No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Characterization of rat intestinal anglotensin II receptors
Abstract
In rat ileum and duodenum 125I-sarcosine1,isoleucine8-angiotensin II labels a single population of binding sites with comparable receptor densities of 98 and 94 fmol/mg protein, respectively. Radioligand binding was dose dependently antagonized by angiotensin II (AII) and related peptides. DuP 753, a selective antagonist for the angiotensin AT1 receptor subtype, potently inhibited radioligand binding in both tissues (Ki: 12.7 and 11.8 nM), while AT2-selective ligands like PD 123.177 or p-amino-phenylalanine6-AII were inactive in concentrations lower than 1 μM. The contractile response to AII (1 μM) in ileal longitudinal and circular smooth muscle preparations amounted to 96 and 16%, respectively, of the response to 100 μM methacholine. The contractile response to AII was inhibited by DuP 753 (pA2 7.53) but unaffected by PD 123.177 (pA2 < 5). The AII effect in longitudinal duodenal preparations amounted to only 24% of the methacholine response and was totally abolished in the presence of 1 μM DuP 753. No contraction due to AII was observed in duodenal circular smooth muscle preparations. The results obtained demonstrate the existence of functional AT1 receptors in the rat ileum and duodenum. In the ileum these receptors are mainly located on the longitudinal smooth muscle and coupled to contraction. In duodenal smooth muscle AII receptors may be either less effectively coupled to contractile elements or involved in another, additional function.
... However, RAS components can play local, tissuelevel functions, as local blood perfusion regulation, tissue remodeling and wound healing, extracellular fluid volume, and electrolyte homeostasis [8].The presence of local RAS in different parts of the gastrointestinal (GI) tract has been demonstrated and its function and regulation are becoming clearer [11]. Locally produced angiotensin II, acting as paracrine regulator, influences the mucosal net fluid and buffer transport [13][14][15][16][17] as well as regulates the GI muscular wall contractility [21,22,25]. The effects of Ang II are mediated by specific G-coupled membrane receptors classified as AT1 and AT2 receptors. ...
... In the GI tract, RAS expression has been previously reported in rodent intestine [17,21,25]; moreover, Pasanen et al. [24] in a recent study characterize in the intestinal RAS expression in young and adult rats, reporting that jejunal AT1R expression was increased in age-related manner. Since AT1 receptors are mainly involved in intestinal motor effects of Ang II in the gut of different species including rodents, we aimed to characterize the response to Ang II in the jejunum of young and old rats, hypothesizing possible differences in the contractile response. ...
... As already observed in different gut preparations from human and animal models [9,21,22,25,29], our data demonstrated that, also in the rat jejunum, Ang II is involved in the modulation of the smooth muscle contractility. In particular, Ang II induced a consistent dose-dependent contractile response regardless of whether the jejunum was from young or old rats. ...
The involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3–6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural blocker ω-conotoxin and by nitric oxide (NO) synthase inhibitor. No difference in the response was observed between young and old groups. AT1 receptor-mediated contractile response was decreased by U-73122, phospholipase C (PLC) inhibitor; or 2-aminoethoxy-diphenylborate (2-APB), inositol triphosphate (IP 3 ) receptor inhibitor; or nifedipine, l -type calcium channel blocker. Age-related changes in the expression of both AT1 receptor subtypes, AT1a and AT1b, and of AT2 receptors were detected. In conclusion, Ang II modulates the spontaneous contractility of rat jejunum via postjunctional AT1 receptors, involving Ca ²⁺ mobilization from intracellular stores, via PLC/IP 3 pathway, and Ca ²⁺ influx from extracellular space, via l -type channels. Prejunctional AT2 receptors would counteract AT1 receptor effects, via NO synthesis. The observed age-related differences in the expression of all AT receptor subtypes are not reflected in the muscular contractile response to Ang II.
... Ang II is also a potent constrictive agent in vascular smooth muscle and, although less investigated, influences intestinal smooth muscle function as well (3,17,39,51,58,61). For example, Fishlock and Gunn (17) reported that Ang II elicited dose-dependent contractions in the human colon. ...
... Ludtke et al. (39) investigated Ang II effects on both gastric and duodenal smooth muscle preparations and also found a dose-dependent contraction. Schinke et al. (51) reported that Ang II, in a dose-dependent fashion, elicited contractions in rat duodenal and ileal longitudinal muscular strips. Additionally, several recent studies demonstrated that Ang II elicits concentrationdependent contractions in human duodenal and jejunal as well as ileal isolated muscle preparations (16,56). ...
... The current view is that the AT 2 R counteracts the "classical" Ang II responses in the cardiovascular and renal systems mediated via the AT 1 R (4). Radioligand binding studies have demonstrated the existence of functional AT 1 R in the rat ileum and duodenum (51). In the ileum these receptors were mainly located on the longitudinal smooth muscle and coupled to contraction (51). ...
Studies have demonstrated that angiotensin II (Ang II) can regulate intestinal fluid and electrolyte transport and control intestinal wall muscular activity. Ang II is also a proinflammatory mediator that participates in inflammatory responses such as apoptosis, angiogenesis, and vascular remodeling; accumulating evidence suggests that this hormone may be involved in gastrointestinal (GI) inflammation and carcinogenesis. Ang II binds to two distinct G protein-coupled receptor subtypes, the AT(1)R and AT(2)R, which are widely expressed in the GI system. Together these studies suggest that Ang II-AT(1)R/-AT(2)R actions may play an important role in GI tract physiology and pathophysiology. Currently it is not known whether miRNAs can regulate the expression of the human AT(1)R (hAT(1)R) in the GI system. PCR and in situ hybridization experiments demonstrated that miR-802 was abundantly expressed in human colon and intestine. Luciferase reporter assays demonstrated that miR-802 could directly interact with the bioinformatics-predicted target site harbored within the 3'-untranslated region of the hAT(1)R mRNA. To validate that the levels of miR-802 were physiologically relevant in the GI system, we demonstrated that miR-802 "loss-of-function" experiments resulted in augmented hAT(1)R levels and enhanced Ang II-induced signaling in a human intestinal epithelial cell line. These results suggest that miR-802 can modulate the expression of the hAT(1)R in the GI tract and ultimately play a role in regulating the biological efficacy of Ang II in this system.
... Recent studies have shown that the peptide AngII has the potential effect of smooth muscle contraction in animals [2,9,11,17,20,27,32], as well as on human gastro-intestinal muscle wall [4,12,14,29]. A previous study from our laboratory showed protein expression of AT1 and AT2 receptors, and gene expression of ACE and angiotensinogen in the human jejunal muscle wall and a concentration-dependent muscle contraction in response to AngII [29]. ...
... AngII may be a more potent activator of smooth muscle contractions, but this study shows that the degradation product AngIV also has the ability to produce an effect on muscle contraction. Differences in sensitivity [14,27] and in the functional significance [4,29] to AngII between the circular and longitudinal musculature have previously been observed. Our in vitro study also showed differences in the contractile significance between the circular and longitudinal musculature to AngIV. ...
... Angiotensin II and [Sar']angiotensin II were the most potent agonists, angiotensin III and [Lys2]angiotensin II being approximately 30-100 fold weaker. While some of these data are comparable with previous studies in guinea-pig ileum (Shimuta et al., 1989), other studies have shown angiotensin II and angiotensin III to be less potent (Robertson et al., 1992;Schinke et al., 1991). Furthermore, differences in the relative potencies of angiotensin II and angiotensin III in different tissues are also evident. ...
... The potency of losartan is close to that previously measured against responses to angiotensin II in other tissues (Barnes et al., 1991;Hawcock et al., 1992;Robertson et al., 1992). Furthermore, these findings agree with previous studies showing AT, receptor-mediated contractile responses to angiotensin II in guinea-pig (Wong et al., 1990) and rat ileum (Schinke et al., 1991). The lack of effect of the AT2 receptor selective antagonist, PD123177 confirms that the neuronal responses to angiotensin II are mediated by the AT, receptor. ...
The contractile responses to angiotensin II, angiotensin III and two synthetic analogues, [Lys ² ] angiotensin II and [Sar ¹ ]angiotensin II, in the guinea‐pig isolated longitudinal muscle preparation of small intestine have been characterized in vitro .
Tachyphylaxis to the angiotensin analogues was reduced by use of a Krebs‐Henseleit solution containing a raised (sub‐contractile) concentration of potassium (11.2 m m ). Under these conditions, reproducible cumulative concentration‐response curves to all agonists were established. The pD 2 estimates for angiotensin II, [Lys ² ]angiotensin II, angiotensin III and [Sar ¹ ]angiotensin II were 9.15 ± 0.14, 7.42 ± 0.06, 7.69 ± 0.18 and 9.50 ± 0.15 respectively and the maximum responses achieved were not significantly different.
The contractile responses to angiotensin II, angiotensin III and [Sar ¹ ]angiotensin II were reduced by greater than 80% by tetrodotoxin (TTX; 0.1 μ m ). However, the responses to [Lys ² ]angiotensin II were reduced by only 63 ± 5%. Atropine (0.1 μ m ) also reduced the responses to angiotensin II, angiotensin III and [Lys ² ]angiotensin II, although its effect was less than that produced by TTX. Furthermore, while responses to these agonists were not significantly modified by the NK 1 receptor antagonist (±)‐CP‐96,345 (30 n m ) alone, the combined pre‐incubation with both atropine and (±)‐CP‐96,345 reduced maximum agonist responses to a level not significantly different from those produced by TTX.
Indirect and direct contractile responses to angiotensin II and [Lys ² ]angiotensin II (in the presence of TTX) respectively were characterized by use of the selective AT 1 receptor antagonist, losartan and the AT 2 receptor antagonist, PD123177. Losartan produced parallel rightward displacement of the concentration‐response curve to angiotensin II and [Lys ² ]angiotensin II, with an estimated p K B of 8.56 (8.42–8.68) and 9.18 (8.63–9.50) respectively. The AT 2 receptor antagonist, PD123177 (3 μ m ) failed to modify the contractile responses to either angiotensin II or [Lys ² ]angiotensin II.
We conclude that two populations of angiotensin II receptors exist in the guinea‐pig longitudinal muscle of small intestine, one located neuronally mediating the release of both acetylcholine and substance P and the other located on the smooth muscle mediating direct contractile responses. The neuronal component provides the major contribution to the agonist responses. Both receptor populations are of the AT 1 receptor subtype.
