Fig. 2. A: Western blot of angiotensin type 1 (AT1) receptor (R) protein expression in PTCs. The 42-kDa protein, corresponding to the reported molecular mass of AT1 receptor protein in the rat kidney (lanes 1 and 2), was inhibited by an AT1 receptor-selective blocking peptide [AT1R antibody (Ab) + blocking peptide (BP); lane 3]. Western blot of -actin on the same membrane after stripping confirmed equal protein loading. B: semiquantitated data of Western blot. **P < 0.01 vs. AT1R Ab only by unpaired t-test.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Intracellular ANG II levels in PTCs and the effect of AT1 receptor-mediated endocytosis of extracellular ANG II. Exposure of PTCs to extracellular ANG II (1 nM) increased intracellular ANG II, whereas losartan (Los, 10 µM) blocked this effect. P < 0.01 vs. control (**) and vs. ANG II (++).

View larger version (28K): [in this window] [in a new window]   Fig. 4. Effect of extracellular ANG II and cell-surface AT1 receptors on peak intracellular calcium concentration ([Ca2+]i) signaling in PTCs. Extracellular losartan completely blocked the cell-surface AT1 receptor-mediated [Ca2+]i response to extracellular ANG II. A: control. B: ANG II (1 nM). C: ANG II + losartan (10 µM). D: quantitative [Ca2+]i levels. Red represents the highest level of calcium signaling (relative fluorescence levels), whereas black is the background. P < 0.01 vs. control (*) and vs. ANG II (#).

View larger version (49K): [in this window] [in a new window]   Fig. 5. A-E: effect of intracellular microinjection of ANG II (1 nM, 70–100 fl) on [Ca2+]i responses in single PTC at baseline (0 s) and 15, 30, 60, and 120 s after microinjection of ANG II in the cells. F: relative levels of [Ca2+]i signaling before and after microinjection of ANG II. Red represents the highest level of [Ca2+]i responses, whereas black is the background. **P < 0.01 vs. basal.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Effect of intracellularly applied AT1 receptor antagonist losartan (10 µM) or AT2 receptor antagonist PD-123319 (PD, 10 µM) on peak [Ca2+]i responses to ANG II microinjection in single PTC. Losartan abolished ANG II-induced [Ca2+]i responses, whereas PD-123319 had no effect. **P < 0.01 vs. control without ANG II microinjection. ++P < 0.01 vs. ANG II microinjection.

View larger version (19K): [in this window] [in a new window]   Fig. 7. A-F: time-dependent [Ca2+]i responses to microinjected ANG II (1 nM, 70–100 fl) in the absence of extracellular Ca2+ in a representative PTC. G: removal of extracellular Ca2+ from the perfusate had no significant effect on peak [Ca2+]i responses to intracellular ANG II. Red represents the highest level of [Ca2+]i responses, whereas black is the background. **P < 0.01 vs. control.

View larger version (11K): [in this window] [in a new window]   Fig. 8. Peak [Ca2+]i responses to microinjected ANG II (1 nM, 70–100 fl) in single PTCs pretreated with thapsigargin (1 µM; A) to deplete intracellular [Ca2+]i stores or with the phospholipase C inhibitor U-73122 (1 µM; B). Thapsigargin and U-73122 abolished [Ca2+]i responses to microinjected ANG II. P < 0.01 vs. control (**) and vs. ANG II (++).

	DISCUSSION

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The present study demonstrates for the first time that microinjection of ANG II directly in single PTCs increases [Ca²⁺]_i levels by activating intracellular AT₁ receptors. Because [Ca²⁺]_i responses to microinjected ANG II were elicited while extracellular losartan was applied to block cell-surface AT₁ receptors, the results indicate that the microinjected ANG II exerted an intracellular effect. Furthermore, because [Ca²⁺]_i responses to microinjected ANG II were abolished by coadministration of losartan but not PD-123319, and because removal of extracellular Ca²⁺ did not affect the intracellular actions of microinjected ANG II, our results suggest that intracellular AT₁ receptors, either internalized from cell membranes or synthesized within the cell, mediate the observed effects via an intracellular mechanism(s). Our results are consistent with previous observations that microinjection of ANG II in VSMCs increased [Ca²⁺]_i (10) and suggest that intracellular ANG II plays an important intracrine role in PTC function.

