RESUMEN
Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1C443S/C443S) mice using the CRISPR/Cas9 system. Zranb1C443S/C443S mice exhibited decreased mRNA expression and MUC2 production. Colonic organoids from Zranb1C443S/C443S mice displayed decreased Muc2 mRNA expression following differentiation into goblet cells. Finally, we analyzed dextran sulfate sodium-induced colitis to understand ZRANB1's role in intestinal inflammation. Zranb1C443S/C443S mice with colitis exhibited significant weight loss, reduced colon length, and worsening clinical and pathological scores, indicating that ZRANB1 contributes to intestinal homeostasis. Together, these results suggest that ZRANB1 regulates MUC2 expression and intestinal inflammation, which may help elucidating the pathogenesis of inflammatory bowel disease and developing new therapeutics targeting ZRANB1.
Asunto(s)
Colitis , Mucosa Intestinal , Proteasas Ubiquitina-Específicas , Animales , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Cisteína/metabolismo , Enzimas Desubicuitinizantes/metabolismo , Sulfato de Dextran/toxicidad , Inflamación/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Mucinas/metabolismo , Moco/metabolismo , ARN Mensajero/genética , Serina/metabolismo , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Inflammatory bowel disease (IBD) comprises two major subtypes, ulcerative colitis (UC) and Crohn's disease, which are multifactorial diseases that may develop due to genetic susceptibility, dysbiosis, or environmental factors. Environmental triggers of IBD include food-borne factors, and a previous nationwide survey in Japan identified pre-illness consumption of isoflavones as a risk factor for UC. However, the precise mechanisms involved in the detrimental effects of isoflavones on the intestinal mucosa remain unclear. The present study employed human colonic organoids (hCOs) to investigate the functional effect of two representative isoflavones, genistein and daidzein, on human colonic epithelial cells. The addition of genistein to organoid reformation assays significantly decreased the number and size of reformed hCOs compared with control and daidzein treatment, indicating an inhibitory effect of genistein on colonic cell/progenitor cell function. Evaluation of the phosphorylation status of 49 different receptor tyrosine kinases showed that genistein selectively inhibited phosphorylation of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR). We established a two-dimensional wound-repair model using hCOs and showed that genistein significantly delayed the overall wound-repair response. Our results collectively show that genistein may exert its detrimental effects on the intestinal mucosa via negative regulation of stem/progenitor cell function, possibly leading to sustained mucosal injury and the development of UC.
RESUMEN
We previously described the discovery of Big angiotensin-25 (Bang-25), an angiotensin-related peptide isolated from human urine. Bang-25 consists of the first 25 amino acids of the N-terminus of angiotensinogen (Aogen), with N-linked glycosylation on the 14th amino acid and a cysteine conjugated to the 18th amino acid. Bang-25 is rapidly converted into angiotensin II (Ang II) by chymase. Because Bang-25 is widely distributed in human tissues, including islet cells in the pancreas and podocytes in the kidney, we hypothesized that it may participate in the Ang II production system in these tissues. To test this hypothesis, we developed a specific assay for Bang-25 and used it to examine the urinary concentrations of Bang-25 in patients with diabetes mellitus (DM). The assay used the Amplified Luminescent Proximity Homogeneous Assay (Alpha)-based ELISA method (AlphaLISA) of PerkinElmer Japan and included antibodies specific to the N-terminus of Ang II and the C-terminus of Bang-25. The AlphaLISA ImmunoAssay specifically recognized Bang-25 and had no cross-reactivity with Aogen or Ang I. Bang-25 was detected in healthy volunteers' urine samples but not in their plasma samples. In patients with DM, the urinary Bang-25 concentration was significantly higher than in healthy volunteers. Moreover, the results indicated that the Bang-25 concentration in the urine may offer a different perspective on disease status from that provided by urinary albumin. This assay could provide a useful tool for determining urinary Bang-25, which may prove an important biomarker for diabetic kidney disease.
