Scientific Contributions

Intracellular Sodium Modulates the Expression of Angiotensin II Subtype 2 Receptor in PC12W Cells

Masaaki Tamura, Yoshio Wanaka, Erwin J. Landon, Tadashi Inagami
https://doi.org/10.1161/01.HYP.33.2.626
Hypertension. 1999;33:626-632
Originally published February 1, 1999

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Abstract

Abstract—Although the angiotensin II subtype 2 receptor (AT2-R) is expressed abundantly in the adrenal medulla, its physiological significance has not yet been determined. To obtain fundamental knowledge of the regulation of AT2-R expression in the adrenal medulla, we investigated the effects of modulating several ion channels on AT2-R expression in PC12W cells. Experiments were performed after 24 hours of serum depletion under subconfluent conditions. After 48 hours of treatment with various agonists or antagonists, the receptor density and mRNA level of AT2-Rs were quantified by 125I-[Sar1,Ile8]angiotensin II binding and Northern blot analysis. Ouabain (10 to 100 nmol/L) and insulin (10 to 100 nmol/L) dose-dependently increased receptor density and mRNA level. Analysis of the binding characteristics revealed that the ouabain-dependent increase in AT2-R levels was due to an increase in binding capacity without a change in the Kd value. These increases were blocked by lowering the Na+ concentration in the medium. A low concentration of the sodium ionophore monensin (10 nmol/L), the K+-channel blocker quinidine (10 μmol/L), and the ATP-sensitive K+-channel blockers tolbutamide (100 μmol/L) and glybenclamide (10 μmol/L) also significantly increased receptor density, but the ATP-sensitive K+-channel agonist cromakalim (100 μmol/L) decreased receptor density significantly (P<0.01). Nifedipine (10 μmol/L) decreased basal receptor density and completely blocked the increase in receptor density caused by these agents. The increase in receptor density caused by an increase in intracellular Na+ was accompanied by an increase in mRNA level, whereas the ATP-sensitive K+-channel blockers did not change mRNA level. Nifedipine slightly decreased mRNA level. These results suggest that AT2-R expression is sensitively regulated by intracellular cation levels. The change in intracellular Na+ level transcriptionally regulates AT2-R expression, whereas the K+-channel blocker–dependent upregulation appears to be at least in part posttranslational.

The renin-angiotensin system plays an important role in the regulation of blood pressure, body fluid and electrolyte homeostasis, the facilitation of adrenergic nerve activity, and drinking behavior.1 2 3 4 These biological actions are initiated through the binding of angiotensin II (Ang II) to specific receptors.1 2 3 4 There are at least two subtypes of Ang II receptor.5 6 7 Most of the known biological effects ascribed to Ang II, such as vasoconstriction, aldosterone release, and cell proliferation, are mediated by the subtype 1 Ang II receptor (AT1-R).1 2 3 4 However, the subtype 2 Ang II receptor (AT2-R) is present in a variety of tissues and is implicated in several biological functions, such as inhibition of cell growth,8 9 pressure natriuresis,10 production of prostaglandins,11 12 13 apoptosis,14 and AT2 regulation of vascular tone.15 16 17 18 In neuronal cells, Ang II regulates K+ currents19 and T-type Ca2+ channels20 and promotes differentiation21 22 through AT2-R. The mechanisms by which these actions occur remain incompletely understood, mainly because of the unclear signaling mechanism(s) and the lower AT2-R expression levels compared with AT1-R levels.

Among several peripheral tissues in adult rats that have been reported to express AT2-Rs, adrenal medulla expresses them most abundantly.6 7 However, no physiological significance has yet been attributed to this AT2-R. In adrenal medulla, Ang II stimulates catecholamine production through AT1-R,23 24 a mechanism for which extracellular Ca2+ entry into the cytoplasm is necessary.25 26 AT2-Rs, however, do not transmit the Ang II signal for intracellular Ca2+ mobilization in adrenal medullary cells.27 If AT2-R expression is regulated in association with intracellular Na+ or K+ levels, AT2-R is anticipated to play a role in signaling that is eventually associated with Na+ or K+ metabolism. Clarification of AT2-R expression, in conjunction with intracellular cation mobilization, may therefore provide us with a clue to the physiological significance of AT2-R in adrenal medulla. We conducted the present study to obtain fundamental knowledge of the potential physiological function of AT2-R using rat pheochromocytoma-derived PC12W cells, which have been reported to express AT2-R exclusively and abundantly.27 28 In the present article, we report on intracellular cation (particularly Na+)–dependent regulation of AT2-R expression in PC12W cells.

