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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania
Received May 17, 2006; accepted September 15, 2006.
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Abstract |
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). For several years, most of the effects of Ang II were attributed to the activation of the AT1-R subtype. Recently, however, an increasing number of reports also suggested the involvement of AT2-R in the actions of Ang II (Siragy et al., 2000; Wu et al., 2001; de Godoy and De Oliveira, 2002; Rattan et al., 2002; de Godoy et al., 2004b).
Internal anal sphincter (IAS) studies in rats suggested that locally generated Ang II contributes in part to the basal tone in the IAS (de Godoy et al., 2004b). A multipronged approach of functional, biochemical, and molecular biology showed the expression of angiotensinogen, renin, and angiotensin-converting enzyme in the IAS at the gene and protein levels (de Godoy and Rattan, 2005). More recently, we showed the presence of the highest levels of renin-angiotensin system components, Ang II and AT1-R, to be in the tonic IAS versus the adjoining phasic smooth muscles of the rectum and anococcygeus (de Godoy and Rattan, 2005).
Although Ang II contracts the IAS via activation of AT1-Rs, there is a physiological brake for this effect. In the rat IAS Ang II produces bimodal effect, a contraction (at lower concentrations) followed by relaxation (at higher concentrations). Experiments with selective antagonists (losartan for AT1-R and PD123319 for AT2-R) further show that contractions are mediated by AT1-R, and relaxation is mediated by AT2-R (de Godoy et al., 2004b). The bimodal effect of Ang II may be explained on the basis of either differences in the receptor affinity for AT1-R versus AT2-R or in the receptor trafficking between the cytosol and the plasma membrane (PM). The first possibility is less likely because of the similar affinity for both subtypes of receptors (De Gasparo et al., 2000). Present studies focus on the hypothesis that once externalized to the membrane, AT2-Rs exert their inhibitory effect on the IAS smooth muscle cells (SMCs).
Using different systems, it was shown previously that short exposures to high concentrations of Ang II lead to internalization of AT1-R (Hein et al., 1997; Olivares-Reyes et al., 2001). We hypothesize here that the internalization of AT1-R with similar exposures of Ang II may release AT2-R from the state of inactivity, causing relaxation of the smooth muscle. To test this hypothesis, we used laser confocal microscopy and biotinylation to determine the location of AT1- and AT2-R in the SMCs isolated from rat IAS. We also used Western blot analysis to investigate the relative distribution of AT1- and AT2-R in the particulate versus cytosolic fractions of the SMCs. Our data show that high concentrations of Ang II cause AT1-R internalization and externalization of AT2-R to the PM. The opposite trafficking of AT1- and AT2-R may be partly responsible for the relaxation in response to the higher concentrations of Ang II.
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Materials and Methods |
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,b). The circular IAS smooth muscle strips (
0.5 x 7 mm) were prepared in oxygenated Krebs' physiological solution (KPS). The composition of KPS was as follows: 118.07 mM NaCl, 4.69 mM KCl, 2.52 mM CaCl2, 1.16 mM MgSO4, 1.01 mM NaH2PO4, 25 mM NaHCO3, and 11.10 mM glucose. The experimental protocol of the study was approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University and was in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.
SMCs from IAS were isolated as described previously (Rattan and Chakder, 1992; Cao et al., 2002; Rattan et al., 2002; Huang et al., 2005). In brief, IAS was cut into small pieces (1-mm cubes) and incubated in oxygenated KPS containing 0.1% collagenase and 0.01% soybean trypsin inhibitor at 37°C for two successive 1-h periods. The mixture was then filtered through a 500-µm Nitex mesh. The tissue trapped on the mesh was rinsed with 25 ml (5 x 5 ml) of collagenase-free KPS. The tissue was incubated in collagenase-free KPS at 37°C, and dispersion of the cells (0–1 h) was monitored periodically by examining a 10-µl aliquot of the mixture microscopically. The SMCs were then harvested by filtration through the Nitex mesh. The filtrate containing the cells was centrifuged at 350g for 10 min at room temperature (RT). The cells in the pellet were resuspended in oxygenated KPS (at 37°C) at a cell density of 3 x 104 cells/ml.
