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1.
Endocrinology ; 163(9)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35863039

RESUMO

Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGß gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.


Assuntos
Neoplasias Hipofisárias , Prolactinoma , Animais , Feminino , Humanos , Cininas , Masculino , Camundongos , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Prolactina/metabolismo , Prolactinoma/genética , Prolactinoma/metabolismo , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Receptores da Bradicinina
2.
J Mol Med (Berl) ; 100(4): 613-627, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35247068

RESUMO

SARS-CoV-2 has evolved to enter the host via the ACE2 receptor which is part of the kinin-kallikrein pathway. This complex pathway is only poorly understood in context of immune regulation but critical to control infection. This study examines SARS-CoV-2-infection and epithelial mechanisms of the kinin-kallikrein-system at the kinin B2 receptor level in SARS-CoV-2-infection that is of direct translational relevance. From acute SARS-CoV-2-positive study participants and -negative controls, transcriptomes of nasal curettages were analyzed. Primary airway epithelial cells (NHBEs) were infected with SARS-CoV-2 and treated with the approved B2R-antagonist icatibant. SARS-CoV-2 RNA RT-qPCR, cytotoxicity assays, plaque assays, and transcriptome analyses were performed. The treatment effect was further studied in a murine airway inflammation model in vivo. Here, we report a broad and strong upregulation of kallikreins and the kinin B2 receptor (B2R) in the nasal mucosa of acutely symptomatic SARS-CoV-2-positive study participants. A B2R-antagonist impeded SARS-CoV-2 replication and spread in NHBEs, as determined in plaque assays on Vero-E6 cells. B2R-antagonism reduced the expression of SARS-CoV-2 entry receptor ACE2, G protein-coupled receptor signaling, and ion transport in vitro and in a murine airway inflammation in vivo model. In summary, this study provides evidence that treatment with B2R-antagonists protects airway epithelial cells from SARS-CoV-2 by inhibiting its replication and spread, through the reduction of ACE2 levels and the interference with several cellular signaling processes. Future clinical studies need to shed light on the airway protection potential of approved B2R-antagonists, like icatibant, in the treatment of early-stage COVID-19. KEY MESSAGES: Induction of kinin B2 receptor in the nose of SARS-CoV-2-positive patients. Treatment with B2R-antagonist protects airway epithelial cells from SARS-CoV-2. B2R-antagonist reduces ACE2 levels in vivo and ex vivo. Protection by B2R-antagonist is mediated by inhibiting viral replication and spread.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , COVID-19/tratamento farmacológico , Epitélio , Humanos , Camundongos , RNA Viral , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo
3.
Oxid Med Cell Longev ; 2022: 1247806, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35154561

RESUMO

OBJECTIVE: The aim of this study was to investigate whether tissue kallikrein (KLK1) can protect the prostate from inflammatory damage and the mechanism involved in it. METHODS: A total of 50 male Wistar rats were used in this study. Initially, 20 rats were sacrificed to obtain the prostate antigen to induce experimental autoimmune prostatitis (EAP), and the remaining 30 rats were randomly divided into 5 experimental groups (normal control group (NC group), NC+KLK1 group (NCK group), EAP group, EAP+KLK1 group (EAPK group), and EAP+KLK1+HOE140 group (EAPKH group); n = 6). It should be explained that KLK1 mainly exerts its biological effects through bradykinin, and HOE140 is a potent and selective bradykinin receptor B2 (BDKRB2) antagonist. EAP was induced by intradermal injection of 15 mg/ml prostate antigen and complete Freund's adjuvant on days 0, 14, and 28. KLK1 was injected via tail vein at a dose of 1.5 × 10-3 PAN U/kg once a day, and HOE140 was administered by intraperitoneal injection at 20 µg/kg once every two days. Rats were sacrificed on day 42. The RNA and protein of the rat prostate were extracted to analyze the expression differences of KLK1, as well as the inflammation-, fibrosis-, and oxidative stress-related genes. The inflammatory cell infiltration and microvessel density of the prostate were also analyzed by pathological examination. In addition, pathological analysis was performed on prostate samples from patients undergoing benign prostate hyperplasia (BPH) surgery. RESULTS: The expression of KLK1 in the prostate decreased in the EAP group as well as BPH patients with obvious inflammation. KLK1 administration significantly inhibited inflammatory cell infiltration and reduced the production of inflammatory cytokines in the EAPK group. Prostate samples from the EAP group showed increased infiltration of T cells and macrophages, as well as gland atrophy, hypoxia, fibrosis, and angiogenesis. KLK1 administration upregulated endothelial nitric oxide synthase (eNOS) expression and suppressed oxidative stress, as well as transforming growth factor ß1 (TGF-ß) signaling pathways and the proangiogenic vascular endothelial growth factor (VEGF) in the EAPK group. However, in the EAPKH group in which HOE140 blocked BDKRB2, the beneficial effects of KLK1 were all cancelled. In addition, KLK1 intervention in normal rats had no obvious side effects. CONCLUSION: The KLK1 expression is inhibited in the inflamed prostates of humans and rats. Exogenous KLK1 restored endothelial function via a BDKRB2-dependent way and then played a role in improving microcirculation and exerted anti-inflammatory, antifibrotic, and antioxidative stress effects in the rat chronic-inflamed prostate.


