INTRODUCTION

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ANG II is a potent regulator of normal and abnormal cardiovascular function. ANG II acts on specific cell membrane receptors for the physiological maintenance of arterial blood pressure (7, 19, 29), sodium and water homeostasis (7, 27, 29), and capillary density (11, 19, 32). In pathophysiological conditions, ANG II acts via the same receptors to modulate ventricular hypertrophy (3), medial-intimal thickening (5, 32), restenosis after angioplasty (15), nephrosclerosis (27, 28), and increased vascular reactivity (6). The functions classically attributed to ANG II such as vasoconstriction, sodium retention, and angiogenesis are mediated by the ANG II type 1 (AT₁) receptor (27). The AT₁ receptor has a broad distribution, consistent with its multiple functions, and has been localized to many cells, including vascular smooth muscle, hepatocyte, adrenal zona glomerulosa, pituitary, testis, spleen, and kidney (22). Although the ANG II type 2 (AT₂) receptor has been found in rat uterus, brain, aorta, and adrenal medulla (27), all the known functions of ANG II have been attributed to the AT₁ receptor until recently. In fact, the AT₂ receptor was thought to be important only during fetal life, when it is more prevalent than it is in maturity and is more common than the AT₁ receptor at that stage of development (31).

Although the actions of the AT₂ receptor have not been solidified, new evidence suggests that the AT₂ receptor mediates functions opposing those of the AT₁ receptor. Scheuer and Perrone (24) demonstrated that in mature rats the AT₂ receptor mediates the depressor phase of the biphasic blood pressure response to ANG II and ANG III infusion, whereas the AT₁ receptor mediates the pressor phase. A similar result was found by Lombard and Huang (18), who demonstrated that, in isolated middle cerebral arteries, specific blockade of the AT₂ receptor potentiated the ANG II-induced vascular smooth muscle cell contraction and depolarization. The vasodilator properties of the AT₂ receptor were further corroborated by Munzenmaier and Greene (19), who revealed that when ANG II was chronically infused with the specific AT₂-receptor antagonist PD-123319, the increase in blood pressure was significantly greater than when AT₁ receptors were activated by ANG II infusion in the absence of AT₂-receptor blockade. In addition to possible vasodilator activity, new studies support a role for the AT₂ receptor in antiproliferation and antiangiogenesis. Stoll et al. (26) showed that ANG II significantly inhibited the proliferation of coronary endothelial cells in a dose-dependent manner. This antiproliferative effect of ANG II was blocked by the AT₂-receptor-specific antagonist PD-123177. Furthermore, antiangiogenesis mediated by the AT₂ receptor was demonstrated in vivo by Munzenmaier and Greene. In that study a significantly greater increase in microvessel density (MVD) was observed when ANG II and PD-123319 were coinfused than in response to a subpressor dose of ANG II alone. The latter study thus demonstrated that the AT₁ and AT₂ receptors are functionally colocalized to the microcirculation.

Despite some functional evidence for the presence of the AT₂ receptor in the normal, adult vasculature, most studies have focused on the AT₂ receptor only in fetal tissue and during pathophysiological conditions (28). In vivo studies have not yet localized AT₂-receptor protein to the vasculature, and in vitro studies report a heterogeneous distribution of AT₂-receptor expression in primary cultures of endothelial cells. Stoll et al. (26) described AT₁- and AT₂-receptor protein in low-passage rat aortic endothelial cells, whereas Pueyo et al. (23) could not detect AT₂-receptor protein with binding studies in rat aortic endothelial cells. Therefore, to study the vascular distribution of the AT₂ receptor, we developed a polyclonal anti-rat AT₂-receptor antiserum. The antiserum was used 1) to demonstrate that AT₂-receptor protein could be detected in skeletal muscle and microdissected blood vessels by Western blot analysis and 2) to determine the microanatomic distribution of the AT₂ receptor in the skeletal muscle microcirculation through the use of immunohistochemistry. Our results indicate that the AT₂-receptor protein is present in the skeletal muscle microcirculation in endothelial and vascular smooth muscle cells.