Asunto(s)
Angiotensina II/orina , Diabetes Mellitus/orina , Ensayo de Inmunoadsorción Enzimática/métodos , Adulto , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Human adrenomedullin (AM) functions as a circulating hormone and as a local paracrine mediator with multiple biological activities. We investigated the metabolism of AM by examining its fragmentation in human serum. Adrenomedullin was rapidly cleaved in human serum, but was relatively stable in plasma. We showed that AM was rapidly digested by thrombin in serum, with AM(13-44) as the main product. On the basis of these data, we prepared AM analogs in which Arg-44 was replaced by Ala, Lys, and D-Arg, respectively. These analogs were resistant to thrombin and showed comparable biological activity to native AM. Furthermore, the bioavailabilities of these peptides were improved after subcutaneous administration in rats. These AM analogs may be promising drug candidates for clinical applications.
Asunto(s)
Adrenomedulina/química , Adrenomedulina/metabolismo , Trombina/metabolismo , Adrenomedulina/síntesis química , Adrenomedulina/farmacocinética , Animales , Células HEK293 , Humanos , Masculino , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacocinética , Proteolisis , Ratas WistarRESUMEN
The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-ß. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1, Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-ß. Surprisingly, addition of TGF-ß induced a set of genes, including Slc28a2, Tubb2a, and Cpe, that were expressed preferentially in fetal IECs. Moreover, TGF-ß significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs.
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Movimiento Celular/genética , Células Epiteliales/patología , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Intestinos/patología , Modelos Biológicos , Factor de Crecimiento Transformador beta/farmacología , Cicatrización de Heridas/genética , Animales , Movimiento Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Feto/efectos de los fármacos , Ratones Endogámicos C57BL , Organoides/efectos de los fármacos , Organoides/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Cicatrización de Heridas/efectos de los fármacosRESUMEN
BACKGROUND: Heart chymase rather than angiotensin (Ang)-converting enzyme has higher specificity for Ang I conversion into Ang II in humans. A new pathway for direct cardiac Ang II generation has been revealed through the demonstration that Ang-(1-12) is cleaved by chymase to generate Ang II directly. Herein, we address whether Ang-(1-12), chymase messenger RNA (mRNA), and activity levels can be differentiated in human atrial tissue from normal and diseased hearts and if these measures associate with various pathologic heart conditions. MATERIALS AND METHODS: Atrial appendages were collected from 11 nonfailing donor hearts and 111 patients undergoing heart surgery for the correction of valvular heart disease, resistant atrial fibrillation, or ischemic heart disease. Chymase mRNA was analyzed by real-time polymerase chain reaction and enzymatic activity by high-performance liquid chromatography using Ang-(1-12) as the substrate. Ang-(1-12) levels were determined by immunohistochemical staining. RESULTS: Chymase gene transcripts, chymase activity, and immunoreactive Ang-(1-12) expression levels were higher in left atrial tissue compared with right atrial tissue, irrespective of cardiac disease. In addition, left atrial chymase mRNA expression was significantly higher in stroke versus nonstroke patients and in cardiac surgery patients who had a history of postoperative atrial fibrillation versus nonatrial fibrillation. Correlation analysis showed that left atrial chymase mRNA was positively related to left atrial enlargement, as determined by echocardiography. CONCLUSIONS: As Ang-(1-12) expression and chymase gene transcripts and enzymatic activity levels were positively linked to left atrial size in patients with left ventricular heart disease, an important alternate Ang II forming pathway, via Ang-(1-12) and chymase, in maladaptive atrial and ventricular remodeling in humans is uncovered.
