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Am J Physiol Regul Integr Comp Physiol 278: R781-R789, 2000;
0363-6119/00 $5.00
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Vol. 278, Issue 3, R781-R789, March 2000

Expression and localization of angiotensin subtype receptor proteins in the hypertensive rat heart

Ryoji Ozono1, Toshiyuki Matsumoto1, Tetsuji Shingu2, Tetsuya Oshima1, Yasuhiro Teranishi3, Masayuki Kambe1, Hideo Matsuura2, Goro Kajiyama2, Zhi-Qin Wang4, Allan F. Moore4, and Robert M. Carey4

1 Department of Clinical Laboratory Medicine, 2 First Department of Internal Medicine and 3 Second Department of Physiology, Hiroshima University School of Medicine, Hiroshima, Japan 734; and 4 Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The cellular localization of the AT2 receptor and the regulation of its expression in hypertrophied left ventricle are not well known. We compared the expression of the cardiac AT1 and AT2 receptor in spontaneously hypertensive rats/Izumo strain (SHR/Izm) and Wistar Kyoto rats/Izumo strain (WKY/Izm), ages 4, 12, and 20 wk, by means of immunohistochemistry and Western blot analysis. In SHR/Izm, compared with WKY/Izm, blood pressure (161 ± 2 vs. 120 ± 2 mmHg at 12 wk, P <=  0.01, and 199 ± 3 vs. 123 ± 3 mmHg at 20 wk, P <=  0.01) and heart-to-body weight ratio (3.76 ± 0.07 vs. 3.06 ± 0.06 mg/g at 12 wk, P <=  0.01, and 3.90 ± 0.08 vs. 3.01 ± 0.12 mg/g at 20 wk, P <=  0.01) were significantly elevated. There was no difference in these values between the two strains at 4 wk of age. Histologically, 20-wk-old SHR/Izm demonstrated myocardial hypertrophy, a thickening of the smooth muscle layer of the intracardiac arteries, and perivascular fibrosis. By immunohistochemistry, the AT2 receptor was localized to cardiomyocytes and vascular endothelial cells, but not in the vascular smooth muscle cells. No major AT2 receptor signal was observed in perivascular fibrosis at any age in either strain of rats. No difference was detected in this localization between the two strains. By Western blotting, a single 44-kDa band for the AT2 receptor and a single 60-kDa band for the AT1 receptor were detected in ventricles from both strains of rats at all ages. Densitometric analysis demonstrated that the AT2 receptor 44-kDa band was decreased by 20% at 12 wk and 32% at 20 wk (P < 0.01) in SHR/Izm compared with WKY/Izm. The intensity of the AT1 receptor 60-kDa band was increased by 57% in 20-wk-old SHR/Izm compared with WKY/Izm (P < 0.05). There was no significant difference in the intensity of the 44- or 60-kDa bands in 4-wk-old animals of either strain. We demonstrated a decrease in the AT2 receptor and an increase in the AT1 receptor protein with no change in their localizations in hypertrophied left ventricular myocytes of SHR/Izm.

immunhistochemistry; AT2 receptor; spontaneously hypertensive rats


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ANG II EXERTS its biological effects by binding to angiotensin receptors. Two major subtypes of angiotensin receptors, AT1 and AT2, have been recognized by ligand binding studies. AT1 receptors mediate most of the well-known ANG II effects in the cardiovascular system. The function of the AT2 receptor, on the other hand, has not been determined, although genomic (14) as well as cDNA (10, 20) sequences encoding the rat AT2 receptor have recently been elucidated. Recent studies have suggested that stimulation of the AT2 receptor has antiproliferative effects on the neointima after vascular injury (22) and in coronary endothelial cells (39). The AT2 receptor also has been demonstrated to mediate inhibition of ANG II induced-hypertrophy in cultured myocytes (4). Furthermore, the AT2 receptor is involved in the induction of apoptosis (49) and activation of tyrosine phosphatase (41). These observations support the hypothesis that the AT2 receptor is coupled to an antigrowth process that counteracts the growth-promoting program initiated by AT1 receptor activation (4).

