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(Hypertension. 1996;27:529-534.)
© 1996 American Heart Association, Inc.

Articles

Regulation of Angiotensin II Type 2 Receptor Gene by the Protein Kinase C–Calcium Pathway

Kazuhisa Kijima; Hiroaki Matsubara; Satoshi Murasawa; Katsuya Maruyama; Naohiko Ohkubo; Yasukiyo Mori; Mitsuo Inada

From the Department of Medicine II, Kansai Medical University, Osaka, Japan.

Correspondence to Hiroaki Matsubara, MD, Department of Medicine II, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570, Japan.

	Abstract

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Abstract In the present study, rat angiotensin II type 2 (AT₂) receptor expression was upregulated in confluence-arrested PC12 cells compared with expression in proliferating cells. Treatment with cycloheximide inhibited the increase in mRNA levels in confluent cells. The state of growth arrest by serum deprivation was associated with increased expression of the AT₂ receptor, which was markedly suppressed by exposure to the active phorbol ester 12-O-tetradecanoylphorbol 13-acetate and the calcium ionophore A23187. Similar inhibitions were also observed in myocytes isolated from neonatal rat heart. The change in AT₂ mRNA levels by serum deprivation was due to the increase in the gene transcription rate. The effect of 12-O-tetradecanoylphorbol 13-acetate was mediated through decreases in gene transcription and mRNA stability, whereas A23187 affected mRNA stability. Vasoactive substances with the protein kinase C–calcium pathway, such as norepinephrine and angiotensin II, also downregulated the AT₂ mRNA level in myocytes. These findings indicate that the expression of AT₂ receptor in PC12 cells is regulated in a growth state–dependent manner, which is involved in confluence-induced new protein synthesis, thus providing a means by which cells can modulate their responsiveness to external angiotensin II stimulus. The activation of protein kinase C or calcium mobilization modifies this regulatory mechanism, suggesting that neurotransmitters or vasoactive substances with the protein kinase C–calcium pathway at least in part affect neuronal activity or blood pressure control by downregulating AT₂ receptor expression.

Key Words: receptors, angiotensin II • protein kinases • calcium • PC12 cells • cardiomyocytes

	Introduction

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The renin-angiotensin system in the brain has an important role in systemic cardiovascular regulation and water balance because centrally injected Ang II has potent pressor and dipsogenic actions.¹ The pressor action of centrally injected Ang II is mediated partly by catecholamines,² and the central catecholamine-rich regions, such as hypothalamus and brain stem, contain Ang II–like material and immunoreactive Ang II fibers and are also rich in Ang II receptors.³ Ang II receptors are separated into two major subtypes, designated AT₁ and AT₂. Most well-known Ang II functions are mediated by the AT₁ receptor, whereas little information exists regarding the physiological roles of the AT₂ receptor.⁴ Although the AT₂ receptor has been suggested to modify phosphotyrosine phosphate activities,^{5 6 7} activate K⁺ currents,⁸ and inhibit the T-type Ca²⁺ current,⁹ the exact signaling pathway of the AT₂ receptor remains to be determined.

Sumners et al^{10 11} reported ₁-adrenergic receptor–mediated downregulation in the densities of Ang II receptors in neuronal cultures from neonatal rats¹⁰ and subsequently showed that the AT₂ receptor is exclusively expressed in these cultured cells.¹¹ These findings suggest that neurotransmitters with the PKC-Ca²⁺ pathway can modify neuronal function by affecting the activities of K⁺ currents or T-type Ca²⁺ current mediated through the AT₂ receptor. We have previously shown that PC12 cells derived from rat pheochromocytoma predominantly express AT₂ receptor.¹² PC12 cells are maintained in the undifferentiated state in the presence of growth medium, and in response to nerve growth factor, the cells differentiate to nonreplicating sympathetic neuronlike cells.¹³ Although serum, growth factors, and cAMP were shown to downregulate the number of AT₂ receptors in R3T3 fibroblasts^{14 15} or PC12W cells,¹⁶ a substrain of PC12 cells, the exact mechanisms by which AT₂ receptor gene expression is regulated remain to be determined.

