Angiotensin peptide synthesis and cyclic nucleotide modulation in sympathetic stellate ganglia.

Chronically elevated angiotensin II is a widely-established contributor to hypertension and heart failure via its action on the kidneys and vasculature. It also augments the activity of peripheral sympathetic nerves through activation of presynaptic angiotensin II receptors, thus contributing to sympathetic over-activity. Although some cells can synthesise angiotensin II locally, it is not known if this machinery is present in neurons closely coupled to the heart. Using a combination of RNA sequencing and quantitative real-time polymerase chain reaction, we demonstrate evidence for a renin-angiotensin synthesis pathway within human and rat sympathetic stellate ganglia, where significant alterations were observed in the spontaneously hypertensive rat stellate ganglia compared with Wistar stellates. We also used Förster Resonance Energy Transfer to demonstrate that administration of angiotensin II and angiotensin 1-7 peptides significantly elevate cyclic guanosine monophosphate in the rat stellate ganglia. Whether the release of angiotensin peptides from the sympathetic stellate ganglia alters neurotransmission and/or exacerbates cardiac dysfunction in states associated with sympathetic over activity remains to be established.

Effect of Xin Mai Jia on atherosclerosis in rats.

We investigated the therapeutic effect of Xin Mai Jia (XMJ) on atherosclerosis (AS) in rats. Rat models of AS were established by peritoneally injecting vitamin D, feeding a high-fat diet, and inducing balloon injuries in rats. The stomachs of the rats were irrigated continuously for 10 weeks with XMJ. Blood lipid- and hemorheology-related indices of blood samples were detected. Pathological changes in the right common carotid arterial tissues were also determined. The protein expression levels of endothelial nitric oxide synthase, angio-tensin-1, and endothelin-1 were determined by western blotting. XMJ reduced cholesterol, trigylecride, and low-density lipoprotein levels as well as blood viscosity, sedimentation, and hematocrit. Furthermore, XMJ alleviated vascular endothelial injury and reduced/eliminated atherosclerotic plaques. In contrast, XMJ significantly increased the endothelium-dependent relaxing response of the AS rat models. The western blotting results showed that XMJ upregulated endothelial nitric oxide synthase but downregulated angiotensin-1 and endothelin-1. XMJ prevented the development of AS by regulating blood lipid levels, hemorheology, and vascular function.

HRP and prorenin: focus on the (pro)renin receptor and vacuolar H+-ATPase.

The function of prorenin, the precursor of renin, remained unknown until the discovery of the (pro)renin receptor ((P)RR). (Pro)renin binding to this receptor allows angiotensin generation and induces signaling. Thus, (P)RR blockade will exert effects beyond angiotensin suppression. Recently, the (P)RR has been identified as an accessory protein of the vacuolar-type H+-ATPase, with important roles in Wnt signaling. In addition, transgenic animals overexpressing prorenin display the consequences of angiotensin generation, whereas transgenic animals overexpressing the (P)RR display an angiotensin-independent phenotype. Finally, both beneficial and deleterious effects have been described following treatment with the (P)RR antagonist 'handle region peptide' (HRP), while a (P)RR knockout in cardiomyocytes is lethal. This review highlights the latest findings in the (P)RR area, focusing on cardiovascular and renal pathology. It critically addresses the possibility that (pro)renin acts as an agonist of this receptor in vivo, and discusses the efficacy of HRP. Conclusions are that convincing evidence for (pro)renin-(P)RR interaction in vivo is currently lacking and, thus, that the concept of HRP exerting beneficial effects by blocking such interaction remains to be proven.

Expression of components of the renin-angiotensin system in proliferating infantile haemangioma may account for the propranolol-induced accelerated involution.

