Department of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson University, Philadelphia, Pennsylvania
Submitted 16 March 2005 ; accepted in final form 22 June 2005
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ABSTRACT |
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smooth muscle tone; internal anal sphincter; rectal smooth muscle; angiotensin-converting enzyme; angiotensinogen
A number of studies has demonstrated that ANG II, a potent contractile agonist of the smooth muscle, may be synthesized locally (1, 11). The studies have important implications. Renin converts angiotensinogen (Angen) into ANG I, the latter via angiotensin-converting enzyme (ACE) is then converted to ANG II (11, 15, 20, 30). This constitutes the renin-angiotensin system (RAS). ANG II once produced and released activates primarily AT1-R, a G protein-coupled receptor (GPCR), causing tonic contraction in the smooth muscle (12, 38). These concepts have been proposed in certain hypertensive smooth muscles under pathophysiological conditions (28). The concept has, however, been not tested in the spontaneously tonic smooth muscles such as the IAS.
Earlier studies in the IAS have demonstrated the presence of ANG II and AT1 receptors (14, 17, 31). However, the major part of the RAS in the IAS SMC has not been investigated.
The aim of the present study was to evaluate the patterns of local generation of ANG II by the SMC from purely tonic smooth muscle of the IAS vs. the phasic smooth muscle of the anococcygeus (ASM). The rectal smooth muscle (RSM), similar to the colon, displays primarily phasic activity with a small component of tonic activity (36). To achieve these objectives, we carried out functional, biochemical, and molecular biology (at the transcriptional and translational levels) studies to examine the presence of RAS and release of ANG II in the SMC from these three types of tissues.
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MATERIALS AND METHODS |
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Male Sprague-Dawley rats (300350 g) were killed by decapitation, and the IAS, RSM, and ASM smooth muscle strips were prepared for the recording of isometric tension as described previously (13, 14, 26). Briefly, circular smooth muscle strips of the IAS and RSM (0.5 mm x 7 mm) and ASM (
1 x 10 mm) were prepared in the oxygenated Krebs physiological solution (KPS). The composition of KPS was as follows (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 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.
Isolation of SMCs and Measurement of Cell Length
SMCs from the above tissues were isolated as described previously (7, 21, 32). Briefly, the smooth muscle tissues were 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) collagenase-free KPS. The tissue was incubated in collagenase-free KPS at 37°C, and dispersion of the cells (01 h) was monitored periodically by examining a 10-µl aliquot of the mixture under microscope. SMCs were harvested by filtration through the Nitex mesh. The filtrate containing the cells was centrifuged at 350 g 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 (19) using a custom-assembled microscope (Olympus, Tokyo, Japan), closed-circuit video camera (model Pulnix MC-7; PULNIX America, Sunnyvale, CA) and personal computer. The images of the cells were stored digitally, and the cell lengths were measured by the Image-Pro Plus version 4.0 program (Media Cybernetics, Silver Spring, MD).
Functional Experiments in Isolated SMC
After the exposure with Angen (10 nM-10 µM) for 2 min, the SMC were fixed with acrolein (final concentration 1%) and transferred onto chrome-alumcoated glass slides (Fisher Scientific, Pittsburgh, PA). The studies were repeated in the presence of selective inhibitors of renin (H-77; 1100 µM) and ACE (captopril; 0.110 µM). The shortening of SMC in each category of experiments was calculated on a percentile bases of the original cell lengths. The studies were repeated in the SMC isolated from at least three animals.
Muscle Bath Studies
Isometric tension was recorded via force transducer (model FT03; Grass Instruments, Quincy, MA) on PowerLab/8SP data-acquisition system (AD Instruments, Castlehill, Australia). After the equilibration period of 90 min, we examined the effects of Angen (1 nM-10 µM) in the absence and presence of H-77 (10 µM) and captopril (1 µM).
In another group of experiments, the effects of H-77 or captopril (0.01100 µM) were tested on the smooth muscles in the basal state. The changes in the smooth muscle tone were expressed as the percent maximal relaxation by EDTA (50 mM) at the end of each experiment (4, 26).
ANG II Determinations
ELISA optimization for ANG II measurements. ELISA method specifically sensitive for the lower range of ANG II values (found in the SMC extracts) was developed for the present studies.