Cardiac hypertrophy is an important risk factor for sudden cardiac death and ischemic heart disease. The heart expresses angiotensinogen (50), renin (3, 6), angiotensin-converting enzyme (37, 50), and both the AT1 (7, 15, 24, 36, 40) and AT2 receptors (17, 36, 40, 44, 50), supporting the concept that the heart possesses its own renin-angiotensin system, which may function in an autocrine/paracrine manner. Of interest, substantial evidence indicates that cardiac hypertrophy is associated with increased local synthesis of ANG II (2, 38) and upregulation of AT1 receptor (7, 13, 15, 17, 40) in cardiac myocytes. The AT2 receptor is also expressed in the heart of rats (17, 18, 24, 36, 40, 44, 48), hamsters (26), rabbits (33), and humans (1, 25, 47), but its cellular localization and physiological or pathological significance still remain unclear. Also, it has not been confirmed whether this AT2 receptor is upregulated (17, 40) or downregulated (25) in cardiac hypertrophy.

The heart consists of myocytes and nonmyocytes, mostly fibroblasts. Pressure overload-induced left ventricular hypertrophy (45) and acute myocardial infarction (19) are associated with remodeling of cardiac structure, with development of perivascular and interstitial fibrosis. Investigators have focused on the effects of ANG II as a mediator of this remodeling process (19, 34, 45). It has been suggested (34, 35) that AT1 receptors play a major role in the transduction of the proliferative signal (18). However, a recent study (26) indicates that AT2 receptors are reexpressed in cardiac fibroblasts in failing heart and contribute to the inhibition of the proliferative process. It has not been studied whether AT2 receptor is expressed in fibroblasts in hypertrophied heart.

Recently, we generated a polyclonal antiserum against the rat AT2 receptor (30, 44) and demonstrated immunohistochemical localization of the receptor subtype in adult rat heart (44). In the present study, we investigated the distribution of the AT2 receptor by immunohistochemistry, for the first time to our knowledge, in the hearts of spontaneously hypertensive rats (SHR) with left ventricular hypertrophy. The cellular localization of this receptor, although providing no direct information on its function, is a necessary first step to begin to clarify the role of the AT2 receptor in the development of cardiac hypertrophy and remodeling. As a model of left ventricular hypertrophy, we used hearts from SHR, which provide an established model of left ventricular hypertrophy associated with systemic hypertension.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. SHR/Izumo strain (SHR/Izm) and their normotensive controls, Wistar Kyoto rats/Izumo strain (WKY/Izm), at ages of 4 (n = 10, each strain), 12 (n = 8, each strain), and 20 (n = 8, each strain) wk, were purchased from Disease Model Cooperative Research Association (Kyoto, Japan). Nara et al. (23) developed these new inbred SHR and WKY strains that have almost identical genetic backgrounds. Systolic blood pressure was measured by the tail cuff method, as described previously (27). Animals were deeply anesthetized with pentobarbital sodium, and the hearts were freshly removed and weighed. The heart weight (mg) divided by the body weight (g) was considered a measure of ventricular hypertrophy.

AT2-receptor antiserum. Polyclonal antiserum was raised against a synthetic peptide sequence derived from the amino terminus of the predicted rat AT2 receptor (MKDNFSFAATSRNITSS) (30, 44). The IgG fraction of the serum was obtained using protein A column as described (29). The protein concentration of the purified serum was 3.1 mg/ml, as determined by the Bradford method. Selectivity of the antiserum to the rat AT2 receptor was fully evaluated and published elsewhere (30, 44). Briefly, the antiserum recognized the AT2 receptor expressed in a stably transfected COS-7 cell line in Western blotting as a single 44-kDa band, whereas no band was observed in the nontransfected cell line. In the immunohistochemistry, the antiserum positively stained the transfected COS-7 cells grown on slides, whereas no signal was observed in nontransfected cells or in the transfected cells when reacted with the antiserum preadsorbed with the pure peptide immunogen (29, 44).

Rabbit anti-AT1 receptor polyclonal antiserum (AB1525), which is directed toward the carboxy terminal of the native receptor, was purchased from Chemicon International. The specificity was evaluated elsewhere (31).

Histological and immunohistochemical analysis. For histological demonstration of cardiac hypertrophy and remodeling, freshly removed ventricles from 20-wk-old SHR/Izm and WKY/Izm were immersion fixed in phosphate-buffered-10% formaldehyde solution and embedded in paraffin. Two-micrometer-thick sections were cut and processed according to an Elastica van Gieson staining protocol, which stains collagen a reddish-purple color. For analysis of ventricular myocyte cross-sectional area, microscopic fields were randomly selected from both epicardial and endocardial portions of ventricles and the images were acquired with a video camera (3 CCD color video camera KYF55B, Victor). The myocyte cross sections were traced, and the area was calculated with National Institute of Health (NIH) Image software program.