Individual cell lengths were measured by micrometry using phase-contrast microscopy on a custom-assembled microscope (Olympus, Tokyo, Japan), close-circuit videocamera (model Pulnix MC-7; PULNIX America, Inc., Sunnyvale, CA), and PC computer. Digital images of the cells were stored, and the cell lengths were measured by the Image-Pro Plus version 4.0 program (Media Cybernetics, Inc., Silver Spring, MD).
Following exposure with Ang II (0.1 nM–10 µM) for 10 min, the SMCs were fixed with acrolein (final concentration 1%) and transferred onto chrome-alum-coated glass slides (Fisher Scientific, Pittsburgh, PA). The studies were repeated in the presence of selective antagonists (losartan for AT1-R and PD123319 for AT2-R, both at 100 nM). The shortening of SMCs in each category of experiments was calculated on the bases of the original cell lengths. The studies were repeated in the SMCs isolated from at least three animals.
Receptor Internalization Assay. To determine the time-course effect of high concentrations of Ang II on the cellular localization of AT1- and AT2-R in the IAS SMCs, freshly isolated cells from the IAS were resuspended in Dulbecco's modified Eagle's medium containing 10 µM amastatin. Cells were aliquoted in groups and exposed to 1 nM to 10 µM Ang II for 0- to 30-min intervals in 5% CO2 humidified atmosphere at 37°C. Another group of cells was exposed to 100 µM bethanechol for 30 min. The cells were then biotinylated on ice in a rocking platform with 0.5 mg/ml Sulfo-NHS-SS-biotin (Pierce Chemical, Rockford, IL) for 30 min as described previously (Huang et al., 1999). The cells were then centrifuged at 1000g and lysed immediately for protein extraction. Biotinylated proteins were affinity-purified from cell lysate with streptavidin-agarose (Invitrogen, Carlsbad, CA) and loaded onto an SDS-10% polyacrylamide gel. Western blot was then performed as described below.
Immunofluorescence and Confocal Microscopy. SMCs were isolated as described above and incubated in a culture medium containing 10 µM amastatin (to inhibit Ang II degradation) for 10 min in the absence or presence of Ang II (100 nM–10 µM). Experiments were repeated in the presence of losartan or 100 nM PD123319 previously incubated for 20 min. Immunocytochemistry of the SMCs was performed by indirect immunofluorescence as described previously (Battish et al., 2000). SMCs were fixed with icecold fixative (4% paraformaldehyde and 0.2% picric acid in PBS, pH 7.4) for 10 min, and then they were thoroughly washed in PBS. The cells were then rinsed with PBS and incubated in a mixture of 1:200 primary antibody (AT1 raised in rabbit and AT2 raised in goat) and diluted in PBS containing 0.5% bovine serum albumin, and 0.2% Triton X-100 overnight at RT in a humid chamber. Cells were then rinsed with PBS and incubated in a mixture of Texas Red- and FITC-labeled secondary antibodies (1:200 in a solution of 2% normal donkey serum and 0.3% Triton X-100 in PBS) raised in donkey against rabbit and goat immunoglobulins, respectively.
The slides containing the cells were incubated for 60 min at RT, rinsed with PBS, air-dried, and coverslipped with Vectashield (Vector Laboratories, Burlingame, CA). Cells were examined using a confocal laser scanning microscope system interfaced to an Axiovert 200 M inverted microscope; Carl Zeiss Microimaging, Inc., Thornwood, NY) at the Kimel Cancer Center's Bioimaging Facility of our institution. The plot profile of the pixel intensities (gray scale, 0–250) was analyzed as described previously (Chiba et al., 2004). In brief, using Image-Pro Plus version 4.0, the pixel intensities over the outer 15% of each cell width were taken as an index of peripheral AT-R, whereas those over the remaining central 70% of the cell width were estimated as cytosolic AT-R. The average peripheral-to-cytosolic ratio of two lines scans in each cell was calculated by dividing the gray value of the highest peak at 15% by the highest peak at 70%. Six randomly selected cells were analyzed for each experimental condition.