Re and associates (5, 23) have supported the concept that intracellular ANG II may play an important intracrine role in hepatocytes and cardiovascular cells. Although intracellular ANG II has been shown to have significant effects in nonrenal cells, it remains unclear if it exerts any biological or physiological role in PTCs. Several lines of evidence suggest that intracellular ANG II may play a role in PTC function. Expression of all necessary components of the RAS in these cells strongly suggests such a possibility (2, 14, 16, 28, 36). Moreover, high levels of ANG II in proximal tubular fluid and interstitial fluid compartments may promote AT₁ receptor-mediated endocytosis of ANG II in PTCs, where it could induce intracellular responses (2, 22, 36). Imig et al. (14) demonstrated the presence of ANG I, ANG II, ACE, and AT_1A receptors in rat renal cortical endosomes; most were likely of the PTC origin. Further studies by us demonstrated increased accumulation of extracellular ANG II in endosomes and intermicrovillar clefts isolated from the renal cortex of rats chronically infused with ANG II (36). However, it has not been shown whether intracellular ANG II, either synthesized intracellularly or internalized from extracellular compartments, exerts a physiological effect. Schelling and Linas (25) demonstrated that, in isolated PTCs, phenylarsine oxide, which inhibits ANG II receptor internalization, also blocks ANG II-stimulated proximal tubule sodium transport. This suggestsindirectly that ANG II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis of ANG II. In the present study, we directly microinjected ANG II in single PTCs and monitored changes in [Ca²⁺]_i signaling. To ensure that [Ca²⁺]_i responses to microinjected ANG II were not mediated by cell-surface AT₁ receptors, we added losartan to the perfusate to block membrane AT₁ receptors while injecting ANG II in the cells, as described by Haller et al. (10). The increases in [Ca²⁺]_i induced by microinjected ANG II thus indicate that it was elicited as a consequence of the intracellular ANG II acting on an intracellular receptor.

The mechanisms by which intracellular ANG II induces [Ca²⁺]_i responses in PTCs are not fully understood. There are two important mechanisms that determine [Ca²⁺]_i levels within the cell (influx of extracellular Ca²⁺ and Ca²⁺ released from intracellular stores). In the present study, we observed that removal of extracellular Ca²⁺ did not alter the time course or the extent of increases in [Ca²⁺]_i responses to microinjected ANG II. Our results suggest that the [Ca²⁺]_i responses to microinjected ANG II do not depend significantly on influx of extracellular Ca²⁺ but are primarily because of Ca²⁺ release from intracellular stores in PTCs. These observations are different from those reported in VSMCs by Haller et al. (10) in that intracellular Ca²⁺ responses to microinjected ANG II were primarily the result of influx of extracellular Ca²⁺ in VSMCs. In that same study, treatment of the cells with EGTA, which removes Ca²⁺ from the medium, or with nitrendipine, an L-type Ca²⁺ channel antagonist, both markedly inhibited [Ca²⁺]_i responses in VSMCs microinjected with ANG II (10). In cardiac (7) or arterial (8) myocytes, dialysis of ANG II in the cells either stimulated or reduced Ca²⁺ channel activity, which may alter [Ca²⁺]_i levels in response to intracellular ANG II. Thus the effects of extracellular Ca²⁺ on the [Ca²⁺]_i responses to microinjected ANG II appear to be dependent on the cells studied. For example, depletion of intracellular Ca²⁺ stores with cyclopiazonic acid or thapsigargin markedly attenuated ANG II-induced constriction of pre- and postglomerular arterioles in the kidney (15, 18).