	METHODS

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Development and validation of an AT₂-receptor antiserum. With use of the sequences published for the AT₂ receptor (2), amino acid sequences of high predicted antigenicity were chosen with the aid of the PEPTIDESTRUCTURE program in GCG computer software (Madison, WI). Peptides corresponding to amino acids 35-46 (NH₂ terminus, QSHKPADKHLEA) and 339-353 (COOH terminus, LQGKRETMSCRKSSS) were synthesized, purified by reverse-phase HPLC, and then conjugated to ovalbumin by carbodiimide reaction (Imject Immunogen EDC conjugation kit, Pierce Chemicals, Rockford, IL) for the COOH terminus and by a maleimide reaction (maleimide conjugation kit, Pierce Chemicals) for the NH₂ terminus. The conjugates were purified by gel filtration, mixed 1:1 with Freund's complete adjuvant, and injected intradermally into 10 sites on the shaved back of anesthetized, adult New Zealand White rabbits. The animals were boosted 21 days later with a similar mixture but using Freund's incomplete adjuvant. Preimmunization serum was obtained from the ear vein before the initial immunization, and blood was collected 3 wk after the booster injection. Blood was withdrawn from the ear vein, and the serum was separated from the whole cells and stored frozen. The serum was evaluated for antigen recognition by testing a series of diluted serum samples on a nitrocellulose membrane that had keyhole limpet hemocyanin-conjugated peptide blotted at several concentrations: 0.6, 0.3, 0.15, and 0.075 mg/dot (Fig. 1A). Serum obtained from a rabbit immunized with the NH₂-terminal peptide conjugate proved the most antigenic and was used in these studies. No cross-reactivity with the AT₁ receptor was demonstrated with an AT₁-receptor-specific antibody purchased from Chemicon International (Temecula, CA). The AT₁- and AT₂-receptor antibodies were diluted 1:2,000.

View larger version (63K): [in this window] [in a new window]   Fig. 1.   ANG II type 2 (AT2)-receptor antibody validation. A: dot blot showing antigenicity against a range of peptide conjugate concentrations (from top to bottom: 0.6, 0.3, 0.15, and 0.075 mg/dot). B: ability of peptide conjugate to compete for native AT2 receptor on Western blot. C: linear behavior of quantitation of AT2-receptor protein obtained from Western blot. OD, optical density. D: multiantisera screen of uniform distribution of adrenal protein on Western blot demonstrating specificity of AT1- and AT2-receptor antisera. 1, AT1-receptor antiserum; 2, AT2-receptor antiserum; P, preimmune serum. Antiserum dilutions are indicated at bottom.

Collection of skeletal muscle tissue and isolated microvessels for use in Western blot analysis. Male, 5-wk-old Sprague-Dawley rats were anesthetized with pentobarbital sodium (50 mg/kg), and the soleus and left cremaster muscles were immediately harvested and flash frozen in liquid nitrogen. The soleus was exposed for harvesting by removing the skin overlaying the calf muscles, severing the Achilles tendon, and reflecting the tendon and gastrocnemius muscle. To isolate microvessels from the cremaster muscle, the right scrotal sac was opened. The cremaster muscle and testicle were tied off securely at the most proximal position, and the cremasteric sac was cut off above the tie. A longitudinal incision was then made in the cremaster muscle, and the testicle was removed. The cremaster muscle was quickly pinned out, using insect pins in a frozen, coated glass petri dish. The muscle was immediately flushed with 20 ml of ice-cold (4°C) normal saline and maintained thereafter in a 100% methanol solution on ice. The cremasteric circulation was viewed at low magnification (×25) under a dissecting microscope (model MZ6, Leica). Microvessels were identified by branching order and carefully dissected manually using special microdissection instruments to remove vessels from the surrounding parenchymal tissue. Dissected vessels were placed in ice-cold storage buffer solution (see below), centrifuged (5 min in a minicentrifuge), washed twice, and frozen at 35°C.

Isolation of RNA from adrenal gland, cremasteric microvessels, bovine aortic endothelial cells, and bovine pulmonary artery endothelial cells. After adrenal glands were harvested and microvessels were hand dissected from cremaster muscle from 8-wk-old Sprague-Dawley rats, the tissue was immediately homogenized in 1 ml of TRIzol (GIBCO Life Technologies, Grand Island, NY). Cultured bovine aortic endothelial cells (BAECs; a generous gift from Dr. Steven Elliot, Medical College of Wisconsin, Milwaukee, WI) and bovine pulmonary artery endothelial cells (BPAECs) were sedimented, and the cellular pellets were immediately homogenized in 1 ml of TRIzol. RNA was extracted and treated with DNase (Amersham Pharmacia Biotech, Piscataway, NJ) for 30 min at 37°C to remove any potential genomic DNA contamination. After DNase treatment the RNA was reextracted using the TRIzol reagent. RNA was resuspended in 50 ml of diethyl pyrocarbonate-treated water and stored at 80°C until use.