Infantile haemangioma is a benign tumour of the microvasculature characterised by excessive proliferation of immature endothelial cells. It typically undergoes rapid proliferation during infancy followed by spontaneous slow involution during childhood often leaving a fibro-fatty residuum. In 2008, propranolol, a non-selective ß-blocker, was serendipitously discovered to induce accelerated involution of a proliferating infantile haemangioma. However, the mechanism by which propranolol causes this dramatic effect is unclear. Using immunohistochemical staining, we show that the CD34+ endothelial progenitor cells of the microvessels in proliferating infantile haemangioma express angiotensin-converting enzyme and angiotensin II receptor-2, but not angiotensin II receptor-1. We have also shown using our in vitro explant model that the cells emanating from proliferating haemangioma biopsies form blast-like structures that proliferate in the presence of angiotensin II. We present here a plausible model involving the renin-angiotensin system that may account for the propranolol-induced accelerated involution of proliferating infantile haemangioma.

Genetic variants of the Renin Angiotensin system: effects on atherosclerosis in experimental models and humans.

The renin angiotensin system (RAS) has profound effects on atherosclerosis development in animal models, which is partially complimented by evidence in the human disease. Although angiotensin II was considered to be the principal effector of the RAS, a broader array of bioactive angiotensin peptides have been identified that have increased the scope of enzymes and receptors in the RAS. Genetic interruption of the synthesis of these peptides has not been extensively performed in experimental or human studies. A few studies demonstrate that interruption of a component of the angiotensin peptide synthesis pathway reduces experimental lesion formation. The evidence in human studies has not been consistent. Conversely, genetic manipulation of the RAS receptors has demonstrated that AT1a receptors are profoundly involved in experimental atherosclerosis. Few studies have reported links of genetic variants of angiotensin II receptors to human atherosclerotic diseases. Further genetic studies are needed to define the role of RAS in atherosclerosis.

Angiotensinogen metabolism in rat aorta: robust formation of proangiotensin-12.

Renin-angiotensin system (RAS) plays important role in the regulation of vessel wall homeostasis. Proangiotensin-12 (proAng-12, Ang-(1-12)) is a newly characterized metabolite of angiotensinogen, formed in array of organs of rats, which may serve as an alternate substrate for local angiotensin production, by-passing the traditional renin-dependent conversion of angiotensinogen to angiotensin I. In this work using LC/MS method we identified proAng-12 as a main product of angiotensinogen metabolism ex vivo, in organ-bath of rat aortic tissue. In this setting, proAng-12 appeared to be not only prevalent metabolite of angiotensinogen, but also served as a substrate for generation of Ang I and subsequently, Ang II. The functional significance of this surprising finding requires further investigation.

Cyclic GMP/PKG-dependent inhibition of TRPC6 channel activity and expression negatively regulates cardiomyocyte NFAT activation Novel mechanism of cardiac stress modulation by PDE5 inhibition.

Increased cyclic GMP from enhanced synthesis or suppressed catabolism (e.g. PDE5 inhibition by sildenafil, SIL) activates protein kinase G (PKG) and blunts cardiac pathological hypertrophy. Suppressed calcineurin (Cn)-NFAT (nuclear factor of activated T-cells) signaling appears to be involved, though it remains unclear how this is achieved. One potential mechanism involves activation of Cn/NFAT by calcium entering via transient receptor potential canonical (TRPC) channels (notably TRPC6). Here, we tested the hypothesis that PKG blocks Cn/NFAT activation by modifying and thus inhibiting TRPC6 current to break the positive feedback loop involving NFAT and NFAT-dependent TRPC6 upregulation. TRPC6 expression rose with pressure-overload in vivo, and angiotensin (ATII) or endothelin (ET1) stimulation in neonatal and adult cardiomyocytes in vitro. 8Br-cGMP and SIL reduced ET1-stimulated TRPC6 expression and NFAT dephosphorylation (activity). TRPC6 upregulation was absent if its promoter was mutated with non-functional NFAT binding sites, whereas constitutively active NFAT triggered TRPC6 expression that was not inhibited by SIL. PKG phosphorylated TRPC6, and both T70 and S322 were targeted. Both sites were functionally relevant, as 8Br-cGMP strongly suppressed current in wild-type TRPC6 channels, but not in those with phospho-silencing mutations (T70A, S322A or S322Q). NFAT activation and increased protein synthesis stimulated by ATII or ET1 was blocked by 8Br-cGMP or SIL. However, transfection with T70A or S322Q TRPC6 mutants blocked this inhibitory effect, whereas phospho-mimetic mutants (T70E, S322E, and both combined) suppressed NFAT activation. Thus PDE5-inhibition blocks TRPC6 channel activation and associated Cn/NFAT activation signaling by PKG-dependent channel phosphorylation.