... Notwithstanding, the establishment of an ACE2-Ang-(1-7)-Mas axis that affects enterocyte glucose uptake has important implications for the control of postprandial glycaemia in diabetes. It is important to note that treatment of diabetic rats with Ang- (1-7) improved glucose tolerance significantly after an oral but not (142) In vitro autoradiography revealed that AngII receptors are most abundant in the colon, followed by the ileum, duodenum and jejunum (33) AT1R and, to a lesser extent, AT2R detected in rat intestine by autoradiography; specific AngII binding sites found to be moderately abundant in jejunal and ileal mucosa (137) Functional AT1R present in rat ileum and duodenum (135). ...
Glucose is the prominent molecule that characterizes diabetes and, like the vast majority of nutrients in our diet, it is absorbed and enters the bloodstream directly through the small intestine, and hence small intestine physiology impacts blood glucose levels directly. Accordingly, intestinal regulatory modulators represent a promising avenue through which diabetic blood glucose levels might be moderated clinically. Despite the critical role of small intestine in blood glucose homeostasis, most physiological diabetes research has focused on other organs, such as the pancreas, kidney, and liver. We contend that an improved understanding of intestinal regulatory mediators may be fundamental for the development of first-line preventive and therapeutic interventions in patients with diabetes and diabetes-related diseases. This review summarizes the major important intestinal regulatory mediators, discusses how they influence intestinal glucose absorption, and suggests possible candidates for future diabe
... for example, a novel data suggest that the aT 1 receptor mediates muscular contractions, that the aT 2 receptor regulates epithelial functions, and that the aT 2 receptors counteract effects mediated by aT 1 receptors in various tissues (Fändriks, 2010). There is evidence that aT 1 receptors are situated directly on the smooth musculature (Romero et al., 1998) and aT 2 receptors are not found in direct association with the smooth muscle cells (Schinke et al., 1991). The signal transduction mechanism for aT 1 receptors is well known. ...
The aim of this study was to analyze in detail and to compare the effects of Angiotensin II (Ang II) and Arginine - vasopressin (AV P) on the contractile activity of smooth muscle strips from different rat gastrointestinal segments by application of time-parameter analysis. Longitudinal muscle strips from the rat stomach and intestine were used for in vitro recording of contraction, iduced by Ang II (10-6 M) and AV P (10-6 M). Amplitude, area under the curve (AUC) and time-parameters of the curves force-vs.-time of the contraction were determined. The colon and rectum responded to Ang II with more powerful contractions (3.43 ± 0.56 g and 4.74 ± 0.65 g, respectively). Jejunum and colon from one side and stomach, duodenum and rectum on other hand showed a similar pattern of contractions and relaxations. The response of the ileum was different. It was shown bilateral symmetry in the responses of the gastrointestinal tract. The differences in the responses of smooth muscle strips on Ang II in the various segments are probably due to unequal distribution of the density and opposite effects of AT1 and AT2- receptors, the presence of local RAS and аctivation of various transduction pathways. AVP induced tonic contractions of the preparations from the stomach. In the intestines, AV P was ineffective. © 2014, Bulgarian Journal of Agricultural Science. All rights reserved.
... Differences between the circular and longitudinal muscle layers were identified with regard to magnitude of contraction, pD2 values and frequency. Differences in sensitivity to Ang II between the circular and the longitudinal musculature have previously been observed but the functional significance remains to be elucidated (Fishlock & Gunn 1970, Schinke et al. 1991). Ewert et al. showed that Ang II elicits concentrationdependent contractions in human duodenal, jejunal as well as ileal isolated muscle preparations (Ewert et al. 2006b). ...
Abstract Aim: Angiotensin II is well known for its contractile effects on smooth muscle cells. This effect is also present in the gut previously shown in animal models. The aim of this study was to clarify expression and localization of angiotensin II receptors in the human small intestine and to explore the pharmacological profile of angiotensin II effects in vitro. Methods: Strips of jejunal muscle wall from 32 patients undergoing bariatric surgery were used to record isometric tension in vitro in response to angiotensin II (10−10–10−5 m) alone and in the presence of PD123319 (10−7 m), losartan (10−7 m), PD123319 (10−7 m) and losartan (10−7 m) in combination, tetrodotoxin (TTX) (10−6 m), atropine (10−6 m) and guanethidine (3 × 10−6 m). Western blot, immunohistochemistry and RT-PCR were performed on corresponding muscle samples to identify expression and localization of key components of the renin–angiotensin system. Results: Angiotensin II elicited concentration-dependent contraction in both longitudinal and circular jejunal muscle wall strips; neither TTX, atropine nor guanethidine affected this action. Losartan alone and in combination with PD123319 shifted the concentration–response curve to the right. Transcription of angiotensinogen, ACE and angiotensin II types 1 and 2 receptor RNA was detected in all patients. Immunohistochemistry detected angiotensin II type 1 receptors in the musculature; both angiotensin II types 1 and type 2 receptors were found in the myenteric plexus. Conclusion: This pharmacological analysis indicates that the contractile action elicited by angiotensin II on jejunal wall musculature is primarily mediated through the angiotensin II type 1 receptor located on the musculature.
... 38 The presence of the AT 1 receptor antagonist losartan shifted the dose-response curve to the right, whereas an AT 2 receptor antagonist was without effect. The results generally confirmed a previous study by Schinke et al., 14 but indicated also that AT 1 receptors are situated directly on the smooth musculature. The latter interpretation could be confirmed by immunohistochemistry also showing that AT 2 receptors were not found in direct association with the smooth muscle cells. ...
The renin-angiotensin system (RAS) is well known for its vital involvement in body fluid homeostasis and circulation. However, very little research has been devoted to the impact of this regulatory system on the gastrointestinal (GI) system. This is surprising because the GI tract is fundamental for the intake and excretion of fluid and electrolytes (and nutrients), and it accommodates a large proportion of bodily haemodynamics and host defence systems. The RAS is well expressed and active in the GI tract, although the exact roles for the key mediator angiotensin II (Ang II) and its receptors in general, and the type 2 (AT( 2)) receptor in particular, are not completely settled. There are several reports showing Ang II regulation of intestinal fluid and electrolyte transport. For example, mucosaprotective duodenal bicarbonate-rich secretion is inhibited by Ang II via type 1 (AT(1)) receptor-mediated facilitation of sympathoadrenergic activity, but this secretory process can also be stimulated by Ang II via AT(2) receptors. Novel data from human oesophagus and jejunum suggest that the AT(1) receptor mediates muscular contractions and that the AT(2) receptor regulates epithelial functions. Data are accumulating suggesting involvement of AT(1) and AT(2) receptors in GI inflammation and carcinogenesis. The picture of the RAS and AT( 2) receptor in the GI tract is, however, far from complete. Much more basic research is needed with regard to GI pathophysiology before concluding clinical significance and potential applicability of pharmacological interferences with the RAS.
... The highest angiotensinconverting enzyme (ACE) mRNA, ACE protein, and activity within the intestine have been found in the brush-border membrane fraction in the proximal to mid region of the rat small intestine(6). Functional AT1Rs have been found in the rat ileum and duodenum(34). Other studies have shown that the AT1R antagonist inhibited the development of intestinal mucosal injury induced by ischemia-reperfusion in rats (40). ...
Although angiotensin II (Ang II) plays a key role in development of organ ischemia-reperfusion injury, it remains unclear whether it is involved in development of intestinal injury following trauma-hemorrhage (T-H). Studies have shown that 17beta-estradiol (E2) administration following T-H improves small intestinal blood flow; however, it is unclear whether Ang II plays a role in this E2-mediated salutary effect. Male Sprague-Dawley rats underwent laparotomy and hemorrhagic shock (removal of 60% total blood volume, fluid resuscitation after 90 min). At onset of resuscitation, rats were treated with vehicle, E2, or E2 and estrogen receptor antagonist ICI 182,780 (ICI). A separate group of rats was treated with Ang II subtype I receptor (AT1R) antagonist losartan. At 24 h after T-H, plasma Ang II, IL-6, TNF-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels, myeloperoxidase (MPO) activity, and AT1R expression were determined. T-H significantly increased plasma and intestinal Ang II, IL-6, TNF-alpha levels, intestinal ICAM-1, CINC-1, CINC-3 levels, MPO activity, and AT1R protein compared with shams. E2 treatment following T-H attenuated increased intestinal MPO activity, Ang II level, and AT1R protein expression. ICI administration abolished the salutary effects of E2. In contrast, losartan administration attenuated increased MPO activity without affecting Ang II and AT1R levels. Thus Ang II plays a role in producing small intestine inflammation following T-H, and the salutary effects of E2 on intestinal inflammation are mediated in part by Ang II and AT1R downregulation.
... However, they probably exert little, if any, direct physiological control in trout. The lack of any significant ANG II effect in fish gut smooth muscle is strikingly different from observations on the mammalian gut, where ANG II, acting predominantly through AT 1 -type receptors, is both directly and indirectly a potent stimulant of smooth muscle contractions (Schinke et al. 1991;Yang et al. 1993). It remains to be determined whether the lack of ANG II potency in fish gut smooth muscle is due to the relative unimportance of the RAS, or whether other key variables or cofactors were not present under the experimental conditions employed. ...
The renin/angiotensin system (RAS) is a tonic anti-drop regulator of arterial blood pressure in many teleosts. In trout, angiotensin II (ANG II) has no direct constrictor effect on large arteries or veins and the identity of specific cardiovascular pressor effectors is unknown. Potential targets of angiotensin activation were examined in the present experiments using perfused organs and isolated tissues from the rainbow trout Oncorhynchus mykiss.
Perfused gill (arches 2 and 3), perfused skeletal muscle-kidney (via the dorsal aorta; PDA) and perfused splanchnic (via the celiacomesenteric; PCM) circulations vasoconstrict in response to salmonid ANG II in a dose-dependent manner. ANG II was significantly (P⩽0.05) more potent in the PCM than in the PDA, and both preparations were more responsive than the gills: pD2=8.0±0.20 (10) for PCM; pD2=7.5±0.07 (13) for PDA; pD2=6.9 ±0.21 (8) for gill arch 3; pD2=6.7±0.23 (8) for gill arch 2; mean ± S.E.M. (N), respectively. Salmonid angiotensin I (ANG I) also produced a dose-dependent constriction of the PDA and PCM. Angiotensin converting enzyme (ACE) activated nearly 100% of ANG I to ANG II in a single pass through the PDA, whereas PCM conversion was estimated to be less than 10%. Inhibitors of adrenergic constriction partially prevented ANG II responses in the PDA but did not affect PCM responses.
