1. Evidence for the role of angiotensin II biosynthesis in the rat internal anal sphincter tone 
Márcio A.F. De Godoy, Stephen Dunn, Satish Rattan 
Gastroenterology 
Volume 127, Issue 1, Pages (July 2004)
DOI: /j.gastro

2. Figure 1
Biosynthesis pathways of Ang II and related peptides. Ang II is produced from Ang I via the action of ACE. Ang II is converted to Ang III via aminopeptidase A (APA) and to Ang IV via aminopeptidase N (APN). Ang II in some systems is also converted to Ang-(1–7) via neutral endopeptidase (NEP). The last pathway may not play a significant role in the IAS.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Comparison of the effects of different angiotensin-related peptides in the rat IAS. All Ang II analogues produce concentration-dependent contraction in the IAS. Ang II causes bimodal effects: a concentration-dependent increase in the basal tone followed by decreases in the higher range. In addition, note the characteristic effects of Ang-(1–7): a trend toward a decrease in the basal tone in the lower concentrations and modest contraction in the higher concentrations. Data represent the mean ± SEM of 4–7 independent determinations.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Time course of IAS contraction by Ang I, II, and III in response to their maximally effective concentrations. (A) Time course followed for 20 minutes for Ang II added at time 0; (B) detailed analysis of the time course for the first 3 minutes for Ang II added at time 0; (C) typical tracings to compare the detailed time course of IAS contraction with Ang I, II, and III for the first 3 minutes, added at the point of the inverted arrow. Note that each of these peptides produces an initial contraction that peaks within the first 40 to 50 seconds, followed by a sustained contraction that lasts for several minutes. However, contraction by Ang II begins earlier than with the other peptides.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Effects of the selective AT1 and AT2 receptor antagonists losartan and PD123,319, respectively, and their combination on IAS contraction by Ang II. Data show 2 components of Ang II effects: contraction at lower concentrations caused via AT1 activation, and inhibition at higher concentrations caused by AT2 activation. The inhibitory component was reversed by PD123,319. Losartan plus PD123,319 caused a parallel rightward shift in the Ang II CRC. Data represent the mean ± SEM of 4–7 independent determinations.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Time course (plotted for ∼3 minutes) of IAS contraction by Ang I (A), II (B), and III (C) before and after losartan and PD123,319. Note that losartan (but not PD123,319) attenuated both the initial and sustained contraction by these peptides. The effects of captopril on Ang I-induced contraction were similar to those of losartan (A).
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7. Figure 6
Effects of the selective Ang-(1–7) receptor antagonist A-779 on Ang-(1–7) and Ang II and effects of the NEP inhibitor thiorphan on Ang II in the IAS. A-779 obliterated the inhibitory effect of Ang-(1–7) without any effect on its contractile effect or that of Ang II. Thiorphan had no significant effect on Ang II effects in the IAS (P > 0.05). Data represent the mean ± SEM of 6–9 independent determinations.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Effect of losartan or PD123,319 on IAS contraction by Ang III. Data show a significant inhibition by losartan (∗P < 0.05; n = 4) but not by PD123,319 (P > 0.05; n = 4–7).
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
(A) Effects of the ACE inhibitor captopril on Ang I-induced contraction in the IAS. (B) Typical tracing to show the profile of basal IAS tone in control vs. after captopril (1 μM), monitored over 1 hour. (C) Quantitative data to show time-dependent decreases in the IAS tone with captopril over 20 minutes (mean ± SE; n = 4).
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 9
Absolute values of Ang I, II, and III in the muscle bath perfusates from the IAS. Data show the highest levels of Ang I, followed by Ang II and Ang III, in the basal state (b). Krebs buffer without the IAS tissues had no detectable levels of any of these peptides (not shown). Levels of Ang I remained unaffected by captopril and amastatin (P > 0.05; n = 3). Captopril (c) caused a significant decrease in the levels of Ang II, and amastatin (a) caused an increase (∗P < 0.05; n = 3). Ang III levels, however, were selectively and significantly reduced by both captopril and amastatin (∗P < 0.05; n = 3).
Gastroenterology  , DOI: ( /j.gastro )

11. Figure 10
(A) An actual chromatogram showing peaks with retention times for Ang II (5.95 minutes), Ang III (7.73 minutes), Ang IV (10.59 minutes), and Ang I (13.55 minutes) as standards (10 μM). (B) Effect of captopril on Ang II levels after Ang I incubation. Captopril (Capt) caused a significant decrease in Ang II (analysis of variance; ∗P < 0.05 compared with basal).
Gastroenterology  , DOI: ( /j.gastro )

12. Figure 11
(A) Inhibitory effect of amastatin on Ang II-induced increases in basal IAS tone (∗P < 0.05; n = 4–7). (B) Amastatin caused a significant inhibition of increases in Ang III levels caused by Ang II incubation (∗P < 0.05; n = 3).
Gastroenterology  , DOI: ( /j.gastro )

13. Figure 12
(A) Significant augmentatory effect of amastatin (Amast) on Ang III-induced increases in the basal IAS tone (∗P < 0.05; n = 4–7). (B) Amastatin caused a significant increase in Ang III levels after exogenous Ang III (∗P < 0.05; n = 3).
Gastroenterology  , DOI: ( /j.gastro )

14. Figure 13
Western blot analyses of AT1, AT2, ACE, APA, APN, and β-actin expression in the IAS smooth muscle. The results show relative distributions of the expected sizes of protein for AT1 and AT2 (40 kilodaltons), ACE (140 kilodaltons), APA (160 kilodaltons), APN (150 kilodaltons), and β-actin (43 kilodaltons). The protein samples (40 μg) were run on a 7.5% SDS-polyacrylamide gel, electrophoresed for 60 minutes, transferred to NCM, and probed by isoform-specific antibodies.
Gastroenterology  , DOI: ( /j.gastro )