Asunto(s)
Angiotensinógeno/metabolismo , Fibrilación Atrial/epidemiología , Quimasas/metabolismo , Atrios Cardíacos/patología , Fragmentos de Péptidos/metabolismo , Accidente Cerebrovascular/epidemiología , Anciano , Angiotensinógeno/análisis , Animales , Fibrilación Atrial/patología , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/cirugía , Quimasas/análisis , Quimasas/genética , Ecocardiografía , Femenino , Perfilación de la Expresión Génica , Atrios Cardíacos/diagnóstico por imagen , Atrios Cardíacos/fisiopatología , Atrios Cardíacos/cirugía , Enfermedades de las Válvulas Cardíacas/patología , Enfermedades de las Válvulas Cardíacas/cirugía , Ventrículos Cardíacos/fisiopatología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/patología , Isquemia Miocárdica/cirugía , Fragmentos de Péptidos/análisis , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Remodelación VentricularRESUMEN
Adrenomedullin (AM) improves colitis in animal models and patients with inflammatory bowel disease. We have developed a PEGylated AM derivative (PEG-AM) for clinical application because AM has a short half-life in the blood. However, modification by addition of polyethylene glycol (PEG) may compromise the function of the original peptide. In this paper, we examined the time course of cAMP accumulation induced by 5 and 60 kDa PEG-AM and compared the activation of calcitonin gene-related peptide (CGRP), AM1 and AM2 receptors by AM, 5 and 60 kDa PEG-AM. We also evaluated the effects of antagonists on the action of 5 and 60 kDa PEG-AM. PEG-AM stimulated cAMP production induced by these receptors; the increase in cAMP levels resulting from application of PEG-AM peaked at 15 min. Moreover, PEG-AM activity was antagonized by CGRP (8-37) or AM (22-52) (antagonists of CGRP and AM receptors, respectively) and the maximal response was not suppressed. These findings indicate that the effects of PEG-AM are similar to those of native AM.
Asunto(s)
Adrenomedulina/farmacología , Proteína 1 Modificadora de la Actividad de Receptores/agonistas , Proteína 2 Modificadora de la Actividad de Receptores/agonistas , Proteína 3 Modificadora de la Actividad de Receptores/agonistas , Adrenomedulina/análogos & derivados , Células HEK293 , Semivida , Humanos , Polietilenglicoles/química , Proteína 1 Modificadora de la Actividad de Receptores/genética , Proteína 1 Modificadora de la Actividad de Receptores/metabolismo , Proteína 2 Modificadora de la Actividad de Receptores/genética , Proteína 2 Modificadora de la Actividad de Receptores/metabolismo , Proteína 3 Modificadora de la Actividad de Receptores/genética , Proteína 3 Modificadora de la Actividad de Receptores/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
NEW FINDINGS: What is the central question of this study? Can chymase inhibition prevent angiotensin I-induced hypertension through inhibiting the conversion of angiotensin I to angiotensin II in the kidney? What is the main finding and its importance? Treatment with TEI-F00806 decreased angiotensin II content of the kidney, renal cortical angiotensinogen protein levels and chymase mRNA expression, and attenuated the development of hypertension. ABSTRACT: The effects of the selective chymase inhibitor TEI-F00806 were examined on angiotensin I (Ang I)-induced hypertension and intrarenal angiotensin II (Ang II) production in salt-treated mice. Twelve-week-old C57BL male mice were given a high-salt diet (4% NaCl + saline (0.9% NaCl)), and divided into three groups: (1) sham + vehicle (5% acetic acid in saline), (2) Ang I (1 µg kg-1 min-1 , s.c.) + vehicle, and (3) Ang I + TEI-F00806 (100 mg kg-1 day-1 , p.o.) (n = 8-10 per group). Systolic blood pressure was measured weekly using a tail-cuff method. Kidney Ang II content was measured by radioimmunoassay. Chronic infusion of Ang I resulted in the development of hypertension (P < 0.001), and augmented intrarenal chymase gene expression (P < 0.05), angiotensinogen protein level (P < 0.001) and Ang II content (P < 0.01) in salt-treated mice. Treatment with TEI-F00806 attenuated the development of hypertension (P < 0.001) and decreased Ang II content of the kidney (P < 0.05), which was associated with reductions in renal cortical angiotensinogen protein levels (P < 0.001) and chymase mRNA expression (P < 0.05). These data suggest that a chymase inhibitor decreases intrarenal renin-angiotensin activity, thereby reducing salt-dependent hypertension.