Detection of AT₂-receptor mRNA using RT-PCR. RT was performed on the DNase-treated, TRIzol-extracted RNA using the first-strand cDNA synthesis kit (Amersham Pharmacia). cDNA was synthesized during a 60-min incubation at 37°C, and the reaction was halted by placing the tubes on ice. Primers for the PCR were purchased from Operon Technologies (Alameda, CA), and the sequences were selected from the published sequences for the rat AT₂-receptor and human -actin cDNA: sense 5'-GCT GTT GTG TTG GCA TTC AT-3' and antisense 5'-CAC TGA AAG CTG GTG GAG TA-3' (AT₂; final PCR product 489 bp) and sense 5'-AGC AGC CGT GGC CAT CTC TTG CTC GAA CTG-3' and antisense 5'-AAC CGC GAG AAG ATG ACC CAG ATC ATG TTT-3' (-actin; final PCR product 350 bp). All PCR reactions were performed in a total volume of 50 ml in the presence of 50 pmol of each primer and 2.5 U of AmpliTaq polymerase (Perkin-Elmer Cetus, Norwalk, CT). The reaction mixtures were first denatured at 96°C for 5 min and then placed at 94°C for an additional 5 min while the AmpliTaq polymerase was added. The reactions were run for 35 cycles between 94°C (denaturation) for 1 min, 55°C (annealing) for 1 min, and 72°C (extension) for 1 min. Samples were incubated for an additional 7 min at 72°C after the completion of the final cycle. For each set of primers, RT-PCR was performed on sterile water to check for contamination, and for each tissue, PCR was conducted on DNase-treated RNA to check for genomic DNA contamination. Ten milliliters of the PCR products were electrophoretically size fractionated on a 1.3% agarose gel, then stained with ethidium bromide for 30 min. The bands were visualized using a FluorImager SI from Molecular Dynamics (Sunnyvale, CA). The molecular weight of the PCR products was compared with the 100-bp ladder (Amersham Pharmacia).

Isolation of protein from tissue samples. Snap-frozen cremaster and soleus muscles, adrenal, kidney, and sedimented BAECs, BPAECs, and PC-12 cells were homogenized in a 1:2 volume of ice-cold protein isolation solution (250 mM sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM MgCl₂, and 10 mM potassium phosphate, pH 7.7) using an overhead homogenizer (PowerGen 700, Fisher Scientific). The homogenate was then centrifuged at 3,500 g (model GS-15R, Beckman) for 15 min at 4°C. The supernatant was removed for ultracentrifugation (Optima Tl, Beckman) at 125,000 g for 30 min at 4°C. The pellet was resuspended in storage buffer (100 mM KPO₄, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 30% glycerol, pH 7.25) and stored at 35°C until use in Western blot analysis.

Western blot analysis to demonstrate the presence of AT₂-receptor protein in the microcirculation. Five micrograms of protein (as determined by protein assay kit, Bio-Rad, Hercules, CA) from each tissue (cremaster, soleus, isolated microvessels, and PC-12 cells for positive control) were separated on an 8% denaturing, polyacrylamide gel. The gels were transferred to a nitrocellulose membrane, which was blocked overnight in 5% nonfat dry milk diluted in Tris-buffered saline (50 mM Tris and 750 mM NaCl, pH 8.0) with 0.08% Tween 20 (Bio-Rad) and then probed with the AT₂-receptor antiserum at a dilution of 1:2,000 for 1.5 h at room temperature. Membranes were incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Sigma Chemical, St. Louis, MO) at a dilution of 1:1,000 for 1 h at room temperature, then subjected to enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL) detection system. Membranes were exposed to X-ray film (Fuji Medical, Stamford, CT) for ~10-25 s and developed using a Kodak M35 X-OMAT processor.

