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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 for publication
February 22, 2006
Accepted
April 26, 2006.
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Abstract |
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). The studies show the expression of key components of the renin-angiotensin system (RAS): angiotensinogen (Angen), the Ang I-generating enzyme renin, and the Ang I-converting enzyme (ACE). Additional studies suggest an autocrine mechanism for Ang II generation in the regulation of the IAS basal tone (De Godoy et al., 2004; De Godoy and Rattan, 2005). These findings are in line with a broad range of reports in different tissues (Dzau et al., 1987, 1988; Eggena et al., 1990; Griendling et al., 1993; Katwa et al., 1996; Bataller et al., 2003). These data expand the role of the RAS from endocrine to local regulation.
The role of Ang II in different organ systems can be determined by the classic ACE inhibitors or by the Ang II type 1 receptor (AT1-R) antagonists, captopril and losartan, respectively. Captopril was first described in 1978 as a chronic antihypertensive agent in hypertensive rats by inhibiting the conversion of Ang I into Ang II by ACE (Rubin et al., 1978). Losartan was introduced later as an alternative to captopril for the treatment of hypertension (Celik et al., 1995).
In previous in vitro studies we showed that inhibition of Ang II generation decreases IAS basal tone (De Godoy et al., 2004; De Godoy and Rattan, 2005), suggesting the potential use of Ang II inhibitors in certain gastrointestinal disorders characterized by the hypertensive IAS. However, a direct role of the RAS and Ang II in hypertensive IAS pressure (IASP) has not been examined.
The goal of the present studies was to evaluate the role of RAS, and the effects of Ang II inhibitors in the IASP of normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). We carried out in vivo and in vitro, functional, biochemical, and molecular biology experiments in these animal models. We also examined the effects of captopril and losartan in the IASP. We systematically compared these parameters in the tonic smooth muscles of the IAS versus those of the rectum (RSM), and the aorta (AoSM). The RSM and AoSM represent phenotypes of phasic smooth muscles.
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Materials and Methods |
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). The catheter assembly was initially introduced into the rectum and then positioned precisely in the IAS via slow pull-through (approximately 7-8 mm from the anal verge). High pressures of the IAS consisted of rhythmic fluctuations superimposed on the steady tone. The IASP was recorded using a PowerLab/8SP recorder and analyzed via the software Chart 4 PowerLab (ADInstruments, Inc., Colorado Springs, CO). Systolic Blood Pressure Measurements. The systolic blood pressure (SBP) was recorded via a catheter (i.d. 0.38 mm; Clay Adams, Parsippany, NJ), attached to a Statham transducer (Medex, Inc., Carlsbad, CA). All recordings and analyses were carried out using Chart 4 PowerLab (ADInstruments, Inc.).
Administration of Agents. IASP and SBP were monitored for 30 min after the additive administration of captopril and losartan (1-50 mg/kg each, over 1 min of infusion). These doses have been shown to be selective in their actions (Celik et al., 1995; Tramontana et al., 2001).
Muscle Bath Studies. Animals were sacrificed by decapitation, and the smooth muscles of IAS, rectum, and thoracic aorta were prepared for the recording of isometric tension as described before (Moummi and Rattan, 1988; De Godoy et al., 2004; Fukada et al., 2005). In brief, circular smooth muscle strips of the IAS and rectum (
1 mm x 10 mm), and aorta rings (5-6 mm in length) were prepared in the oxygenated Krebs' physiological solution.
Isometric tension was recorded via force transducer (model FT03; Grass Instruments, Quincy, MA) on a PowerLab/8SP data acquisition system (ADInstruments, Inc.). After the equilibration period of 90 min, we measured the development of tone in the WKY and SHR groups in the basal state. The basal tone in the IAS was calculated with reference to the baseline determined at the end of each experiment with 50 mM EDTA (Biancani et al., 1985; Moummi and Rattan, 1988).