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Presión Sanguínea/efectos de los fármacos , Quimasas/antagonistas & inhibidores , Hipertensión/tratamiento farmacológico , Sistema Renina-Angiotensina/efectos de los fármacos , Angiotensina I , Angiotensina II/metabolismo , Animales , Quimasas/metabolismo , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Ratones , Peptidil-Dipeptidasa A/metabolismoRESUMEN
Adrenomedullin (AM) is a potent hypotensive peptide that exerts a powerful variety of protective effects against multiorgan damage through the AM type 1 receptor (AM1 receptor), which consists of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2). Two ß-arrestin (ß-arr) isoforms, ß-arr-1 and ß-arr-2, play a central role in the agonist-induced internalization of many receptors for receptor resensitization. Notably, ß-arr-biased agonists are now being tested in phase II clinical trials, targeting acute pain and acute heart failure. Here, we examined the effects of ß-arr-1 and ß-arr-2 on human AM1 receptor internalization. We constructed a V5-tagged chimera in which the cytoplasmic C-terminal tail (C-tail) of CLR was replaced with that of the ß2-adrenergic receptor (ß2-AR), and it was transiently transfected into HEK-293 cells that stably expressed RAMP2. The cell-surface expression and internalization of the wild-type or chimeric receptor were quantified by flow cytometric analysis. The [125I]AM binding and the AM-induced cAMP production of these receptors were also determined. Surprisingly, the coexpression of ß-arr-1 or -2 resulted in significant decreases in AM1 receptor internalization without affecting AM binding and signaling prior to receptor internalization. Dominant-negative (DN) ß-arr-1 or -2 also significantly decreased AM-induced AM1 receptor internalization. In contrast, the AM-induced internalization of the chimeric AM1 receptor was markedly augmented by the cotransfection of ß-arr-1 or -2 and significantly reduced by the coexpression of DN-ß-arr-1 or -2. These results were consistent with those seen for ß2-AR. Thus, both ß-arrs negatively control AM1 receptor internalization, which depends on the C-tail of CLR.
Asunto(s)
Adrenomedulina/metabolismo , Proteína Similar al Receptor de Calcitonina/metabolismo , Receptores de Adrenomedulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismo , Células HEK293 , HumanosRESUMEN
Ligand-dependent activation of Notch signaling is required to maintain the stem-cell niche of normal intestinal epithelium. However, the precise role of Notch signaling in the maintenance of the intestinal tumor stem cell niche and the importance of the RBPJ-independent non-canonical pathway in intestinal tumors remains unknown. Here we show that Notch signaling was activated in LGR5+ve cells of APC-deficient mice intestinal tumors. Accordingly, Notch ligands, including Jag1, Dll1, and Dll4, were expressed in these tumors. In vitro studies using tumor-derived organoids confirmed the intrinsic Notch activity-dependent growth of tumor cells. Surprisingly, the targeted deletion of Jag1 but not RBPJ in LGR5+ve tumor-initiating cells resulted in the silencing of Hes1 expression, disruption of the tumor stem cell niche, and dramatic reduction in the proliferation activity of APC-deficient intestinal tumors in vivo. Thus, our results highlight the importance of ligand-dependent non-canonical Notch signaling in the proliferation and maintenance of the tumor stem cell niche in APC-deficient intestinal adenomas.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Neoplasias Intestinales/metabolismo , Proteína Jagged-1/genética , Receptores Notch/metabolismo , Células Madre/citología , Adenoma/metabolismo , Animales , Proliferación Celular , Factor de Crecimiento Epidérmico/metabolismo , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Ligandos , Ratones , Microscopía Fluorescente , Células Madre Neoplásicas/citología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
