Renan Paulo Martin

Angiotensin II Binding to Angiotensin I–Converting Enzyme Triggers Calcium Signaling

Angiotensin (Ang) I–converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.

Functional expression of kinin B1 and B2 receptors in mouse abdominal aorta

Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg(9)BK (DBK). Our aim was to determine the potential expression of kinin B(1) and B(2) receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD(2)) calculated from the curves was 7.0 +/- 0.1 for BK and 7.3 +/- 0.2 for DBK. The efficacy was 51 +/- 2% for BK and 30 +/- 1% for DBK when compared to 1 microM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 microM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B(1) and B(2) subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.

Evidence that kinin B2 receptor expression is upregulated by endothelial overexpression of B1 receptors

Bradykinin (BK) and des-Arg(9)-bradykinin (DBK) of kallikrein-kinin system exert its effects mediated by the B2 (B2R) and B1 (B1R) receptors, respectively. It was already shown that the deletion of kinin B1R or of B2R induces upregulation of the remaining receptor subtype. However studies on overexpression of B1R or B2R in transgenic animals have supported the importance of the overexpressed receptor but the expression of another receptor subtype has not been determined. Previous study described a marked vasodilatation and increased susceptibility to endotoxic shock which was associated with increased mortality in response to DBK in thoracic aorta from transgenic rat overexpressing the kinin B1R (TGR(Tie2B1)) exclusively in the endothelium. In another study, mice overexpressing B1R in multiple tissues were shown to present high susceptibility to inflammation and to lipopolysaccharide-induced endotoxic shock. Therefore the role of B2R was investigated in the thoracic aorta isolated from TGR(Tie2B1) rats overexpressing the B1R exclusively in the vascular endothelium. Our findings provided evidence for highly increased expression level of the B2R in the transgenic rats. It was reported that under endotoxic shock, these rats exhibited exaggerated hypotension, bradycardia and mortality. It can be suggested that the high mortality during the pathogenesis of endotoxic shock provoked in the transgenic TGR(Tie2B1) rats could be due to the enhanced expression of B2R associated with the overexpression of the B1R.

Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC

This study characterized pharmacologically the functional responses to agonists angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label at the N-terminal (TOAC1-AngII and TOAC0-BK) and internal (TOAC3-AngII and TOAC3-BK) positions of these vasoactive peptides. Affinity constants of the ligands for AT1 and B2 receptors were evaluated in vitro by binding assays and biological effects by extracellular acidification rates and in vivo by blood pressure responses. In contrast to internally labeled analogues (TOAC3-AngII or TOAC3-BK), the TOAC1-AngII and TOAC0-BK derivatives dose-dependently increased the extracellular acidification rate in adherent cultured Chinese hamster ovary (CHO) cells expressing AT1 or B2 receptors, respectively. In addition, TOAC(1)-AngII induced an increase in blood pressure when injected intravenously in awaken rats although with a potency four times smaller when compared to native AngII. Similarly to BK, TOAC0-BK dose-dependently decreased blood pressure when injected intra-arterially in rats with a lower potency when compared to the native peptide. On the contrary, TOAC3-AngII or TOAC3-BK did not provoke any alteration in blood pressure levels. In summary, our results confirmed that the insertion of TOAC-probe in the N-terminal region of peptides does not significantly modify the affinity or biological activity in vitro and in vivo conditions and could be an important tool to evaluate peptide-receptor interaction mechanism. Conversely, possibly due to the unique bend-inducing property of the cyclic TOAC probe, its insertion at position 3 in both AngII and BK structures seems to restrict the interaction and the activation of the AT1 and B2 receptors.

Cross talk between kinin and angiotensin II receptors in mouse abdominal aorta.