Immunohistochemistry was performed in frozen sections as described previously (29, 30, 44). Briefly, the hearts from the 4-, 12-, and 20-wk-old animals were immediately immersion fixed in 2% paraformaldehyde in PBS for 1-2 h. The tissue was cryoprotected overnight at 4°C in 30% sucrose in PBS, and frozen sections (6-8 mm) were cut. The sections were stored at -80°C until use. For the AT1 receptor staining, the endogenous peroxidase was quenched with 1% H2O2 in methanol, then the nonspecific binding sites of 1) avidin, 2) biotin, and 3) secondary goat antibody were blocked with 1) avidin solution for 15 min, 2) biotin solution for 15 min (avidin biotin blocking kit, Vector Lab), and 3) 10% normal goat serum and 1% nonfat dry milk in PBS for 45 min, respectively. For the AT2 receptor staining, the endogenous peroxidase was quenched with 0.3% H2O2 in methanol, then the nonspecific binding sites of secondary goat antiserum were blocked with 3% normal goat serum and 2% nonfat dry milk in PBS for 45 min. The sections for both AT1 and AT2 receptors were then incubated overnight at 4°C with one of the following: 1) AT1 or AT2 receptor antiserum, 2) the IgG fraction of the preimmune serum, or 3) the antiserum against the AT2 receptor preadsorbed with its pure peptide antigen. Sera were diluted at 1:500 in 1.5% normal goat serum and 1% nonfat dry milk in PBS for AT2 receptor and 1:1,000 in 10% normal goat serum and 1% nonfat dry milk for AT1 receptor. In the preadsorption of the AT2 receptor antiserum, the serum was incubated for 24-48 h at 4°C with the immunizing peptide at 10-fold molar excess. Staining was visualized with the avidin-biotin immunoperoxidase reaction (Vectastain ABC Kit) using diaminobenzidine (Fast DAB tablets, Sigma) according to the manufacturer's instructions.

Western blot analysis of AT1 and AT2 receptor protein expression. AT2 receptor protein expression in the heart was compared between SHR/Izm and WKY/Izm by Western blot analysis. Samples were prepared as previously described (29, 30, 44). Tissues were homogenized with Polytron in buffer A (10% glycerol, 20 mM Tris · HCl, 100 mM NaCl, 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM EGTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A). The homogenate was centrifuged at 30,000 g for 30 min at 4°C. The pellet was resuspended in buffer B (buffer A with 1% Nonidet P-40), stirred for 1 h at 4°C, and centrifuged again at 30,000 g. The supernatant was used for the analysis. The solubilized samples were subjected to SDS-PAGE (10% running gel). For comparison of the AT1 and AT2 receptor protein expression level between SHR/Izm and WKY/Izm, precisely 50 µg protein was loaded per gel. The protein concentration was determined by the bicinchoninic acid method. The resolved proteins were transferred onto a nitrocellulose membrane [Hi-bond enhanced chemiluminescence (ECL), Amersham] by electroblotting at 15 V for 20 min (Transblot SD DNA, Bio-Rad). The nitrocellulose membrane was soaked in Tris-buffered saline (TBS: 10 mmol/l Tris · HCl, 150 mmol/l NaCl) containing 5% nonfat dry milk (Skim Milk, Snow Brand) and 0.1% polyoxyethylene-sorbitan monolaurate (Tween 20) overnight at 4°C to block nonspecific sites, and then incubated with the AT1 or AT2 receptor antiserum (1:1,000 dilution in TBS with 5% nonfat dry milk and 0.1% Tween 20 ) for 2 h, and reacted with a peroxidase-conjugated donkey anti-rabbit secondary antibody (1:5,000 dilution) for 1 h. Immunoreactivity was visualized with an ECL Western Blotting Detection Kit (Amersham). NIH Image software program analyzed the intensity of the band. The bands were area traced, the size and the mean density were analyzed, and the product of the density by the area was defined as a band intensity that reflects the amount of protein and was expressed as arbitrary units.