Localization of AT₂ receptor to the microcirculation by immunohistochemistry. Five- to six-wk-old Sprague-Dawley rats were anesthetized with pentobarbital sodium (50 mg/kg), and skeletal muscles (cremaster and soleus) were removed. The soleus muscle was embedded in Tissue-Tek (OCT compound, Sakura Finetek, Torrance, CA), immediately snap frozen in a slurry of dry ice-isopentane, and then sectioned to 6 µm thickness (cryocut 1800, Reichert-Jung). After removal of the cremaster muscle, specific regions in the cremaster muscle containing first-, second-, and third-order arterioles were rapidly selected and positioned strategically in the folded cremaster muscle to optimize sectioning of these vessels in a transverse orientation. The cremaster muscle was embedded in Tissue-Tek, immediately snap frozen in a slurry of dry ice-isopentane, and sectioned to 6 µm thickness. The cryostat sections were processed for immunohistochemical localization of antigen (AT₂ receptor) in combination with Griffonia simplicifolia I lectin to identify blood vessels (9). The sections were lightly fixed with 8% Formalin, rinsed, blocked with 1.0% BSA (Sigma Chemical), and then exposed to the primary antibody (1:250 dilution) for 1 h at room temperature. After the section was rinsed with PBS (Sigma Chemical), the FITC-conjugated goat anti-rabbit IgG (1:150 dilution; Sigma Chemical) was applied in combination with tetramethylrhodamine B isothiocyanate (TRITC)-labeled G. simplicifolia I lectin (1:100 dilution; Sigma Chemical) for 1 h at room temperature. The samples were observed by digital epifluorescence microscopy (Nikon Optiphot) and confocal microscopy (model 600, Bio-Rad) using specific filters to separate FITC and TRITC labeling.

Colocalization of AT₂ receptor to vascular smooth muscle cells. Tissues were harvested, frozen, sectioned, fixed, and blocked as described above. AT₂-receptor antiserum (1:250 dilution) was used in combination with TRITC-conjugated, monoclonal smooth muscle anti--actin (1:300 dilution; Sigma Chemical), an actin isoform that is specific for smooth muscle cells. Sections were incubated with the primary antibodies for 1 h in the dark and then kept covered. After they were rinsed with PBS, the sections were incubated for 1 h at room temperature with FITC-conjugated goat anti-rabbit IgG (1:150 dilution; Sigma Chemical) and visualized as described above. Colocalization was defined as cells with TRITC and FITC labeling.

	RESULTS

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Validation of the antigenicity and specificity of the AT₂-receptor antibody. Of the six rabbits immunized, serum from one passed all the initial screens for antigenicity and specificity, including specific reactivity to the synthesized peptide, recognition of a single protein band in electrophoresed microsomal fractions obtained from PC-12 cells, and no detectable immunoreactivity against late-passage BAEC protein extract. This potential AT₂-receptor antiserum was further tested as shown in Fig. 1. The dot blot in Fig. 1A demonstrates the sensitivity of the AT₂-receptor antiserum against the synthesized AT₂-receptor peptide fragment used to generate the antiserum. A range of peptide conjugate concentrations (0.6, 0.3, 0.15, and 0.075 mg/dot) was tested with three different dilutions of antiserum (1:1,000, 1:2,000, and 1:10,000) showing the dose-dependent reactivity of peptide to antiserum. Figure 1B displays the ability of the specific peptide conjugate to compete in a dose-dependent fashion with the antiserum for the binding of the native AT₂ receptor in rat adrenal extract. In immunohistochemical sections the peptide fully competed off AT₂-receptor binding in the adrenal, cremaster, and soleus tissues (data not shown). Similarly, Fig. 1C demonstrates a linear increase of densitometrically quantified band intensity that is directly proportional to the quantity of adrenal protein used in the immunoblot. Finally, no cross-reactivity between the AT₁-receptor antiserum (which does not discriminate between the AT_1A and AT_1B subtypes) and the AT₂-receptor antiserum is visible in Fig. 1D. Figure 1D also shows that there is no reactivity in the preimmune serum with the AT₂ receptor.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Specificity of AT2-receptor antibody. A: 6-µm frozen section of adrenal gland from a Sprague-Dawley rat with fluorescently labeled AT2 receptors. Four fields of view show AT2-receptor-positive sites extending from capsule to inner medullary cells (as depicted in line drawing at top). B: Western blot revealing AT2-receptor-negative cell lines, bovine aortic endothelial cells (BAEC) and bovine pulmonary artery endothelial cells (BPAEC), and AT2-receptor-positive adrenal gland. Molecular mass markers (79 and 42) in kDa.