First, we titrated the optimal concentrations of primary and secondary antibodies as follows. Aliquots of 100 µl ANG II (1 µM) were prepared in PBS containing amastatin (1 µM), transferred onto a 96-well React-Bind NeutrAvidin-coated ELISA plate (Pierce, Rockford, IL) and incubated for 1 h at RT. Nonspecific binding was blocked with blocking buffer (PBS, BSA, and 0.05% Tween-20) for 30 min at RT. Then, each well was washed three times with washing buffer (PBS and 0.05% Tween-20) and incubated for 30 min at RT with 100 µl rabbit anti-ANG II primary antibody (Peninsula Laboratories, San Carlos, CA) diluted in blocking buffer. Wells were washed three times and incubated for 30 min at RT with the donkey anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer. Wells were washed, and the plate was flicked and slapped to avoid any remaining washing buffer in the wells. The 1-Step TMB Substrate (Pierce) was added to each well followed by 30 min of incubation at RT. Finally, the reaction was stopped by 2 M H2SO4, and the plate was read at 450 nm in an ELISA plate reader.
ANG II measurements. Our preliminary experiments determined optimal conditions to be primary antibody concentration at 1:1,000 and secondary antibody concentration at 1:2,000 in PBS with 0.1% BSA and 0.05% Tween-20. This method provides a standard linear curve for ANG II in the concentrations expected in the cellular and tissue extracts. In such concentrations, the cross-reactivity with Angen or ANG I is insignificant (Fig. 1A). To eliminate the cross-reactivity with ANG III, the samples were prepared in the presence of 1 µM amastatin (14). This has been documented by the data in Fig. 1B.
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RT-PCR.
Total RNA was isolated and purified from different tissues by the acid guanidine-phenol-chloroform method (9) and quantified by measurement of absorbance at 260 nm in a spectrophotometer. Total RNA (2.0 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 Angen, renin, ACE, and -actin cDNA were designed as shown on Table 1. PCR was performed in a Promega 2x Master Mix (M750B, Promega) in a final volume of 25 µl, using a Perkin-Elmer Thermal Cycler (PerkinElmer Life and Analytical Sciences). The PCR cycle consisted of 94°C for 5 min (for the denaturation phase), 57°C for 30 s (for the annealing phase), and 72°C for 1 min (for the elongation phase); this was repeated for 35 cycles. The PCR conditions for Angen, renin, and
-actin were identical except in the case of ACE, in which the annealing phase temperature was set at 60°C for better results. The PCR products were separated on 1.5% (wt/vol) agarose gels containing ethidium bromide and were visualized with ultraviolet light. The relative densities of Angen, renin, and ACE were calculated by normalizing the integrated optical density (IOD) of each blot with that of
-actin.
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The NCM was then incubated with the specific primary antibodies (mouse IgG, 1:500 for Angen and renin; goat IgG, 1:500 for ACE) for 2 h at RT. After being washed with TBS-T, 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) and Hyperfilm MP (Amersham Bioscience, NJ).
NCMs were then stripped of antibodies using Restore Western Blot Stripping Buffer (Pierce) 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 corresponding to different proteins were scanned (SnapSacn.310; Agfa, Ridgefield Park, NJ), and the respective areas and 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.
Immunocytochemistry of SMCs.
Immunocytochemistry of SMC was performed by the indirect immunofluorescence as described before (3). To examine the presence of Angen, renin, and ACE, the SMC from IAS, RSM, and ASM were fixed with ice-cold fixative (4% paraformaldehyde and 0.2% picric acid in PBS, pH 7.4) for 10 min and then thoroughly washed in PBS. The cells were rinsed with PBS and incubated in a mixture of 1:400 primary antibody (Angen and renin raised in mouse and ACE raised in goat) diluted in PBS containing 0.5% BSA 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 (TR)-labeled secondary antibodies (1:200 in a solution of 2% normal donkey serum and 0.3% Triton X-100 in PBS) raised in donkey. The SMC were identified by their incubation with FITC-labeled antibody against -actin (1:800). The slides were incubated for 60 min at RT, rinsed with PBS, air dried, and placed on coverslips with Vectashield (Vector Labs, Burlingame, CA). Cells were observed with fluorescent microscopy using the appropriate filters and photographed by a Leica DC350F Digital Camera (Leica Microsystems Digital Imaging, Cambridge, UK). All groups of cells were exposed to the same exposure time, binning, brightness, and contrast.
Data analysis. Results are expressed as means ± SE. Agonist concentration response curves (CRCs) were fitted using a nonlinear interactive fitting program (GraphPad Prism 3.0, Graph Pad Software Incorporated). Maximum responses and potencies of different agents were expressed as maximum effect elicited by the agonist (Emax), maximum effect elicited by the inhibitor (Imax), negative log of the molar concentration of agonist producing 50% of the maximum response (pEC50), and negative log of the molar concentration of the inhibitor producing 50% of maximal inhibition (pIC50), respectively. Statistical significance was tested by using the one-way ANOVA followed by the Newman-Keuls post hoc test when three or more different groups were compared. To compare two different groups of data from the same tissue sample, we used paired Student's t-test, and at other times, we used unpaired Student's t-test. A P value <0.05 was considered to be statistically significant.