ANG II did not affect paced rings of ventricular muscle in the presence of high or low [Ca2+] or epinephrine concentrations, nor did it have any inotropic or chronotropic effects in the in situ perfused heart. Red blood cell swelling was unaffected by ANG II. Similarly, the effects of ANG II on gut, urinary bladder and gall bladder smooth muscle were negligible or non-existent; thus, an increase in splanchnic resistance due to extravascular compression can be discounted.
These results indicate that, in trout, the systemic microcirculation is the major cardiovascular effector of angiotensin-mediated pressor responses. In addition, the RAS has little direct effect on non-vascular smooth muscle or the heart. From an evolutionary perspective, the initial site of direct systemic RAS action appears to be the vascular microcirculation.
... rats did not alter the characteristics of intestinal responses to bradykinin and induced the shift to the right of the dose-response curve for angiotensin II in the ileum. Bradykinin and angiotensin II evoked responses in the rat intestinal smooth Ž muscle via intracellular pathways Zagorodnyuk et al., . Ž 1998 mediated by different receptor types Schinke et al., . 1991;Calixto, 1995 . Therefore, our data may be due to Ž some changes in cell membrane properties such as decrease in the number andror affinity of angiotensin II . receptors rather than to alterations in the contractile apparatus of these muscles. No analysis of this machinery was done in the present study, although the gross structure of th ...
The following study is an investigation of the changes in the contractile reactivity of visceral muscles in response to agonists and alterations in metabolic parameters after neonatal rat treatment with monosodium-L-glutamate. This treatment markedly sensitizes ileum and colon preparations to adenosine-5'-triphosphate (ATP) stimulation and also increases the colon activity to acetylcholine (p<0.05). Response to bradykinin remained unchanged, while ileum activity to angiotensin II was characterized by a reduction in the maximal tension (E(max)) and an increase in the EC(50) (p<0.05) value. The responses of nonintestinal muscle preparations from monosodium-glutamate-treated rats to both ATP and bradykinin did not show a significant difference when compared to the controls. This treatment diminished food intake, feces excretion and increased plasma insulin, nonesterified fatty acids and triglyceride concentrations (p<0.001). These results suggest that the changes in intestinal muscle activity, in response to agonists, can be due to metabolic alterations as well as the monosodium glutamate action on enteric neurons and/or smooth muscle receptors.
... drenergic inhibition of duodenal mucosal alkaline secretion, are mediated by activation of AT 1 receptors, which are present in most organs including the intestine (4)(5)(6)22). ...
The aims of this study were to elucidate the distribution of angiotensin receptors (AT(1) and AT(2)) in the duodenal wall and to investigate whether AT(2) receptors are involved in the regulation of duodenal mucosal alkaline secretion, which is of importance for the mucosal defense against gastric acid. Immunohistochemistry was used to locate AT(1) and AT(2) receptors in chloralose-anesthetized rats. Duodenal mucosal alkaline output was measured by use of in situ pH-stat titration. Immunohistochemistry demonstrated a distinct staining for both AT(1) and AT(2) receptors in the lamina propria of the villi and also for AT(1) receptors in the muscularis interna. When angiotensin II was infused in the presence of the AT(1) receptor antagonist losartan, mucosal alkaline secretion increased by ~50%. This response was inhibited by the AT(2) receptor antagonist PD-123319. The AT(2) receptor agonist CGP-42112A increased mucosal alkaline secretion by ~50%. This increase was absent in the presence of PD-123319 but not in the presence of losartan or the local anesthetic lidocaine. We conclude that angiotensin II stimulates duodenal mucosal alkaline secretion by activation of AT(2) receptors located in the duodenal mucosa/submucosa.
... Angiotensin II (ANG II) receptors exist as subtype 1 (AT 1 ) and subtype 2 (AT 2 ) and are known to play a major role in the cardiovascular and renovascular systems that mediate inflammation, cell growth, fibrosis, and vascular tone. There has been considerable interest in the presence of a local angiotensin system in the gastrointestinal tract, including the demonstration of ANG II binding sites, angiotensin-converting enzyme, and AT 1 and AT 2 receptors in the rat jejunum [8][9][10][11] and, more recently, in a human intestinal cell line (B.A.C.-F., G.A.C.B., and R.L.G., personal communication). The AT 1 receptor is present throughout the body in most species that have been studied to date [12]. ...
Angiotensin II (ANG II) has been described in the regulation of intestinal secretion and absorption via angiotensin subtype
1 (AT1) and AT2 receptors, respectively, in rats. We investigated the role that ANG II plays in the rabbit ileal‐loop model of Clostridium difficile infection. Expression of AT1, the more abundant ANG II receptor, was demonstrated in ileal loops, and an AT1 receptor blocker, losartan, inhibited hypersecretion induced by C. difficile toxin A (mean volume&rcolon;length ratio, 0.27±0.06 vs. 0.60±0.06 mL/cm in controls). Losartan also decreased production of ANG II in the ileum (0.48±0.06 vs. 0.87±0.12 pg/mg in controls), raising the possibility that ANG II may participate in a positive feedback loop involving the hypersecretory
response. Our findings suggest that ANG II plays a significant role in the pathogenesis of C. difficile toxin–induced diarrhea.
... These observations can be explained, in part, by a pathological redistribution of blood flow giving rise to hypoxic microcirculatory units next to well-perfused or even overperfused normoxic units678 . Even in the absence of systemic hypotension, blood flow and capillary perfusion distribution in both endotoxin and focal models of sepsis can be highly heterogeneous between and within organ systems such as skeletal muscle and the small bowel mucosa [7,91011121314. Studies in critical care support a reduction in the red blood cell (RBC) transfusion threshold [15] and the use of recombinant human erythropoietin (rHuEPO) treatment to reduce transfusion requirements161718. However, besides the regulation of erythropoiesis [19], recent studies indicate that this hormone exerts complex actions promoting the maintenance of homeostasis of the organism under stressors such as oxidation induced during ischemic-reperfusion injury20212223. ...
The relationship between oxygen delivery and consumption in sepsis is impaired, suggesting a microcirculatory perfusion defect. Recombinant human erythropoietin (rHuEPO) regulates erythropoiesis and also exerts complex actions promoting the maintenance of homeostasis of the organism under stress. The objective of this study was to test the hypothesis that rHuEPO could improve skeletal muscle capillary perfusion and tissue oxygenation in sepsis.
Septic mice in three experiments received rHu-EPO 400 U/kg subcutaneously 18 hours after cecal ligation and perforation (CLP). The first experiment measured the acute effects of rHuEPO on hemodynamics, blood counts, and arterial lactate level. The next two sets of experiments used intravital microscopy to observe capillary perfusion and nicotinamide adenine dinucleotide (NADH) fluorescence post-CLP after treatment with rHuEPO every 10 minutes for 40 minutes and at 6 hours. Perfused capillary density during a three-minute observation period and NADH fluorescence were measured.
rHuEPO did not have any effects on blood pressure, lactate level, or blood cell numbers. CLP mice demonstrated a 22% decrease in perfused capillary density compared to the sham group (28.5 versus 36.6 capillaries per millimeter; p < 0.001). Treatment of CLP mice with rHuEPO resulted in an immediate and significant increase in perfused capillaries in the CLP group at all time points compared to baseline from 28.5 to 33.6 capillaries per millimeter at 40 minutes; p < 0.001. A significant increase in baseline NADH, suggesting tissue hypoxia, was noted in the CLP mice compared to the sham group (48.3 versus 43.9 fluorescence units [FU]; p = 0.03) and improved with rHuEPO from 48.3 to 44.4 FU at 40 minutes (p = 0.02). Six hours after treatment with rHuEPO, CLP mice demonstrated a higher mean perfused capillary density (39.4 versus 31.7 capillaries per millimeter; p < 0.001) and a lower mean NADH fluorescence as compared to CLP+normal saline mice (49.4 versus 52.7 FU; p = 0.03).
rHuEPO produced an immediate increase in capillary perfusion and decrease in NADH fluorescence in skeletal muscle. Thus, it appears that rHuEPO improves tissue bioenergetics, which is sustained for at least six hours in this murine sepsis model.
... AT 2 receptor has been shown to be involved in absorption/secretion of water and electrolytes (47,48),and to induce mucosal release of nitric oxide (29). However, this receptor does not influence smooth muscular contractions (49,50). Since Ang II has similar affinity to both of its receptor subtypes it is suggested in most cases the AT 1 receptor counteracts AT 2 receptor-mediated effects. ...
We previously demonstrated that angiotensin II (Ang II) receptor signaling is involved in azoxymethane-induced mouse colon tumorigenesis. In order to clarify the role of Ang II in COX-2 expression in the intestinal epithelium, the receptor subtype-specific effect on COX-2 expression in a rat intestinal epithelial cell line (RIE-1) has been investigated. Ang II dose- and time-dependently increased the expression of COX-2, but not COX-1 mRNA and protein. This stimulation was completely blocked by the AT(1) receptor antagonist but not the AT(2) receptor antagonist. Ang II and lipopolysaccharide (LPS) additively induced COX-2 protein in RIE-1 cells, whereas the LPS-induced COX-2 expression was significantly attenuated by low concentrations of Ang II or the AT(2) agonistic peptide CGP-42112A only in AT(2) over-expressed cells. These data indicate that Ang II bi-directionally regulates COX-2 expression via both AT(1) and AT(2) receptors. Control of COX-2 expression through Ang II signaling may have significance in cytokine-induced COX-2 induction and colon tumorigenesis.