Western Blot Analysis. SMCs were treated with Ang II (1 nM–10 µM) for 10 min as described above. Western blot analyses were performed to determine the relative distribution of AT1- and AT2-R following the approach described previously (Rattan et al., 2002). In brief, the SMCs were homogenized in lysis buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol, and 1 mM sodium orthovanadate) and centrifuged for 10 min at 100,000g at 4°C (Optima TLX ultracentrifuge; Beckman Coulter, Fullerton, CA), and the supernatant was collected as the cytosolic fraction. The precipitate (the particulate or membrane fraction) was dissolved in 1% SDS-containing lysis buffer, and the respective protein contents were determined by the method of Lowry et al. (1951). The proteins were then separated by gel electrophoresis followed by their transfer to the nitrocellulose membrane (NCM) by electrophoresis at 4°C.
The NCM was then incubated with the specific primary antibodies (rabbit IgG; 1:500) for 2 h at RT. After washing with Tris-buffered saline/Tween 20, the NCMs were incubated with horseradish peroxidase labeled-secondary antibody (1:10,000) for 1 h at RT. The corresponding bands were visualized with enhanced chemiluminescence substrate using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical) and Hyperfilm MP (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).
NCMs were then stripped of antibodies using Restore Western Blot Stripping Buffer (Pierce Chemical) for 15 min at RT. NCMs were reprobed for 
-actin using the specific primary (mouse IgG, 1:10,000 for -actin) and secondary (1:10,000) antibodies. Bands of interest were scanned (SnapSacn.310; Agfa, Ridgefield Park, NJ), and the respective areas and integrated optical density (IOD) were determined using Image-Pro Plus 4.0. The relative densities were calculated by normalizing the IOD of each blot with that of -actin.
Reverse Transcription-Polymerase Chain Reaction. IAS SMCs were exposed to 10 µM Ang II for 30 min. Total RNA was isolated and purified by the acid guanidine-phenol-chloroform method (Chomczynski and Sacchi, 1987) and quantified by measurement of absorbance at 260 nm in a spectrophotometer. Total RNA (2 µg) was subjected to first-strand cDNA synthesis using oligo(dT) primers (Promega, Madison, WI) and Omniscript RT kit (QIAGEN, Germantown, MD) in a final volume of 20 µl at 42°C for 60 min. PCR primers specific for AT1-R, AT2-R, and 
-actin cDNA were designed as shown in Table 1. PCR was performed in a Promega 2x Master Mix (M750B; Promega) in a final volume of 25 µl, using a PerkinElmer thermal cycler (PerkinElmer Life and Analytical Sciences, Boston, MA). The PCR conditions consisted of 94°C for 5 min (for the initial denaturation phase) followed by 35 cycles of 94°C for 30 s (denaturation), 57°C for 30 s (annealing), and 72°C for 1 min (extension), with a final extension at 72°C for 7 min. The PCR products were separated on 1.5% (w/v) agarose gel containing ethidium bromide and were visualized with UV light. The relative densities were calculated by normalizing the IOD of each blot with that of -actin.
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Data Analysis. Results were expressed as means ± S.E.M. Concentration-response curves were analyzed using a nonlinear interactive fitting program (GraphPad Prism 3.0; Graph Pad Software Inc., San Diego, CA). Agonist potencies and maximal responses were expressed as negative logarithm of the molar concentration of agonist producing 50% of the maximal response (pD2) and maximal effect elicited by the agonist (Emax), respectively, calculated from the concentration-response curves. Biotinylation data for AT1- and AT2-R were expressed on the basis of their respective percentages of maximal IOD at the cell surface. Statistical significance was tested by the one-way analysis of variance (ANOVA) followed by the Dunnett's post hoc test when three or more different groups were compared. The unpaired Student's t test was used to compare only two different groups. A p value less than 0.05 was considered to be statistically significant.
Drugs and Antibodies. Ang II, amastatin, and PD123319 were from Sigma-Aldrich (St. Louis, MO). Losartan was a gift from Merck (Rahway, NJ). All antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All PCR primers were from MWG Biotech (High Point, NC).