Statistical analysis. Results were expressed as means ± SE. Change within a group was analyzed by ANOVA for repeated measures. Comparisons between SHR/Izm and WKY/Izm in the same age were made with two-tailed unpaired Student's t-test. A value of P < 0.05 was accepted as statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Validation of hypertension and left ventricular hypertrophy in SHR/Izm. As shown in Table 1, systolic blood pressure and the ratio of heart weight to body weight were significantly increased in 12- and 20-wk-old SHR/Izm compared with age-matched WKY/Izm, whereas no difference was detected in 4-wk-old animals. Histologically (Fig. 1), the ventricles of 20-wk-old SHR/Izm demonstrated characteristic signs of left ventricular hypertrophy, including increased cardiomyocyte diameter, perivascular fibrosis, and thickening of the vascular smooth muscle cell layer of the small coronary arteries. The mean values of ventricular myocyte cross-sectional areas in 20-wk-old SHR/Izm and WKY/Izm were 349 ± 12 and 276 ± 8 µm2, respectively (P < 0.01). Interstitial fibrosis, which characterizes decompensated cardiac dysfunction, was present but not severe (Fig. 1). Similar, but less marked, histological findings were also observed in 12-wk-old SHR/Izm (data not shown).

                              
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  		Table 1.   Body weight, heart weight, and blood pressure in 4-, 12-, and 20-week-old SHR/Izm and WKY/Izm



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  		Fig. 1.   Light photomicrograph of paraffin section of left ventricles from 20-wk-old spontaneously hypertensive rat/Izumo strain (SHR/Izm; A, C) and Wistar Kyoto/Izumo strain (WKY/Izm; B, D) stained by Elastica van Gieson method, which stains connective tissues a reddish-purple color selectively. A, left ventricle from SHR/Izm (×100); B, left ventricle from WKY/Izm (×100); C, high-power magnification of left ventricle from SHR/Izm (×400), demonstrating myocyte cross sections; D, high-power magnification of ventricle from WKY/Izm (×400), demonstrating myocyte cross sections. In SHR/Izm (A), vascular smooth muscle cell layer is markedly thickened and mild hyperplasia of perivascular and interstitial connective tissue is noted.

Immunohistochemistry of the AT2 receptor protein. In both SHR/Izm and WKY/Izm at all ages, the AT2 receptor immunohistochemical signal was detected throughout the myocardium of left ventricle (Figs. 2 and 3), right ventricle, and atria (data not shown). The myocardial staining was homogeneous. Intracardiac small vessels were positively stained, but it was indistinguishable whether the staining was from vascular endothelium or vascular smooth muscle (data not shown). On the other hand, the vascular smooth muscle layer in the relatively large coronary artery (Fig. 3, broken arrow) and ascending aorta (data not shown) was clearly negative. In such large coronary arteries, the endothelium was positively stained (Fig. 3, arrowhead). The fibrous tissues were not remarkably stained, but a positive signal was observed in some perivascular fibrotic areas (Figs. 2 and 3, arrow). The preadsorption and nonimmune serum controls for all of these immunohistochemical studies were negative. There was no difference in the distribution pattern or the intensity of the staining in the cardiomyocyte, connective tissue, and vascular endothelium between SHR/Izm and WKY/Izm at any age.


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  		Fig. 2.   Light photomicrographs of frozen sections of ventricles from 20-wk-old SHR/Izm (A, B) and WKY/Izm (C, D) treated with anti-AT2 receptor serum or negative control serum diluted at 1:500. Magnification ×200. A: coronary artery, perivascular connective tissue, and myocardium of left ventricle from 20-wk-old SHR/Izm, demonstrating positive staining in myocytes and vascular endothelium (arrowhead). Perivascular fibrosis (arrow) and thickening of the vascular smooth muscle layer (broken arrow) is noted. There is a very light signal in fibrotic tissue, but no staining in vascular smooth muscle layer. B: preadsorption control for A. C: coronary artery, perivascular connective tissue, and myocardium of left ventricle from 20-wk-old WKY/Izm, demonstrating positive staining in myocytes and vascular endothelium. In WKY/Izm, the arterial wall (broken arrow) is not thickened and perivascular connective tissue (arrow) does not show hyperplasia. It is not distinguishable whether staining in blood vessel is from endothelium or smooth muscle. D: preimmune control for C.