Use of RT-PCR to confirm AT₂-receptor-negative cell lines and to demonstrate the presence of AT₂-receptor mRNA in isolated microvessels. The negative results of our AT₂-receptor antibody for the BAEC and BPAEC lines as determined by Western blot analysis (Fig. 2B) were verified by RT-PCR. Figure 3A confirms that no mRNA for the AT₂ receptor was detectable with PCR amplification of RNA isolated from BAECs and BPAECs using AT₂-specific primers (lanes 9 and 13, respectively). Detection of -actin in lanes 10 and 14 for the BAECs and BPAECs, respectively, suggests that the negative result obtained for the AT₂ receptor was not due to degradation of RNA. Additionally, the water controls for the AT₂-receptor-specific and -actin-specific primers (lanes 1 and 2, respectively) revealed no contamination of the primers themselves or the diethyl pyrocarbonate-treated water or other reagents used in the RT and PCR reactions.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Two independent techniques for confirming presence of AT2 receptor in isolated cremasteric vessels. A: RT-PCR of adrenal gland (Adr), hand-dissected cremasteric microvessels (Vessels), BAECs, and BPAECs. Lane 1, water control for AT2-receptor primers; lane 2, water control for -actin primers; lane 3, AT2-receptor mRNA detected in adrenal gland; lane 4, -actin mRNA detected in adrenal gland; lane 5, AT2-receptor mRNA detected in cremasteric microvessels; lane 6, -actin mRNA detected in cremasteric microvessels; lane 7, PCR of DNase-treated RNA (no RT) from cremasteric microvessels using AT2-receptor-specific primers; lane 8, PCR of DNase-treated RNA (no RT) from cremasteric microvessels using -actin-specific primers; lane 9, no detection of AT2-receptor mRNA in BAECs; lane 10, -actin mRNA detected in BAECs; lane 11, PCR of DNase-treated RNA (no RT) from BAECs using AT2-receptor-specific primers; lane 12, PCR of DNase-treated RNA (no RT) from BAECs using -actin-specific primers; lane 13, no detection of AT2-receptor mRNA in BPAECs; lane 14, -actin mRNA detected in BPAECs; lane 15, PCR of DNase-treated RNA (no RT) from BPAECs using AT2-receptor-specific primers; lane 16, PCR of DNase-treated RNA (no RT) from BPAECs using -actin-specific primers. M, 100-bp markers; b, blank. B: Western blot showing expression of AT2-receptor protein in cremaster muscle (C), kidney (K), soleus muscle (S), cremasteric microvessels (V), and PC-12 cells (P). M, molecular mass standards in kDa.

Detection of AT₂-receptor protein in skeletal muscle and isolated microvessels. The ability of our antiserum to detect AT₂-receptor protein in soleus and cremaster muscles by immunoblotting is demonstrated in Fig. 3B. The Western blot shows a single, strong band for the AT₂-receptor protein at approximately the correct molecular mass for a 363-amino acid protein (16). It is important to note that the single band eliminates potential complications of nonspecific binding when the antiserum is used in immunohistochemistry. AT₂-receptor protein was also detected in manually dissected microvessels, thus confirming the vascular localization of the receptor and corroborating our result from RT-PCR (Fig. 3A). The PC-12 cells used as a positive control and the microvessels exhibited a second band that was not seen in the other tissues. This band is the correct size for a dimerization product of the AT₂ receptor. Alternatively, the second band could be due to a varied amount of N-glycosylation that occurs in AT₂ receptors of different tissues, as reported by Servant et al. (25). Although no function has been attributed to the different glycosylation products of the AT₂ receptor, the amount of glycosylation appears to be very tissue dependent (25) and could represent differences in receptor stability, regulation, membrane targeting, intracellular trafficking, or a tissue-specific receptor function. Finally, no immunoreactivity was detected using preimmune serum as a negative control (Fig. 1D).