In this study, we report that the activation of PKC and intracellular calcium mobilization downregulate AT₂ receptor expression by affecting AT₂ receptor mRNA stability as well as the AT₂ receptor gene transcription rate. In addition, we also studied the effects of norepinephrine and Ang II on the PKC-Ca²⁺ pathway¹⁷ using myocytes isolated from neonatal rat heart that express both AT₁ and AT₂ receptors,¹⁸ because ₁-adrenergic receptor or AT₁ receptor–mediated signals induce a hypertrophic change in neonatal cardiomyocytes¹⁹ and AT₂ receptor expression in the heart is increased in the remodeling heart, such as in cardiac hypertrophy²⁰ and infarction.²¹

	Methods

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Cell Culture
PC12 cells were generously provided by Dr Eva J. Neer (Cardiology, Brigham and Women's Hospital, Boston, Mass). PC12 cells were grown in DMEM (GIBCO-BRL) containing 10% (vol/vol) fetal calf serum (GIBCO-BRL), 5% (vol/vol) horse serum (GIBCO-BRL), penicillin (100 U/mL), and streptomycin (100 µg/mL) with the use of plates precoated with poly-D-lysine (1%, Sigma Chemical Co) as previously reported.¹² Cells were fed every 3 days, passed weekly, and maintained at 37°C in a humidified atmosphere of 5% CO₂/95% air. Myocyte-rich cultures were prepared from 1-day-old neonatal rat hearts by the preplating method as previously described.¹⁸ Myocytes were cultured with DMEM containing 10% fetal calf serum for 2 days after dissociation and then exposed to the compounds in the serum-depleted DMEM, in which the contamination of fibroblasts was shown to be about 5% to 10% by the use of anti-desmin monoclonal antibody.¹⁸

Preparation of Membranes and ¹²⁵I–Ang II Binding
Receptor binding assays were performed with membrane fractions as previously described.¹² Briefly, cells were treated with acid buffer (50 mmol/L glycine, 150 mmol/L NaCl, pH 3.0) on ice for 10 minutes to remove Ang II bound to its receptors, washed twice, homogenized in ice-cold buffer containing 5 mmol/L Tris, 5 mmol/L EDTA, 10 µmol/L leupeptin, 10 µmol/L pepstatin A, and 100 µmol/L phenylmethylsulfonyl fluoride, and centrifuged for 10 minutes at 600g. The supernatant was then centrifuged at 48 000g for 30 minutes at 4°C and resuspended in the assay buffer. Binding assays were performed in 250 µL of 50 mmol/L Tris-HCl (pH 7.4) containing 1 mmol/L EDTA, 0.2% bovine serum albumin, 0.06% bacitracin, 10 µmol/L leupeptin, 10 µmol/L antipain, 10 µmol/L chymostatin, 100 µmol/L phenylmethylsulfonyl fluoride, ¹²⁵I-labeled Ang II (2000 µCi/mmol), 10 to 30 µg membrane protein, and 1 µmol/L CV-11974 (Takeda Pharmaceutical, an AT₁ receptor antagonist). B_max and K_d values were determined by Scatchard analyses.^{12 17}

Northern Blotting
Total cellular RNA (30 µg) isolated by the CsCl-centrifugation method was denatured with 6% formaldehyde, fractionated by 1% agarose gel electrophoresis, transferred to a nylon filter, and hybridized at 42°C for 12 to 16 hours to rat AT₂ receptor cDNA and GAPDH cDNA probes labeled with [³²P]dCTP.²⁰ After hybridization, the filter was washed in 0.1x SSC plus 0.1% sodium dodecyl sulfate at 60°C and exposed with a intensifying screen. The obtained mRNA signals were counted by a densitometer.

Transcript Stability Analysis
AT₂ receptor mRNA stability was measured by incubation with actinomycin D (5 µg/mL) to block transcription.^{18 21} Serum-depleted confluent PC12 cells were incubated with TPA or A23187 at concentrations of 1 µmol/L in the presence of actinomycin D. After various incubation times (6, 12, and 24 hours), total RNA was isolated from individual dishes, and the disappearance of mRNA abundance was determined by Northern blotting.

