Angiotensin Peptides Research Articles

Circulating angiotensin peptides levels in Acute Respiratory Distress Syndrome correlate with clinical outcomes: A pilot study.

RationaleWe propose renin angiotensin system (RAS) peptides are critical in wound reparative processes such as in acute respiratory distress syndrome (ARDS). Their role in predicting clinical outcomes in ARDS has been unexplored; thus, we used a targeted metabolomics approach to investigate them as potential predictors of outcomes.MethodsThirty-nine ARDS patients were enrolled within 24 hours of ARDS diagnosis. Plasma RAS peptide levels were quantified at study entry and 24, 48 and 72 hours using a liquid chromatography-mass spectrometry based metabolomics assay. RAS peptide concentrations were compared between survivors and non-survivors, and were correlated with clinical and pulmonary measures.Measurements and main resultsAngiotensin I (Ang-I or A(1–10)) levels were significantly higher in non-survivors at study entry and 72 hours. ARDS survival was associated with lower A(1–10) concentration (OR 0.36, 95% CI 0.18–0.72, p = 0.004) but higher A(1–9) concentration (OR 2.24, 95% CI 1.15–4.39, p = 0.018), a biologically active metabolite of A(1–10) and an agonist of angiotensin II receptor type 2. Survivors had significantly higher median A(1–9)/A(1–10) and A(1–7)/A(1–10) ratios than the non-survivors (p = 0.001). Increased A(1–9)/A(1–10) ratio suggests that angiotensin converting enzyme II (ACE2) activity is higher in patients who survived their ARDS insult while an increase in A(1–7)/A(1–10) ratio suggests that ACE activity is also higher in survivors.ConclusionA(1–10) accumulation and reduced A(1–9) concentration in the non-survivor group suggest that ACE2 activities may be reduced in patients succumbing to ARDS. Plasma levels of both A(1–10) and A(1–9) and their ratio may serve as useful biomarkers for prognosis in ARDS patients.

Metrologically traceable quantification of trifluoroacetic acid content in peptide reference materials by 19F solid-state NMR

Although solution-state NMR is frequently used in metrologically-traceable quantification studies, this is not the case for solid-state NMR. However, solid-state NMR allows quantification of substances without the need of dissolution, providing a truly non-destructive approach, and extending metrologically-traceable quantitative NMR to sample classes that are difficult to characterize in solution. In this contribution we present a thorough and rigorous protocol for 19F quantitative solid-state NMR employing a certified reference material as external calibrant to provide metrological traceability to absolutely quantify the content of trifluoroacetic acid (TFA) in a peptide sample, typically the major impurity in synthetic peptides. The protocol includes determining the quantitative volume of the solid-state NMR sample holder (rotor), the ERETIC (electronic reference to access in vivo concentrations) method (Akoka et al 1999 Anal. Chem. 71 2554) to compensate for variations in the sensitivity of the radio frequency resonant circuit when an external calibrant is used, and the EASY (elimination of artefacts in NMR spectroscopy) method (Jaeger and Hemmann 2014 Solid State Nucl. Magn. Reson. 57–58 22) to effectively suppress the 19F NMR background signal from the probe head. We applied the protocol to quantify the amount of TFA in a candidate NRC certified reference material of the peptide angiotensin II. The results obtained by 19F quantitative solid-state NMR are in excellent agreement with those obtained by quantitative NMR in solution employing an internal calibrant.

Differential Effects of Doxazosin on Renin-Angiotensin-System- Regulating Aminopeptidase Activities in Neuroblastoma and Glioma Tumoral Cells.