The present study shows that, in PTCs, the [Ca²⁺]_i responses to microinjected ANG II are mediated primarily by activating AT₁ receptors inside PTCs. This conclusion is supported by previous observations that AT₁ receptors predominate in proximal tubules (36, 37) and that concurrent microinjection of losartan and ANG II almost completely abolished [Ca²⁺]_i responses, whereas the AT₂ receptor blocker PD-123319 had little effect. The present results are consistent with the reported [Ca²⁺]_i or cell membrane voltage-operated Ca²⁺ channel response to intracellular ANG II that was abolished by candesartan in VSMCs (10) or arterial myocytes (8). However, they contrast with the effect of losartan in cardiac myocytes, where losartan did not block intracellular ANG II-induced changes in membrane calcium currents (7). The reason for the differences between cardiac myocytes and PTCs is not clear. It is well documented that AT₁ receptors are internalized in the endosomal compartments after exposure to extracellular ANG II in VSMCs (1, 9) and renal epithelial cells (13, 32, 33). Moreover, AT_1A receptors have been shown to colocalize with ANG II in isolated rat renal cortical endosomes derived primarily from PTCs (14, 36). Although it remains to be determined how intracellular ANG II interacts with the AT₁ receptor to induce [Ca²⁺]_i responses, our results suggest that intracellular AT₁ receptors are activated by intracellular ANG II to increase [Ca²⁺]_i through mobilization of intracellular Ca²⁺ stores.

Our results further show that activation of PLC may be involved in intracellular ANG II-induced [Ca²⁺]_i responses in PTCs. In many ANG II-targeted cells (for instance, VSMCs), biological effects of extracellular ANG II in PTCs are often mediated by activation of PLC, followed by the hydrolysis of phosphatidylinositol 4,5-bisphosphate to form IP₃ and diacylglycerol (6, 9, 26). Increases in IP₃ and diacylglycerol in turn induce intracellular [Ca²⁺]_i mobilization and activation of protein kinase C (9, 26, 34). Together with inhibition of adenylyl cyclase to decrease cAMP production, these intracellular signaling pathways play an important role in ANG II-regulated proximal sodium and fluid transport. In the present study, we found that intracellular ANG II-induced increases in [Ca²⁺]_i could be prevented by pretreating the cells with the PLC inhibitor U-73122. Interestingly, Schelling et al. (26) reported that inhibition of PLC with U-73122 decreased apical ANG II-induced sodium flux in isolated rat PTCs. It is therefore likely that activation of PLC is required for both extracellular and intracellular ANG II-induced calcium signaling and sodium transport in PTCs.

In summary, we have demonstrated for the first time that intracellular microinjection of ANG II in single PTCs increases [Ca²⁺]_i and that these responses do not depend significantly on influx of extracellular Ca²⁺ but instead require activation of intracellular AT₁ receptors and PLC. Our results suggest that intracellular ANG II, either derived from endocytosis of extracellular ANG II or synthesized within the cell, exerts an important intracrine role in [Ca²⁺]_i signaling in PTCs. However, it remains unclear how the increase in [Ca²⁺]_i in response to intracellular ANG II stimulation is related to the known effects of ANG II on proximal tubular sodium and fluid transport. Early studies by Taylor and Windhager (30) showed that marked elevation of [Ca²⁺]_i was associated with reduced transepithelial sodium transport. By contrast, small increases in [Ca²⁺]_i have been reported to stimulate sodium flux in cultured renal epithelial cells (30). Moreover, the complex intracellular mechanisms by which microinjected ANG II increases [Ca²⁺]_i and regulates sodium transport in PTCs remain to be investigated. Extracellular ANG II has been shown to increase [Ca²⁺]_i by promoting extracellular Ca²⁺ influx or by [Ca²⁺]_i release from intracellular stores in renal epithelial cells (4, 34); intracellular ANG II may mobilize [Ca²⁺]_i primarily via an intracellular mechanism(s).

	GRANTS

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This work was supported by National Institutes of Health Grants RO1DK-067299 (J. L. Zhuo), HL-28982 (O. A. Carretero and J. L. Garvin), and HL-26371 (G. Navar), American Heart Association Grant-in-Aid 0355551Z (J. L. Zhuo), and a National Kidney Foundation of Michigan Grant-in-Aid (J. L. Zhuo).

	ACKNOWLEDGMENTS

 
Parts of this work were presented as an abstract at the 57th Annual Fall Conference and Scientific Sessions of the Council for High Blood Pressure Research (Washington, DC, September 23–26, 2003); the Gordon Research Conference on Angiotensin (Ventura, CA, February 29–March 5, 2004); and the FASEB Summer Research Conference on Renal Microcirculatory and Tubular Dynamics (Pine Mountain, GA, June 26–July 1, 2004).

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. L. Zhuo, Division of Hypertension and Vascular Research, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202 (e-mail: jzhuo1{at}hfhs.org)

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.

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