The (pro)renin receptor: a new addition to the renin-angiotensin system?

The renin-angiotensin system is still incompletely understood. In particular, the function of prorenin, the inactive precursor of renin, is unknown. Yet, prorenin levels are >10-fold higher than renin levels, and prorenin increases even further in subjects with diabetes mellitus displaying microvascular complications. The recent discovery of a (pro)renin binding receptor may shed light on the role of prorenin. This review discusses the possibility that prorenin binding to this receptor results in prorenin activation, thereby allowing angiotensin generation, and that prorenin simultaneously acts as an agonist of this receptor, inducing angiotensin-independent effects. Transgenic animals overexpressing the receptor, as well as a receptor antagonist are now available, and future studies should reveal to what degree this concept is applicable to humans as well.

The renin-angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition.

BACKGROUND: The renin-angiotensin aldosterone system (RAAS) is a hormonal cascade that functions in the homeostatic control of arterial pressure, tissue perfusion, and extracellular volume. Dysregulation of the RAAS plays an important role in the pathogenesis of cardiovascular and renal disorders. OBJECTIVES: To review the role of the RAAS in the development of hypertensive cardiovascular disease and related conditions and provide an overview of the classes of pharmacologic agents that inhibit this system. RESULTS: The RAAS is initiated by the regulated secretion of renin, the rate-limiting enzyme that catalyzes the hydrolysis of angiotensin (Ang) I from the N-terminus of angiotensinogen. Ang I is in turn hydrolyzed by angiotensin-converting enzyme (ACE) to form Ang II, a potent vasoconstrictor and the primary active product of the RAAS. Recent evidence has suggested that other metabolites of Ang I and II may have biological activity, particularly in tissues. Development of agents that block the RAAS (e.g., beta blockers, ACE inhibitors [ACE Is], and angiotensin receptor blockers [ARBs]) began as a therapeutic strategy to treat hypertension. Preclinical and clinical studies have indicated important additional cardiovascular and renal therapeutic benefits of ACE Is and ARBs. However, blockade of the RAAS with these agents is incomplete. CONCLUSION: Therapeutic approaches that target more complete inhibition of the RAAS may offer additional clinical benefits for patients with cardiovascular and renal disorders. These approaches may include dual blockade using ACE Is and ARBs in combination, or new therapeutic modalities such as direct renin inhibition with aliskiren, recently approved for the treatment of hypertension.

Prorenin is the endogenous agonist of the (pro)renin receptor. Binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor.

OBJECTIVE: Mannose 6-phosphate receptors (M6PR) bind both renin and prorenin, and such binding contributes to renin/prorenin clearance but not to angiotensin generation. Here, we evaluated the kinetics of renin/prorenin binding to the recently discovered human (pro)renin receptor (h(P)RR), and the idea that such binding underlies tissue angiotensin generation. METHODS AND RESULTS: Vascular smooth muscle cells from control rats and transgenic rats with smooth muscle h(P)RR overexpression were incubated at 4 or 37 degrees C with human renin or prorenin. Incubation at 37 degrees C greatly increased binding, suggesting that (pro)renin-binding receptors cycle between the intracellular compartment and the cell surface. Blockade of the M6PR reduced binding by approximately 50%. During M6PR blockade, h(P)RR cells bound twice as much prorenin as control cells, while renin binding was unaltered. Incubation of h(P)RR (but not control) cells with prorenin + angiotensinogen yielded more angiotensin than expected on the basis of the activity of soluble prorenin, whereas angiotensin generation during incubation of both cell types with renin + angiotensinogen was entirely due to soluble renin. The renin + angiotensinogen-induced vasoconstriction of isolated iliac arteries from control and transgenic rats was also due to soluble renin only. The recently proposed (P)RR antagonist 'handle region peptide', which resembles part of the prosegment, blocked neither prorenin binding nor angiotensin generation. CONCLUSIONS: H(P)RRs preferentially bind prorenin, and such binding results in angiotensin generation, most likely because binding results in prorenin activation.