It has been described that doxazosin, an antihypertensive drug, also promotes glioblastoma cells death by inhibiting cell proliferation, arresting cell cycle and inducing apoptosis. Doxazosin has also demonstrated several modulator effects on renin-angiotensin system (RAS)- regulating aminopeptidase activities, which are highly involved in tumor growth in experimental glioma. Therefore, it remains to elucidate if the anti-tumoral effects of doxazosin could also be mediated by the proteolytic regulatory components of the RAS. To analyze the effects of doxazosin on cell growth and on RAS-regulating proteolytic regulatory aspartyl aminopeptidase (ASAP), aminopeptidase A (APA), aminopeptidase N (APN), aminopeptidase B (APB) and insulin-regulated aminopeptidase (IRAP) specific activities in the human neuroblastoma NB69 and astroglioma U373-MG tumoral cell lines. Human neuroblastoma NB69 and astroglioma U373-MG cell lines were treated with doxazosin 50-500 μM for 24h or 48h. The effects on cell growth and on RAS-regulating aminopeptidase specific activities were analyzed. Doxazosin treatments promote a concentration-dependent inhibition on cell growth in both NB69 and U373-MG cells, being NB69 cells more sensitive to the drug than U373-MG cells. However, its effects on RAS-regulating aminopeptidase specific activities depend on the concentration used, the duration of the treatment and the cell type. These data confirm the existence of a different dynamic progression of RAS cascade in each tumoral cell line as a consequence of the treatment with doxazosin and time of action, which also implies a very dynamic metabolism of the peptides which participate in each step of RAS cascade. Our results indicate that doxazosin modifies the proteolytic regulatory enzymes of RAS cascade, modulating the bioactive efficacy of the different angiotensin peptides, and therefore, of their functional roles as initiators/promoters of cell proliferation as autocrine/paracrine mediators.

ACE Insertion/Deletion Polymorphism (rs4646994) Is Associated With the Increased Risk of Multiple Myeloma.

Introduction: The insertion (I allele) deletion (D allele) polymorphism of ACE gene (rs4646994) may influence the etiopathogenesis of multiple myeloma (MM). ACE gene is expressed in bone marrow cells and encodes angiotensin converting enzyme (ACE). It converts angiotensin I to active peptide angiotensin II, which stimulates proliferation of hematopoietic stem cells. This suggests possible association of ACE I/D gene polymorphism with MM. The aim of our study was to check possible impact of this polymorphism on risk of development and outcome of MM, as well as, sensitivity to bortezomib in cell cultures derived from MM patients.Objects and Methods: Genomic DNA from 98 newly diagnosed MM patients and 100 healthy blood donors were analyzed by PCR method. Chromosomal aberrations were detected by use of cIg-FISH. In a subgroup of 40 MM patients nucleated bone marrow cells were treated with bortezomib in vitro.Results: The Hardy-Weinberg equilibrium test showed that genotypic frequencies diverged significantly from the equilibrium. The differences between I and D allele frequencies in control and study population were significant (p = 0.046). We observed the association between DD genotype and more than 2-fold risk of MM - OR = 2.69; p < 0.0001. We did not detect any significant differences among studied genotypes regarding clinical and laboratory parameters. Moreover, we did not observe the association between survival of MM patients and I/D genotypes. Bortezomib increased number of apoptotic and necrotic cells, but the only statistically significant differences were observed in the number of viable cells at 1 nM between ID and DD genotypes (p = 0.026).Conclusion: Presented results confirmed the significant relationship between ACE (I/D) polymorphism and risk of MM development. We did not observe the association of ACE I/D polymorphism with disease outcome and bortezomib in vitro sensitivity.

Acute intrarenal angiotensin (1-7) infusion decreases diabetes-induced glomerular hyperfiltration but increases kidney oxygen consumption in the rat.