Abstract Bradykinin (BK) is a vasorelaxant, algesic and inflammatory agent. Angiotensin II (AngII) is known to control vascular tone and promote growth, inflammation and artherogenesis. There is evidence for cross talking between BK and AngII receptors. Therefore, the effect of lack of kinin receptors was assessed in mice with genetic disruption of B1 or B2 and both receptors. Responsiveness of abdominal aortic rings to BK and AngII as well as the receptor gene expression of both peptides were analysed. Although no specific phenotype was displayed in the normotensive and healthy mice lacking the kinin receptors, a decreased expression level of the remaining kinin receptor mRNA was observed. AT1 receptor mRNA level was also reduced, indicating that kinin receptors regulate AngII receptors. Downregulation of the receptors was well correlated with reduction in the reactivity of both agonists to induce contraction of aortic rings, but other signal regulations must be sought in these transgenic mice. We conclude that cross talk between kinin and AngII receptors occurs in mouse abdominal aorta and that both peptides may regulate the initiation and progression of important pathophysiological processes, such as hypertension and inflammation.

New mutations in SERPING1 gene of Brazilian patients with hereditary angioedema.

Abstract Hereditary Angioedema is an autosomal dominant inherited disease leading to oedema attacks with variable severity and localization predominantly caused by C1-INH deficit. More than 400 mutations have been already identified, however no genetic analysis of a Brazilian cohort of HAE patients with C1-INH deficiency has been published. Our aim was to perform genetic analysis of C1-INH gene (SERPING1) in Brazilian HAE patients. We screened the whole SERPING1 coding region from 30 subjects out of 16 unrelated families with confirmed diagnosis of HAE due to C1-INH deficiency. Clinical diagnosis was based on symptoms and quantitative and/or functional analysis of C1-INH. We identified fifteen different mutations among which eight were not previously described according to databases. We found five small deletions (c.97_115del19; c.553delG; c.776_782del7; c.1075_1089del15 and c.1353_1354delGA), producing frameshifts leading to premature stop codons; seven missense mutations (c.498C>A; c.550G>C; c.752T>C; c.889G>A; c.1376C>A; c.1396C>T; c.1431C>A); one nonsense mutation (c.1480C>T), and two intronic alterations (c.51+1G>T; c.51+2T>C). Despite the small number of participants in this study, our results show mutations not previously identified in SERPING1 gene. This study represents the first Brazilian HAE cohort evaluated for mutations and it introduces the possibility to perform genetic analysis in case of need for differential diagnosis.

The correlation between CRB1 variants and the clinical severity of Brazilian patients with different inherited retinal dystrophy phenotypes

Inherited retinal dystrophies are characterized by progressive retina degeneration and mutations in at least 250 genes have been associated as disease-causing. CRB1 is one of many genes analyzed in molecular diagnosis for inherited retinal dystrophy. Crumbs homolog-1 protein encoded by CRB1 is important for cell-to-cell contact, polarization of epithelial cells and the morphogenesis of photoreceptors. Pathogenic variants in CRB1 lead to a huge variety of phenotypes ranging from milder forms of inherited retinal dystrophy, such as retinitis pigmentosa to more severe phenotypes such as Leber congenital amaurosis. In this study, seven novel likely-pathogenic variants were identified: four missense variants (p.Leu479Pro, p.Ala921Pro, p.Cys948Arg and p.Asp1031Asn), two frameshift deletions (c.2536_2542del7 and c.3460_3461delTG) and one frameshift indel variant (c.276_294delinsTGAACACTGTAC). Furthermore, two patients with cone-rod dystrophy due to mutations in CRB1 were reported, supporting previous data, in which mutations in CRB1 can also cause cone-rod dystrophy. Finally, our data suggested there was a direct relation between phenotype severity and the mutation effect on protein functionality in 15 Brazilian CRB1 patients.