Nuclear Runoff Assay
The preparation of nuclei and runoff assays was performed as described previously.^{18 21} Confluent PC12 cells were incubated with TPA or A23187 at concentrations of 1 µmol/L for 24 hours in serum-depleted medium. Nuclei were incubated for 20 minutes at 30°C in the presence of 50 mmol/L Tris (pH 7.9); 100 mmol/L KCl; 12.5% glycerol; 6 mmol/L MgCl; 0.2 mmol/L EDTA; 0.5 mmol/L dithiothreitol; 4 mmol/L each of ATP, GTP, and CTP; 1 U/µL RNasin; and 200 µCi of [-³²P]UTP. After RNase-free DNase I and proteinase K digestion, the reaction products were extracted with guanidium isothiocyanate (4 mmol/L) and phenol/chloroform, and unincorporated [-³²P]UTP was removed by trichloroacetic acid precipitation and filtration. The radiolabeled RNA (4x10⁶ cpm) was hybridized at 42°C for 48 hours with linearized pGEM vector containing rat AT₂ receptor cDNA (15 µg) or GAPDH cDNA (5 µg) fragments. After the membrane was washed in 2x SSC plus 0.1% sodium dodecyl sulfate at 65°C for 1 hour, 0.2x SSC plus 0.1% sodium dodecyl sulfate at room temperature for 30 minutes twice, and 0.2x SSC plus 10 µg/mL RNase at room temperature for 15 minutes, the bound radioactivity was determined by scintillation counting.

Reagents and Statistical Methods
All reagents were purchased from Sigma Chemical Co unless otherwise indicated. The results are expressed as mean±SE. ANOVA and Fisher's protected least significant difference tests were used for multigroup comparisons. Differences at a value of P<.05 were considered significant.

	Results

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AT₂ Receptor mRNA Levels in Proliferating and Confluent PC12 Cells
We initially examined AT₂ receptor expression in proliferating (approximately 30% and 80% confluent) or 100% confluent cells cultured with serum-supplemented medium because AT₂ receptor expression in R3T3 fibroblasts has been reported to be upregulated in the confluent phase.^{14 15} Northern blot analyses indicated that PC12 cells express a single size (approximately 3.5 kb) of AT₂ receptor mRNA, as previously reported in PC12W cells,^{5 22} and that AT₂ receptor mRNA levels in the confluent cells increased 2.3-fold compared with levels in proliferating cells (Fig 1). Increased mRNA levels were maintained even after the confluent cells were cultured for the subsequent 24 and 48 hours in the same medium, indicating that AT₂ receptor expression in PC12 cells is regulated in a cell density–dependent manner, as in R3T3 cells. Incubation with cycloheximide for 24 hours downregulated AT₂ receptor mRNA levels in the confluent cells to the levels in proliferating cells, suggesting that de novo protein synthesis is important in inducing the increase in the AT₂ receptor mRNA level observed in the confluent cells.

View larger version (35K): [in this window] [in a new window]   Figure 1. Northern blot analyses of AT2 receptor (AT2-R) mRNA levels in proliferating (approximately 30% and 80% confluent) and 100% confluent PC12 cells cultured with serum-supplemented medium and effect of cycloheximide (5 µg/mL, CHX). Confluent cells were exposed to cycloheximide for 24 hours in the presence of serum. AT2 receptor mRNA levels are expressed relative to GAPDH mRNA levels; a relative value in 30% confluent cells was arbitrarily normalized to 1 for easy comparison. Results are mean±SE of four separate experiments. Exposure times of AT2 receptor and GAPDH signals, 24 and 12 hours, respectively. *P<.01 vs 30% confluent cells; P<.01 vs 100% confluent cells.

Regulation of AT₂ Receptor mRNA Level by Phorbol Esters and Calcium Ionophore
Since the inhibitory effect of serum on AT₂ receptor protein expression was shown in R3T3 fibroblasts,^{14 15} we examined whether a similar effect of serum is observed in PC12 cells. As shown in Fig 2, serum deprivation of confluent cells for 24 hours caused a marked increase (2.6-fold) in AT₂ receptor mRNA levels compared with levels in serum-supplemented cells, and coincubation with cycloheximide completely inhibited this increase (data not shown). Next, we examined the effect of TPA or A23187 on AT₂ receptor mRNA level using serum-depleted confluent cells (Fig 2). The increase in AT₂ receptor mRNA levels by serum deprivation was markedly inhibited by TPA and A23187, and after 24 hours of exposure, the mRNA level was decreased to less than the level in proliferating cells (Fig 2). Similar inhibitory actions were observed even in the presence of serum and a phorbol ester that does not stimulate PKC; 4--phorbol did not elicit any change (data not shown). The action of TPA was blocked by H-7, indicating that stimulation of PKC is involved in this regulation.