Common kidney alterations early after the onset of insulinopenic diabetes include glomerular hyperfiltration, increased oxygen consumption and tissue hypoxia. Increased activity of the renin-angiotensin-aldosterone system (RAAS) has been implicated in most of these early alterations. The RAAS peptide angiotensin (1-7) has the potential to modulate RAAS-mediated alterations in kidney function. Thus, the aim of the present study was to determine the acute effects of angiotensin (1-7) in the kidney of insulinopenic type 1 diabetic rat and the results compared to that of normoglycaemic controls. Renal haemodynamics and oxygen homeostasis were measured 3 weeks after administration of streptozotocin before and after acute intrarenal infusion of angiotensin (1-7) at a dose of 400ngmin-1 . Arterial pressure and renal blood flow were similar between groups and not affected by exogenous angiotensin (1-7). Diabetics presented with glomerular hyperfiltration, increased urinary sodium excretion and elevated kidney oxygen consumption. Angiotensin (1-7) infusion normalized glomerular filtration, increased urinary sodium excretion, decreased proximal tubular reabsorption, and elevated kidney oxygen consumption even further. The latter resulting in tubular electrolyte transport inefficiency. Angiotensin (1-7) did not affect tissue oxygen tension and had no significant effects in controls on any of the measured parameters. Diabetes results in increased responsiveness to elevated levels of angiotensin (1-7) which is manifested as inhibition of tubular sodium transport and normalization of glomerular filtration. Furthermore, elevated angiotensin (1-7) levels increase kidney oxygen consumption in the diabetic kidney even further which affects tubular electrolyte transport efficiency negatively.

The renin-angiotensin system: going beyond the classical paradigms

Thirty years ago, a novel axis of the renin-angiotensin system (RAS) was unveiled by the discovery of angiotensin-(1−7) [ANG-(1−7)] generation in vivo. Later, angiotensin-converting enzyme 2 (ACE2) was shown to be the main mediator of this reaction, and Mas was found to be the receptor for the heptapeptide. The functional analysis of this novel axis of the RAS that followed its discovery revealed numerous protective actions in particular for cardiovascular diseases. In parallel, similar protective actions were also described for one of the two receptors of ANG II, the ANG II type 2 receptor (AT2R), in contrast to the other, the ANG II type 1 receptor (AT1R), which mediates deleterious actions of this peptide, e.g., in the setting of cardiovascular disease. Very recently, another branch of the RAS was discovered, based on angiotensin peptides in which the amino-terminal aspartate was replaced by alanine, the alatensins. Ala-ANG-(1−7) or alamandine was shown to interact with Mas-related G protein-coupled receptor D, and the first functional data indicated that this peptide also exerts protective effects in the cardiovascular system. This review summarizes the presentations given at the International Union of Physiological Sciences Congress in Rio de Janeiro, Brazil, in 2017, during the symposium entitled “The Renin-Angiotensin System: Going Beyond the Classical Paradigms,” in which the signaling and physiological actions of ANG-(1−7), ACE2, AT2R, and alatensins were reported (with a focus on noncentral nervous system-related tissues) and the therapeutic opportunities based on these findings were discussed.

Structural basis for the specificity of renin-mediated angiotensinogen cleavage

The renin–angiotensin cascade is a hormone system that regulates blood pressure and fluid balance. Renin-mediated cleavage of the angiotensin I peptide from the N terminus of angiotensinogen (AGT) is the rate-limiting step of this cascade; however, the detailed molecular mechanism underlying this step is unclear. Here, we solved the crystal structures of glycosylated human AGT (2.30 Å resolution), its encounter complex with renin (2.55 Å), AGT cleaved in its reactive center loop (RCL; 2.97 Å), and spent AGT from which the N-terminal angiotensin peptide was removed (2.63 Å). These structures revealed that AGT undergoes profound conformational changes and binds renin through a tail-into-mouth allosteric mechanism that inserts the N terminus into a pocket equivalent to a hormone-binding site on other serpins. These changes fully extended the N-terminal tail, with the scissile bond for angiotensin release docked in renin's active site. Insertion of the N terminus into this pocket accompanied a complete unwinding of helix H of AGT, which, in turn, formed key interactions with renin in the complementary binding interface. Mutagenesis and kinetic analyses confirmed that renin-mediated production of angiotensin I is controlled by interactions of amino acid residues and glycan components outside renin's active-site cleft. Our findings indicate that AGT adapts unique serpin features for hormone delivery and binds renin through concerted movements in the N-terminal tail and in its main body to modulate angiotensin release. These insights provide a structural basis for the development of agents that attenuate angiotensin release by targeting AGT's hormone binding pocket.