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  		Fig. 3.   Light photomicrographs of intracardiac coronary arteries in left ventricles of 20-wk-old SHR/Izm (A, B) and WKY/Izm (C, D). Frozen sections were treated with anti-AT2 receptor serum or nonimmune serum diluted at 1:500. Magnification ×400. A: coronary artery of 20-wk-old SHR/Izm. Vascular smooth muscle layer (broken arrow) is markedly thickened, and perivascular fibrosis (arrow) is enhanced. Note that AT2 receptor staining is observed in endothelium (arrowhead) and cardiomyocytes, but not in vascular smooth muscle and fibroblasts. B: preimmume serum control for A. C: coronary artery of 20-wk-old WKY/Izm. AT2 receptor staining is observed in endothelium (arrowhead) and cardiomyocytes, but not in vascular smooth muscle cells (broken arrow) and fibrotic tissue (arrow). Compared with SHR/Izm, vascular smooth muscle layer is thinner and perivascular fibrosis is less marked, but localization of AT2 receptor staining is similar. D: preimmune serum control for C.

Figure 4 shows AT1-receptor staining in the left ventricles of 20-wk-old SHR/Izm and WKY/Izm. AT1 receptor was observed in cardiomyocytes, the vascular smooth muscle cells (Fig. 4, broken arrows), and perivascular tissue (Fig. 4, arrows) in both strains at all ages. No significant difference in the intensity or distribution of the signal was detected between the two strains.


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  		Fig. 4.   Light photomicrographs of frozen sections of right ventricle from 20-wk-old SHR/Izm (A, B) and WKY/Izm (C, D) demonstrating AT1 receptor staining. Magnification ×200. A: left ventricle and intracardiac coronary artery of 20-wk-old SHR/Izm treated with anti-AT1 receptor antiserum diluted at 1:1,000. AT1 receptor staining is observed in cardiomyocytes, vascular smooth muscle layer of coronary artery (broken arrow), and in fibroblasts in perivascular connective tissue (arrow). B: nonimmune serum control for A. C: left ventricle and intracardiac coronary artery of 20-wk-old WKY/Izm treated with anti-AT1 receptor antiserum diluted at 1:1,000. AT1 receptor staining is observed in cardiomyocytes, vascular smooth muscle layer of coronary artery (broken arrow), and in fibroblasts (arrow). D: nonimmune serum control for C.

Western blot analysis. A single 44-kDa band was observed in Western blots of the AT2 receptor transfected COS-7 cells, but not in the nontransfected COS-7 cells (Fig. 5A). The same molecular weight band was seen in the ventricles at every age and strain. The approximate molecular mass of the AT2 receptor was consistent with the calculated mass based on the molecular sequence and with that previously reported by our group (30, 44) and others (32). Densitometric analysis of the 44-kDa band (Fig. 5B) demonstrated that the band intensity was smaller in SHR/Izm compared with WKY/Izm by 1 ± 7% at 4 wk, 20 ± 5% at 12 wk, and 32 ± 4% (P <=  0.01) at 20 wk (Table 2). When only the right ventricles from 20-wk-old animals were used, a similar decrease in the 44-kDa band in SHR/Izm compared with WKY/Izm was observed (n = 2). The amount of AT2 receptor signal for SHR/Izm was significantly smaller at 20 wk of age than at 4 wk of age (P < 0.05). In WKY/Izm, the intensities of the bands were not significantly changed by the age (Table 2).


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  		Fig. 5.   Western blot analysis of rat AT2 receptor in ventricles from 4-, 12-, and 20-wk-old-SHR/Izm and WKY/Izm and in AT2 receptor transfected or nontransfected COS-7 cells. Approximate molecular mass of AT2 receptor is 44 kDa. A: lane 1, 4-wk-old SHR/Izm; 2, 4-wk-old WKY/Izm; 3, 12-wk-old SHR/Izm; 4, 12-wk-old WKY/Izm; 5, 20-wk-old SHR/Izm; 6, 20-wk-old WKY/Izm; 7, COS-7 cells transfected with AT2 receptor; 8, COS-7 cells without transfection. Note that band intensity is larger in 12-wk-old and 20-wk-old WKY/Izm compared with SHR/Izm. B: bar graphs showing intensity of 44-kDa band (AT2 receptor) in Western blot analysis in 4-, 12-, and 20-wk-old SHR/Izm in comparison with WKY/Izm. Band intensity was calculated by multiplying band area and its mean density. Numbers are given in percentage of SHR/Izm relative to WKY/Izm at same age. ** P < 0.01 vs. WKY/Izm at same age, dagger  P < 0.05 vs. 4-wk-old SHR/Izm.