Preclinical Research Human adrenomedullin (hAM), a hypotensive peptide, also has anti-inflammatory effects. hAM can reduce the severity of the dextran sulphate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in animal models. Furthermore, in a clinical study, hAM treatment reduced the Disease Activity Index in ulcerative colitis. However, these therapeutic effects required continuous administration of hAM as the half-life of native hAM is quite short in blood. To resolve this problem, hAM N-terminal was conjugated with two kinds of polyethylene glycol (PEG); 5 kDa PEG or 60 kDa PEG (5 kDa PEG-hAM and 60 kDa PEG-hAM respectively). In a previous study, 5 kDa PEG-hAM stimulated cAMP production and prolonged the plasma half-life compared with native hAM. Herein we examine the effect of PEG-hAM in the DSS colitis model. Treatment with both PEG-hAM preparations reduced the total inflammation score. In addition, the plasma half-life of 60 kDa PEG-hAM was much longer than 5 kDa PEG-hAM. In summary, a single subcutaneous administration of 60 kDa PEG-hAM reduced the total inflammation score in mice with DSS-induced colitis. Therefore, these results suggest that 60 kDa PEG-hAM is a possible therapeutic agent for the treatment of inflammatory bowel disease. Drug Dev Res 78 : 129-134, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Adrenomedulina/administración & dosificación , Antiinflamatorios/administración & dosificación , Colitis Ulcerosa/tratamiento farmacológico , Sulfato de Dextran/efectos adversos , Polietilenglicoles/química , Adrenomedulina/química , Adrenomedulina/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/metabolismo , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Semivida , Humanos , Inyecciones Subcutáneas , Masculino , RatonesRESUMEN
Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [(125)I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or ß2-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449-453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM1 receptor and further determined the region of the CLR C-tail responsible for this GRK function.
Asunto(s)
Membrana Celular/metabolismo , Quinasa 4 del Receptor Acoplado a Proteína-G/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Receptores de Adrenomedulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Regulación hacia Abajo/fisiología , Células HEK293 , HumanosRESUMEN
The present study attempted to identify T helper epitope long peptides capable of inducing cytotoxic T lymphocytes (CTL) from Lck antigen (p56(Lck) ), the src family tyrosine kinase, which is known to be aberrantly expressed in metastatic cancers cells, in order to develop a long peptide-based cancer vaccine for HLA-A2(+) cancer patients. Based on the biding motif to the HLA-DR and HLA-A2 alleles, 94 peptides were prepared from the Lck antigen. These peptides were screened for their reactivity to immunoglobulin G (IgG) from plasma of cancer patients, followed by testing of their ability to induce both CD4(+) and CD8(+) T lymphocytes showing not only peptide-specific IFN-γ production but cytotoxicity against HLA-A2(+) cancer cells from peripheral blood mononuclear cells (PBMC) of HLA-A2(+) cancer patients. Among 94 peptides tested, the three T helper epitope long peptides and their inner CTL epitope short peptides with HLA-A2 binding motifs were frequently recognized by IgG of cancer patients, and efficiently induced both CD4(+) IFN-γ(+) and CD8(+) IFN-γ(+) T lymphocytes. Patients' PBMC stimulated with these long peptides showed cytotoxicity against HLA-A2(+) Lck(+) cancer cells in HLA-class I and HLA-class II dependent manners. These three peptides might be useful for long peptide-based vaccines for HLA-A2(+) cancer patients with Lck(+) tumor cells.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Epítopos de Linfocito T/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Neoplasias/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Antígeno HLA-A2/inmunología , Humanos , Péptidos/inmunologíaRESUMEN
Lessons learned from the characterization of the biological roles of Ang-(1-7) [angiotensin-(1-7)] in opposing the vasoconstrictor, proliferative and prothrombotic actions of AngII (angiotensin II) created an underpinning for a more comprehensive exploration of the multiple pathways by which the RAS (renin-angiotensin system) of blood and tissues regulates homoeostasis and its altered state in disease processes. The present review summarizes the progress that has been made in the novel exploration of intermediate shorter forms of angiotensinogen through the characterization of the expression and functions of the dodecapeptide Ang-(1-12) [angiotensin-(1-12)] in the cardiac production of AngII. The studies reveal significant differences in humans compared with rodents regarding the enzymatic pathway by which Ang-(1-12) undergoes metabolism. Highlights of the research include the demonstration of chymase-directed formation of AngII from Ang-(1-12) in human left atrial myocytes and left ventricular tissue, the presence of robust expression of Ang-(1-12) and chymase in the atrial appendage of subjects with resistant atrial fibrillation, and the preliminary observation of significantly higher Ang-(1-12) expression in human left atrial appendages.