In the first part of this article, the role of intestinal epithelial tight junctions (TJs), together with gastrointestinal dopaminergic and renin–angiotensin systems, are narratively reviewed to provide sufficient background. In the second part, the current experimental data on the interplay between gastrointestinal (GI) dopaminergic and renin–angiotensin systems in the regulation of intestinal epithelial permeability are reviewed in a systematic manner using the PRISMA methodology. Experimental data confirmed the copresence of DOPA decarboxylase (DDC) and angiotensin converting enzyme 2 (ACE2) in human and rodent enterocytes. The intestinal barrier structure and integrity can be altered by angiotensin (1-7) and dopamine (DA). Both renin–angiotensin and dopaminergic systems influence intestinal Na+/K+-ATPase activity, thus maintaining electrolyte and nutritional homeostasis. The colocalization of B0AT1 and ACE2 indicates the direct role of the renin–angiotensin system in amino acid absorption. Yet, more studies are needed to thoroughly define the structural and functional interaction between TJ-associated proteins and GI renin–angiotensin and dopaminergic systems.
Das Renin-Angiotensin-System (RAS) mit seinem Effektorpeptid Angiotensin II (Ang II) als zirkulierendem Hormon wird seit langem als ein klassisches endokrines System angesehen. Die Entdeckung des Renins geht auf das Jahr 1898 zurück, als Tigerstedt und Bergmann die Beobachtung machten, daß durch Injektion eines Kaninchennierenextrakts eine Blutdrucksteigerung in Kaninchen erzielt werden kann (277). In den folgenden Jahrzehnten wurde die Existenz des Vasopressors Renin kontrovers diskutiert, und erst 1934 wurde die Bedeutung von Renin für die Blutdruckregulation von Goldblatt erneut erkannt (99). Seiner Forschungsgruppe gelang es, einen Zusammenhang zwischen Reninfreisetzung und renaler Ischämie nachzuweisen. Nur wenige Jahre später, 1937, diskutierten Blalock und Levy die Abhängigkeit der Reninfreisetzung vom renalen Perfusionsdruck (20) und kamen zu dem Schluß, daß renale Barorezeptoren dabei eine Rolle spielen mußten (279). Die physiologische und biochemische Charakterisierung von Renin wurde in den folgenden Jahren vervollständigt. Die enzymatischen Eigenschaften von Renin wurden von Page und Helmer (204) sowie von Braun-Menendez et al. (26) beschrieben. Zur selben Zeit identifizierten Page et al. (205) das Renin-Substrat Angiotensinogen als ein Plasmaprotein und deuteten damit das Renin-Angiotensin-System als eine funktionelle Einheit. In jenen Tagen herrschte die Meinung vor, daß das Spaltprodukt von Angiotensinogen, das Dekapeptid Angiotensin I (Ang I), für die Vasokonstriktion verantwortlich sei.
A reliable and sensitive radioreceptor assay based on rat lung homogenate as receptor preparation was developed to determine the angiotensin-II antagonistic profile of losartan and its main active metabolite EXP 3174 as well as its congeners exemplified by UP 269-6 and SL 91.0102-90 DL. This method proved to be precise with an intra- and interday variability of less than 10% and a limit of quantification ≤1 ng ml−1. The analysis of the Ki values in protein-free Hepes-buffer versus blank human or rat plasma revealed the distinct high plasma–protein binding of EXP 3174 which consequently caused a dramatic drop of potency from 10–15-fold in the buffer to only about 2-fold in control plasma, when compared to the parent compound losartan and the two congeners investigated. Upon evaluation of clinical samples by both the reported radioreceptor assay (RRA) and the established high-performance liquid chromatography (HPLC), the correlation of the normalized data pairs (concentration equivalents) suggested the contribution of active metabolites to the angiotensin-II antagonistic effect of SL 91.0102-90 DL, but not to the effect of UP 269-6. In the context of an extended preclinical study in rats, the correlation of RRA with the respective HPLC concentration equivalents of losartan and its main active metabolite EXP 3174 confirmed previous findings that only losartan and EXP 3174 exert the angiotensin-II-AT1 receptor blockade without the contribution of other metabolites (P.C. Wong, W.A. Price, A.T. Chiu et al., J. Pharmacol. Exp. Ther. 255 (1990) 211–217).
The existence of a local renin/angiotensin system in the intestine of mammals is speculative despite the known importance of angiotensin II for water and electrolyte homeostasis. We demonstrate the presence of ren-2 transcripts in the small intestine of DBA/2 mice. The marked expression of the ren-2 gene is blunted tissue-specifically by starvation, corroborating a local renin/angiotensin system in this organ.
Cardiac hypertrophy results in an increased deposition of the extracellular matrix (ECM) proteins fibronectin and collagen. Recent evidence indicates that angiotensin II (Ang II) might have an important role in the development of myocardial fibrosis accompanying cardiac hypertrophy. We sought to determine whether fibroblasts of cardiac origin (isolated from neonatal and adult animals) express receptors for Ang II and to examine the ability of this peptide to regulate fibronectin and collagen gene expression in a cultured adult cardiac fibroblast cell preparation.
Binding of 125I-Ang II to both neonatal and adult cardiac fibroblasts in culture was specific, reversible, and saturable, with the receptor evenly distributed over the cell population. Competition binding experiments with receptor-specific antagonists indicate that Ang II receptors found on both fibroblast types were of the AT1 subtype. Analysis of mRNA levels for the AT1 receptor indicates that adult cardiac fibroblasts express higher levels of the message than neonatal fibroblasts or cardiac myocytes. Addition of 10(-9) mol/L Ang II to adult cardiac fibroblasts resulted in an induction of ECM proteins above control levels, as determined through Northern blots and total collagen assays.
Results from this study indicate that neonatal and adult rat cardiac fibroblasts in culture express AT1 receptors for Ang II. Ang II stimulation of AT1 receptors results in an increased gene expression for ECM proteins. These data suggest that Ang II might have important regulatory roles over cardiac fibroblast function under normal and pathological conditions.
Thanks to the recent discovery of angiotensin II (ANG II) receptor subtypes linked to different signalling pathways, research in the different areas related to this peptide has regained a strong interest. In the following review, we first describe the biochemistry and actions of angiotensin peptides formed both in the circulation and locally at the tissue and organ level. Evidence for the existence and distribution of ANG II receptor subtypes in mammalian as well as in nonmammalian species and lower organisms is presented. The changes in receptor subtype expression during development and disease are described. The signal transduction mechanisms and biological actions of ANG II mediated by the recently cloned AT1 receptor are reviewed and the recent data concerning the signalling pathways linked to the AT2 receptor are discussed. Finally, based upon their molecular pharmacology, we present evidence and also speculate upon the physiological function of the ANG II receptor subtypes.
1. The contractile responses to angiotensin II, angiotensin III and two synthetic analogues, [Lys2]angiotensin II and [Sar1]angiotensin II, in the guinea-pig isolated longitudinal muscle preparation of small intestine have been characterized in vitro. 2. Tachyphylaxis to the angiotensin analogues was reduced by use of a Krebs-Henseleit solution containing a raised (sub-contractile) concentration of potassium (11.2 mM). Under these conditions. reproducible cumulative concentration-response curves to all agonists were established. The pD2 estimates for angiotensin II, [Lys2]angiotensin II, angiotensin III and [Sar1]angiotensin II were 9.15 +/- 0.14, 7.42 +/- 0.06, 7.69 +/- 0.18 and 9.50 +/- 0.15 respectively and the maximum responses achieved were not significantly different. 3. The contractile responses to angiotensin II, angiotensin III and [Sar1]angiotensin II were reduced by greater than 80% by tetrodotoxin (TTX; 0.1 microM). However, the responses to [Lys2]angiotensin II were reduced by only 63 +/- 5%. Atropine (0.1 microM) also reduced the responses to angiotensin II, angiotensin III and [Lys2]angiotensin II, although its effect was less than that produced by TTX. Furthermore, while responses to these agonists were not significantly modified by the NK1 receptor antagonist (+/-)-CP-96,345 (30 nM) alone, the combined pre-incubation with both atropine and (+/-)-CP-96,345 reduced maximum agonist responses to a level not significantly different from those produced by TTX. 4. Indirect and direct contractile responses to angiotensin II and [Lys2]angiotensin II (in the presence of TTX) respectively were characterized by use of the selective AT1 receptor antagonist, losartan and the AT2 receptor antagonist, PD123177. Losartan produced parallel rightward displacement of the concentration-response curve to angiotensin II and [Lys2]angiotensin II, with an estimated pKB of 8.56(8.42-8.68) and 9.18 (8.63-9.50) respectively. The AT2 receptor antagonist, PD123177 (3 microM) failed to modify the contractile responses to either angiotensin II or [Lys2]angiotensin II.5. We conclude that two populations of angiotensin II receptors exist in the guinea-pig longitudinal muscle of small intestine, one located neuronally mediating the release of both acetylcholine and substance P and the other located on the smooth muscle mediating direct contractile responses. The neuronal component provides the major contribution to the agonist responses. Both receptor populations are of the AT1 receptor subtype.
A role for endogenous angiotensin II and its AT1 and AT2 receptor subtypes for mediating drinking elicited by eating was examined in adult male Sprague-Dawley rats. The ability of pharmacological antagonism of AT1 and/or AT2 receptors to abolish drinking elicited by exogenous angiotensin II was established first. The s.c. injection of the AT1 antagonist losartan (DuP 753) was sufficient to abolish drinking elicited by s.c. angiotensin II. The ICV injection (through a surgically implanted chronic cannula) of losartan inhibited drinking elicited by ICV angiotensin II; the combined ICV injection of losartan plus the AT2 antagonist PD123319 was sufficient to abolish drinking elicited by ICV angiotensin II. For rats drinking and eating after 24-h food deprivation, s.c. losartan plus PD123319 inhibited water to food ratio, but ICV losartan and/or PD123319 failed to inhibit food-related drinking. For nondeprived rats eating a small cracker, s.c. losartan and/or PD123319 attenuated water intake, but only ICV losartan produced statistically significant inhibition of drinking elicited by ingestion of cracker. The IG infusion (through a surgically implanted gastric catheter) of 2 ml 600 or 900 mOsm/kg NaCl, a treatment that is subthreshold for increase in systemic plasma osmolality at the initiation of drinking, elicited drinking that was attenuated by s.c. losartan and/or PD123319 and attenuated by ICV losartan only. The IG infusion of 2 ml 1800 mOsm/kg NaCl, a treatment that is above threshold for increase in systemic plasma osmolality at the initiation of drinking, elicited drinking that was not inhibited by S or ICV losartan and/or PD123319. These results demonstrate that peripheral AT1 and AT2 and central AT1 receptors for angiotensin II contribute to drinking elicited by eating and the gastrointestinal osmotic consequences of eating. These findings extend the evidence demonstrating a renal renin-angiotensin contribution to food-related drinking in rats.