Novel Complex ABCA4 Alleles in Brazilian Patients With Stargardt Disease: Genotype-Phenotype Correlation

Purpose To analyze the presence of complex alleles of the ABCA4 gene in Brazilian patients with Stargardt disease and to assess the correlation with clinical features. Methods This was an observational cross-sectional study. Patients with a diagnosis of Stargardt disease who presented three pathogenic variants of the ABCA4 gene or who had variants previously described as complex alleles were included. The relatives of these probands were evaluated in the segregation analysis. The patients were evaluated based on age at symptom onset and visual acuity, and the clinical characteristics were classified according to the findings observed on autofluorescence examination. Results Among the 47 families analyzed, approximately 30% (14/47) presented complex alleles. The segregation analysis in 14 families with cases of Stargardt disease identified three novel complex alleles and one previously described complex allele. The known complex allele p.[Leu541Pro; Ala1038Val] was identified in two families. The novel complex alleles identified were p.[Leu541Pro; Arg1443His] in five families, p.[Ser1642Arg; Val1682_Val1686del] in seven families, and p.[Pro1761Arg; Arg2106Cys] in one family. Furthermore, four new variants (p.Lys22Asn, p.Asp915Asn, p.Glu1447Val, and p.Pro1761Arg) were identified in the second allele of the ABCA4 gene. Conclusions Segregation analysis is important in order to confirm the molecular diagnosis of patients with Stargardt disease, given the frequency of complex alleles in the ABCA4 gene. The various pathogenic variation combinations observed in this study were associated with different phenotypes.

Distinct binding mode of 125I-AngII to AT1 receptor without the Cys18-Cys274 disulfide bridge

Previous studies on angiotensin II (AngII) AT(1) receptor function have revealed that the N-terminal residues of AngII may modulate receptor activation by binding at the receptor extracellular site. A remarkable feature of this site is an insertion of 8 amino acids in the middle of the EC-3 loop including the Cys(274) residue that supposedly makes a disulfide bond with N-terminal Cys(18). As demonstrated by assays with Del(267-275)AT(1), the role of the Cys(18)-Cys(274) disulfide bridge is to keep a conformation of the inserted residues that allows a normal binding of the AngII N-terminal residues. C18S AT(1) receptor mutant, supposedly having a dissociated disulfide bridge, but an intact residue insertion, is constitutively activated and can less efficiently bind AngII. Similar results were observed when the S-S disulfide bond was disrupted in (C18S,C274S) AT(1) receptor. The importance of the free N-terminal amino group of Asp(1) and of the Arg(2) guanidino group for the binding of AngII to C18S mutant with EC-3 loop insertion was investigated by means of assays using AngII peptide analogues bearing a single mutation of Asp(1) for Sar(1) or Arg(2) for Lys(2), as ligands. This study showed that like AngII, [Sar(1)]-AngII can bind the C18S mutant receptor with low affinity whereas [Lys(2)]-AngII binding is still more reduced. Interestingly, when (125)I-AngII instead of (3)H-AngII was used, no significant binding of this mutant was observed although wild type AT(1) receptor was shown to bind all AngII analogues.

PROM1 gene variations in Brazilian patients with macular dystrophy.

ABSTRACT Background: Although the pathogenicity of the prominin-1 (PROM1) gene has already been described as associated with autosomal dominant Stargardt disease, little is known about sequence variations in this gene. Purpose: The aim of this study was to evaluate PROM1 gene sequence variations in patients with macular dystrophy. Material and methods: This retrospective study evaluated variations in the PROM1 gene detected by next-generation sequencing test in patients with macular dystrophy and Stargardt disease. Results: Of 25 medical records of patients with Stargardt disease, three records of patients with PROM1 gene sequence variations were selected for the study. The p.Asp776Val and p.Asp829Asn variants were detected in cases 1 and 2, respectively, and predicted to be pathogenic; they were probably responsible for macular dystrophy in these patients. Case 3 showed a p.Ala643Gly variant in the PROM1 gene and a single variation in the ABCA4 gene, but molecular testing results were inconclusive. Conclusions: In cases of Stargardt disease, where molecular testing results are inconclusive for pathogenic variations in the ABCA4 gene, variations in the PROM1 gene may occur and be considered responsible for the disease in the molecular analysis. This study described three cases in which variations in PROM1 gene may play a role in the pathogenesis of macular dystrophy or be associated with both autosomal recessive and autosomal dominant inheritance.