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Angiotensina II/fisiología , Secuencia de Aminoácidos , Angiotensinógeno/química , Angiotensinógeno/fisiología , Animales , Humanos , Datos de Secuencia Molecular , RoedoresRESUMEN
Our current recognition of the renin-angiotensin system is more convoluted than originally thought due to the discovery of multiple novel enzymes, peptides, and receptors inherent in this interactive biochemical cascade. Over the last decade, angiotensin-converting enzyme 2 (ACE2) has emerged as a key player in the pathophysiology of hypertension and cardiovascular and renal disease due to its pivotal role in metabolizing vasoconstrictive/hypertrophic/proliferative angiotensin II into favorable angiotensin-(1-7). This review addresses the considerable advancement in research on the role of tissue ACE2 in the development and progression of hypertension and cardiac and renal injury. We summarize the results from recent clinical and experimental studies suggesting that serum or urine soluble ACE2 may serve as a novel biomarker or independent risk factor relevant for diagnosis and prognosis of cardiorenal disease. We also review recent proceedings on novel therapeutic approaches to enhance ACE2/angiotensin-(1-7) axis.
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Angiotensina II/metabolismo , Angiotensina I/metabolismo , Cardiopatías/metabolismo , Hipertensión/metabolismo , Enfermedades Renales/metabolismo , Fragmentos de Péptidos/metabolismo , Peptidil-Dipeptidasa A/fisiología , Enzima Convertidora de Angiotensina 2 , Animales , Enfermedades Cardiovasculares/metabolismo , Humanos , Peptidil-Dipeptidasa A/metabolismoRESUMEN
The classical view of biochemical pathways for the formation of biologically active angiotensins continues to undergo significant revision as new data uncovers the existence of important species differences between humans and rodents. The discovery of two novel substrates that, cleaved from angiotensinogen, can lead to direct tissue angiotensin II formation has the potential of radically altering our understanding of how tissues source angiotensin II production and explain the relative lack of efficacy that characterizes the use of angiotensin converting enzyme inhibitors in cardiovascular disease. This review addresses the discovery of angiotensin-(1-12) as an endogenous substrate for the production of biologically active angiotensin peptides by a non-renin dependent mechanism and the revealing role of cardiac chymase as the angiotensin II convertase in the human heart. This new information provides a renewed argument for exploring the role of chymase inhibitors in the correction of cardiac arrhythmias and left ventricular systolic and diastolic dysfunction.
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Angiotensina II/metabolismo , Quimasas/metabolismo , Hipertensión/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Corazón/fisiopatología , Humanos , Especificidad por SustratoRESUMEN
Ghrelin is a peptide hormone with various important physiological functions. The unique feature of ghrelin is its serine 3 acyl-modification, which is essential for ghrelin activity. The major form of ghrelin is modified with n-octanoic acid (C8:0) by ghrelin O-acyltransferase. Various acyl modifications have been reported in different species. However, the underlying mechanism by which ghrelin is modified with various fatty acids remains to be elucidated. Herein, we report the purification of bovine, porcine, and equine ghrelins. The major active form of bovine ghrelin was a 27-amino acid peptide with an n-octanoyl (C8:0) modification at Ser3. The major active form of porcine and equine ghrelin was a 28-amino acid peptide. However, porcine ghrelin was modified with n-octanol (C8:0), whereas equine ghrelin was modified with n-butanol (C4:0) at Ser3. This study indicates the existence of structural divergence in ghrelin and suggests that it is necessary to measure the minor and major forms of ghrelin to fully understand its physiology.