Cumulative concentration-response curves for Ang II (0.1 nM-10 µM) in the IAS were obtained before and after 100 nM losartan, both in the background of 10 µM captopril. The changes in IAS basal tone were expressed as the percentage of maximal contraction by 10 µM bethanechol. The losartan concentration used in this study was previously shown to be effective (Fan et al., 2002; De Godoy et al., 2004). The experimental protocols for in vivo and in vitro studies were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University and were in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care.
Ang II Determinations. Arterial blood (2 ml) was rapidly collected into a chilled (4°C) tube containing 1 mg/ml EDTA and 1 µM amastatin (inhibitor of the enzymes that degrade Ang II) (De Godoy et al., 2004). The blood was centrifuged at 4000g for 10 min, and the plasma (1 ml) was immediately applied to an octadecasilyl (C8)-silica cartridge (Waters Corp., Milford, MA). Samples were also collected as 2 ml of the muscle bath perfusates (MBPs) after incubation of the IAS, RSM, and AoSM tissues for 30 min in the presence of 1 µM amastatin and immediately applied to C8 cartridges. All samples were washed with 10 ml of 0.1% trifluoroacetic acid. The peptides were eluted with 6 ml of methanol-water-trifluoroacetic acid (80: 19:1, v/v/v). The eluate was dried in a vacuum centrifuge and stored at 4°C until the further procedures (Bagby et al., 1979; Aihara et al., 1999). The eluate was dissolved in 300 µl of PBS, transferred onto a 96-well React-Bind NeutrAvidin-coated enzyme-linked immunosorbent assay plate (Pierce, Rockford, IL), and incubated for 1 h at room temperature. Nonspecific binding was blocked with blocking buffer (PBS, 0.1% bovine serum albumin, and 0.05% Tween 20) for 30 min at room temperature. Then, each well was washed 3 times with washing buffer (PBS and 0.05% Tween 20) and incubated for 60 min at room temperature with 100 µl of rabbit anti-Ang II antibody diluted in blocking buffer. Wells were washed and incubated for 60 min at room temperature with the donkey adsorbed anti-rabbit secondary antibody in blocking buffer. Wells were washed and exposed to the 1-Step TMB Substrate (Pierce) for 30 min at room temperature. Finally, the reaction was stopped by 2 M H2SO4, and the plate was read at 450 nm in an enzyme-linked immunosorbent assay plate reader. Our preliminary experiments determined optimal conditions to be 1:1000 and 1:2000 for primary and secondary antibodies (De Godoy and Rattan, 2005). The levels of Ang II in plasma and MBP were determined in the basal state and in the presence of captopril or losartan (50 mg/kg and 10 µM each in the case of plasma and MBP, respectively).
RT-PCR. Total RNA was isolated and purified from different tissues 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 an Omniscript RT Kit (Qiagen, Germantown, MD) in a final volume of 20 µl at 42°C for 60 min. PCR primers specific for Angen, renin, ACE, AT1-R, and 
-actin cDNA were designed as shown in Table 1. PCR was performed in a Promega 2x Master Mix (Promega) in a final volume of 25 µl, using a PerkinElmer Thermal Cycler (PerkinElmer Life and Analytical Sciences, Boston, MA). The PCR conditions (in the case of Angen, renin, and -actin) 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). In the end, a final extension at 72°C for 7 min was allowed. In the case of ACE and AT1-R, the annealing temperature was set at 60°C. The PCR products were separated on 1.5% (w/v) agarose gel containing ethidium bromide and were visualized with UV light. The relative densities of Angen, renin, ACE, and AT1-R were calculated by normalizing the integrated optical density (IOD) of each blot with that of -actin.
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). In brief, the tissues isolated and dissected as described above were subjected to homogenization, protein extraction, and determination by the method of Lowry et al. (1951). The proteins (20 µg) were then separated by gel electrophoresis and transferred onto a nitrocellulose membrane (NCM) at 4°C. The NCM was then incubated with the specific primary antibodies (mouse IgG, 1:1000 for Angen and renin; goat IgG, 1:1000 for ACE; and rabbit IgG 1:1000 for AT1-R) for 2 h at room temperature. After washing with Tris-buffered solution-Tween 20, the NCMs were incubated with horseradish peroxidase-labeled secondary antibody (1: 10,000) for 1 h at room temperature. The corresponding bands were visualized with enhanced chemiluminescence substrate using the SuperSignal West Pico Chemiluminescent Substrate (Pierce) and Hyperfilm MP (Amersham Biosciences Inc., Piscataway, NJ).