Dysregulation of the circulating and tissue-based renin-angiotensin system in preeclampsia.

The renin-angiotensin system (RAS) participates in preeclampsia; however, the relative contributions from the circulating RAS and the tissue-based, uteroplacental RAS are unknown. We hypothesized that the tissue-based uteroplacental RAS is dysregulated in preeclampsia. We performed microarray and gene expression studies and confirmed the findings on the protein level by immunohistochemistry in ureteroplacental units from 10 preeclamptic women and 10 women with uneventful pregnancies. All of the women were delivered by cesarean section. We also analyzed plasma renin activity and circulating agonistic angiotensin II type 1 (AT1) receptor autoantibodies. In preeclampsia, we found that the angiotensin II AT1 receptor gene was 5-fold upregulated in decidua (maternal origin). We also found AT1 autoantibodies in preeclamptic women and in their offspring by neonatal cardiomyocyte bioassay compared with women with normal pregnancies and their infants (mother: 17.5+/-2.2 versus 0.05+/-0.4; fetus: 14.5+/-1.8 versus 0.5+/-0.5 Deltabpm). Gene expressions for renin (35.0-fold), angiotensin-converting enzyme (2.9-fold), and angiotensinogen (8.9-fold) were higher in decidua than placenta (fetal origin) in both control and preeclamptic women, whereas the AT1 receptor was expressed 10-fold higher in placenta than in decidua in both groups. Our findings elucidate the ureteroplacental unit RAS in preeclamptic and normal pregnancies. We found that, in preeclampsia, the AT1 receptor expression is particularly high in decidua, combined with pregnancy-specific tissue RAS involving decidual angiotensin II production and AT1 autoantibodies. We also showed that AT1 autoantibodies cross the ureteroplacental barrier. These components could participate in the pathophysiology of preeclampsia.

Local renin angiotensin expression regulates human mesenchymal stem cell differentiation to adipocytes.

Clinical and experimental evidence suggest that the renin-angiotensin system (RAS) plays a role in metabolic syndrome. Adipogenesis is suggested to modulate obesity and obesity-related consequences, such as metabolic syndrome. Although mesenchymal stem cells (MSCs) are a major source of adipocyte generation, the influence of RAS on MSC differentiation to adipocyte is unknown. We evaluated the expression of endogenous RAS in human MSCs during its differentiation to adipocytes and studied the effects of angiotensin II (Ang II), Ang II type 1 receptor blocker Valsartan, and type 2 (AT(2)) receptor blocker PD123319. Our data showed that differentiation was associated with an increase in cellular renin and AT(2) receptor expression and a concomitant decrease in angiotensinogen and angiotensin-converting enzyme expression. The net effect is an increase in endogenous cellular angiotensin II production. Incubation with Ang II (exogenous) inhibited adipogenesis. Combined treatment of exogenous Ang II and Valsartan further inhibited adipogenesis, whereas combined treatment of Ang II and PD123319 completely abolished the inhibition of adipogenesis, suggesting an important role for the AT(2) receptor. Blockade of endogenous angiotensin II effect by incubation with Valsartan alone inhibited adipogenesis, whereas PD123319 alone promoted adipogenesis, confirming the data using exogenous Ang II. The combination of Valsartan and PD123319 had no net effect. Our data demonstrate an important role of the expression of the local RAS in the regulation of human MSC differentiation to adipocytes. Elucidation of the molecular mechanism should provide important insight into the pathophysiology of the metabolic syndrome and the development of future therapeutics.

[Effect of peroxisome proliferator-activated receptor-alpha agonist on adipokines expression in rats fed with high-fat diet].