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Results |
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Effect of Ang II (10 µM) on AT1-R Internalization Using Biotinylation. For the quantitation of the cell surface population of Ang II receptors, we performed biotinylation assays following a time course (0–30 min) after pretreatment of the cells with 10 µM Ang II. The IAS SMCs were isolated, biotinylated with Sulfo-NHS-SS-biotin, and lysed for protein extraction. Biotinylated proteins were purified by the streptavidin-agarose method, and AT1-R and AT2-R (Fig. 2A) were monitored by Western blot analysis followed by densitometry analysis. Ang II (10 µM) produced a significant decrease in AT1-R (suggesting internalization) or increase in AT2-R (suggesting externalization) at the IAS SMC surface, in a time-dependent manner that plateaued at 10 min (*, p < 0.05; Fig. 2B). Bethanechol (100 µM) produced no significant change in the density of Ang II receptors on the cell surface (data not shown).
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By contrast with the AT1-R, most of the AT2-R-IR were found to be distributed evenly in the cytosol of the IAS SMCs (Fig. 4Aa). Higher concentrations of Ang II increased the AT2-R-IR in the PM while almost eliminating them from the cytosol (Fig. 4A, b and c). Losartan produced no significant effect in the basal state (Fig. 4Ad) but inhibited AT2-R migration to PM (Fig. 4A, e and f). PD123319 produced no significant effect under any of the conditions tested (Fig. 4A, g–i). The semiquantitation analysis by plot profile revealed trafficking of AT2-Rs from cytosol to PM by Ang II (Fig. 4B).
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Relative Distribution of AT2-R in the Particulate versus Cytosolic Fractions of the IAS SMC Extracts. The typical bands for AT2-R expression were at 50 to 70 kDa. The relative density of AT2-R was higher in the cytosolic compared with the particulate fraction in the basal state. Higher concentration of Ang II caused a significant decrease in the AT2-R density in the cytosolic fraction (Fig. 5B) without a significant change in the density of 50 to 70 kDa AT2-R in the particulate fraction. However, there was a significant increase in the density of 105 to 120 kDa AT2-R. The latter represents a homodimer of AT2-R (two units physically bound) as has already been shown in previous studies in different systems (Lazard et al., 1994).
Effects of Ang II on AT2 and AT1 Expression. We evaluated the effects of Ang II in the overall transcriptional and translational expression of AT1-R and AT2-R in the total cell extracts using RT-PCR and Western blot analyses after exposure to 10 µM Ang II for 30 min. Western blot analysis revealed no significant change in the expression of either AT1-R or AT2-R following the pretreatment with Ang II (p > 0.05; n = 3; Fig. 6, A and B).
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These results were confirmed by the transcriptional expression via RT-PCR (p > 0.05; n = 3; Fig. 7, A and B). Longer incubation times (up to 180 min) did not produce any significant change in the transcriptional and translational expression of AT1-Rs and AT2-Rs (data not shown).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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,b; Fukada et al., 2005) has been known for some time. These studies provide a mechanism for that bimodal effect of Ang II in the IAS smooth muscle using functional and molecular approaches in the isolated SMC.
Functional studies in the IAS SMC show that concentrations of Ang II lower than 100 nM induce contraction, whereas higher concentrations produce relaxation (Fig. 1). These results in the isolated SMC are conceptually similar to those obtained in the intact IAS smooth muscle strips (de Godoy et al., 2004b), except for quantitative differences in the amplitude of the responses. This may be because of differences in the responses of the IAS SMCs when they are in situ conditions of basal tone (for the smooth muscle strips experiments) compared with when they are examined under isolated conditions (as is the case in the present studies). In support of that concept, chemomechanical studies in the arterial SMCs (Yang et al., 2003) have shown that the absence of tension imbalances the electrolyte behavior, protein phosphorylation, and protein-protein interactions in the SMCs.
Ang II has a similar affinity for both AT1-R and AT2-R (De Gasparo et al., 2000). Experiments with the selective antagonists (losartan for AT1-R and PD123319 for AT2-R) show that activation of AT1-R induces contractions and activation of AT2-R produces relaxation of the IAS SMCs. Lack of overlapping effects of the antagonists suggests that the AT1-R are activated in the lower concentrations and that AT2-R, in contrast, are activated at the higher ranges of Ang II concentrations.