NCMs were then stripped of antibodies using the Restore Western Blot Stripping Buffer (Pierce) for 5 min at room temperature and reprobed for -actin using the specific primary (mouse IgG 1:10,000 for -actin) and secondary (1:10,000) antibodies. Bands corresponding to different proteins were scanned (SnapSacn.310; Agfa, Ridgefield Park, NJ), and the IODs were determined using Image-Pro Plus 4.0. 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/dose-response curves were analyzed using a nonlinear interactive fitting program (Prism 3.0; GraphPad Software Inc., San Diego, CA). Agonist potencies and maximal responses were expressed as pEC50 (negative logarithm of the molar concentration of agonist producing 50% of the maximal response) and Emax (maximal effect elicited by the agonist), respectively. Inhibitor potencies and maximal inhibition were expressed as log ID50 (logarithm of the milligram per kilogram dose of inhibitor producing 50% of the maximal inhibition) and Imax (maximal inhibition elicited by the inhibitor), respectively. Statistical significance was tested by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test when three or more different groups were compared. To compare only two different groups, the unpaired Student's t test was used. A p value < 0.05 was considered to be statistically significant.
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Results |
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Effects of Captopril and Losartan on the IASP. Captopril and losartan significantly (p < 0.05) reduced the IASP in both strains of rats (Figs. 2 and 3). However, these agents were more efficacious and potent in the SHR (Table 2). For example, 5 mg/kg captopril produced a significantly greater fall in the IAS in the SHR versus WKY, 6.7 ± 0.8 and 1.5 ± 0.3 mm Hg, respectively (*, p < 0.05; n = 4) (Fig. 2). Interestingly, the final IASP for WKY after 50 mg/kg captopril was significantly lower (7.9 ± 0.4 mm Hg) (Fig. 4A) than that in the SHR (9.4 ± 0.4 mm Hg) (Fig. 4B). An actual defecation reflex was observed after captopril (50 mg/kg) in one of four WKY, in which the pressures from the IAS fell down to levels not significantly different from those of the rectum (3.5 ± 2.5 mm Hg). This suggests that higher doses of captopril may compromise IAS tone, one of the components of rectoanal continence.
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Likewise, 5 mg/kg losartan produced a significantly greater fall in the IASP in SHR versus WKY, 7.0 ± 0.6 and 3.3 ± 0.3 mm Hg (*, p < 0.05; n = 4) (Fig. 3). It is noteworthy that in SHR although captopril and losartan were more potent in causing the fall in the IASP in absolute terms, these agents simply normalized the IASP to the levels not significantly different from those in the WKY group (Table 2).
Effects of Captopril and Losartan on SBP. Captopril and losartan caused a significant fall in SBP of SHR but not WKY (Fig. 5). However, the final values for SHR, even after the higher doses of these agents, were not significantly different from those of WKY. The final SBPs after 50 mg/kg captopril and losartan for SHR was 122.2 ± 2.3 and 123.0 ± 6.3 mm Hg, respectively, and these values in the case of WKY rats before the administration of any of these agents were 114.3 ± 2.3 and 117.1 ± 5.7 mm Hg, respectively (p > 0.05; n = 4). Therefore, both the ACE inhibitor and AT1-R antagonist caused similar normalization of elevated SBP and IASP in the SHR.
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Comparison of Basal IAS Tone and Ang II Levels in the MBPs from the IAS, RSM, and AoSM in WKY and SHR. In agreement with the in vivo data, we observed a significant increase in the basal tone in the IAS from SHR versus WKY. The IAS tone in these groups of animals was 0.065 ± 0.013 and 0.025 ± 0.009 g/mg of the tissue weight, respectively (*, p < 0.05; n = 6) (Fig. 7A).