Antisense oligonucleotides targeting angiotensinogen: insights from animal studies.

Angiotensinogen (AGT) is the unique substrate of all angiotensin peptides. We review the recent preclinical research of AGT antisense oligonucleotides (ASOs), a rapidly evolving therapeutic approach. The scope of the research findings not only opens doors for potentially new therapeutics of hypertension and many other diseases, but also provides insights into understanding critical physiological and pathophysiological roles mediated by AGT.

Effects of inhibitors of the renin-angiotensin system on reducing blood pressure and expression of inflammatory factors in CHD patients: A network meta-analysis.

The renin-angiotensin system (RAS) is an ever-evolving endocrine system with considerable checks and balances on the production and catabolism of angiotensin peptides most likely due to the manifold effects of angiotensins. We aimed to explore the effects of different inhibitors of RAS on blood pressure and expression of inflammatory factors in patients with coronary heart disease (CHD). We initially searched PubMed, EMBASE and Cochrane Library electronic databases with nine eligible randomized controlled trials enrolled. Direct and indirect evidence was combined to calculate the weighted mean difference value and draw surface under the cumulative ranking curves. The results demonstrated that, compared with placebo and enalapril, ramipril had a better effect on reducing systolic blood pressure after short-term usage of drugs (<12 months), while perindopril had better effects on reducing diastolic blood pressure and C-reactive protein expression. Furthermore, after long-term usage of drugs (≥12 months), there was no significant difference among olmesartan, quinapril and candesartan in the treatment of patients with CHD. Perindopril and ramipril had better effects on inhibiting blood pressure and expression of inflammatory factors among eight inhibitors after short-term usage of drugs (<12 months); while quinapril had better effects on reducing blood pressure and expression of inflammatory factor after long-term usage of drugs, and there was little difference in the effects between olmesartan and candesartan (≥12 months). Perindopril may have better short-term effects on reducing blood pressure and expression of inflammatory factor, while quinapril may have better long-term effects on reducing blood pressure and expression of inflammatory factor.

Effect of novel GPCR ligands on blood pressure and vascular homeostasis.

Maintenance of normal blood pressure under conditions of drug treatment is a measure of system-wide neuro-hormonal controls and electrolyte/fluid volume homeostasis in the body. With increased interest in designing and evaluating novel drugs that may functionally select or allosterically modulate specific GPCR signaling pathways, techniques that allow us to measure acute and long-term effects on blood pressure are very important. Therefore, this chapter describes techniques to measure acute and long-term impact of novel GPCR ligands on blood pressure regulation. We will use the angiotensin type 1 receptor, a powerful blood pressure regulating GPCR, in detailing the methodology. Normal blood pressure maintenance depends upon dynamic modulation of angiotensin type 1 receptor activity by the hormone peptide angiotensin II. Chronic activation of angiotensin type 1 receptor creates hypertension and related cardiovascular disease states which are treated with angiotensin type 1 receptor blockers (ARBs). Thus, a prototype for evaluation of blood pressure control under experimental evaluation of novel drugs.

Comparative Expression of Renin-Angiotensin Pathway Proteins in Visceral Versus Subcutaneous Fat.