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Ghrelina , Animales , Ghrelina/metabolismo , Ghrelina/química , Caballos , Bovinos , Porcinos , Secuencia de Aminoácidos , Acilación , Caprilatos/metabolismoRESUMEN
Angiotensin-(1-12) [ANG-(1-12)], a new member of the renin-angiotensin system, is recognized as a renin independent precursor for ANG II. However, the processing of ANG-(1-12) in the circulation in vivo is not fully established. We examined the effect of angiotensin converting enzyme (ACE) and chymase inhibition on angiotensin peptides formation during an intravenous infusion of ANG-(1-12) in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). WKY and SHR were assigned to a short ANG-(1-12) infusion lasting 5, 15, 30, or 60 min (n = 4-10 each group). In another experiment WKY and SHR were assigned to a continuous 15-min ANG-(1-12) infusion with pretreatment of saline, lisinopril (10 mg/kg), or chymostatin (10 mg/kg) (n = 7-13 each group). Saline or lisinopril were infused intravenously 15 min before the administration of ANG-(1-12) (2 nmol·kg(-1)·min(-1)), whereas chymostatin was given by bolus intraperitoneal injection 30 min before ANG-(1-12). Infusion of ANG-(1-12) increased arterial pressure and plasma ANG-(1-12), ANG I, ANG II, and ANG-(1-7) levels in WKY and SHR. Pretreatment with lisinopril caused increase in ANG-(1-12) and ANG I and large decreases in ANG II compared with the other two groups in both strains. Pretreatment of chymostatin had no effect on ANG-(1-12), ANG I, and ANG II levels in both strains, whereas it increased ANG-(1-7) levels in WKY. We conclude that ACE acts as the primary enzyme for the conversion of ANG-(1-12) to smaller angiotensin peptides in the circulation of WKY and SHR and that chymase may be an ANG-(1-7) degrading enzyme.
Asunto(s)
Angiotensinógeno/metabolismo , Hipertensión/metabolismo , Fragmentos de Péptidos/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Angiotensina I/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Angiotensinógeno/administración & dosificación , Angiotensinógeno/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Quimasas/antagonistas & inhibidores , Quimasas/efectos de los fármacos , Quimasas/metabolismo , Modelos Animales de Enfermedad , Hipertensión/fisiopatología , Infusiones Intravenosas , Lisinopril/farmacología , Masculino , Oligopéptidos/farmacología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/farmacología , Peptidil-Dipeptidasa A/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
The renin-angiotensin system (RAS), including angiotensin II (Ang II), plays an important role in the regulation of blood pressure and body fluid balance. Consequently, the RAS has emerged as a key target for treatment of kidney and cardiovascular disease. In a search for bioactive peptides using an antibody against the N-terminal portion of Ang II, we identified and characterized a novel angiotensin-related peptide from human urine as a major molecular form. We named the peptide Big angiotensin-25 (Bang-25) because it consists of 25 amino acids with a glycosyl chain and added cysteine. Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. The peptide is abundant in human urine and is present in a wide range of organs and tissues. In particular, immunostaining of Bang-25 in the kidney is specifically localized to podocytes. Although the physiological function of Bang-25 remains uncertain, our findings suggest it is processed from angiotensinogen and may represent an alternative, renin-independent path for Ang II synthesis in tissue.
Asunto(s)
Angiotensina II/metabolismo , Angiotensina II/orina , Secuencia de Aminoácidos , Glicosilación , Humanos , Datos de Secuencia Molecular , Péptidos/metabolismo , Péptidos/orina , Distribución TisularRESUMEN
Adrenomedullin (AM) is a peptide with pleiotropic physiological functions that attenuates intestinal mucosal inflammation. However, the mechanism underpinning mucosal protection by AM is not fully understood, and its effect on intestinal epithelial cells remains unclear. Here, we investigated the effects of AM on junctional molecules in primary-cultured murine intestinal epithelial cells and discovered that AM upregulates claudin-4 expression. In a mouse model of dextran sulfate sodium-induced colitis, AM administration also enhanced claudin-4 expression and accelerated mucosal regeneration. Furthermore, AM reversed TNFα-mediated downregulation of claudin-4 and loss of cell-cell adhesion of the HCT116 human intestinal epithelial cell line in vitro. These results indicate that AM may enhance intestinal epithelial integrity by upregulating claudin-4 expression.