This article has no abstract; the first 100 words appear below.
Angiotensins are peptide hormones derived from the protein precursor angiotensinogen by the sequential actions of proteolytic enzymes (Figure 1). The classic pathway of angiotensin synthesis includes a reaction catalyzed by angiotensin-converting enzyme (ACE), which occurs not only in plasma but also in the kidneys, brain, adrenal glands, ovaries, and possibly other tissues.¹ The intrarenal renin–angiotensin system affects glomerular filtration, as discussed below, but the importance of angiotensin synthesis in other tissues is not known. Angiotensin II, the principal effector of the renin–angiotensin cascade, can also be synthesized by a pathway that does not require ACE.² Angiotensin II stimulates a variety . . .
Supported in part by the Department of Veterans Affairs and the National Institutes of Health.
We are indebted to Ms. Kathryn Kleckner for assistance with the figures, to Ms. Susi Nehls for editorial assistance, and to Drs. Ernesto Schiffrin and Tamas Balla for helpful suggestions.
Source Information
From the William S. Middleton Memorial Veterans Hospital and the Departments of Medicine and Pharmacology, School of Medicine, University of Wisconsin, Madison (T.L.G., M.E.E.); and the Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. (K.J.C.).
Address reprint requests to Dr. Goodfriend at the William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terr., Madison, WI 53705.
The present studies assessed the levels of [125I][Sar1,ILE8]angiotensin II-labelled angiotensin AT1 and AT2 receptor recognition sites in homogenates of various brain areas (including caudate nucleus, putamen, substantia nigra, hippocampus, frontal cortex, temporal cortex and cerebellum) from patients with clinically diagnosed Parkinson's disease, Huntington's disease and Alzheimer's disease and those from age-, sex- and post-mortem delay-matched neurologically and psychiatrically normal patients. Radiolabelled angiotensin AT1 receptor recognition site levels were significantly decreased by approximately 70%, 70% and 90% in the caudate nucleus, putamen and substantia nigra, respectively, from patients with Parkinson's disease relative to matched controls. Furthermore, radiolabelled angiotensin AT2 receptor levels were decreased by some 60% in the caudate nucleus of patients with Parkinson's disease relative to control patients. In brain tissue homogenates from patients with Huntington's disease, the angiotensin AT1 receptor recognition site levels were decreased by approximately 30% in putamen relative to the control patients whilst angiotensin AT2 receptor levels were increased by some 90% in the caudate nucleus relative to the control patients. In brain tissue homogenates from patients with Alzheimer disease, the angiotensin AT2 receptor recognition site levels were significantly increased by approximately 200% in the temporal cortex relative to the control patients. The present results indicate that the reduction of angiotensin AT1 and/or AT2 receptor recognition site levels in the caudate nucleus, putamen and substantia nigra correlates with the principal neuropathology associated with Parkinson's disease and as such indicates that at least a significant population of angiotensin AT1 and AT2 receptors are located on the human dopaminergic nigrostriatal pathway. In addition, the marked increase in the levels of angiotensin AT2 receptor recognition sites in temporal cortex from patients with Alzheimer's disease correlates with some other markers associated with the renin-angiotensin system previously investigated in tissue from patients with this neurological disease.
The results obtained in the present work have shown that [125I]motilin bound specifically to basolateral (BL) membrane but it did not bind to the brush border (BB) membrane of the rabbit jejunum enterocyte. The [125I]motilin dissociation constant (Kd) was 95.58 +/- 15.0 pM and the receptor density (Bmax) was 2.54 +/- 0.40 fmol/mg protein. The binding of [125I]motilin to BL membrane was competitively inhibited by both unlabeled motilin and erythromycin. The IC50s were (2.1 +/- 0.4) 10(-8) M and (1.3 +/- 0.1) 10(-6) M for motilin and erythromycin, respectively, and the Ki were (6.83 +/- 1.3) 10(-9) M for motilin and (4.32 +/- 0.33) 10(-7) M for erythromycin. Saturation and competition binding studies showed interaction at only one class of binding sites in BL membrane.
Chronically elevated blood pressure results from pathological alterations in control systems. Current approaches to elucidate the underlying etiology strongly emphasize the (patho)physiological significance of the Renin-Angiotensin-Aldosterone System (RAAS) which interestingly interacts with the sympathetic, the cholinergic and purinergic systems. While the angiotensin-II-receptor subtype 1 (AT1), which mediates the blood-pressure-related effects of angiotensin II (All), has so far been extensively investigated, the physiological relevance of the other angiotensin-II-receptor subtypes-in particular of the AT2-receptor subtype-is about to be evolved by analysis of the various signal transduction mechanisms and by evaluation of transgenic animals, e.g. the knock-out mice, following disruption of the single A-II-receptor subtypes. Based on the clinical success of ACE inhibitors, the blockade of the Renin-Angiotensin-Aldosterone System in many different ways has been recognized as a successful strategy to effectively lower blood pressure.
A reliable and sensitive radioreceptor assay based on rat lung homogenate as receptor preparation was developed to determine the angiotensin-II antagonistic profile of losartan and its main active metabolite EXP 3174 as well as its congeners exemplified by UP 269-6 and SL 91.0102-90 DL. This method proved to be precise with an intra- and interday variability of less than 10% and a limit of quantification < or = 1 ng ml-1. The analysis of the Ki values in protein-free Hepes-buffer versus blank human or rat plasma revealed the distinct high plasma-protein binding of EXP 3174 which consequently caused a dramatic drop of potency from 10-15-fold in the buffer to only about 2-fold in control plasma, when compared to the parent compound losartan and the two congeners investigated. Upon evaluation of clinical samples by both the reported radioreceptor assay (RRA) and the established high-performance liquid chromatography (HPLC), the correlation of the normalized data pairs (concentration equivalents) suggested the contribution of active metabolites to the angiotensin-II antagonistic effect of SL 91.0102-90 DL, but not to the effect of UP 269-6. In the context of an extended preclinical study in rats, the correlation of RRA with the respective HPLC concentration equivalents of losartan and its main active metabolite EXP 3174 confirmed previous findings that only losartan and EXP 3174 exert the angiotensin-II-AT1 receptor blockade without the contribution of other metabolites (P.C. Wong, W.A. Price, A.T. Chiu et al., J. Pharmacol. Exp. Ther. 255 (1990) 211-217).
Ca2+ pathways activated by angiotensin II and carbachol were evaluated in the circular muscle of the guinea-pig ileum by recording mechanical and electrical activities. Transient contractions induced by angiotensin II were greatly reduced by Ca2+ removal from the medium whereas carbachol-induced responses were not significantly altered. Nifedipine had no effect on the responses to both agonists. A high concentration of tetrodotoxin (0.1 microM) inhibited angiotensin II-induced contractile responses without affecting the depolarization, whereas 1 mM Ni2+ inhibited the mechanical and electrical effects. Neither tetrodotoxin nor Ni2+ affected carbachol-induced effects. These results indicate that angiotensin II-induced phasic contractions depend on extracellular Ca2+ but not on voltage-dependent L-type Ca2+ channels. It is suggested that angiotensin II activates Ni2+-sensitive Na+ and non-specific cationic channels, whereas the responses to carbachol are dependent on receptor-activated Ca2+ release. Furthermore the different response of the longitudinal and circular muscles to the inhibitory effects of tetrodotoxin and Ni2+ on the angiotensin II- and carbachol-induced contractions indicates that these agonists exert their own myogenic effects on each layer and are able to trigger different Ca2+ mobilization pathways.
Since the discovery 100 years ago by Tigerstedt and Bergman of renin, an acid protease generating angiotensin peptide, numerous discoveries have advanced our understanding of the renin-angiotensin system (RAS). The recent cloning of angiotensin receptors and the availability of specific receptor ligands have allowed characterization of angiotensin-receptor-mediated actions, and an increasing number of studies using biochemical, pharmacological and molecular biological methods has focused on the many different physiological actions of the RAS in various tissues. Angiotensin II, the main effector peptide of the RAS, exerts most of its known actions in blood pressure control and body fluid homeostasis via the AT, receptor. AT, receptors not only play a role in growth control and cell differentiation but have been implicated in apoptosis and tissue regeneration. This review focuses on the extrarenal functions of angiotensin, especially in neuronal cells and the nervous system, and on recent advances in angiotensin receptor research.
To study the role of mast cell chymase in the inflammatory processes of human chronic gastritis. Experimental studies have shown that mast cell chymase stimulates inflammatory cell accumulation, and contributes to angiotensin II formation.
Tissue sections from human stomachs with Helicobacter pylori-associated gastritis (surgery/autopsy n = 20; biopsy n = 16) and normal stomachs (n = 10) were studied using immunohistochemical single and double labelling techniques. Monoclonal antibodies used were directed against mast cell chymase, tryptase, neutrophils (CD66b, elastase, and myeloperoxidase), macrophages, T-lymphocytes, and interleukin (IL)-4. The expression of angiotensin-converting enzyme and angiotensin II type 1 receptor was investigated using immunohistochemical analysis and the reverse transcription-polymerase chain reaction. The number of chymase-positive mast cells was significantly higher (P < 0.0001) in H. pylori-associated gastritis than in normal stomachs. Increased expression of chymase in inflamed mucosa was closely related to an increase in the accumulation of neutrophils, macrophages, T-lymphocytes, and IL-4-positive cells. The expression of angiotensin-converting enzyme and angiotensin II type 1 receptor was not altered in gastritis specimens.
These observations suggest that mast cell chymase may be an important mediator in the inflammatory processes of human H. pylori-associated gastritis.