OBJECTIVE: To explore the effect of peroxisome proliferator-activated receptor-alpha ( PPAR-alpha) agonist fenofibrate on adipokines expression in high-fat diet fed SD rats and its relationship to insulin resistance (IR). METHODS: Rats were randomized into three groups (n = 10) : HD group, fed with high-fat diet; HDF group, fed with high fat diet and treated with fenofibrate; and control group, fed with normal diet. Animals were sacrificed after 4-week follow-up. Plasma lipids, fasting plasma insulin, free fatty acids (FFA), and insulin sensitivity were detected. Reverse transcription-polymerase chain reaction was used to semi-quantitatively determine the mRNA expression of adipokines including tumor necrosis factor-alpha (TNF-alpha) , interleukin-6 (IL-6), angiotensinogen (AGT), angiotensin 11 type 1 receptor (AT1R), and adiponectin in brown fat. RESULTS: The plasma level of FFA, TG, and homeostatic model approach-IR index were (2. 37+/-0. 60) vs (1. 59+/-0. 30) vs (1. 33+/-0. 34 ) mmol/L, (0. 48+/-0. 11) vs (0. 30+/-0. 04) vs (0. 36+/-0. 07) mmol/L, and 12. 30+/-3. 97 vs 5. 03 +/-1. 88 vs 4. 17+/-1. 27 in the HD group, HDF group, and control group after 4 weeks of treatment with fenofibrate, respectively. The mRNA expressions of TNF-alpha and adiponectin were 1. 726+/-1. 408 vs 0. 713+/-0. 711 vs 0. 593+/-0. 382 and 0. 660+/-0. 192 vs 0. 949+/-0. 35 vs 0. 936+/-0. 130 in these three groups, which showed significant difference between HD group and HDF group (P < 0. 05 ) , while no significant difference between HDF group and control group (P > 0. 05). The mRNA expressions of AGT, AT1 R, and IL-6 had no significant difference among these three groups (P > 0. 05 ). CONCLUSION: PPAR-alpha agonist fenofibrate may reverse high-fat diet induced lipid abnormalities, improve insulin sensitivity, and regulate the mRNA expressions of TNF-alpha and adiponectin in adipose tissues.

Biosynthesis and physio-pharmacological actions of angiotensin peptides: 1. Synthetic enzymes.

The present work introduces a brief review of the actual knowledge concerning the enzymes involved in the biosynthesis of the active angiotensins, followed by a presentation of their main physio-pharmacological actions. The enzymatic pathways that generate active ang. II (1-8) are complemented with data concerning its transformation into angiotensin III (2-8), ang. IV (3-8), ang. V (1-5) and ang. 1-7. Besides the classic renin of renal origin, the tissue isorenins, represented by tonin and cathepsins D and G, inactive angiotensin-I-forming are also reviewed. Furthermore, chymase and the new angiotensin-converting enzyme 2 (ACE2), which generates angiotensin 1-7, having opposite properties from the mother-substance (Ang. II) are discussed at length. The presentation of properties of angiotensin-generating enzymes is followed by the presentation of the action of angiotensinases (aminopetidases, carboxypeptidase and endopeptidases), which are involved both in the generation of biologically active angiotensin peptides and in their inactivation.

The renin-angiotensin system in the mammalian central nervous system.

The brain renin-angiotensin system enables the formation of different biological active forms of angiotensins within the brain. All enzymes and peptides necessary for the biosynthesis of these angiotensins have been recognized within the central nervous system. Since there are considerable mismatches concerning the localization of the different enzymes, this system is not fully understood. Moreover, since alternative pathways of the angiotensin biosynthesis exists, localization and generation, especially of the short forms of biologically active angiotensins, are largely enigmatic. The brain renin-angiotensin system mediates several classic physiological effects including body water balance, maintenance of blood pressure, sexual behaviors, and regulation of pituitary gland hormones. Beside these classic functions, the brain renin-angiotensin system has more subtle functions involving complex mechanisms such as learning and memory. The mechanisms of action seem to differ depending on the utilized different bioactive angiotensin fragments, which are formed by the action of a variety of enzymes. This phenomenon appears to represent an important mechanism for neuromodulation. Moreover, there is evidence to suggest that the renin-angiotensin system is involved in neurological disorders, as e.g. Alzheimer's or Parkinson's disease.

Characteristics of attenuated endothelium-dependent relaxation seen in rabbit intrapulmonary vein following chronic nitroglycerine administration.