Body fat distribution contributes to obesity-related metabolic and cardiovascular disorders. Visceral fat is more detrimental than subcutaneous fat. However, the mechanisms underlying visceral fat-mediated cardiometabolic dysregulation are not completely understood. Localized increases in expression of the renin angiotensin system (RAS) in adipose tissue (AT) may be implicated. We therefore investigated mRNA and protein expression of RAS components in visceral versus subcutaneous AT using paired samples from individuals undergoing surgery (N = 20, body mass index: 45.6 ± 6.2 kg/m2, and age: 44.6 ± 9.1 years). We also examined RAS-related proteins in AT obtained from individuals on renin angiotensin aldosterone system (RAAS) targeted drugs (N = 10, body mass index: 47.2 ± 9.3 kg/m2, and age: 53.3 ± 10.1 years). Comparison of protein expression between subcutaneous and visceral AT samples showed an increase in renin (p = 0.004) and no change in angiotensinogen (p = 0.987) expression in visceral AT. Among proteins involved in angiotensin peptide generation, angiotensin converting enzyme (p = 0.02) was increased in subcutaneous AT while chymase (p = 0.001) and angiotensin converting enzyme-2 (p = 0.001) were elevated in visceral fat. Furthermore, visceral fat expression of angiotensin II type-2 receptor (p = 0.007) and angiotensin II type-1 receptor (p = 0.031) was higher, and MAS receptor (p < 0.001) was lower. Phosphorylated-p53 (p = 0.147), AT fibrosis (p = 0.138) and average adipocyte size (p = 0.846) were similar in the two depots. Nonetheless, visceral AT showed increased mRNA expression of inflammatory (TNFα, p < 0.001; IL-6, p = 0.001) and oxidative stress markers (NOX2, p = 0.038; NOX4, p < 0.001). Of note, mRNA and protein expression of RAS components did not differ between subjects taking or not taking RAAS related drugs. In summary, several RAS related proteins are differentially expressed in subcutaneous versus visceral AT. This differential expression may not alter AngII but likely increases Ang1-7 generation in visceral fat. These potential differences in active angiotensin peptides and receptor expression in the two depots suggest that localized RAS may not be involved in differences in visceral vs subcutaneous AT function in obese individuals. Our findings do not support a role for localized RAS differences in visceral fat-mediated development of cardiovascular and metabolic pathology.

A17284 The modulation of angiotensin peptide levels by RAS blockade in spontaneously hypertensive rats

Objectives: There are only sparse data on dual (or single) RAS blockade effect on angiotensin peptides. We aimed to investigate the effects of single/combined RAS blockade on angiotensin peptides in spontaneously hypertensive rats (SHR). Methods: 8 groups of SHR were investigated for 4 weeks: aliskiren (Ali), lisinopril (Lis), olmesartan (Olm) or their dual combinations along with SHR and Wistar-Kyoto (WKY) rats. Instant and equilibrated RAS peptide levels in plasma and equilibrated levels in the kidney and left ventricle (LV) were determined along with blood pressure (BP), LV weight and myocardial echocardiography. Results: Compared to WKY, the SHR had increased BP, LV hypertrophy, reduced renin activity and higher circulatory ACE activity. While Ang II was reduced in the LV, it was increased in the kidneys of SHR. Ali achieved only modest BP reduction, while the reduction by Olm, Lis and Olm + Ali were more pronounced. The combinations Olm + Lis and Lis + Ali achieved highest BP reductions. Hypertrophy correlated with BP. While Ali suppressed the RAS, Olm and Lis led to increased Ang I production, which in case of Olm resulted in increased Ang II levels. Both Lis and Olm increased Ang 1–7 equilibrium levels. A discrepancy between instant and equilibrium levels of Ang II and Ang 1-7 suggested their sequestration/degradation in the periphery. Further, levels of both peptides were increased in the kidneys, which was reduced by Olm. All combination treatments reduced the circulatory levels of Ang 1–7 compared to Lis or Olm monotherapy. Conclusion: The circulatory RAS strongly influences the peripheral RAS levels. Dual RAS blockade compromises the potentially protective Ang 1-7 increase. These results provide rationale for the novel therapeutic strategies, combinations and personalized treatment schemes. (VEGA 1/127/17)

Function of AT1 and AT2 receptors in atrial contractions from spontaneous hypertensive and diabetic-induced streptozotocin rats.