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Interestingly, in agreement with the data from the plasma samples, losartan pretreatment (10 µM) caused an increase in the levels of Ang II in the MBPs from the IAS tissues of SHR but not WKY, from 72.7 ± 11.8 to 115.7 ± 6.0 pg/ml (*, p < 0.05) (Fig. 7C). However, pretreatment of the IAS tissues with captopril (10 µM), as expected, caused a significant reduction in the levels of Ang II in both WKY and SHR.
Western Blots to Determine the Relative Expression of Angen, Renin, ACE, and AT1-R in WKY and SHR. Angen was observed as a doublet: 65- and 55-kDa bands (Fig. 8A). As previously demonstrated in rat pancreas (Leung et al., 1999) and in rat IAS (De Godoy and Rattan, 2005), the higher molecular weight band represents the proAngen, and the lower molecular weight band represents the renin substrate Angen. SHR samples expressed significantly (p < 0.05) higher levels of Angen (but not proAngen) than WKY samples (Fig. 8A). Expression of renin was also significantly higher in the IAS of SHR versus WKY (*, p < 0.05) (Fig. 8B), but not in the RSM and AoSM (p > 0.05).
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Discussion |
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There is an abundance of literature to show the relationship between the up-regulation of the RAS and the development of cardiovascular (CV) hypertension (Bagby et al., 1979; Hubner et al., 1995; Bolterman et al., 2005) in the SHR. In all of these studies normotensive WKY rats have been used as controls. Considering the recent concept of the role of the RAS in IAS tone (De Godoy and Rattan, 2005; Rattan, 2005), the present studies provide important information on the role of the RAS in the pathophysiology of IAS tone in the SHR. SHR develop significantly higher IASP (on anorectal manometry for the intraluminal pressures in in vivo studies) and IAS tone (in in vitro studies), suggesting a relationship between CV and IAS hypertension. A similar relationship has been reported in certain patients (Celik et al., 1995) with CV hypertension associated with hypertensive IAS and constipation. These symptoms disappeared after treatment with antihypertensive agents. The above changes in IAS tone in SHR appear to be selective because no such hyperactivity was observed in the RSM in these animals. Such data provide important future directions to explore the effects of ACE and AT1-R inhibitors in motility disorders characterized by IAS hypertension.
Since their advent, captopril and losartan have been used extensively as antihypertensive agents because of their ability to inhibit Ang II generation and interaction with the AT1-R, respectively (Rubin et al., 1978; Celik et al., 1995). The present data also demonstrate the normalization of the hypertensive IAS with both captopril (Fig. 2) and losartan (Fig. 3) in SHR on a par with the basal IASP in WKY. Similar results were also observed in parallel, showing normalization of SBP of SHR after these inhibitors (Fig. 5). Beyond normalization, these agents do not produce any hypotensive effect in either CV or IAS hypertension in SHR. This is in agreement with previous reports showing the absence of adverse effects of captopril and losartan in normotensive rats and humans (Lee et al., 1991; Horita et al., 2004; Cosentino et al., 2005).
Present data support the previous concept that the basal tone in the IAS of normal rats is partially mediated by the interaction of Ang II with the AT1-R (De Godoy et al., 2004; De Godoy and Rattan, 2005). We also observed in one of four normotensive animals that the higher dose of captopril induces a defecation reflex, resulting in the expulsion of fecal pellets (Fig. 4A). This effect was not encountered in any of the SHR (Fig. 4B) in which higher doses of captopril or losartan do not reduce the basal IASP below the normotensive levels. Such observations may provide an explanation for the lack of symptomatic anal incontinence after captopril specifically in hypertensive patients.
The above symptomatic changes in the IAS of normotensive rats were not observed with losartan. A possible mechanism for the distinct effects of captopril in the normotensive animals may be a potentiation effect by captopril on bradykinin. Accordingly, in addition to ACE inhibition, captopril is known to inhibit the degradation of bradykinin, a circulating peptide that produces smooth muscle relaxation (Engel et al., 1972). In support of this notion, we have observed that bradykinin is selectively and significantly more potent in producing IAS relaxation in WKY in comparison with SHR (data not included).