1 This study was undertaken to determine whether long-term in vivo administration of nitroglycerine (NTG) downregulates the endothelium-dependent relaxation induced by acetylcholine (ACh) in the rabbit intrapulmonary vein and, if so, whether the type 1 angiotensin II receptor (AT(1)R) blocker valsartan normalizes this downregulated relaxation. 2 In strips treated with the cyclooxygenase inhibitor diclofenac, ACh induced a relaxation only when the endothelium was intact. A small part of this ACh-induced relaxation was inhibited by coapplication of two Ca(2+)-activated K(+)-channel blockers (charybdotoxin (CTX)+apamin) and the greater part of the response was inhibited by the nitric-oxide-synthase inhibitor N(omega)-nitro-L-arginine (L-NNA). 3 The endothelium-dependent relaxation induced by ACh, but not the endothelium-independent relaxation induced by the nitric oxide donor NOC-7, was significantly reduced in NTG-treated rabbits (versus those in NTG-nontreated control rabbits). The attenuated relaxation was normalized by coapplication of valsartan with the NTG. 4 In the vascular wall, both the amount of localized angiotensin II and the production of superoxide anion were increased by in vivo NTG treatment. These variables were normalized by coapplication of valsartan with the NTG. 5 It is suggested that long-term in vivo administration of NTG downregulates the ACh-induced endothelium-dependent relaxation, mainly through an inhibition of endothelial nitric oxide production in the rabbit intrapulmonary vein. A possible role for AT(1)R is proposed in the mechanism underlying this effect.

Angiotensin and bradykinin: targets for the treatment of vascular and neuro-glial pathology in diabetic retinopathy.

The renin-angiotensin system (RAS) and kallikrein-kinin system (KKS) are complex pathways linked by a number of molecules that participate in both systems. Apart from modulating a variety of normal physiological processes, both the RAS and KKS are up-regulated following tissue injury where they influence vascular function, inflammation, cell growth and differentiation and angiogenesis. The RAS exerts its effects by the generation of a family of bioactive angiotensin peptides in which angiotensin II (ANG II) and the angiotensin type 1 (AT1) and angiotensin type 2 (AT2) receptors are most well characterised. In the KKS, bradykinin (BK) and kallidin and their carboxypeptidase metabolites, des-Arg(9)-BK and des-Arg(10)-kallidin, are the effector peptides exerting their actions via BK type 1 (BK-B1) and BK type 2 (BK-B2) receptors. Emerging evidence suggests that an ocular RAS is activated in diabetic retinopathy and may contribute to progressive alterations to retinal cells such as pericytes, endothelial cells, neurons and glia. Less well studied is the retinal KKS, however recent studies indicate effects on retinal electrophysiology and angiogenesis. The pathogenetic actions of the RAS and KKS in many tissues and possibly the diabetic retina are mediated by specific growth factors such as vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF). This review will examine the roles of the RAS and KKS in both retinal vascular and neuro-glial dysfunction in diabetic retinopathy, and the potential of blockade of these systems for the prevention and treatment of this serious diabetic complication.

Differential modulation of baroreflex control of heart rate by neuron- vs. glia-derived angiotensin II.

We developed transgenic mice with targeted expression of human renin (hREN) and human angiotensinogen (hAGT) to either neurons (N-AII mice) or glia (G-AII mice) to test the hypothesis that neuronal and glial ANG II may have differential function. Since baseline blood pressure (BP) did not differ between the models (109 +/- 3 vs. 114 +/- 4 mmHg), we stressed the BP regulatory pathway by measuring the heart rate (HR) (baroreflex) response to phenylephrine- and nitroprusside-induced changes in arterial BP. The midpoint of the baroreflex curve (BP50) was reset to a significantly higher BP in N-AII mice (131 +/- 5 mmHg) compared with littermate controls (115 +/- 3 mmHg). Baroreflex gain (slope of BP-HR relation) was similar in N-AII and control mice (12 +/- 1 vs. 14 +/- 2 beats x min(-1) x mmHg(-1)). In contrast, G-AII mice exhibited less of an increase in BP50 (125 +/- 5 mmHg) but a larger decrease in baroreflex gain (8 +/- 1 beats x min(-1) x mmHg(-1)) compared with both control and N-AII mice. Differences in BP50 and gain between N-AII, G-AII, and control mice persisted after parasympathetic blockade with atropine but were eliminated after sympathetic blockade with propranolol, indicating the effects of ANG II were selective for cardiosympathetic arm of the reflex. ANG II-like immunoreactivity was observed more prominently around the paraventricular nucleus and nucleus tractus solitarii in G-AII mice but more prominently in the ventrolateral medulla in N-AII mice. We conclude that ANG II differentially modulates baroreflex control of HR in mice producing ANG II in neurons vs. glia, and its differential function may reflect regional differences in the production of ANG II in cardiovascular control nuclei of the brain.