Diabetes mellitus and hypertension are diseases that are strongly correlated. A major factor in this correlation is the renin-angiotensin system (RAS), with the peptide angiotensin II being a key component. This study analyzed the impact of Angiotensin Type 1 receptor (AT1R) and Angiotension Type 2 receptor (AT2R) in atrial function. MAIN METHODS: To perform the experiments, Wistar Kyoto rats (WKY), diabetic streptozotocin-induced WKY rats and spontaneously hypertensive rats (SHR) were used, and stimulation of cardiovascular function was done by means of the following drugs: angiotensin II, novokinin and the antagonists losartan and PD123177. We also measured the systolic blood pressure (SBP). RESULTS: An increase in AT1R function was observed in diabetic and hypertensive rats (18% in right atria [RA] and 11% in left atria [LA]). We also observed an increase in calcium release from the endoplasmic reticulum in right atria of diabetic rats (31%) and in right atria of hypertensive rats (35%). On the other hand, a decreased response of AT2R in diabetic and hypertensive rats was observed, this decreased response was greater in hypertensive rats (RA, 10%; LA, 12%). These results have demonstrated a dysfunction of the RAS that may contribute to the common dysfunctions of the cardiovascular system in diabetic and hypertensive rats.

Retinal Renin-Angiotensin System Modulators: A Recent Implication for Therapy in Glaucomatous Patients

Glaucoma is the ultimate commonly acquired optic neuropathy. It signifies a public health challenge since it causes an irreversible blindness. The single known treatment of the disease is decreasing of intraocular pressure (IOP), which has been revealed to lessen glaucoma progress in a diversity of large proportions of clinical trials. Herein in this literature, we briefly define the optical Renin Angiotensin system (RAS) signaling pathway and define the most essential components, physiological actions of major angiotensin peptides, and the Renin Angiotensin system blockers. And discuss the potential implications of their modulators as a new therapeutic target in glaucoma. The literature has shown that the individual RAS modulators including, Angiotensin converting enzymes 1(ACE1) inhibitors, Angiotensin converting enzymes 2 (ACE2) Activators, Angiotensin receptor-1 (AT-1) blocker, and renin inhibitors have a potentials role in modulation of aqueous humour homodynamic, by neuroprotection of the retinal ganglion cells (RGC) and acceleration of the aqueous humour outflow. In conclusion, RAS modulators have an imperious role in lowering IOP, these compounds will pave the approach for prospect innovation, improvement, and publicizing of novel drugs to treat glaucoma and therefore, aid save vision for millions of people suffering with this slow progressive optic neuropathy.

Retinal angiotensin II and angiotensin-(1-7) response to hyperglycemia and an intervention with captopril.

Hypothesis:Hyperglycemia decreases angiotensin-(1-7), the endogenous counter-regulator of angiotensin II in the retina.Materials and methods:The distribution and levels of retinal angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) were evaluated by confocal imaging and quantitative immunohistochemistry during the development of streptozotocin-induced diabetes in rats.Results:In the nondiabetic eye, Ang II was localized to the endfeet of Müller cells, extending into the cellular processes of the inner plexiform layer and inner nuclear layer; Ang-(1-7) showed a wider distribution, extending from the foot plates of the Müller cells to the photoreceptor layer. Eyes from diabetic animals showed a higher intensity and extent of Ang II staining compared with nondiabetic eyes, but lower intensity with a reduced distribution of Ang-(1-7) immunoreactivity. Treatment of the diabetic animals with the angiotensin-converting enzyme inhibitor (ACEI) captopril showed a reduced intensity of Ang II staining, whereas increased intensity and distribution were evident with Ang-(1-7) staining.Conclusions:These studies reveal that pharmacological inhibition with ACEIs may provide a specific intervention for the management of the diabetes-induced decline in retinal function, reversing the profile of the endogenous angiotensin peptides closer to the normal condition.