Methods

Chemicals and Reagents

Ang II and [Sar1,Ile8]Ang II were purchased from Peninsula Laboratories. Ouabain, insulin, monensin, quinidine, nifedipine, tolbutamide, glybenclamide, and cromakalim were from Sigma Chemical Co. Losartan was a gift from DuPont-Merck. [125I]-Na was purchased from DuPont New England Nuclear. Dulbecco’s modified Eagle’s medium (DMEM), the ingredients of minimum essential medium (MEM), and fetal bovine serum (FBS) were purchased from GIBCO-BRL. All other chemicals were of analytical grade.

Cell Culture

The rat pheochromocytoma cell line PC12 was derived from clonal isolation of an adrenal chromaffin cell tumor.29 A substrain, PC12W, was the generous gift of Drs. K.H. Kim and R.C. Speth (Washington State University, Pullman). PC12W cells were grown in DMEM supplemented with 10% FBS at 37°C under a humidified atmosphere of 95% air/5% CO2. For determination of receptor density, cells were plated in 24-well plates at a density of 2.5×104/well and cultured for 2 to 3 days. The medium was changed to serum-free DMEM 24 hours before the binding assay, which was performed when the culture was approximately 80% to 90% confluent. When the effect of extracellular Na+ concentration on AT2-R expression levels was studied, PC12W cells were cultured in DMEM supplemented with 10% FBS for 2 to 3 days. They were then cultured further in a modified DMEM consisting of 145 mmol/L NaCl, 1.8 mmol/L KCl, 1.2 mmol/L KH2PO4, 5 mmol/L NaHCO3, 0.5 mmol/L MgSO4, 1.8 mmol/L CaCl2, 25 mmol/L glucose, 4% MEM amino acid solution (50×), 4% MEM vitamin solution (100×), 1% MEM nonessential amino acid solution (100×), 0.5% FBS, and 0.5 mg/mL bovine serum albumin (BSA) (pH 7.4) at 37°C in a CO2 incubator. The modified DMEM with low Na+ (35 mmol/L NaCl) was prepared by substituting 110 mmol/L NaCl with choline chloride.

Vascular smooth muscle cells (VSMCs) were obtained from thoracic aorta tissue of male spontaneously hypertensive rats as described previously.30 VSMCs were cultured for 5 to 6 days in the same medium and under the same conditions as described for PC12W cells. COS-7 cells stably expressing AT2-R were prepared by the electroporation transfection method with pRC/CMV (Invitrogen) containing a 2.9-kb insert derived from AT2 cDNA as described previously.31 The cells were cultured in DMEM supplemented with 10% FBS under the same conditions as described for PC12W cells for 2 to 3 days before the experiment.

125I-[Sar1,Ile8]Ang II Binding Assay

The radioligand receptor binding assay was performed using intact cultured cells and 125I-[Sar1,Ile8]Ang II. 125I-[Sar1,Ile8]Ang II was prepared from [Sar1,Ile8]Ang II and 125I-Na by the lactoperoxidase method. Subconfluent PC12W cells in 24-well plates were washed twice with Hanks’ balanced salt solution (HBSS) and incubated with 0.5 nmol/L 125I-[Sar1,Ile8]Ang II with or without 1 μmol/L unlabeled [Sar1,Ile8]Ang II for 3 hours at 4°C in the presence of 1 μmol/L losartan and 0.5 mg/mL BSA. Unbound ligand was thoroughly washed out with HBSS at 4°C. Cells were solubilized with 0.5 mol/L NaOH, and the remaining radioactivity was counted. Specific binding was estimated by subtracting the nonspecific binding obtained in the presence of 1 μmol/L unlabeled ligand from the total binding. An aliquot of the solubilized cells was subjected to protein assay (BCA protein assay method, Pierce Chemical Co). Specific binding was normalized by protein quantity per well. Saturation isotherm data were analyzed according to the Scatchard method.32

Northern Blot Analysis

The total RNA in PC12W cells was isolated by the acid guanidinium-phenol-chloroform extraction method.33 Twenty micrograms of the total RNA was then electrophoresed on a 1.0% agarose/1.0% formaldehyde gel and transferred to a Hybond N+2 membrane (Amersham). A full-length cDNA of the mouse AT2 gene was labeled with 32P by a Prime It kit (Stratagene) and used as a probe after heat denaturation. The filter was then exposed to Kodak X-OMAT film at −70°C. The hybridized filter was stripped and hybridized to a 32P-labeled GAPDH probe to obtain a reference for the amount of applied RNA. Autoradiographic analysis was performed by an image scanner (ES-800C scanner, Epson America, Inc) and a computer program (Image 1.59, National Institutes of Health).