                              
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  		Table 2.   Intensities of 60-kDa (AT1) and 44-kDa (AT2) receptor band

The AT1 receptor was also detected by use of anti- AT1 receptor antiserum (Fig. 6A). The approximate molecular mass was 60 kDa, consistent with the reported value (13, 21, 31). The densitometric analysis revealed that the band intensity was greater in SHR/Izm by 29 ± 6% at 12 wk and 57 ± 12% (P <=  0.05) at 20 wk than WKY/Izm at the same age (Fig. 6B), whereas no significant difference was detected in 4-wk-old animals (Table 2).


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  		Fig. 6.   Western blot analysis of AT1 receptor of ventricles from SHR/Izm and WKY/Izm. Approximate molecular mass of AT1 receptor is 60 kDa. A: lane 1, 20-wk-old SHR/Izm; 2, 20-wk-old WKY/Izm; 3, 12-wk-old SHR/Izm; 4, 12-wk-old WKY/Izm. Note that band intensity is greater in 12- and 20-wk-old SHR/Izm than in WKY/Izm. B: bar graphs showing intensity of 60-kDa band (AT1 receptor) in Western blot analysis in 4-, 12-, and 20-wk-old SHR/Izm in comparison with WKY/Izm. Band intensity was calculated by multiplying band areas by their mean density. Numbers are given in percentage of SHR/Izm relative to WKY/Izm at same age. * P < 0.05 vs. WKY/Izm at same age.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrated that 1) SHR/Izm, at ages of 12 and 20 wk, had left ventricular hypertrophy accompanied by remodeling of the connective tissue; 2) the expression of AT2 receptor protein in ventricular myocytes was decreased, whereas that of the AT1 receptor was increased in 12- and 20-wk-old SHR/Izm compared with WKY/Izm at the same age; and 3) there was no remarkable AT2 receptor staining in perivascular and interstitial connective tissues, and the staining intensity was not changed during the development of the cardiac remodeling. There had been no immunohistochemical studies localizing both AT1 and AT2 receptors in diseased heart. Recent studies indicated that AT2 receptors are locally reexpressed in the sites of cellular hyperplasia in the failing heart (26, 47) or in wound healing (42). In left ventricular hypertrophy in SHR/Izm, however, such a local accumulation of the receptor subtype was not observed until at least 20 wk of age.

It is tempting to speculate that the balance of the expression of two receptor subtypes may determine the overall activity of ANG II in the heart, and the decrease in the AT2 receptor may contribute to development of left ventricular hypertrophy in SHR. However, we cannot conclude this from our present observations, because we only examined change in the receptor number but not change in receptor-agonist interaction or receptor-mediated signal transduction. Up- or downregulation of the receptor does not always parallel the change in the activity of the receptor system. In addition, whether the change in the ratio of AT1/AT2 receptor can modulate development of left ventricular hypertrophy also needs to be established by functional studies.

The mechanism for the regulation of AT1 and AT2 receptor expression is not well understood. Mechanical stretch of myocardium (13, 34) causes enhanced ANG II production and upregulation of AT1 receptor. The latter finding was consistent with our observations. Wang et al. (43) recently reported that administration of ANG II downregulated AT1 receptors but had no effect on AT2 receptors in the kidney. It is unclear whether the changes in the AT1 and AT2 receptors observed in the present study were caused by pressure overload or by other humoral factors including ANG II, because the mechanism of the left ventricular hypertrophy in SHR is multifactorial. However, we (28) recently observed that the AT2 receptor was uniformly downregulated in left ventricular hypertrophy regardless of the cause of hypertrophy, including coarctation of aorta, deoxycorticosterone-acetate salt hypertension, and two kidney, one-clip hypertension, indicating that the pressure overload is likely to be the major mechanism whereby this receptor subtype is downregulated.

As to the subcellular mechanism, the gene expression of the AT2 receptor is also regulated by multiple factors. Increase in intracellular calcium level by ionophore (13) and activation of protein kinase C (PKC) by phorbol ester (13) and cAMP analog (16) downregulated the AT2 receptor mRNA or AT2 receptor binding in PC12 cells. Norepinephrine and ANG II, which elevate Ca2+ levels and activate PKC, downregulate the AT2 receptor in cardiac myocytes (12). Growth factors, including epidermal growth factor, nerve growth factor, and platelet-derived growth factor, also downregulate AT2 receptor mRNA expression in PC12 cells (12) and R3T3 cells (8). On the other hand, Ichiki et al. (8, 9) and Kambayashi et al. (11) reported that AT2 receptor mRNA is upregulated by interleukin-1beta , insulin, and insulin-like growth factor. According to these observations, it is more conceivable that AT2 receptor is downregulated in left ventricular hypertrophy as observed in the present study, because PKC, cAMP, and growth factors are all increased in left ventricular hypertrophy.