View larger version (32K): [in this window] [in a new window]   Figure 2. Northern blot analyses of effects of serum deprivation, TPA, and A23187 on AT2 receptor (AT2-R) mRNA levels. Confluent PC12 cells were exposed to TPA or A23187 at concentrations of 1 µmol/L in serum-depleted medium for 6 and 24 hours. H-7 (5 µmol/L) was added 30 minutes before exposure to TPA and incubated for 24 hours. AT2 receptor mRNA levels are expressed relative to GAPDH mRNA levels; a relative value in serum-supplemented cells (0 time point) was arbitrarily normalized to 1. Results are mean±SE of four separate experiments. Exposure times of AT2 receptor and GAPDH signals, 72 and 24 hours, respectively. *P<.01 vs values at 0 time in serum (-).

Changes in AT₂ Receptor mRNA Stability, Gene Transcription, and Protein Expression
We examined the stability of AT₂ receptor mRNA by inhibiting new mRNA transcription with actinomycin D. A half-life of 18.4±0.2 hours for AT₂ receptor mRNA was obtained from serum-depleted confluent cells; this value was not significantly different from that in the presence of serum (18.8±0.2 hours, n=5). The half-life periods of serum-depleted cells were significantly (P<.01) decreased to 8.4±0.1 and 5.6±0.1 hours by treatment with TPA (n=6) and A23187 (n=6), respectively. We studied the effects on AT₂ receptor gene transcription level by means of a nuclear runoff assay as previously described.^{18 21} The results of runoff assays were normalized to the transcription rate of the GAPDH gene, which was unchanged by these stimulations. As shown in Fig 3, the relative rate of AT₂ receptor gene transcription in serum-supplemented confluent cells was assigned to the value 1 for easy comparison. The rate of AT₂ receptor gene transcription was increased 2.5-fold by serum deprivation compared with that in serum-supplemented cells. Treatment with TPA significantly (64%) inhibited the increase in serum-depleted cells, whereas A23187 did not affect the transcriptional level of the AT₂ receptor gene.

View larger version (46K): [in this window] [in a new window]   Figure 3. Nuclear runoff assays of effects of serum deprivation, TPA, and A23187 on the transcription rate of rat AT2 receptor (AT2-R) gene. Confluent PC12 cells were treated with TPA or A23187 at concentrations of 1 µmol/L for 24 hours in serum-depleted medium, and nuclei were isolated. Radiolabeled RNA was hybridized with linearized pGEM vector alone (15 µg, negative control), pGEM containing rat AT2 receptor cDNA (15 µg), or GAPDH (10 µg), and bound radioactivities were determined by scintillation counting. AT2 receptor transcription rates are expressed relative to those of GAPDH gene after their background levels (pGEM vector alone) were reduced; the relative values in serum-supplemented confluent cells are normalized to 1. Results are mean±SE of three separate experiments. *P<.01 vs values in serum-supplemented cells; P<.01 vs values in serum-depleted cells.

We have previously shown that serum-supplemented confluent PC12 cells exclusively express the AT₂ receptor with a single binding site and high binding affinity.¹² As shown in the Table, serum deprivation upregulated the densities by 2.7-fold, whereas exposure to TPA or A23187 significantly inhibited this increase. The receptor affinity was not significantly changed by these stimulations. Activities of lactate dehydrogenase and creatine kinase were not detectable in the medium in which PC12 cells were cultured for 24 hours in the presence of TPA or A23187, and GAPDH mRNA levels were unchanged by these compounds (Fig 2), indicating that the actions of TPA and A23187 are specific to the AT₂ receptor rather than their toxic effects on PC12 cells.

View this table: [in this window] [in a new window]   Table 1. Effects of TPA and A23187 on AT2 Receptor Density

Effects on Myocytes Isolated From Neonatal Rat Heart
We have previously reported that myocytes isolated from neonatal rat heart express substantial amounts of AT₁ and AT₂ receptor.¹⁸ We examined the effects of norepinephrine and Ang II on AT₂ receptor expression using the myocytes because norepinephrine and AT₁ receptor–mediated signals activate the PKC-Ca²⁺ pathway⁴ and induce myocyte hypertrophy.^{19 23} As shown in Fig 4, AT₂ receptor mRNA levels, determined by competitive reverse transcription–polymerase chain reaction, were increased 3.2-fold by 24 hours of serum deprivation, and this increase was markedly suppressed by the addition of norepinephrine, Ang II, TPA, and A23187. We previously showed that fibroblasts prepared from 1-day-old rat hearts exclusively express AT₁ receptor.¹⁸ In addition, we could not detect AT₂ receptor mRNA accumulation in fibroblasts by reverse transcription–polymerase chain reaction (data not shown). Therefore, although myocyte-rich cultures were contaminated with 5% to 10% fibroblasts, the contribution of fibroblasts to AT₂ receptor expression was considered to be negligible. Myocytes cultured with serum-supplemented medium were actively beating, and incubation with serum-free medium for 24 hours slightly decreased the beating rate. Myocytes continued beating in the presence of norepinephrine, Ang II, and A23187, whereas exposure to TPA markedly suppressed the beating rate of myocytes.