Biochemical Effects of Intraliposomal Angiotensins on Isolated Vascular Smooth Muscle Cells

The intracellular renin-angiotensin effectors (peptides, enzymes, receptors) and their effects are intriguing for a lot of systems. That�s why we aimed the effects of intracellularly-administered angiotensins (angiotensin II, angiotensin III, angiotensin IV, angiotensin fragment 1-7), angiotensinogen, CGP-42112A, apelin, and angiotensin receptors blockers (losartan, PD123319), by the means of liposomes, on apoptosis of cultured isolated rat aortic vascular smooth muscle cells. We evidenced that CGP-42112A (a potent AT2 angiotensin II receptor agonist), administered intracellularly, induced the apoptosis of the cultured isolated vascular smooth muscle cells in a much higher proportion than other agonists and antagonists of angiotensin system: CGP-42112A ] angiotensin II] angiotensin III@ angiotensinogen. Moreover, losartan (an AT1 angiotensin II receptor antagonist), administered intracellularly, induced an important degree of apoptosis of cultured isolated vascular smooth muscle cells. Losartan, administered as concomitant treatment for other angiotensin peptides and CGP-42112A, did not significantly modified the apoptotic effects of these peptides. On the other hand, PD123319 (an AT2 angiotensin II receptor antagonist) was able to significantly reduce the losartan effects when administered as co-treatment for 24 h. The same effects were obtained when LY294002, a PI3K/Akt signaling inhibitor, was administered as a co-treatment. We can conclude an involvement of an AT2 angiotensin II receptor and PI3K/Akt signaling in these apoptotic effects induced by some angiotensin peptides and losartan on cultured isolated rat aortic vascular smooth muscle cells.

Critical role of the chymase/angiotensin-(1–12) axis in modulating cardiomyocyte contractility

BackgroundAngiotensin-(1–12) [Ang-(1–12)] is a chymase-dependent source for angiotensin II (Ang II) cardiac activity. The direct contractile effects of Ang-(1–12) in normal and heart failure (HF) remain to be demonstrated. We assessed the hypothesis that Ang-(1–12) may modulate [Ca2+]i regulation and alter cardiomyocyte contractility in normal and HF rats. Methods and resultsWe compared left ventricle (LV) myocyte contractile and calcium transient ([Ca2+]iT) responses to angiotensin peptides in 16 SD rats with isoproterenol-induced HF and 16 age-matched controls. In normal myocytes, versus baseline, Ang II (10−6 M) superfusion significantly increased myocyte contractility (dL/dtmax: 40%) and [Ca2+]iT (29%). Ang-(1–12) (4 × 10−6 M) caused similar increases in dL/dtmax (34%) and [Ca2+]iT (25%). Compared with normal myocytes, superfusion of Ang II and Ang-(1–12) in myocytes obtained from rats with isoproterenol-induced HF caused similar but significantly attenuated positive inotropic actions with about 42% to 50% less increases in dL/dtmax and [Ca2+]iT. Chymostatin abolished Ang-(1–12)-mediated effects in normal and HF myocytes. The presence of an inhibitory cAMP analog, Rp-cAMPS prevented Ang-(1–12)-induced inotropic effects in both normal and HF myocytes. Incubation of HF myocytes with pertussis toxin (PTX) further augmented Ang II-mediated contractility. ConclusionsAng-(1–12) stimulates cardiomyocyte contractile function and [Ca2+]iT in both normal and HF rats through a chymase mediated action. Altered inotropic responses to Ang-(1–12) and Ang II in HF myocytes are mediated through a cAMP-dependent mechanism that is coupled to both stimulatory G and inhibitory PTX-sensitive G proteins.

Chemical Analysis of Complex Surface-Adsorbed Molecules and Their Reactions by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry.