Statistical Analysis

Data obtained from the binding assay were averaged and are presented as mean±SE. Significant differences between groups were evaluated by one-way analysis of variance with the Student-Newman-Keuls test. A value of P<0.05 was considered significant.

Results

The cardiac glycoside ouabain is a well-known Na+ pump–specific inhibitor and has been widely used for the modulation of intracellular Na+ levels. As illustrated in Figure 1⇓, low concentrations of ouabain (10 to 100 nmol/L) dose- and time-dependently increased AT2-R expression in PC12W cells. A maximal increase (50%) was observed at a concentration of 50 nmol/L ouabain in the medium. PC12W cells easily detach from culture plates when the conditions are serum-free for longer than 24 hours. Therefore, ouabain was added to the 10% serum-containing medium, and the cells were initially cultured for 24 hours under these conditions before changing to the serum-free medium for an additional 24 hours. This ouabain effect was also studied with serum in the medium (0.5% or 10% serum in the DMEM) for 48 hours, but the AT2-R stimulation pattern by ouabain was consistent (data not shown).

Figure 1.
Figure 1.

Dose dependency (A) and time dependency (B) of ouabain effect on AT2-R expression in PC12W cells. A, After 2 days of culture, cells were treated for 24 hours with various ouabain concentrations in DMEM containing 10% FBS, followed by an additional 24 hours in serum-free DMEM in the presence of ouabain. B, For the time course study initiation time for ouabain treatment (50 nmol/L) within this 48-hour period was adjusted so that all time point samples could be assayed together. 125I-[Sar1,Ile8]Ang II binding assay was performed in HBSS containing 1 μmol/L losartan and 0.5 mg/mL BSA at 4°C as described in Methods. Values were normalized by cell protein quantities. Each value represents mean±SE of 6 incubations. *P<0.05 compared with basal receptor density level.

Since PC12W cells express AT2-R exclusively,27 28 the receptor subtype specificity of the ouabain effect was investigated using VSMCs, which predominantly express AT1-Rs (no detectable AT2-Rs were expressed), and COS-7 cells, which contained permanently transfected AT2-R coding regions. In VSMCs, AT1-R expression was slightly decreased by low concentrations of ouabain (10 and 25 nmol/L, Figure 2A⇓). In COS-7 cells, AT2-R expression was significantly increased at low concentrations of ouabain (25 and 50 nmol/L, Figure 2B⇓). These results, together with the results shown in Figure 1⇑, clearly demonstrate that the ouabain effect on Ang II receptors is specific to AT2-R. The involvement of intracellular Na+ level in AT2-R expression was further examined using insulin and a mixture of insulin and ouabain. Insulin alone dose-dependently (10 to 100 nmol/L) increased AT2-R expression (Figure 3⇓). When insulin and ouabain were added together, they showed additive effects on the increase in AT2-R expression in PC12W cells (Figure 3⇓). To clarify whether the ouabain-dependent increase in receptor density is due to an increment in the receptor density or induction of another type of receptor protein, we analyzed 125I-[Sar1,Ile8]Ang II binding characteristics using intact whole cells. Data in Figure 4⇓ indicate that AT2-R in PC12W cells possesses two types of binding sites (Kd=1.7 nmol/L, high-affinity site; Kd=17.4 to 19.1 nmol/L, low-affinity site). Ouabain treatment increased the binding maximum of both high- and low-affinity sites by approximately 55%. These data suggest that the intracellular Na+–dependent increase in AT2-R levels is due to an increment in receptor density.

Figure 2.
Figure 2.

Ouabain effect on Ang II receptor expression in VSMCs (A) and AT2-transfected COS-7 cells (B). Both cell types were cultured until confluence and then were treated with various concentrations of ouabain as indicated for 48 hours in serum-free DMEM. 125I-[Sar1,Ile8]Ang II binding assay was performed in HBSS containing 0.5 mg/mL BSA and 1 μmol/L PD123319 for AT1-R (A) or 1 μmol/L losartan for AT2-R (B) at 4°C. Values were normalized by cell protein quantities. Each value represents mean±SE of 6 incubations. *P<0.05 compared with basal receptor density level.