View larger version (44K): [in this window] [in a new window]   Figure 4. Competitive reverse transcription–polymerase chain reaction analyses of effects of norepinephrine (NA), Ang II, TPA, and A23187 on AT2 receptor (AT2-R) mRNA levels in myocytes isolated from 1-day-old rat hearts. Myocytes were isolated and cultured for 48 hours in serum-supplemented medium (10% fetal calf serum) and subsequently exposed to norepinephrine, Ang II, TPA, and A23187 at concentrations of 1 µmol/L for 24 hours in serum-depleted medium. Total RNA (1 µg) digested with RNase-free DNase I was reverse-transcribed with deletion mutated cRNA (AT2 receptor, 0.1 pg), and the resultant cDNA mixtures were amplified by polymerase chain reaction in the presence of [32P]dCTP, as previously described.21 GAPDH autoradiographic signals (7 µg total RNA) from Northern blots are shown. Incorporated 32P counts in AT2 receptor signals were normalized to those in AT2 receptor and GAPDH autoradiographic counts that were measured with densitometry. A normalized value in serum-supplemented cells was arbitrarily expressed as 1. Values are mean±SE (n=4). **P<.01, *P<.05 vs values in serum-supplemented cells; P<.01 vs values in serum-depleted cells.

	Discussion

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AT₂ receptor is abundantly and widely expressed in fetal tissues,^{24 25} and its expression is activated in skin wounds,²⁶ neointima after vascular injury,²⁷ and cardiac remodeling.^{20 21} These expression patterns suggest an important role of AT₂ receptor in growth and development. In vitro binding studies including ours¹² have shown that PC12,¹² PC12W,¹⁶ and R3T3¹⁴ cells abundantly express the AT₂ receptor. Dudley and Summerfelt¹⁴ and Horiuchi et al¹⁵ have reported that AT₂ receptor expression is upregulated in confluent R3T3 cells compared with expression in proliferating cells. The present study extended their studies and indicated that the same growth-dependent expression occurs in PC12 cells and that the increase of AT₂ receptor expression in the confluent state is at least partly attributable to confluence-induced new protein synthesis. This study also showed that serum deprivation causes a marked increase in AT₂ receptor densities in confluent PC12 cells, as observed in R3T3 cells,^{14 15} and that this effect is mediated through the increase on the gene transcription level and requires new protein synthesis. Similar regulation by serum deprivation was observed in the receptors for platelet-derived growth factor-ß²⁸ or insulin²⁹ in BALB/c-3T3 cells. Growth factors are reported to cause a decrease in AT₂ receptor expression in R3T3¹⁴ and PC12W¹⁶ cells. Thus, cell proliferation and growth-stimulating substances suppressed AT₂ receptor expression, which is in contrast with in vivo observations that AT₂ receptor expression is increased in tissue remodeling situations such as neointima after vascular injury,²⁷ skin wounds,²⁶ or cardiac remodeling.^{20 21} Other factors that upregulate AT₂ receptor expression may be induced in such pathological lesions and determine expression patterns of the AT₂ receptor in vivo.

Exposure to TPA or A23187 reduced the increased level of AT₂ receptor mRNA by serum deprivation to the level in proliferating cells, suggesting that activation of PKC and mobilization of intracellular calcium are involved in the regulation of AT₂ receptor expression by serum deprivation as well as in confluent cells. The effect of PKC was mediated through the changes in gene transcription level and mRNA stability, whereas the action of A23187 was due to the change in mRNA stability. Kobayashi et al³⁰ showed that the rat AT₂ receptor gene contains activator protein-1 binding sites in the promoter region. Proto-oncoproteins such as c-fos or c-jun activated by the PKC-Ca²⁺ pathway may bind this cis-regulatory element to induce gene transcription of rat AT₂ receptor.³¹ Since this study shows that newly synthesized protein, inhibited by cycloheximide, plays an important role in the increase of AT₂ receptor expression in serum-deprived or confluent, quiescent PC12 cells, it is conceivable that the PKC-Ca²⁺ pathway affects the synthesis of this protein and indirectly inhibits AT₂ receptor expression. Recently, Horiuchi et al¹⁵ have shown that the increased synthesis of interferon regulatory factor-1 protein, induced in confluent R3T3 cells, activates transcription of the mouse AT₂ receptor gene. Thus, the PKC-Ca²⁺ pathway directly or indirectly affects the regulatory mechanism for AT₂ receptor gene expression, and various neurotransmitters mediating this pathway likely affect AT₂ receptor expression.

