Márcio A. F. de Godoy

Translocation of AT1- and AT2-Receptors by Higher Concentrations of Angiotensin II in the Smooth Muscle Cells of Rat Internal Anal Sphincter

Previous studies have reported bimodal effects by angiotensin II (Ang II) in the rat internal anal sphincter (IAS), a concentration-dependent contraction (at lower concentrations) and relaxation (at higher concentrations). The experiments suggest the above-mentioned responses are the result of Ang II subtype I receptor(s) (AT1-R) and subtype II receptor(s) (AT2-R) activation, respectively. These studies determined the role and mechanism of AT2-R-induced relaxation of the smooth muscle cells (SMCs) from the IAS in response to Ang II. Laser confocal microscopy showed that in the basal state, the AT1-Rs reside in the plasma membrane, whereas AT2-Rs are present in the cytosol. Higher concentrations of Ang II caused movement of AT1-R and AT2-R in opposite directions to the cytosol and the membrane, respectively. Losartan (AT1-R antagonist) but not S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid (PD123319; AT2-R antagonist) selectively inhibited these movements. These results are based on biotinylation assays, confocal images, and Western blot analyses of the densities of AT1-Rs and AT2-Rs in the plasma membrane versus cytosolic fractions of the IAS SMCs. Ang II in higher concentrations did not change the total contents of Ang II receptors. These data combined with the functional data using measurements of IAS SMC lengths suggest that internalization of AT1-R and externalization of AT2-R may be responsible for the activation of the AT2-R, which leads to the relaxation of the IAS with higher concentrations of Ang II.

Cross-talk between AT(1) and AT(2) angiotensin receptors in rat anococcygeus smooth muscle.

Schild regressions for the selective AT1 and AT2 receptor antagonists, losartan and PD123319 (S-[+]-1-[(4-dimethylamino]-3-methylphenyl)methyl]-5-[diphenylacetyl]-4,5,6,7-tetrahydro-1H-imidazol[4,5-c]pyridine-6-carboxilic acid), respectively, were calculated to analyze the heterogeneity of receptor populations in the rat anococcygeus muscle. For a one-receptor system, the Schild regression has a slope of unity and an intercept of K B for competitive antagonists. However, in a two-receptor system, a deviation from the single-receptor plot will occur. This is predicated on the assumption that the secondary receptor is less sensitive to the antagonist than the primary receptor. Results showed that the Schild regression for losartan did not produce a slope of unity, and PD123319 did not produce any effect. However, tissue incubation with losartan plus PD123319 resulted in a Schild regression that has a slope of unity and a pK B of 9.32. In the presence of prazosin, an α1-adrenoceptor antagonist, losartan did not produce any effect. Conversely, PD123319 enhanced the angiotensin II (Ang II)-induced contraction in a concentration-dependent fashion, suggesting an inhibitory AT2-mediated effect. This effect was confirmed with assays that showed a relaxant response induced by Ang II on precontracted tissues incubated with prazosin. PD123319 andN G-nitro-l-arginine methyl ester [nitric-oxide (NO) synthase inhibitor)] markedly inhibited the relaxant response of Ang II. In contrast, losartan did not produce any significant effect. Consequently, results show that the mechanism underlying the AT2-mediated effect is highly dependent on NO generation. Results indicate the presence of a heterogeneous angiotensin receptor population in the rat anococcygeus muscle following a negative cross-talk relationship between the AT1 and AT2 subtypes.

Angiotensin-Converting Enzyme and Angiotensin II Receptor Subtype 1 Inhibitors Restitute Hypertensive Internal Anal Sphincter in the Spontaneously Hypertensive Rats

The present study determined the effects of the angiotensin-converting enzyme (ACE) inhibitor captopril and angiotensin II receptor subtype 1 (AT1-R) antagonist losartan on the internal anal sphincter pressures (IASP) in spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto rats (WKY). The SHR had significantly higher IASP (21.7 ± 0.8 mm Hg) than the WKY (14.7 ± 0.9 mm Hg), which was associated with the higher levels of angiotensin II (Ang II) in plasma (50.3 ± 0.9 pg/ml) and in muscle bath perfusates (72.7 ± 11.8 pg/ml) compared with the WKY (p < 0.05). Captopril and losartan decreased the IASP in SHR and WKY, but they were more potent in SHR. Captopril and losartan normalized the IASP in the SHR, whereas these agents may compromise rectoanal continence in the WKY. Reverse transcriptase-polymerase chain reaction and Western blots showed higher levels of angiotensinogen, renin, ACE, and AT1-R in the internal anal sphincter (IAS) of SHR. Ang II caused concentration-dependent contraction of IAS smooth muscle strips from WKY (pEC50 = 8.5 ± 0.1) and SHR (pEC50 = 8.6 ± 0.2). Losartan (100 nM) significantly (p < 0.05) inhibited this effect. From these data, we conclude that 1) hypertensive IAS in SHR is primarily the result of renin-angiotensin system up-regulation, 2) ACE inhibitors and AT1-R antagonists simply relieve the hypertensive IAS, and 3) the differential effect of these inhibitors in the hypertensive versus the normotensive IAS may explain the lack of incontinence as a side effect in hypertensive patients receiving ACE inhibitors and AT1-R antagonists.