Distribution of AT₂ receptor in skeletal muscle microcirculation. Immunohistochemical staining was used for the specific, cellular localization of AT₂-receptor protein. Figure 4 shows a section of the cremaster muscle displaying a lectin-positive arteriole in cross section. AT₂-receptor-positive cells colocalize with the vessel. AT₂-receptor-positive cells appeared to be found in the endothelium (arrows) and abluminally in the vascular wall (arrowheads). Figure 5A shows a cross section of the soleus muscle revealing that the capillaries were also AT₂-receptor positive. Although most capillaries have an AT₂-receptor-positive cell associated with them, not all the AT₂-receptor-positive cells are endothelial cells. Figure 5B is a ×4 zoom of the boxed area in Fig. 5A. The arrowhead points to an AT₂-receptor-positive abluminal endothelial cell. Juxtaposed to the capillary is an AT₂-receptor-positive cell associated with the capillary (arrow). To approximate the number of AT₂-receptor-positive endothelial cells among the total AT₂-receptor-positive sites in a capillary-dense region, 10 fields of view from soleus muscle in cross section were randomly selected. Each field of view was magnified with a ×100 objective and contained no vessels larger than capillaries. The total number of AT₂-receptor-positive sites was counted and divided into AT₂-receptor-positive capillary endothelial or nonendothelial cells. The percentage of endothelial or nonendothelial AT₂-receptor-positive cells was determined from the total AT₂-receptor binding in the capillary-dense region. A capillary endothelial cell was defined as any AT₂-receptor-positive cell that was exactly colocalized with the lectin-stained capillary. A capillary nonendothelial cell was considered to be any AT₂-receptor-positive cell that was adjacent to, but not directly on top of, the lectin-stained capillary. Thirty-three percent of the total AT₂-receptor-positive sites in the capillary-only regions were determined to be endothelial cells, and 67% were not endothelial cells.

View larger version (141K): [in this window] [in a new window]   Fig. 4.   Section (6 µm) of cremaster muscle (×400) displaying ~35-µm-diameter vessel in cross section. Red fluorescence, rhodamine-labeled Griffonia simplicifolia I lectin staining basement membrane of vessel wall; green fluorescence, AT2-receptor-positive sites labeled with FITC-conjugated goat anti-rabbit IgG. Arrows point to AT2-receptor-positive endothelial cells; arrowheads point to AT2-receptor-positive sites in smooth muscle of vascular wall.

View larger version (106K): [in this window] [in a new window]   Fig. 5.   A: cross section of soleus muscle using a ×100 objective. AT2-receptor-positive cells fluorescently labeled with FITC-conjugated goat anti-rabbit IgG. B: ×4 zoom of boxed area in A displaying AT2-receptor-positive capillary endothelial cell (arrowhead) and an AT2-receptor-positive capillary, nonendothelial cell (arrow).

Colocalization of the AT₂ receptor to vascular smooth muscle cells. A monoclonal antibody to smooth muscle -actin was used to identify vascular smooth muscle cells. Figure 6 shows a section of the cremaster muscle with three larger microvessels in cross section. Dual-labeled cells demonstrate AT₂ receptor colocalized to vascular smooth muscle cells. Non--actin-positive, AT₂-receptor-positive cells were also seen in the vessel lumen and in the vascular wall. The luminal cells are thought to be endothelial cells, whereas the "other" cells of the vascular wall could represent several different cell types. Interestingly, there were also -actin-positive, non-AT₂-receptor-positive cells, indicating a heterogeneity of protein expression on vascular smooth muscle cells. Figure 7 quantitates the percentage of the total AT₂-receptor-positive cells that are endothelial cells, vascular smooth muscle cells, or other cells. Unexpectedly, ~40% of the total AT₂-receptor-positive cells found in noncapillary microvessels were other cells.

View larger version (178K): [in this window] [in a new window]   Fig. 6.   Cryostat section of cremaster muscle (×400) displaying 3 large microvessels in cross section. Red fluorescence, smooth muscle -actin staining for vascular smooth muscle cells; green fluorescence, AT2-receptor-positive cells. Arrowheads point to AT2-receptor-positive endothelial cells; yellow represents AT2-receptor-positive cells colocalized with -actin; arrows mark nonendothelial, nonvascular smooth muscle cells positive for AT2 receptors in vascular wall.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Ten randomly chosen fields of dual-labeled (stained for -actin and AT2 receptors) skeletal muscle arterioles viewed using a ×40 objective. All AT2-receptor-positive luminal cells were considered to be endothelial cells. Dual-labeled cells were considered to be vascular smooth muscle cells, and nonendothelial, non-smooth-muscle, AT2-receptor-positive vascular wall cells were classified as "other." Data are expressed as percentage of total AT2-receptor-positive cells. Error bars, SD calculated from mean of 10 fields.