Desorption/ionization induced by neutral SO2 clusters (DINeC) is used for mass spectrometry (MS) of surface-adsorbed molecules. The method is shown to be a surface-sensitive analysis tool capable of detecting molecular adsorbates in a wide range of molecular weights as well as their reactions on surfaces, which are otherwise difficult to access. Two different surface/adsorbate systems prepared by means of electrospray ion beam deposition (ES-IBD) were investigated: For the peptide angiotensin II on gold, intact molecules were desorbed from the surface when deposited by soft landing ES-IBD. By comparison to the well-controlled amount of substance deposited by ES-IBD, the sensitivity of DINeC-MS was shown to be on the order of 0.1% of a monolayer coverage, corresponding to femtomoles of analyte. Depending on deposition and sample conditions, the original state of charge of the molecules could be retrieved. Reaction of the adsorbed molecules both with surface atoms as well as with coadsorbed D2O was monitored. Rhodamine 6G was also desorbed as an intact molecule when deposited with kinetic energies below 50 eV. For higher deposition energy, fragmentation of the dye molecules was observed by means of DINeC-MS.

Anti-Inflammatory and Antitumor Effects of Hydroxytyrosol but Not Oleuropein on Experimental Glioma In Vivo. A Putative Role for the Renin-Angiotensin System.

Functional roles of the angiotensin peptides of the renin-angiotensin system (RAS) cascade can be analyzed through their corresponding proteolytic regulatory enzymes aspartyl aminopeptidase (ASAP), aminopeptidase A (APA), aminopeptidase B (APB), aminopeptidase N (APN) and insulin-regulated aminopeptidase (IRAP). These enzyme activities generate active or inactive angiotensin peptides that alter the ratios between their bioactive forms, regulating several important processes such as the regulation of cardiovascular functions, body water regulation, normal memory consolidation and retrieval, but also cell growth, differentiation and apoptosis or the inflammatory response. We have previously described that the treatment with hydroxytyrosol but not with oleuropein or with the mixture of both compounds led to the significant inhibition of tumor growth in an in vivo glioma model by mechanisms not only related to redox balance. Using this glioma model, here we analyze the effects of the phenolic compounds oleuropein and hydroxytyrosol in circulating RAS-regulating ASAP, APA, APN, APB and IRAP specific activities and the pro-inflammatory cytokines IL-6 and TNFα to understand the relationship between the antitumor and anti-inflammatory effects of hydroxytyrosol, but not oleuropein, and the components of the RAS. We found that oleuropein increased all the activities analyzed and promoted a pro-inflammatory status, whereas hydroxytyrosol only modified ASAP and IRAP activities and promotes an anti-inflammatory status. When administrated together, oleuropein overrode the effects of hydroxytyrosol. Our results suggest a role for angiotensin III and angiotensin 1-7 in both tumor growth inhibition and anti-inflammatory response promoted by hydroxytyrosol.

Role of intracellular angiotensin II.

It has become clear that the vasoactive peptide angiotensin II, like other so-called intracrines, can act in the intracellular space. Evidence has accumulated indicating that such angiotensin II activity can be upregulated in disease states and cause pathology. Indeed, other intracrines appear to be involved in disease pathogenesis as well. At the same time, nitric oxide, potentially a cell protective factor, has been shown to be upregulated by intracellular angiotensin II. Recently data have been developed indicating that other potentially protective factors are directly upregulated at neuronal nuclei by angiotensin II. This led to the suggestion that intracellular angiotensin II is cell protective and not pathological. Here, the data on both sides of this issue and a possible resolution are discussed. In summary, there is evidence for both protective and pathological actions of intracellular angiotensin, just as there is abundant evidence derived from whole animal physiology to indicate that angiotensin-driven signaling cascades, including angiotensin II type 2 receptor- and Mas receptor-mediated events, can mitigate the effects of the angiotensin II/angiotensin II type 1 receptor axis (25). This mitigation does not negate the physiological and pathological importance of angiotensin II/angiotensin II type 1 receptor action but does expand our understanding of the workings of both intracellular and extracellular angiotensin II.