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RESULTS |
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Effect of Angiotensinogen (Angen) on the SMC. Angen produced concentration-dependent shortening of the SMC isolated from the IAS as well as from the RSM and ASM (Fig. 2). Because Angen must be converted into ANG II for biological activity, these results suggest that the SMC investigated possess the ability to convert Angen into ANG II. Enzymatic conversion system for Angen to ANG II appears to be functionally more relevant in the IAS. Angen was more potent in the IAS SMC compared with the other tissues. For the respective pEC50 values, see Table 2. The lengths of the cells isolated from different tissues in the basal state varied depending on the tissue (for absolute values, please refer to Table 2).
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Effects of H-77 and captopril on basal SMC lengths. Interesting differences emerged when we compared the effects of H-77 and captopril on the basal cell lengths from three different regions (Fig. 3). Data show that H-77 and captopril produced significantly greater relaxation of the SMC from the IAS vs. RSM. Percent maximal relaxation of the IAS SMC with captopril was 13.8 ± 0.7 vs. the RSM and ASM 5.3 ± 1.2, and 0.4 ± 0.7, respectively (P < 0.05; n = 4). These data suggest that renin and ACE are functionally present especially in the IAS SMC.
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Effects of Angen, H-77, and captopril on the basal smooth muscle tone. A majority of the spontaneous activity in the IAS was of tonic in nature, in contrast to that in the RSM that was primarily phasic. ASM, on the other hand, had no spontaneous activity (Table 3). Similar to experiments in isolated cells, Angen produced concentration-dependent contraction of the smooth muscles with highest efficacy and potency in the IAS (Fig. 4, Table 3).
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H-77 (0.1100 µM) and captopril (0.1100 µM) also caused a significant and concentration-dependent fall in the basal tone of the IAS (P < 0.05, n = 4; Fig. 5) but not in the RSM and ASM, which might be related to the absence of tone in these tissues. Values of pIC50 show that captopril is more potent than H-77 in inducing relaxation of IAS as seen in isolated cells. In addition, analysis of Hill slope shows that the CRC of captopril is steeper than the CRC of H-77 (Table 4). In some experiments, the tension in the RSM and ASM was artificially raised (by 100 µM bethanechol) to further assess the effect, because these tissues do not have significant tone in the basal state. Even under these experimental conditions, H-77 and captopril failed to exert any significant effect (Fig. 6).
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Presence of Angen, Renin, and ACE
Western blot data. Western blot data for Angen, renin, and ACE is summarized in Fig. 8. Higher levels of Angen, renin, and ACE were found in the SMC of IAS vs. the ASM. Interestingly, Angen was observed as a doublet, comprising 65- and 55-kDa bands. Larger molecular weight bands represent proangiotensinogen, and the smaller molecular weight bands represent the processed isoform, as demonstrated in rat pancreas (24). It is noteworthy that Angen and renin determinations with Western Blot followed the same trend as with RT-PCR (IAS > RSM > ASM).
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Higher levels of RAS in the IAS (at the message and translational level) combined with the functional data suggest that RAS may have a role in the genesis of basal tone in the IAS.
Immunocytochemistry data.
The identity of SMC from different tissues was first confirmed by indirect immunofluorescence staining of the cells by -actin (green) under the FITC filter (Fig. 10, A-C). Incubation of the cells with primary antibody against Angen, renin, and ACE led to positive signals (red) when present. Immunocytochemical presence of Angen, renin, and ACE-IR (labeled with Texas red) was demonstrated in the respective panels of Fig. 10.
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DISCUSSION |
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ANG II generation requires Angen, a rate-limiting aspartyl protease enzyme renin (which converts Angen into ANG I), and ACE, which finally converts ANG I into ANG II (1, 11). First, we focused our studies on the demonstration of Angen in the SMC. Among different tissues examined, Angen was primarily present in the SMC of the IAS. This was demonstrated at the message level by RT-PCR and at the translational level by Western blot studies. The studies also demonstrate the actual presence of Angen-IR by immunocytochemistry studies, primarily within the IAS SMC.
To further determine the presence and significance of Angen within the IAS SMC, we carried out two additional types of studies: 1) the effects of Angen on the SMC, before and after renin and ACE inhibitors, and 2) handling of Angen by the SMC. Renin inhibitor H-77 blocks conversion of Angen to ANG I, whereas ACE inhibitor captopril blocks conversion of ANG I into ANG II.