The AT₂ receptor is expressed in limited tissues in adults, including adrenal medulla,³² heart,²¹ specific brain regions,³³ uterus,³⁴ and ovarian follicles.³⁵ PC12 cells are derived from pheochromocytoma in the adrenal gland and differentiate to neurons in the presence of nerve growth factor.¹³ AT₂ receptor is abundantly expressed in neuronal cells isolated from neonatal rat brain¹¹ and modulates the voltage-sensitive K⁺ currents⁸ or T-type Ca²⁺ current.⁷ The pressor action of centrally injected Ang II is mediated by catecholamines,² and the central catecholamine-rich regions are also rich in Ang II receptors.³ Sumners et al³⁶ showed that Ang II receptors in neuronal cultures from neonatal rat brains are downregulated by the stimulation of ₁-adrenergic receptor, whereas treatment with TPA upregulates the Ang II receptor sites. Although they did not examine the characterization of Ang II receptor subtypes in their studies,^{10 36} the discrepant result about the effect of TPA may be due to the difference in the cell types used in the experiments (neuronal versus PC12 cells). Similar interactions between the PKC-Ca²⁺ pathway and AT₂ receptor have also been observed in myocytes isolated from neonatal rat heart, in which AT₂ receptor is abundantly expressed compared with cardiac fibroblasts,¹⁸ raising the possibility that changes in AT₂ receptor expression caused by the PKC-Ca²⁺ pathway modulate the action potentials of excitable cells such as neurons or myocytes by affecting voltage-sensitive K⁺ or Ca²⁺ channels. Recently, it has been shown that in mice lacking AT₂ receptor, blood pressure is elevated³⁷ and the vasopressor response to Ang II infusion is enhanced,^{37 38} suggesting that AT₂ receptor plays an important role in lowering blood pressure and that vasoactive substances with the PKC-Ca²⁺ pathway, such as norepinephrine, Ang II, or vasopressin, enhance their vasoconstrictive actions by downregulating AT₂ receptor expression. Thus, the present findings may point to a novel mechanism for vasoactive substances with the PKC-Ca²⁺ pathway controlling blood pressure through the AT₂ receptor.

This study also demonstrated that exposure of myocytes to Ang II causes a decrease in AT₂ receptor mRNA levels. Since the addition of Ang II to R3T3 cells¹⁴ or ovarian granulosa cells,³³ expressing the AT₂ receptor predominantly, upregulates the expression of the AT₂ receptor itself and both AT₁ and AT₂ receptors are expressed in myocytes from neonatal rat hearts,¹⁸ the Ang II–induced decrease in the AT₂ receptor mRNA level in myocytes may be caused by the activation of the AT₁ receptor–mediated PKC-Ca²⁺ pathway. We previously found that AT₂ receptor expression is upregulated in the remodeling heart, such as cardiac hypertrophy²⁰ or infarction,²¹ whereas the present study demonstrates that norepinephrine and Ang II, known to induce a hypertrophic change,^{19 23} cause a decrease in AT₂ receptor expression in myocytes. Since Sadoshima et al³⁹ reported that mechanical stretch causes Ang II release from myocytes, it may be possible that some stimuli, such as a stretch-induced hypertrophic response, downregulate AT₂ receptor expression through enhancing local Ang II production from myocytes. Thus, the interaction between hypertrophic signals and cardiac AT₂ receptor expression appears to be more complicated than we speculated, and further studies, including the elucidation of AT₂ receptor signal transduction, will be required to understand the role of AT₂ receptor in the heart.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1, AT2 = angiotensin type 1, type 2 DMEM = Dulbecco's modified Eagle's medium PKC = protein kinase C TPA = 12-O-tetradecanoylphorbol 13-acetate

	Acknowledgments

 
This study was supported in part by research grants from the Ministry of Education, Science, and Culture, Japan; the Study Group of Molecular Cardiology; The Naito Foundation; and the Clinical Pharmacology Foundation in Japan.

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