Evidence for the role of angiotensin II biosynthesis in the rat internal anal sphincter tone.

BACKGROUND & AIMS: The internal anal sphincter tone is important for anorectal continence. This study examined the role of angiotensin II as a neurohumoral signal for the myogenic tone in the internal anal sphincter. METHODS: We determined the effect of angiotensin I, II, III, and IV and angiotensin-(1-7) on the basal tone of the rat internal anal sphincter smooth muscle before and after selective receptor antagonists and biosynthesis inhibitors. Selective pharmacological tools used were losartan (for the AT(1) receptor), PD123,319 (for AT(2)), A-779 [for angiotensin-(1-7)], captopril (for angiotensin-converting enzyme), and amastatin (for aminopeptidases A and N). Angiotensins were measured by using high-performance liquid chromatography/UV. Western blot studies were used to determine AT(1) and AT(2) receptors, ACE, and aminopeptidases A and N. RESULTS: Angiotensin I, II, and III produced concentration-dependent contraction in the internal anal sphincter mediated by AT(1) receptors. However, in the higher concentrations (from 100 nM to 10 microM), angiotensin II showed an inhibitory effect via AT(2) receptors. Captopril (1 microM) inhibited the biosynthesis of angiotensin II in the internal anal sphincter, antagonized the contractile effects of angiotensin I, and, importantly, caused a decrease in the basal tone. Amastatin inhibited the effects of angiotensin II while augmenting those of angiotensin III. In contrast, angiotensin-(1-7) and angiotensin IV had only minor effects in the internal anal sphincter. Angiotensin I, II, and III; angiotensin-converting enzyme; aminopeptidase A and aminopeptidase n; at(1); and at(2) receptors were shown to be present in the internal anal sphincter. CONCLUSIONS: Locally produced angiotensin II may partially regulate basal tone in the internal anal sphincter.

Prorenin uptake in the heart: a prerequisite for local angiotensin generation?

Interference with locally generated angiotensin II most likely underlies the beneficial effects of renin-angiotensin system blockers in cardiac disorders. Since renin is not synthesized in the heart, this enzyme must be sequestered from the circulation in order to allow angiotensin generation at cardiac tissue sites. This review addresses the various ways through which circulating (i.e., kidney-derived) renin may reach cardiac tissue sites, considering in particular the possibility that prorenin, the inactive precursor of renin, is involved in cardiac angiotensin generation, as the plasma concentrations of prorenin are tenfold higher than those of renin. Renin and prorenin diffuse into the cardiac interstitial space and bind to cardiac (pro)renin receptors/renin-binding proteins. One of these receptors is the mannose 6-phosphate/insulin-like growth factor II receptor. This receptor not only binds mannose 6-phosphate-containing ligands like renin and prorenin, it also internalizes these enzymes, and activates prorenin intracellularly. This process possibly represents (pro)renin clearance, since intracellular angiotensin generation could not be demonstrated following (pro)renin uptake by cardiomyocytes. Angiotensin II-mediated myocyte proliferation did occur when incubating cardiomyocytes with prorenin plus angiotensionogen. The effects of prorenin plus angiotensinogen were comparable to those of 100nmol/l angiotensin II, although the angiotensin II levels in the medium during exposure of the cells to prorenin plus angiotensinogen were <1nmol/l. This suggests that cardiac angiotensin II generation by circulating renin occurs predominantly on the cell surface. The presence of ACE and/or renin on the cell membrane, in the microenvironment of angiotensin receptors, would allow maximal efficiency of local angiotensin II generation, i.e., immediate binding of angiotensin II to its receptors with minimal loss into the extracellular space.