Previously, Suzuki et al. (40) reported that both AT1 and AT2 receptor binding capacity and mRNAs detected by RT-PCR were increased in the heart with left ventricular hypertrophy from renovascular hypertensive rats as well as in SHR at ages 20 and 24 wk. Similarly, the same group (13) reported that mechanical stretch for several days in cultured cardiomyocytes upregulated AT1 and AT2 receptors via signals involving stretch-activated tyrosine kinases. In addition, Lopez et al. (17), using a ligand binding technique, observed an increase in the proportion of AT2 receptor number relative to that of AT1 receptor after 4 wk of treatment with aortic banding. However, Wolf et al. (48) reported that neither AT1 nor AT2 receptor message was affected by aortic banding for several weeks. Nozawa et al. (25), again using ligand binding technique, reported a downregulation of AT2 receptor in left ventricular hypertrophy in humans. Therefore, it has not been conclusively determined whether AT2 receptor is upregulated or downregulated in left ventricular hypertrophy. The discrepancy may be explained by differences in species of animals, pathological stage of left ventricular hypertrophy, and technique to detect the AT2 receptor. More studies are needed to determine whether AT2 receptor is increased or decreased in left ventricular hypertrophy.

It is known that left ventricular hypertrophy, in its late stage, progresses into heart failure associated with interstitial fibrosis, a loss of myocardial contractility, and an increase in cardiac diameter (5). It is possible that our observation in 20-wk-old SHR/Izm is, in part, related to heart failure rather than hypertrophy. However, the histological observation in 20-wk-old SHR/Izm demonstrates no evidence of transition of the hypertrophy to heart failure, including severe interstitial fibrosis and decrease in myocardial wall thickness. In addition, recent studies (1, 46) have demonstrated that the AT1 receptor was downregulated in end-stage heart failure. In the present study, the AT1 receptor was upregulated by ~50%. These observations suggest that the 20-wk-old SHR was in the stage of ventricular hypertrophy that preceded heart failure.

There is a substantial difference in the genetic backgrounds of conventional SHR and WKY. To circumvent this disadvantage of SHR, in the present study, we used a newly developed inbred strain of SHR/Izm and WKY/Izm, in which it has been established that the matching of the genetic backgrounds has been markedly improved (23). Similarly, observed changes in AT1 and AT2 receptors could be associated with a genetic defect in the regulation of the renin-angiotensin system that is unique to SHR, but has nothing to do with the hypertension. To study this possibility, we compared the change in AT2 receptor density in other pressure overload models in rats (28) and obtained the same finding as in this study. Furthermore, in the present study, there was no difference in either the AT1 and AT2 receptor between the strains in prehypertensive 4-wk-old animals. Therefore, it is unlikely that the change in AT1 and AT2 receptors during the development of cardiac hypertrophy is independent of systemic hypertension.

In summary, we localized AT1 and AT2 receptors in the heart of SHR/Izm with left ventricular hypertrophy. The AT2 receptors were observed only in cardiomyocytes regardless of the hemodynamic and structural changes associated with the left ventricular hypertrophy. Also, we found that left ventricular hypertrophy in SHR/Izm was associated with the downregulation of the AT2 receptor and upregulation of AT1 receptor in cardiomyocytes. These findings provide important information for the investigation of the functional role of angiotensin receptors in left ventricular hypertrophy.


    ACKNOWLEDGEMENTS

The authors thank Dr. Tadashi Inagami at Vanderbilt University for providing the AT2 receptor transfected cell line, Sachiko Hidaka for technical support, and Yuko Omura and Yumi Tsujimura for secretarial assistance.


    FOOTNOTES

This study was supported by grants-in-aid for Scientific Research (nos. 08457639, 07407065, and 11470518 to T. Oshima and 11771511 to R. Ozono).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. Ozono, Dept. of Clinical Laboratory Medicine, Hiroshima Univ. School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima, Japan 734 (E-mail: ozono{at}mcai.med.hiroshima-u.ac.jp).

Received 19 April 1999; accepted in final form 24 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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