COX-1 vs. COX-2 as a determinant of basal tone in the internal anal sphincter

Prostanoids, produced endogenously via cyclooxygenases (COXs), have been implicated in the sustained contraction of different smooth muscles. The two major types of COXs are COX-1 and COX-2. The COX subtype involved in the basal state of the internal anal sphincter (IAS) smooth muscle tone is not known. To identify the COX subtype, we examined the effect of COX-1- and COX-2-selective inhibitors, SC-560 and rofecoxib, respectively, on basal tone in the rat IAS. We also determined the effect of selective deletion of COX-1 and COX-2 genes (COX-1(-/-) and COX-2(-/-) mice) on basal tone in murine IAS. Our data show that SC-560 causes significantly more efficacious and potent concentration-dependent decreases in IAS tone than rofecoxib. In support of these data, significantly higher levels of COX-1 than COX-2 mRNA were found in the IAS. In addition, higher levels of COX-1 mRNA and protein were expressed in rat IAS than rectal smooth muscle. In wild-type mice, IAS tone was decreased 41.4 +/- 3.4% (mean +/- SE) by SC-560 (1 x 10(-5) M) and 5.4 +/- 2.2% by rofecoxib (P < 0.05, n = 5). Basal tone was 0.172 +/- 0.021 mN//mg in the IAS from wild-type mice and significantly less (0.080 +/- 0.015 mN/mg) in the IAS from COX-1(-/-) mice (P < 0.05, n = 5). However, basal tone in COX-2(-/-) mice was not significantly different from that in wild-type mice. We conclude that COX-1-related products contribute significantly to IAS tone.

Arachidonic acid metabolites follow the preferential course of cyclooxygenase pathway for the basal tone in the internal anal sphincter

Present studies determined the roles of the cyclooxygenase (COX) versus the lipoxygenase (LO) pathways in the metabolic pathway of arachidonic acid (AA) in the internal anal sphincter (IAS) tone. Studies were performed in the rat IAS versus the nontonic rectal smooth muscle (RSM). Indomethacin, the dual COX inhibitor, but not nordihydroguaiaretic acid (NDGA), the LO inhibitor, produced a precipitous decrease in the IAS tone. However, when added in the background of indomethacin, NDGA caused significant reversal of the IAS tone. These inhibitors had no significant effect on the RSM. To follow the significance of COX versus LO pathways, we examined the effects of AA and its metabolites. In the IAS, AA caused an increase in the IAS tone (Emax=38.8+/-3.0% and pEC50=3.4+/-0.1). In the RSM, AA was significantly less efficacious and potent (Emax=11.3+/-3.5% and pEC50=2.2+/-0.3). The AA metabolites (via COXs) PGF2alpha and U-46619 (a stable analog of thromboxane A2) produced increases in the IAS tone and force in the RSM. Conversely, AA metabolites (via 5-LO) lipoxin A4, 5-HETE, and leukotriene C4 decreased the IAS tone. Finally, the contractile effects of AA in the IAS were selectively attenuated by the COX-1 but not the COX-2 inhibitor. Collectively, the specific effects of AA and the COX inhibitor, the Western blot and RT-PCR analyses showing specifically higher levels of COX-1, suggest a preferential role of the COX (specifically COX-1) pathway versus the LO in the regulation of the IAS tone.

Biosynthetic Pathways and the Role of the Mas Receptor in the Effects of Angiotensin-(1–7) in Smooth Muscles

Ang-(1–7) is produced via degradation of Ang II by the human angiotensin converting enzyme, also known as ACE2. In the cardiovascular system, Ang-(1–7) has been shown to produce effects that are opposite to those of Ang II. These include smooth muscle relaxation and cardioprotection. While the roles of Ang-(1–7) in other systems are currently topic of intense research, functional data suggest a relaxation action in gastrointestinal smooth muscles in a way that corroborates the results obtained from vascular tissues. However, more studies are necessary to determine a relevant role for Ang-(1–7) in the gastrointestinal system. The Ang-(1–7) actions are mediated by a distinct, functional, Ang-(1–7) receptor: the Mas receptor as shown by diverse studies involving site-specific binding techniques, selective antagonists, and targeted gene deletion. This paper provides an overview of the functional role and the molecular pathways involved in the biosynthesis and activity of Ang-(1–7) in diverse systems.

Effects Induced by Tityus serrulatus Scorpion Venom and Its Toxins TsTX-I and TsTX-V on the Rat Isolated Retractor Penis Muscle

The aims of the present study were to investigate the pharmacological effects induced by Tityus serrulatus venom (TsV) and its fractions and to compare with the effects induced by pure α (TsTX-V) and β (TsTX-I) toxins isolated from TsV on rat retractor penis muscle (RPM). TsV, fractions X, XI, XIIa, XIIb (0.01–100 µg/ml) and TsTX-V (1 nmol/l–10 µmol/l) induced concentration-dependent contractions. Prazosin and guanethidine or tetrodotoxin (TTX, 5 µmol/l, 30 min) completely abolished these contractions, suggesting complete dependence on sympathetic nerves. TsV or fractions X, XI, XIIa, XIIb (0.01– 100 µg/ml), TsTX-I and TsTX-V (1 nmol/l–10 µmol/l) induced concentration-dependent relaxations in the precontracted RPM. TTX or Nω-nitro-L-arginine methyl ester (L-NAME, 100 µmol/l, 30 min) completely abolished the relaxations. Our results suggest that most of TsV-derivated toxins induce contraction and relaxation on RPM by sympathetic and NANC nitrergic nerve stimulation. Noteworthy, TsTX-I only induces relaxation on RPM suggesting that this protein selectively acts on inhibitory nerves.