Since the first identification of renin by Tigerstedt and Bergmann in 1898, the renin-angiotensin system (RAS) has been extensively studied. The current view of the system is characterized by an increased complexity, as evidenced by the discovery of new functional components and pathways of the RAS. In recent years, the pathophysiological implications of the system have been the main focus of attention, and inhibitors of the RAS such as angiotensin-converting enzyme (ACE) inhibitors and angiotensin (ANG) II receptor blockers have become important clinical tools in the treatment of cardiovascular and renal diseases such as hypertension, heart failure, and diabetic nephropathy. Nevertheless, the tissue RAS also plays an important role in mediating diverse physiological functions. These focus not only on the classical actions of ANG on the cardiovascular system, namely, the maintenance of cardiovascular homeostasis, but also on other functions. Recently, the research efforts studying these noncardiovascular effects of the RAS have intensified, and a large body of data are now available to support the existence of numerous organ-based RAS exerting diverse physiological effects. ANG II has direct effects at the cellular level and can influence, for example, cell growth and differentiation, but also may play a role as a mediator of apoptosis. These universal paracrine and autocrine actions may be important in many organ systems and can mediate important physiological stimuli. Transgenic overexpression and knock-out strategies of RAS genes in animals have also shown a central functional role of the RAS in prenatal development. Taken together, these findings may become increasingly important in the study of organ physiology but also for a fresh look at the implications of these findings for organ pathophysiology.
Angiotensin II (Ang II) is a well-known activator of smooth muscle in the vasculature but has been little explored with regard to intestinal wall muscular activity. This study investigates pharmacological properties of Ang II and expression of its receptors in small-intestinal smooth muscle from rats and humans.
Isometric recordings were performed in vitro on small intestinal longitudinal muscle strips. Protein expressions of Ang II typ 1 (AT1R) and typ 2 (AT2R) receptors were assessed by Western blot.
Ang II elicited concentration-dependent contractions of rat jejunal and ileal muscle preparations. The concentration-response curve (rat ileum, EC(50): 1.5 +/- 0.9 x 10(-8) M) was shifted to the right by the AT1R receptor antagonist losartan (10(-7) M) but was unaffected by the AT2R antagonist PD123319 (10(-7) M) as well as by the adrenolytic guanethidine (3 x 10(-6) M) and the anticholinergic atropine (10(-6) M). Human duodenal, jejunal and ileal longitudinal muscle preparations all contracted concentration-dependently in response to Ang II. The concentration-response curve (human jejunum, EC(50): 1.5 +/- 0.8 x 10(-8) M) was shifted to the right by losartan (10(-7) M) but was unaffected by PD123319 (10(-7) M). Both AT1R and AT2R were detected in all segments of the rat small intestinal wall musculature, whereas only AT1R was readily detectable in the human samples.
Ang II elicits contractions of small-intestinal longitudinal muscle preparations from the small intestine of rats and man. The pharmacological pattern and protein expression analyses indicate mediation via the AT1R.
Angiotensin II is well known for its contractile effects on smooth muscle cells. This effect is also present in the gut previously shown in animal models. The aim of this study was to clarify expression and localization of angiotensin II receptors in the human small intestine and to explore the pharmacological profile of angiotensin II effects in vitro.
Strips of jejunal muscle wall from 32 patients undergoing bariatric surgery were used to record isometric tension in vitro in response to angiotensin II (10(-10)-10(-5) M) alone and in the presence of PD123319 (10(-7) M), losartan (10(-7) M), PD123319 (10(-7) M) and losartan (10(-7) M) in combination, tetrodotoxin (TTX) (10(-6) M), atropine (10(-6) M) and guanethidine (3 x 10(-6) M). Western blot, immunohistochemistry and RT-PCR were performed on corresponding muscle samples to identify expression and localization of key components of the renin-angiotensin system.
Angiotensin II elicited concentration-dependent contraction in both longitudinal and circular jejunal muscle wall strips; neither TTX, atropine nor guanethidine affected this action. Losartan alone and in combination with PD123319 shifted the concentration-response curve to the right. Transcription of angiotensinogen, ACE and angiotensin II types 1 and 2 receptor RNA was detected in all patients. Immunohistochemistry detected angiotensin II type 1 receptors in the musculature; both angiotensin II types 1 and type 2 receptors were found in the myenteric plexus.
This pharmacological analysis indicates that the contractile action elicited by angiotensin II on jejunal wall musculature is primarily mediated through the angiotensin II type 1 receptor located on the musculature.
The activity of angiotensin converting enzyme (ACE) has been studied on functional parameters of intact isolated preparations of extrapulmonary tissues. The conversion of angiotensin I (A I) to angiotensin II (A II) and the cleavage of bradykinin (BK) were used as indicators of ACE activity. Captopril was employed as a specific inhibitor of ACE.
Captopril augmented the BK‐induced contractions of the rat isolated uterus, the BK‐ and substance P‐induced contractions of the guinea‐pig ileum, and the BK‐induced venoconstriction in the isolated perfused ear of the rabbit. Degradation of BK by ACE was calculated to be 52% in the rat uterus and 75% in the rabbit perfused ear.
Captopril inhibited the A I‐induced contractions of the rat isolated colon, the A I‐induced vasoconstriction in the isolated perfused ear of the rabbit and the rise in blood pressure induced by i.a. injections of A I in pithed rats. Conversion of A I to A II was calculated to be 13% in the rat colon and 26% in the rabbit perfused ear.
From estimations of the A II activity (bioassay on the rat colon) in the effluent of the perfused ear of the rabbit after injections of A I into the arterial inflow cannula it was calculated that approximately one tenth of A I was converted to A II during a single passage through the ear (less than 15 s).
The present experiments suggest that the high activity of ACE in endothelium of blood vessels of extrapulmonary tissues may provide an additional (endothelium‐dependent) local vasoconstrictor mechanism by the rapid formation of A II and inactivation of BK. The ACE activity in non‐vascular smooth muscles, other than those of blood vessels, may also affect the physiological functions of these tissues.
Previous studies have documented that high affinity binding of [125I]angiotensin II to adrenal cortex receptors was modulated by guanine nucleotides. Since in other receptor systems, similar properties of hormone-receptor interactions were shown to be specific for agonists, we studied the differential binding characteristics of agonists and antagonists to this receptor using a new radiolabeled antagonist [125I] [Sar1,Ile8] angiotensin II. Receptor saturation studies indicate that the antagonist is binding to a homogeneous population of sites with a Kd of 0.6-2.0 nM and with a receptor density around 1 pmol/mg of protein. Competition curves using unlabeled antagonists are characterized by a slope factor of 1 and a single Kd of 1-3 nM. Addition of guanylylimidodiphosphate to the assay is absolutely without effect on radiolabeled antagonist binding. In contrast, competition curves using the full agonists angiotensin II, [Sar1]angiotensin II, angiotensin III, and [des-Arg]angiotensin III display slope factors of 0.79, 0.87, 0.70, and 0.84, respectively. These curves can be explained by two apparent forms of the receptor having high and low affinity for the agonist. The higher affinity form associated with these four agonists is characterized by a Kd of 1.2 nM, 0.25 nM, 0.8 nM, and 3 microM, and corresponds to 60, 56, 42, and 25% of angiotensin II-binding sites, respectively. The other form displays 13- to 33-fold lower affinity. Addition of guanine nucleotide to the assay results in a 2-4-fold shift to the right and a steepening (slope factor 0.9-1.0) of agonist competition curves. Angiotensin II receptors, occupied by the full agonist [131I] [Sar1] angiotensin II or by the antagonist [125I] [Sar1, Ile8]angiotensin II, were then solubilized with the nonionic detergent octylglucoside. Dissociation of the agonist [131I] [Sar1] angiotensin II from solubilized receptors is enhanced by guanylylimidodiphosphate or sodium acetate, while dissociation of the antagonist [125I] [Sar1, Ile8]angiotensin II displays little sensitivity towards guanine nucleotides or increased ionic strength. Inclusion of bile salts in the solubilization medium preferentially destabilizes receptor-bound agonist, presumably by interfering with protein-protein interactions required for high affinity agonist binding. Separation of radiolabeled agonist and antagonist-occupied solubilized receptor complexes by steric exclusion high performance liquid chromatography reveals that the agonist-occupied receptor complex behaves as a larger protein than the antagonist-occupied receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)
1. The Dose-Response Relation.- The Dose-Response Relation.- Methods of Plotting Dose-Response Curves.- Drug Antagonism.- Use of Dose-Response Curves.- Enhancement of Drug Action.- References.- 2. Functions and Relations.- Mathematical Symbols and Conventions.- Relations and Functions.- The Linear Function.- Equations in Linear Form: Scatchard and Lineweaver-Burk Plots.- Power Functions.- Exponential Functions: Half-Life.- Logarithms and Logarithmic Functions: The Henderson-Hasselbach Equation.- Rate of Change and Drug Action.- Integration.- References.- Additional Readings.- 3. Kinetics of Drug-Receptor Interaction: Interpreting Dose-Response Data.- Pharmacological Receptor.- Formation of the Drug-Receptor Complex.- Classical Theory.- Modification of Classical Theory.- Dissociation Constants of Competitive Antagonists.- Dissociation Constants of Agonists: Method of Partial Irreversible Blockade.- Dissociation Constants of Agonists: Method of Partial Agonists.- Perturbation Methods.- Allosteric Theory.- Rate Theory.- References.- 4. Construction of Dose-Response Curves: Statistical Considerations.- Mean Dose and Mean Response.- Mean and Standard Deviation.- Samples and Populations.- Distributions.- Normal Distribution.- Estimation.- Tests of Significance.- Linear Regression.- Parallel Lines-Assays and Antagonism.- Quantal Dose-Response Relation.- Probit Diagram.- References.- Additional Readings.- 5. Drug Binding and Drug Effect.- Receptor Interaction and Effect.- Binding Constants and Dissociation Constants.- Desensitization.- Molecularity and Order.- Pharmacokinetic Considerations.- In Vivo Considerations.- Protein Binding.- Receptor Status and Disease States.- References.- 6. Isolated Preparations: Dose-Response Data.- Rabbit Thoracic Aorta.- Guinea Pig Ileum.- Isolated Taenia Ceca.- Ductus Deferens Preparation of the Guinea Pig and Rat.- Rat Fundus Strip.- Phrenic Nerve Diaphragm Preparation of the Rat.- Rat Uterus Preparation.- Frog Rectus Abdominus.- Isolated Rabbit Heart.- References.- Appendix A. Mathematical Tables.- Appendix B. Molecular Weights of Selected Drugs and Composition of Solutions.- Appendix C. Calculus.