Figure 3.
Figure 3.

Effects of insulin and insulin plus ouabain on AT2-R expression levels in PC12W cells. Experimental conditions were identical to conditions described in Figure 1⇑. *P<0.05 compared with basal receptor density level.

Figure 4.
Figure 4.

A, Binding characteristics of 125I-[Sar1,Ile8]Ang II to untreated (○) or ouabain-treated (•) PC12W cells. Subconfluent cells were treated either with or without ouabain (50 nmol/L) for 48 hours. Each data point represents two experiments performed in duplicate determinations. Saturation curves drawn are obtained from a computer-assisted nonlinear regression analysis. B, Scatchard transformation of the same data.

The above experiments suggest that intracellular Na+ level is an important factor in the regulation of AT2-R expression. To clarify this hypothesis, we manipulated intracellular Na+ level with the sodium ionophore monensin. A very low concentration of monensin (10 nmol/L) significantly increased AT2-R expression by approximately 75%. Although a higher concentration of monensin (≥100 nmol/L) is known to be cytotoxic, the cytotoxicity of monensin at 10 nmol/L for 48 hours of treatment was not significant. This result confirms that intracellular Na+ level is tightly associated with the regulation of AT2-R expression. Since an increase in intracellular Na+ level was shown to upregulate AT2-R expression, we studied the reverse effect by lowering extracellular Na+ concentration. In this experiment, osmolarity in the low-sodium-modified medium was maintained at the same level as in the high-sodium-modified medium by substituting the removed NaCl with choline chloride. When PC12W cells were cultured in modified DMEM containing 0.5% FBS for 48 hours, the increase in AT2-R expression caused by ouabain or insulin in the 145 mmol/L Na+ medium was abolished by lowering the Na+ concentration in the medium to 35 mmol/L (Figure 5⇓). These results again suggest that increased intracellular Na+ upregulates AT2-R expression.

Figure 5.
Figure 5.

Effect of extracellular Na+ concentration on AT2-R expression levels in PC12W cells in modified DMEM containing 0.5% FBS, as described in Methods. Each value represents mean±SE (n=8) as a percentage of individual untreated control in medium containing 145 or 35 mmol/L NaCl. *P<0.05 compared with basal receptor density level.

In addition to investigating AT2-R expression in PC12W cells by manipulating intracellular Na+ level, we investigated such expression by using a K+-channel agonist and a variety of antagonists. The ATP-sensitive K+-channel blockers tolbutamide and glybenclamide dose-dependently increased AT2-R expression, whereas the ATP-sensitive K+-channel agonist cromakalim significantly downregulated receptor density (Figure 6A⇓). Although the nonspecific K+-channel blockers tetraethylammonium and 4-aminopyridine increased receptor level significantly, both blockers required much higher concentrations (higher than millimoles per liter) than the specific channel blockers to increase the receptor level in PC12W cells (data not shown). However, another type of nonspecific K+-channel blocker, quinidine (10 μmol/L), significantly increased AT2-R expression over a narrow concentration range (Figure 6B⇓). The Ca2+-sensitive K+-channel blocker charybdotoxin (≈1 μmol/L) did not show any effect on AT2-R expression levels (data not shown). These results demonstrate that the changing K+ currents are also an important factor affecting AT2-R expression levels in PC12W cells.

Figure 6.
Figure 6.

Effects of ATP-sensitive K+-channel blockers tolbutamide (Tol) and glybenclamide (Gly) and agonist cromakalim (Cro) (A) and nonspecific K+-channel blocker quinidine (Qui) (B) on AT2-R expression levels. Each value represents mean±SE of 6 incubations. *P<0.05, **P<0.01 compared with basal receptor density level.

K+-channel blockade and Na+ pump inhibition promote decreased membrane polarization in neuronal-type cells such as PC12W cells. This in turn activates the L-type voltage-dependent Ca2+ channels, which could in turn account for AT2-R upregulation. Nifedipine (10 μmol/L), a specific blocker of the L-type Ca2+ channel, slightly but significantly lowered basal AT2-R expression level and almost abolished the effect of 50 nmol/L ouabain, 10 μmol/L quinidine, or 0.5 mmol/L tolbutamide (Figure 7⇓).