	DISCUSSION

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This study provides the first direct evidence for the cellular localization of the AT₂ receptor in the microcirculation and is the first to use immunoblotting to detect AT₂-receptor protein in the microcirculation. We developed a polyclonal AT₂-receptor-specific antibody to use for the immunohistochemical and Western blot studies. Sensitivity and specificity of the antibody were carefully validated. The adrenal gland was used as a positive control (Fig. 2A), whereas BPAEC and BAEC lines were used as negative controls (Fig. 2B). RT-PCR was also used as a non-antibody-based technique to confirm that these two cell lines were negative for AT₂-receptor mRNA (Fig. 3A). Briefly, the AT₂ receptor was widely distributed throughout the microcirculation and was seen in large and small vessels and capillaries. It was localized to endothelial and vascular smooth muscle cells, and it was also seen in other vessel-associated cells.

The presence of AT₂ receptors in the microcirculation has important physiological implications. Until now, only functional evidence (14, 19, 24) consistently indicated the presence of AT₂-receptor protein in the vasculature. There are differing reports on the expression of AT₂ receptors in primary cultures of endothelial and smooth muscle cells (23, 26, 28). The AT₁ receptor has been localized to a variety of tissues, including adrenal gland, kidney, aorta, and vascular smooth muscle cells (22). This receptor is known to be an important modulator of peripheral resistance through its vasoconstrictor actions on vascular smooth muscle cells (7, 19, 29). Recent evidence suggests that the AT₂ receptor may oppose the vasoconstrictor activity of the AT₁ receptor (10, 14, 19, 24). The present study provides histological corroboration of the AT₂-receptor distribution for studies demonstrating functional colocalization of the two ANG II receptors on the basis of their opposing actions on blood pressure and reveals the anatomic distribution of the AT₂ receptor in the skeletal muscle microcirculation.

The vasodilator properties of the AT₂ receptor remain somewhat controversial. Munzenmaier and Greene (19) demonstrated AT₂-receptor-dependent vasodilation in vivo, such that when the AT₂-receptor-specific antagonist was chronically infused with ANG II, the mean arterial pressure was significantly elevated compared with ANG II infusion alone. Furthermore, Ichiki et al. (14) and Hein et al. (10) disrupted the embryonic AT₂-receptor gene located on the X chromosome, creating two separate strains of AT₂-receptor "knock-out" mice. In the study by Ichiki et al., the mice displayed significantly elevated blood pressure, suggesting that the AT₂ receptor is important in the day-to-day maintenance of vascular tone. However, the results from Hein et al. showed no change in blood pressure. Nevertheless, both groups demonstrated an increased sensitivity to ANG II infusion, suggesting a potentiation of the constrictor AT₁-receptor actions or a loss of AT₂-receptor dilator activity. Finally, in a study in which the AT₂-receptor-specific antagonist PD-123319 was chronically administered with ANG II, Levy et al. (17) saw no increase in blood pressure above subcutaneous ANG II infusion alone. This result disagrees with the findings of Ichiki et al. and Munzenmaier and Greene, although the differences could have been due to differences in the protocol, such as the method of blood pressure measurement, the manner in which blood pressure was reported, the method of drug administration, and the length of the study. Nevertheless, taken together, these functional studies support the hypothesis that both receptor types are expressed in the vasculature and may mediate opposite effects.