Angiotensin receptor binding interactions of the angiotensin agonist, 125I-angiotensin II (125I-AII), and antagonist, 125I-[sarcosine1,leucine8]angiotensin II (125I[Sar1,Leu8]AII), are differentially affected by sodium ion concentration. 125I-AII binding to calf cerebellar cortex or adrenal cortex is increased 25 or 2.5 fold respectively when sodium ion concentration is increased from 10 to 150 mM. In brain membranes increasing sodium concentration accelerates the association and slows the dissociation of 125I-AII. 125I[Sar1,Leu8]AII binding to these tissues is much less sensitive to changes in sodium ion concentration. In rabbit uterine homogenates, neither 125I-AII nor 125I[Sar1,Leu8]AII binding is significantly altered by changes in sodium ion concentration. The sodium elicited increase in 125I-AII binding to calf cerebellum is correlated with cationic size and is not an ionic strength effect. The effect of sodium on potencies of angiotensin analogues in competing for 125I[Sar1,Leu8]AII binding does not correlate with agonist or antagonist properties, but is largest for peptides with aspartic acid at position one in the peptide structure.
A theoretical analysis has been made of the relationship between the inhibition constant (KI) of a substance and the (I50) value which expresses the concentration of inhibitor required to produce 50 per cent inhibition of an enzymic reaction at a specific substrate concentration. A comparison has been made of the relationships between KI and I50 for monosubstrate reactions when noncompetitive or uncompetitive inhibition kinetics apply, as well as for bisubstrate reactions under conditions of competitive, noncompetitive and uncompetitive inhibition kinetics. Precautions have been indicated against the indiscriminate use of I50 values in agreement with the admonitions previously described in the literature. The analysis described shows KI does not equal I50 when competitive inhibition kinetics apply; however, KI is equal to I50 under conditions of either noncompetitive or uncompetitive kinetics.
When studied on isolated rat mesenteric arteries perfused with Tyrode's solution, angiotensin I and angiotensin II (1 ng/ml), a synthetic tetradecapeptide renin substrate, and a purified hog renin substance (50-100 ng/ml) potentiated vasoconstrictor responses to sympathetic nerve stimulation and to injected norepinephrine without altering basal pressure. These agents produced a greater augmentation of the vasoconstrictor responses to nerve stimulation than to injected norepinephrine. The potentiation of vasoconstrictor responses to sympathetic nerve stimulation and injected norepinephrine which was elicited by renin substrate and angiotensin I was abolished by an inhibitor of angiotensin I-converting enzyme, SQ 20,881, and by an angiotensin II receptor antagonist, [Sar1-Ile8]angiotensin II. In contrast, the potentiating effect of angiotensin II was blocked only by the latter compound. We conclude that utilization of renin substrate within the vascular wall by renin or renin-like enzymes results in the formation of angiotensin I, which is converted to angiotensin II. Angiotensin in turn potentiates the vasoconstrictor responses to adrenergic stimuli presumably by augmenting release of the adrenergic transmitter and inhibiting its neuronal reuptake as well as by increasing vascular reactivity to norepinephrine.
Angiotensin II receptor binding sites in rat liver and PC12 cells differ in their affinities for a nonpeptidic antagonist, DuP 753, and p-aminophenylalanine6 angiotensin II. In liver, which primarily contains the sulfhydryl reducing agent-inhibited type of angiotensin II receptor, which we refer to as the AII alpha subtype, DuP 753 displays an IC50 of 55 nM, while p-aminophenylalanine6 angiotensin II displays an IC50 of 8-9 microM. In PC12 cells, which primarily contain the angiotensin II receptor type whose binding affinity is enhanced by sulfhydryl reducing agents (AII beta), DuP 753 displays an IC50 in excess of 100 microM, while p-aminophenylalanine6 angiotensin II displays an IC50 of 12 nM. p-Aminophenylalanine6 angiotensin II binding affinity in liver is decreased in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) suggesting that this analogue is an agonist.
[125I]Sar1, Ile8 angiotensin II labeled two distinct binding sites in rat brain. The displacement potencies of WL-19, a selective ligand for the angiotensin II subtype 2 receptor, angiotensin II and related peptides indicated that one binding site in the rat brain is the same as the adrenal angiotensin subtype 2 receptor. The second binding site in rat brain was displaced by the selective angiotensin II subtype 1 receptor antagonist DuP-753; however, the displacement potencies of angiotensin II, angiotensin III and Ile7-angiotensin III were significantly less than at the adrenal angiotensin subtype 1 receptor. The data suggests that this binding site in rat brain may represent an angiotensin II receptor subtype which shares some characteristics with the adrenal angiotensin subtype 1 receptor.
The purpose of this study was to map the distribution of angiotensin II (ANG II) receptors and ANG I-converting enzyme (ACE) in rat intestine. ANG II binding sites were visualized by in vitro autoradiography using iodinated [Sar1, Ile8]ANG II. The distribution of ACE was mapped using an iodinated derivative of lisinopril. Male Sprague-Dawley rats were killed and the interior of the whole intestine washed with ice-cold saline. Segments of duodenum, jejunum, ileum, and colon were quickly frozen in a mixture of isopentane and dry ice. Twenty-micron frozen sections were thaw-mounted onto gelatin-coated slides, incubated with either ligand, and exposed to X-ray film. After exposure and subsequent development, the films were quantitated by computerized densitometry. ANG II receptors were most dense in the colon, followed by the ileum, duodenum, and jejunum. Within each segment of intestine, specific ANG II binding sites were localized exclusively to the muscularis. In contrast, ACE was present in both the mucosa and the muscularis. The colocalization of ANG II receptors and ACE may suggest a role for locally generated ANG II in the control of intestinal function. The luminal orientation of ACE in the mucosa of the small intestine may suggest that at this site ACE serves primarily to hydrolyze dietary peptides.
We have demonstrated the existence of two distinct subtypes of the angiotensin II receptor in the rat adrenal gland using radioligand binding and tissue section autoradiography. The identification of the subtypes was made possible by the discovery of two structurally dissimilar, nonpeptide compounds, DuP 753 and EXP655, that show reciprocal selectivity for the two subtypes. In the rat adrenal cortex, DuP 753 inhibited 80% of the total AII binding with an IC50 value on the sensitive sites of 2 x 10(-8) M, while EXP655 displaced only 20%. In the rat adrenal medulla, EXP655 gave 90% inhibition of AII binding with an IC50 value of 3.0 x 10(-8) M, while DuP 753 was essentially inactive. The combination of the two compounds completely inhibited AII binding in both tissues.
A growing body of evidence suggests that angiotensin II, the effector protein of the renin-angiotensin system, is intimately involved with cell growth in target tissues. Most recently, evidence has been provided to indicate that angiotensin II is capable of inducing a hypertrophic response in cultured arterial smooth muscle cells. At the same time, considerable evidence has been developed to indicate that local analogs of the systemic renin-angiotensin system exist in multiple tissues and, in particular, in the vascular wall and the heart. Finally, data have accumulated to indicate that local growth regulatory factors, in many instances operating through regulation of proto-oncogene transcription, are involved in the hypertrophic and hyperplastic sequelae of hypertension. Included amongst these growth factors is angiotensin II. Thus, accumulating data indicate that angiotensin II is a growth factor with potential implications for the development of the sequelae of hypertension. In addition, studies from this laboratory and others suggest that angiotensin acts at least partially through what we have called an "intracrine" mechanism to produce its effects. In these multiple actions, angiotensin may provide a paradigm for other peptide growth factors and hormones.
Two angiotensin II receptor subtypes (A and B) are described from rat and human tissues. They have been characterised using specific peptidic and non-peptidic ligands with affinities differing by 1000 fold or more. These subtypes are present in adrenal glomerulosa of both species. Human uterus contains only subtype A, whereas both subtypes are found in rat uterus. Vascular smooth muscle cells in culture express only subtype B. Dithio-threitol totally inhibits binding to subtype B, but enhances the affinity to subtype A. There is a good correlation between the affinities of the selected agonists and antagonists for the two subtypes in the various tissues tested which is a usual requirement for receptor classification.
Antagonist binding to rat pancreatic muscarinic receptors was relatively slow at 25 degrees (tracer dissociation half-life, 50 to 60 min). We, therefore, chose this system to investigate the errors induced by nonequilibrium incubations on the estimates of receptor capacity and selectivity, in binding studies. We took advantage of the fact that muscarinic antagonists recognize only one receptor subtype in rat pancreatic homogenates and that association and dissociation kinetics conform to the law of mass action to analyze quantitatively the binding kinetics of [3H]N-methylscopolamine and of several unlabeled progressive to these receptors. We observed no correlation between the affinities of drugs for muscarinic receptors and their dissociation rate constants. As a result, the apparent receptor specificity (based on relative affinities for different antagonists) varied markedly with the incubation period. We, therefore, strongly recommend that in general competition curves established for receptor classification should be compared at different incubation periods to ensure that equilibrium is attained. The association rate constants of muscarinic antagonists for rat pancreas receptors were remarkably low, when compared with other ligand-receptor systems. This suggests that the antagonist-pancreatic muscarinic receptor association reaction included a rate-limiting conformational change of the drug-receptor complex. This isomerization step was not directly detectable in our kinetic studies, due to the very low affinity and rapid dissociation rate of the initial nonisomerized complex.
Specific [ ¹²⁵ I]‐angiotensin II (AII) and [ ¹²⁵ I]‐bradykinin (Bk) binding sites have been identified within epithelial membranes from rat jejunum and descending colon.
These high affinity intestinal sites exhibited K D values of 0.64 ± 0.16 nM for [ ¹²⁵ I]‐AII and 0.69 ± 0.13 nM for [ ¹²⁵ I]‐Bk, which were similar to those for [ ¹²⁵ I]‐AII (0.85 nM) and [ ¹²⁵ I]‐Bk binding sites (1.03 nM) previously identified in renal cortex epithelia.
Specific [ ¹²⁵ I]‐AII binding capacity was only 19.77 ± 2.74 fmol mg ⁻¹ in small intestine and 11.31 ± 2.66 fmol mg ⁻¹ in descending colon epithelia while a larger population, 332.0 ± 72.9fmol‐mg ⁻¹ of specific [ ¹²⁵ I]‐Bk sites were identified in epithelial membranes from small intestine.