Quantitative determination of AT1- and AT2-R in the PM via biotinylation studies after different exposure times with 10 µM Ang II (Emax concentration) show migration of AT1-Rs from the PM, whereas that of AT2-Rs to the PM plateaus at 10 min (Fig. 2). Confocal microscopy studies confirm these findings (Figs. 3 and 4). Losartan, but not PD123319 (Figs. 3B and 4B), inhibits this response, which suggests that AT1-R activation causes the trafficking of AT1- and AT2-R in the opposite direction. Losartan by itself produces a significant increase in the AT1-R density on the PM of IAS SMCs, suggesting that inactivation of AT1-R prevents constitutive internalization of the receptor.
As discussed above, a major mechanism for the switch from AT1- to AT2-R activation is the activation of AT1-R. In addition, reports show that Ang II receptor trafficking and desensitization depend on -arrestin recruitment (Turu et al., 2006) and on G protein-coupled receptor kinases activities such as the G protein-coupled kinase-5 and the -adrenoceptor kinase 1 (Rockman et al., 1996; Kim et al., 2005). The specific role of G protein-coupling in the switch from AT1- to AT2-R in the IAS smooth muscle remains to be identified. However, this switch seems to be specific to AT1-R activation because bethanechol (a muscarinic agonist that also produces smooth muscle contraction via the activation of Gq-coupled signaling) does not modify the cellular distribution of AT1- and AT2-R in the IAS SMCs.
Western blot experiments confirm Ang II concentration dependence for the trafficking of Ang II receptor subtypes. The higher concentrations of Ang II cause the movement of AT1-R from the PM to the cytosol (Fig. 5A), whereas reverse is the case for AT2-R. Higher concentrations of Ang II decrease the density of AT2-R in the cytosolic fraction (Fig. 5B) and promote its migration toward the periphery of the SMCs. Our studies demonstrate specific increase in the relative density of 105- to 120-kDa bands for AT2-R in the particulate fraction of the SMC with high concentrations of Ang II. In agreement with this concept, earlier studies in the human myometrium SMC have reported a similar increase in AT2-R homodimers in the PM (Lazard et al., 1994). RT-PCR analyses further show that relaxation of the SMC with higher concentrations of Ang II is associated with the cross-translocation of AT1-R and AT2-R rather than an overall change in their population density (Fig. 7). In addition, our preliminary studies suggest that short exposures (10 min) to higher concentrations of Ang II are appropriate for the changes in AT1- and AT2-R trafficking, since there was no significant difference in this trafficking pattern when the cells are exposed to Ang II for longer periods. These observations are in agreement with the earlier data in other systems (Ullian and Linas, 1990; Hein et al., 1997).
In summary, present studies provide significant insight into the mechanism for the bimodal effect of Ang II in the smooth muscle. The contractile effect of Ang II occurs by the activation of AT1-R (situated mostly in the PM) and the relaxation component via AT2-R (normally situated in the cytosol). Higher concentrations of Ang II cause internalization of the AT1-R to the cytosol and externalization of AT2-R to the PM. This process exposes AT2-R for the activation. However, the molecular events leading up to the above-mentioned pattern of movement of Ang II receptors remain to be determined.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: Ang II, angiotensin II; AT1-R, angiotensin receptors subtype I; AT2-R, angiotensin receptor subtype II; IAS, internal anal sphincter; PD123319, S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid; PM, plasma membrane; SMC, smooth muscle cell; KPS, Krebs' physiological solution; RT, room temperature; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; NCM, nitrocellulose membrane; IOD, integrated optical density; NHS, N-hydroxysuccinimide; RT-PCR, reverse transcriptasepolymerase chain reaction; PCR, polymerase chain reaction; ANOVA, analysis of variance; IR, immunoreactivity.
Address correspondence to: Dr. Satish Rattan, Jefferson Medical College, Thomas Jefferson University, 1025 Walnut St., Room 901 College, Philadelphia, PA 19107. E-mail: satish.rattan{at}jefferson.edu
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