Figure 7.
Figure 7.

Effect of nifedipine (Nif) on basal AT2-R expression and expression stimulated by ouabain (Oua), quinidine (Qui), or tolbutamide (Tol) in PC12W cells. Experimental conditions were identical to conditions described in Figure 1⇑. Nifedipine was added to medium 15 minutes before stimuli. Each value represents mean±SE of 6 incubations. *P<0.05, **P<0.01 compared with basal or corresponding chemical-stimulated receptor density level.

Receptor protein expression is a summation of transcriptional and translational regulation and protein degradation. To evaluate the involvement of transcriptional regulation in AT2-R expression, we estimated AT2-R mRNA levels using Northern blot analysis. Treatment with ouabain (50 nmol/L), monensin (10 nmol/L), quinidine (10 μmol/L), or insulin (100 nmol/L) for 48 hours significantly increased AT2-R mRNA levels in PC12W cells (Figure 8⇓), whereas the ATP-sensitive K+-channel blocker tolbutamide and the K+-channel agonist cromakalim (100 μmol/L) at levels effective for receptor upregulation did not change mRNA levels in PC12W cells. Nifedipine (10 μmol/L) slightly decreased mRNA level. These results suggest that an increase in intracellular Na+ may stimulate transcriptional regulation of AT2-R expression, whereas a K+-channel blocker–dependent increase in AT2-R expression may be due to posttranslational regulation.

Figure 8.
Figure 8.

Effects of various ion channel inhibitors and chemicals on AT2-R mRNA expression in PC12W cells. Cells were grown to subconfluence for 2 days and then treated with various chemicals for 48 hours as described in Figure 1⇑. Total RNA was isolated, and 20 μg was subjected to Northern blot analysis (A). AT2-R mRNA was detected as described in Methods. AT2-R mRNA expression level with respect to GAPDH was estimated (B). Data are representative of 3 experiments.

Discussion

Although AT2-R is expressed abundantly in rat adrenal medulla, its physiological significance has not yet been clarified. The present study focused on determining the basic regulation mechanism of AT2-R expression in adrenal medullary cells. PC12W cells are a substrain of the rat pheochromocytoma cell line and are capable of differentiating to neuronal cells under appropriate culture conditions.34 PC12W cells do not produce all of the catecholamines.35 However, this cell line is a good model for the study of the regulation of AT2-R expression because these cells express high levels of AT2-R exclusively.27 28

In the present study intracellular Na+ level was manipulated by inhibition of the Na+ pump with ouabain, by activation of sodium entry with insulin, or by treatment with the sodium ionophore monensin; then AT2-R level was evaluated. In these three types of experiments, AT2-R expression was upregulated (see Figures 1⇑ and 3⇑ and Results). Thus, AT2-R expression appears to be upregulated when intracellular Na+ level is increased. This result was supported by another experiment, in which the intracellular Na+-dependent AT2-R upregulation caused by ouabain was abolished by lowering extracellular Na+ level (Figure 5⇑). In agreement with the present study, insulin has been reported to increase AT2-R expression in R3T3 cells.36

Changes in intracellular Na+ influence membrane potential. Since increases in intracellular Na+ sensitively upregulated AT2-R expression, changes in K+ currents were also anticipated to regulate AT2-R expression. Indeed, our study demonstrated that manipulation of K+ currents by a variety of K+-channel blockers increased AT2-R expression. Among many K+-channel blockers tested, the ATP-sensitive K+-channel blockers tolbutamide and glybenclamide and the classic nonspecific K+-channel blocker quinidine most effectively upregulated AT2-R expression. The ATP-sensitive K+-channel agonist cromakalim dose-dependently downregulated AT2-R expression. These results indicate that as with Na+, changes in K+ currents in the cell membrane regulate AT2-R expression (Figure 6⇑).

K+ channels and the Na+ pump alter membrane polarization and Ca2+ entry through voltage-dependent Ca2+ channels. Neuronal cells possess voltage-dependent Ca2+ channels,37 so it is of interest to evaluate the effect of intracellular Ca2+ on AT2-R expression. In the experiment with the voltage-dependent Ca2+-channel blocker nifedipine, lowering Ca2+ entry from extracellular sources decreased basal AT2-R expression as well as the intracellular Na+- or K+-current–dependent upregulation of AT2-R (Figure 7⇑). Nifedipine (10 μmol/L) treatment for 48 hours also decreased AT2-R mRNA level by approximately 25%. These results suggest that there could be intracellular Ca2+–dependent regulation of AT2-R downstream of the intracellular Na+ and K+ current–dependent regulation mechanism. However, since all of the chemicals used for the present study are known to modulate membrane potential, modification of the membrane potential may contribute to the regulation of AT2-R expression.