Additional functional significance of finding AT₂ receptors in the microcirculation resides in the suggested antiproliferative actions of the AT₂ receptor, which would oppose the proliferative effects of the AT₁ receptor. Munzenmaier and Greene (19) showed that the AT₂ receptor counterbalances the angiogenic effects of the AT₁ receptor in vivo. In that study a significantly greater increase in MVD was observed when ANG II and PD-123319 were coinfused (AT₁-receptor stimulation only) than when a subpressor dose of ANG II was infused alone (AT₁- and AT₂-receptor stimulation). A potential mechanism to explain the AT₂-receptor-induced loss of blood vessels is suggested in a study by Yamada et al. (33), which demonstrated that the AT₂ receptor could mediate apoptosis. In another study, Nakajima et al. (20) demonstrated that the overexpression of the AT₂ receptor antagonized the growth effect of the AT₁ receptor in rat carotid artery after balloon injury and in cultured vascular smooth muscle cells. This group also showed that the AT₂ receptor slows DNA synthesis in the fetal aorta as measured by bromodeoxyuridine uptake. Furthermore, Stoll et al. (26) used thymidine incorporation to demonstrate that the AT₁ receptor increased DNA synthesis in coronary endothelial cells and that the AT₂ receptor suppressed DNA synthesis. However, a study by Levy et al. (17) reports that the cross-sectional area, media thickness, collagen content, and elastin content in the thoracic aorta increase with ANG II and losartan coadministration compared with control levels. This result suggests that the AT₂ receptor is mediating the well-characterized hypertrophy that occurs in elevated ANG II conditions and disagrees with the finding from Nakajimi et al. revealing that the function of the AT₂ receptor is still disputed. Altogether, the bulk of the evidence in the literature suggests that the AT₁ and AT₂ receptors have opposing actions on vascular tone, cell proliferation, and vessel density. The present study histologically localizes the AT₂ receptors to the same cellular locations in the vasculature as the AT₁ receptor.

Colocalization with smooth muscle -actin (Fig. 6) demonstrated that AT₂ receptors are found on vascular smooth muscle cells. This location places them side by side with the AT₁ receptors, which may have implications for the net action of ANG II. This finding has two additional implications. First, not all the -actin-positive cells are also AT₂-receptor positive, suggesting a heterogeneity of protein expression on the smooth muscle cells. Heterogeneity of smooth muscle cells in uninjured, large vessels, such as the aorta, has been established (1, 30), but neither the distribution of the AT₂ receptor nor the heterogeneity of protein expression in the microcirculation is well documented. Furthermore, the occurrence of distinct classes of smooth muscle cells in large vessels has important consequences for atherosclerotic plaque formation and restenosis (1, 30). Second, our observations indicate that the distribution of AT₂-receptor-positive vascular smooth muscle cells varies with vessel order. Although the aim of this study was not to quantify these differences, this finding is intriguing and worthy of further investigation. Thus the present study is the first evidence for heterogeneity of smooth muscle cells in small vessels. Additional studies are necessary to determine whether a heterogeneous distribution of smooth muscle cells plays a role in the pathological changes in the microcirculation, such as rarefaction, or whether this heterogeneity creates functional differences between vessels of different orders.

Another important finding in this study was that 35% of the total AT₂-receptor-positive cells in noncapillary microvessels were colocalized with -actin, whereas 27% of the total AT₂-receptor-positive cells in these vessels were endothelial cells as determined by morphology and studies (Fig. 7). Endothelial cell localization of the AT₂ receptor is consistent with the receptor's vasodilator properties (14, 19, 24) and its hypothesized transduction through the release of endothelium-derived relaxing factor (8). Additional studies with a specific marker for individual endothelial cells are necessary to determine whether there is also a heterogeneous expression of AT₂-receptor protein on endothelial cells, as there is on vascular smooth muscle cells. Also, ~40% of the AT₂-receptor-positive cells in noncapillary microvessels were nonendothelial, non-smooth-muscle cells and are fibroblasts, mast cells, macrophages, or pericytes.

Furthermore 67% of the total AT₂-receptor-positive cells associated with the capillaries were not endothelial cells. Additional colocalization studies with cell-type-specific markers are necessary to identify these AT₂-receptor-positive cell types. Possibilities include mast cells, macrophages, pericytes, and mesenchymal cells. Because of the close association of pericytes with all capillaries (12), we hypothesized that the nonendothelial, AT₂-receptor-positive capillary cells may be pericytes. Interestingly, loss of pericytes has been implicated in proliferative vascular disorders (12, 13), suggesting that pericytes have an inhibitory effect on vessel growth. This would be consistent with the evidence for antiproliferative (20, 26) and antiangiogenic (19) effects attributed to the AT₂ receptor.

In conclusion, the AT₂ receptor was detected throughout the skeletal muscle microcirculation. It is present in endothelial cells, vascular smooth muscle cells, and other vessel-associated cells. In all cases, the AT₁ and AT₂ receptors are situated in the microcirculation, such that they could directly oppose the actions of one another.