In the first type of studies, we theorized the contractile effect of Angen on SMC will be evidence in favor of the intracellular machinery for the conversion of Angen into ANG I and then into ANG II. This theory was based on the fact that neither Angen nor ANG I (1, 11) have any activity of their own and that their effects are because of their conversion into ANG II. As shown by the data, Angen causes concentration-dependent contraction of the SMC that is attenuated by H-77 and captopril. In the second type of study, Angen causes an increase in the concentrations of ANG II in the SMC, and these increases are selectively blocked by renin and ACE inhibitors. Additionally, H-77 and captopril cause a concentration-dependent fall in the basal tone of the IAS. The fall in the IAS tone by H-77 and captopril correspond to the decrease in the levels of ANG II. These data provide strong evidence in favor of the presence of constituents of RAS, Angen, renin, and ACE in the IAS SMC.
Expression of Angen, the precursor of angiotensin peptides, has been observed in a number of adult cells, including aortic smooth muscle cells (16), fibroblasts (22), and human colonic mucosa (20). Data also identify the nonprocessed form of Angen (65 kDa) and the Angen (55 kDa) by Western blot studies in the SMC extracts. This confirms the ability of the SMC to generate Angen, as shown before in other tissues (24).
The intrinsic expression of renin in structures other than juxtaglomerular cells has not been well recognized (29). Present studies show that renin is particularly present in higher amounts in the IAS than in the RSM and ASM. It is of interest that renin was virtually absent in the purely phasic smooth muscle of ASM. In addition, potencies of Angen-induced contractions were IAS > RSM > ASM. These data combined with the dramatically lower levels of Angen in the ASM and RSM suggest functional significance of RAS in the IAS. In other studies (unrelated to the smooth muscle tone), the expression of renin in large intestine and stomach of rats (30), human colonic mucosa (20), and human stellate cells has been shown (2). Additionally, renin-binding proteins have been described in the rat vasculature (5). These membrane-bound proteins bind circulating renin and contribute to the local pool of the protein in tissues not expressing renin gene (5). Whether such renin-binding proteins may in part explain the presence of low levels of renin in the RSM and ASM remains to be determined.
The studies also demonstrate the presence and significance of ACE in the IAS tone as follows. ACE was found to be selectively in higher levels in the IAS compared with the ASM and RSM. Additionally, the ACE inhibitor caused a potent fall in the basal tone in the IAS, such an effect was not demonstrable in the ASM and RSM because these tissues have no tone. To further examine this issue, we contracted these tissues with bethanechol and examined the effects of H-77 and captopril. Even in these experiments, these inhibitors have no inhibitory effect. The presence of RAS, including ACE, has been demonstrated in the rat intestine (15), the rat valvular interstitial cells (22), and the vascular SMC (18).
Angen, renin, and ACE expression were characteristically higher in the IAS SMC. To further elucidate the ability of IAS, RSM, and ASM to locally generate ANG II, we measured ANG II levels in the freshly isolated SMC. ANG II levels measured in the SMC from IAS are several fold higher compared with those in the RSM and ASM. These values in the isolated SMC from the IAS should not be compared with the higher levels reported in the intact smooth muscle strips (14). The measurements reported earlier reflect ANG II levels released from the entire cellular components of the IAS. We speculate that the local release of ANG II by IAS SMC activates AT1-R to exert autocrine control in the IAS tone. However, an additional paracrine and endocrine control of ANG II may also play a role.
H-77 and captopril attenuate ANG II levels in the IAS SMC and produce relaxation of the IAS smooth muscle and the SMC. In addition, a specific increase in the levels of ANG II following Angen pretreatment and higher expression of RAS enzymes in the IAS SMC vs. other tissues suggest physiological relevance of RAS in the genesis of basal tone in the IAS.
In summary, present data combined with the potent contractile effect of ANG II (17, 27, 33), presence of AT1-R (35), the corresponding signal transduction cascade (31, 34) in the IAS SMC, and fall in the IAS tone by ANG II biosynthesis inhibitors suggest that RAS, in part, may regulate basal tone in the IAS. The other extracellular signals for the balance of the basal tone may be prostanoids (6, 7). The effects of captopril in the IAS pressures in vivo studies are not known. However, its negative effects on the IAS may not negate the importance of ACE, because increased kinin levels (23) (with excitatory effects) may cancel the expected inhibitory effect of captopril on the basal tone.
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GRANTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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