Significant hydrolysis of both free [ ¹²⁵ I]‐AII and [ ¹²⁵ I]‐Bk was observed while membrane bound peptides remained relatively resistant to degradation. Whilst no corrections have been made to the observed values of K D and B max quoted above, one may assume that the calculated reductions in the free hormone concentration will result in a decrease of the K D value for both peptides. Loss of membrane bound peptide, particularly of [ ¹²⁵ I]‐AII, may indicate that the calculated B max value is an underestimation.
Despite the rapid degradation of unbound [ ¹²⁵ I]‐AII and [ ¹²⁵ I]‐Bk during incubations the kinetics of specific peptide binding were reversible and highly selective. The order of potency for specific [ ¹²⁵ I]‐AII binding was [Sar ¹ , Leu ⁸ ]‐AII > [Sar ¹ , Thr ⁸ ]‐AII > AII > [Sar ¹ , Ile ⁸ ]‐AII > [Des, Asp ¹ , Ile ⁸ ] AII > AIII. Specific [ ¹²⁵ I]‐Bk binding was also highly selective, the order of potency being Phe ⁸ ‐Bk>Tyr ⁸ ‐Bk>Lys‐Bk>Des, Arg ¹ ‐Bk. AII exhibited an IC 50 of > ImM for specific [ ¹²⁵ I]‐Bk binding and likewise Phe ⁸ ‐Bk for specific [ ¹²⁵ I]‐AII binding.
The effects of various modulators (cations, Gpp(NH)p) of hormone-receptor interaction were tested on agonist [( 125I]angiotensin II) and antagonist (125I-[Sar1,Ala8]angiotensin II) binding to membrane particles from the rat adrenal zona glomerulosa and uterine smooth muscle. The two radioiodinated peptides labeled the same population of binding sites. Sodium ion (140 mM) induced a 2 fold increase in the affinity of adrenal angiotensin II receptors for the agonist (Ka = 2.15 nM-1, vs. 1.01 nM-1 for controls), but decreased antagonist binding by reducing the number of available receptors by up to 50% in both adrenal and uterine membrane particles. Potassium ion only inhibited antagonist binding. Calcium and magnesium ions (0-10 mM) increased agonist binding and decreased antagonist binding to adrenal and uterine angiotensin II receptors, an effect mediated by changes in both affinity and number of receptors for the two peptides. The non-hydrolyzable GTP analog, Gpp(NH)p (10(-9) - 10(-4) M) decreased the affinity of angiotensin II receptors for the agonist by up to 50%, but did not affect antagonist binding to the receptor. Thus, there were marked differences in the sensitivity of agonist and antagonist peptides of angiotensin II to the modulatory effect of cations and guanyl nucleotides on ligand-receptor interaction. It is suggested that these differences may be important in determining the activatory/inhibitory properties of angiotensin peptides.
The brain contains its own angiotensin II (AII) system. To better understand the role of central AII in cardiovascular regulation, we used 125I-[Sar1, Ile8]-AII (125I-SI-AII), radioactive AII antagonist, to autoradiographically localize putative AII receptor binding in many parts of the central nervous system of the spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. With 125I-SI-AII binding on brain membrane preparations. Scatchard analysis indicated that Kd values were from 0.10 +/- 0.04 nM to 0.13 +/- 0.05 nM, whereas Bmax values (femtomol/mg protein) were found to be from 6.95 +/- 1.60 to 15.52 +/- 4.99 among brain regions studied. Various SI-AII receptor binding activities among brain regions revealed in this study were therefore most likely due to differences in AII receptor density with high affinity binding of 125I-AII. Using 125I-SI-AII, specific binding for SI-AII was found in the nucleus tractus solitarius (NTS), paraventricular hypothalamic nucleus (PVN), subfornical organ (SFO), suprachiasmatic nucleus (SCN), area postrema, the dorsal motor nucleus of the vagus (DMX), and the nucleus of spinal tract of the trigeminal system (NSV). With quantitative receptor autoradiography in conjunction with radioactive standards, we have observed that the NTS possesses the highest SI-AII binding, followed by the PVN, SFO, NTS, DMX, and NSV. No significant differences were observed between the SHR and WKY rats in the SI-AII binding within the SFO, PVN and NTS. However, SHR at early hypertensive (7 weeks) and established hypertensive (16 weeks) stages contained significantly higher SI-AII bindings in the NSV, as compared to age-matched WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Specific, high-affinity (Kd approximately equal to 0.6 nM), and saturable (3.3 fmol/mg of tissue, wet weight) binding of 125I-labeled [Sar1,Ile8]angiotensin II to rat ovarian membranes was observed. Displacement of 125I-labeled [Sar1,Ile8]angiotensin II binding to rat ovarian membranes by angiotensin II analogs and fragments resembled the potency order of these compounds on angiotensin II receptors in other tissues: [Sar1,Ile8]angiotensin II greater than angiotensin II greater than des-Asp1-angiotensin II greater than angiotensin I greater than des-Asp1,Arg2-angiotensin II. Several unrelated peptides, including follicle-stimulating hormone at 10 microM, did not displace ovarian 125I-labeled [Sar1,Ile8]angiotensin II binding. Autoradiograms of 125I-labeled [Sar1,Ile8]angiotensin II binding to ovarian sections indicated that the angiotensin II receptor binding sites were localized exclusively to a subpopulation of follicles, occurring on the granulosa and theca interna cells. Other follicles were devoid of 125I-labeled [Sar1,Ile8]angiotensin II binding sites. Angiotensin II immunoreactive material was also identified in the ovary. The concentration of ovarian Ang II immunoreactivity was 8- to 75-fold greater than that of plasma, was not reduced in bilaterally nephrectomized rats, and was shown by high-pressure liquid chromatographic analysis to be the native angiotensin II octapeptide. The presence of angiotensin II and its receptor binding sites in the ovary suggests a role for angiotensin II as a regulator of ovarian function.
The radiolabeled angiotensin II (ANG II) antagonist, [N 125I]-sar1,ile8-ANG II, was used to study brain ANG II receptors by both homogenate binding and in vitro autoradiography. In homogenate preparations of the hypothalamus, thalamus, septum and midbrain (HTSM), [125I]-sar1,ile8-ANG II bound to a single class (Hill slope 0.84 +/- 0.05) of high affinity binding sites (KD 0.42 +/- 0.03 nM, Bmax 5.98 +/- fmol/mg protein). Competition for the [125I]-sar1,ile8-ANG II binding site in HTSM membranes demonstrated a rank order potency characteristic of binding to the ANG II receptor, with the unlabeled antagonist being slightly more potent than ANG II (Ki 0.22 +/- 0.03 vs 0.95 +/- 0.06 nM, respectively). Brain slices from the region of the rostral third ventricle were incubated with 0.5 nM[125I]-sar1,ile8-ANG II in the presence or absence of 1 microM ANG II and exposed to LKB Ultrofilm. Autoradiographic images of [125I]-sar1,ile8-ANG II binding revealed that structures situated within the anterior wall of the third ventricle, i.e. the lamina terminalis, were heavily labeled; including the subfornical organ, median preoptic nucleus and organum vasculosum laminae terminalis. These results show the utility of [125I]-sar1,ile8-ANG II as a probe to study brain ANG II receptors and provides pharmacological evidence for the rostral third ventricle as a possible site for central ANG II actions.
The concentration of renin substrate (RS) was measured in rat mesenteric artery tissue. The concentration of this substrate both in arterial tissue and in plasma was markedly higher in rats 1 day after bilateral nephrectomy than in sham-operated controls, the percentage difference being higher in plasma than in arterial RS. Conversely, the decrease apparently induced 3 days after adrenalectomy (i.e., the difference in RS concentration from sham-operated rats) was greater in arterial tissue than in plasma. This finding may be explained by changes in RS concentrations induced by the sham operation. Sham surgery itself increased plasma RS after 1 day (but not after 3 days) and arterial RS after 3 days (but not after 1 day). There was a positive correlation between arterial and plasma renin substrate concentration for the overall results but not within individual groups. As renin and angiotensin-converting enzyme activity are also present in arterial tissues, all the necessary components for local generation of angiotensin II have now been shown to be present within the wall of resistance vessels.
G proteins: transducers of receptor-generated signals Prints, 1'486, Localization of central angiotensin il receptors with [~zsl]-Sar',ile~-angioten -sin ll: periventricular sites of the anterior third ventricle
- A G Gilman
Gilman, A.G., 1987, G proteins: transducers of receptor-generated signals, Ann. Rev. Biochem. 56, ~15. Healey, D.P., A.R. Maciejewski and M.P. Prints, 1'486, Localization of central angiotensin il receptors with [~zsl]-Sar',ile~-angioten -sin ll: periventricular sites of the anterior third ventricle, Neu-roendocrinolog~ 44, 15.
Quantitative autoradiography of IZSl-[Sarl,lle~]-angiotensin II binding in the brain of spontaneously hypertensive rats Demonstration of extrapulmonary activity of angiotensin-converting enzyme in intact tissue preparations
- B H Hwang
- J W Harding
- D K Liu
- L S Hibbard
- C M Wiczorek
- J Y Wu
Hwang, B.H., J.W. Harding, D.K. Liu, L.S. Hibbard, C.M. Wiczorek and J.Y. Wu, 1986, Quantitative autoradiography of IZSl-[Sarl,lle~]-angiotensin II binding in the brain of spontaneously hypertensive rats, Brain Res. Bull. 16, 75. Lembeck, F., T. Griesbacher and M. Eckhardt, 1990, Demonstration of extrapulmonary activity of angiotensin-converting enzyme in intact tissue preparations, Br. J. Pharmacol. 100, 49. Lowry, H.O., N.J. Rosebmugh, A.L. Farr and R.J. Randell, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265.
Two angiotensin II binding sites in rat brain revealed using ~251-SarL lleS-angiotensin II and selective nonpeptide antagonists Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 per cent inhibition (IC5.) of an enzymatic reaction
- R S L Chang
- V J Lotti
- T B Chen
- K A Faust
Chang, R.S.L., V.J. Lotti, T.B. Chen and K.A. Faust, 1990, Two angiotensin II binding sites in rat brain revealed using ~251-SarL lleS-angiotensin II and selective nonpeptide antagonists, Biochem. Biophys. Res. Commun. 171,813. Cheng, Y.C. and W.H. Prusoff, 1973, Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 per cent inhibition (IC5.) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099.
The hepatic angiotensin 11 receptor
- Crane