In agreement with the earlier concept of the autocrine control of RAS in the IAS (De Godoy and Rattan, 2005), the levels of Ang II in the IAS MBP were significantly higher compared with those in the RSM and AoSM in normotensive WKY (Fig. 7, B and C). These levels in the IAS increase severalfold further in SHR. The authenticity of Ang II measurements in the MBP and in the plasma (Fig. 6) was ascertained by the precipitous fall in these levels in the presence of an ACE inhibitor.
In contrast to the effect of captopril on Ang II levels in the plasma and MBP, losartan increases Ang II levels in the SHR. These findings may be explained on the bases of Ang II displacement from the receptor binding sites by competition with the AT1-R antagonist (Zhu et al., 2004).
Higher levels of Ang II in SHR were accompanied by up-regulation of the RAS in all tissues examined. Interestingly, however, AoSM does not express RT-PCR (Fig. 10B) and Western blot products (Fig. 8B) for renin. In contrast to the clear demonstration of the RAS up-regulation at the translational level in SHR, at the transcriptional level, all tissues show lower levels of Angen gene transcripts, and no significant differences in renin and ACE (shown by RT-PCR data). These observations suggest that up-regulation of the RAS occurs at the post-transcriptional level (Figs. 8, 9, 10). Up-regulation of the AT1-R in SHR, however, may occur at both the transcriptional and translational levels (Figs. 9B and 10D).
Previous studies from our laboratory have shown that Ang II causes direct contraction of the rat IAS via AT1-R (Fan et al., 2002; De Godoy and Rattan, 2005). In the present studies we compared the effects of different concentrations of Ang II in the basal tone of WKY and SHR IAS after treatment with captopril. Captopril was used to eliminate interference of endogenous Ang II production. The data reveal no significant difference in the potencies of Ang II between WKY and SHR (Fig. 11) despite the increase in the number of AT1-R in SHR. The maximal biological response may be achieved at agonist concentrations lower than those required to occupy all of the available receptors (Zhu, 1993). Because of this, the above findings in the IAS do not negate the physiological relevance of an increase in the number of AT1-R in SHR. Based on the theory of spare receptors (Zhu, 1993) we hypothesized that if increases in the numbers of AT1-R are of functional importance, less Ang II would be necessary to compete with losartan to produce 50% of the maximal response in SHR than in WKY. Our observations are in agreement with this concept (Fig. 11; Table 3).
In summary, the present data demonstrate hypertensive IAS in the SHR associated with the up-regulation of the RAS in the IAS. These data, along with the medical report associating CV hypertension with the hypertensive IAS, suggest a link between the two. The ability of captopril and losartan to normalize elevated IASP in the hypertensive SHR suggests the potential use of such agents in certain anorectal disorders associated with hypertensive IAS (Sun et al., 1992; Cook et al., 2001; Azpiroz and Whitehead, 2002). Based on the data in the SHR, normalization of the IAS tone to the levels of normotensive patients (without lowering it further) may provide a plausible explanation for the lack of anorectal incontinence in hypertensive patients receiving ACE and AT1-R inhibitors.
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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; IAS, internal anal sphincter; RAS, renin-angiotensin system; Angen, angiotensinogen; Ang I, angiotensin I; ACE, angiotensin-converting enzyme; AT1-R, Ang II receptor subtype 1; IASP, internal anal sphincter pressure; SHR, spontaneously hypertensive rat(s); WKY, Wistar-Kyoto rat(s); RSM, rectum smooth muscle; AoSM, aorta smooth muscle; SBP, systolic blood pressure; MBP, muscle bath perfusate; PBS, phosphate-buffered solution; RT, reverse transcriptase; PCR, polymerase chain reaction; IOD, integrated optical density; NCM, nitrocellulose membrane; HOE-140, D-arginyl-L-arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-thyenyl)-L-alanyl-L-seryl-D-1,2,2,4-tetrahydro-3-isoquinolinecarbonyl-L-(2a,3b,7ab)-octahydro-1H-indole-2-carbonyl-L-arginine; ANOVA, analysis of variance; CV, cardiovascular.
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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