The binding characteristics of radiolabeled Ang II or its analogues to AT2-R have been studied in many types of cells.27 31 38 39 40 Many of these studies have reported that AT2-R contains a single, saturable binding site.27 31 38 39 The present study, however, revealed that AT2-R in PC12W cells contains distinct high- and low-affinity binding sites (Figure 4⇑). In agreement, Siemens et al40 have reported that neuroblastoma cells (N1E-115) possess two distinct AT2-Rs. Despite the use of different cells and different procedures (Siemens et al used solubilized membrane fractions, whereas the present study used intact cells), the Kd values from their experiment and from the present study are almost identical (≈1.7 nmol/L). The discrepancy between the present study and other studies, which have reported a single binding site on AT2-R, can be explained by the following. In the present study, saturation curves were drawn using a wide range of 125I-[Sar1,Ile8]Ang II concentrations (0 to 15 nmol/L), whereas most other reports have used only a very narrow concentration range of the radiolabeled ligands (<5 nmol/L). The present study showed a low-affinity binding site that emerged at approximately 4 nmol/L 125I-[Sar1,Ile8]Ang II (Figure 4A⇑), so most of the other reports would not have been able to detect this low-affinity binding site. The results of the present study clearly indicate that the binding capacities at both binding sites were increased to almost an identical extent by ouabain treatment. This may suggest that the two binding sites belong to a single molecule.

AT1-R and AT2-R have been shown to be derived from different genes located on different chromosomes.41 Their mRNA and protein expressions are apparently regulated by different mechanisms. In the present study, AT1-R expression in VSMCs was not upregulated by ouabain (Figure 2⇑), suggesting that the intracellular cation level or an alteration of the membrane potential is not a signal for the regulation of AT1-R expression. Although nonspecific K+-channel blockers such as tetraethylammonium have been reported to inhibit agonist-induced receptor desensitization in seven transmembrane–type receptors,42 it has been shown that AT2-R is not internalized by Ang II.27 38 43 In the present study, K+-channel blocker–dependent increases in AT2-R expression were not accompanied by an increment in mRNA level. These results may suggest that AT2-R upregulation by K+-channel blockers is in part due to a decrease in the receptor turnover rate.

Protein expression is the sum result of transcription and translation. In the present study we measured mRNA levels by Northern blot analysis. We also determined the relative and functional quantities of the expressed protein by measuring ligand-receptor binding. The results from both determinations suggest that an increase in intracellular Na+ sensitively upregulates AT2-R expression. The mechanism underlying this upregulation is possibly transcriptional regulation, whereas a K+-channel blocker–dependent increase in AT2-R level is postulated to be at least in part translational/posttranslational regulation. The results provide a potential approach to determination of the physiological significance of AT2-R–mediated signals. It is of interest to determine whether similar mechanisms occur in vivo. To the best of our knowledge, the present study is the first to demonstrate that AT2-R is regulated by intracellular cations, particularly the sodium ion.

Acknowledgments

This work was supported by National Institutes of Health grants HL-14192 (T.I.) and HL-35323 (T.I.), a pilot project grant from the Clinical Nutrition Research Unit of Vanderbilt University (DK26657, M.T.), and a Grant-in-Aid from the American Heart Association (9750624N, M.T.). We are indebted to Trinita Fitzgerald and Eric F. Howard (Department of Biochemistry, Vanderbilt University) for their excellent technical assistance. We are grateful to Pamela J. Tamura (Department of Chemistry, Vanderbilt University) for critical reading and constructive comments during the preparation of the manuscript.

  • Received July 30, 1998.
  • Revision received August 28, 1998.
  • Accepted October 1, 1998.

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  • Intracellular Sodium Modulates the Expression of Angiotensin II Subtype 2 Receptor in PC12W Cells
    Masaaki Tamura, Yoshio Wanaka, Erwin J. Landon and Tadashi Inagami
    Hypertension. 1999;33:626-632, originally published February 1, 1999
    https://doi.org/10.1161/01.HYP.33.2.626
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