Assuntos
Doenças Autoimunes/complicações , Doenças Autoimunes/tratamento farmacológico , Células Endoteliais/metabolismo , Próstata/patologia , Prostatite/complicações , Prostatite/tratamento farmacológico , Substâncias Protetoras/administração & dosagem , Receptor B2 da Bradicinina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Calicreínas Teciduais/administração & dosagem , Calicreínas Teciduais/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Doenças Autoimunes/metabolismo , Doença Crônica , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Hiperplasia/cirurgia , Masculino , Pessoa de Meia-Idade , Prostatite/metabolismo , Ratos , Ratos Wistar , Estudos Retrospectivos , Calicreínas Teciduais/genética
4.
Nat Commun ; 13(1): 714, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35132089

RESUMO

The type 2 bradykinin receptor (B2R) is a G protein-coupled receptor (GPCR) in the cardiovascular system, and the dysfunction of B2R leads to inflammation, hereditary angioedema, and pain. Bradykinin and kallidin are both endogenous peptide agonists of B2R, acting as vasodilators to protect the cardiovascular system. Here we determine two cryo-electron microscopy (cryo-EM) structures of human B2R-Gq in complex with bradykinin and kallidin at 3.0 Å and 2.9 Å resolution, respectively. The ligand-binding pocket accommodates S-shaped peptides, with aspartic acids and glutamates as an anion trap. The phenylalanines at the tail of the peptides induce significant conformational changes in the toggle switch W2836.48, the conserved PIF, DRY, and NPxxY motifs, for the B2R activation. This further induces the extensive interactions of the intracellular loops ICL2/3 and helix 8 with Gq proteins. Our structures elucidate the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins coupling of B2R.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Receptor B2 da Bradicinina/química , Sequência de Aminoácidos , Sítios de Ligação , Bradicinina/química , Bradicinina/metabolismo , Microscopia Crioeletrônica , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Calidina/química , Calidina/metabolismo , Ligantes , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptor B2 da Bradicinina/metabolismo
5.
Int Immunopharmacol ; 105: 108523, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35086057

RESUMO

PHA-022121 is a novel small molecule bradykinin B2 receptor antagonist, in clinical development for the treatment and prevention of hereditary angioedema attacks. The present study describes the in vitro pharmacological characteristics of PHA-022121 and its active metabolite, PHA-022484 (M2-D). In mammalian cell lines, PHA-022121 and PHA-022484 show high affinity for the recombinant human bradykinin B2 receptor with Ki values of 0.47 and 0.70 nM, respectively, and potent antagonism of the human bradykinin B2 receptor with Kb values of 0.15 and 0.26 nM, respectively (calcium mobilization assay). Antagonist potency at the recombinant cynomolgus monkey bradykinin B2 receptor is similarly high (Kb values of 1.42 and 1.12 nM for PHA-022121 and PHA-022484, respectively), however, potency at rat, mouse, rabbit and dog bradykinin B2 receptors is at least 100-fold lower than the potency at the human receptor for both compounds. In the human umbilical vein contractility assay, both PHA-022121 and PHA-022484 show a potent, surmountable and reversible B2 antagonist activity with pA2 values of 0.35 and 0.47 nM, respectively. The in vitro off-target profile of PHA-022121 and PHA-022484 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B1 receptor. It is concluded that PHA-022121 is a novel, low-molecular weight, competitive antagonist of the human bradykinin B2 receptor with high affinity, high antagonist potency, and high selectivity. It is about 20-fold more potent than icatibant at the human bradykinin B2 receptor as assessed using recombinant or endogenously expressed receptors.


Assuntos
Antagonistas dos Receptores da Bradicinina , Bradicinina , Animais , Ligação Competitiva , Bradicinina/metabolismo , Antagonistas dos Receptores da Bradicinina/metabolismo , Antagonistas dos Receptores da Bradicinina/farmacologia , Cães , Macaca fascicularis/metabolismo , Mamíferos , Camundongos , Coelhos , Ratos , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Veias Umbilicais/metabolismo
6.
Eur J Pharmacol ; 912: 174591, 2021 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-34710369

RESUMO

Dry cough has been reported in patients receiving statin therapy. However, the underlying mechanism or other possible alterations in the airways induced by statins remain unknown. Thus, the aim of this study was to evaluate whether simvastatin promotes alterations in airways, such as bronchoconstriction and plasma extravasation, as well as the mechanism involved in these events. Using methods to detect alterations in airway resistance and plasma extravasation, we demonstrated that simvastatin [20 mg/kg, intravenous (i.v.)] caused plasma extravasation in the trachea (79.8 + 14.8 µg/g/tissue) and bronchi (73.3 + 8.8 µg/g/tissue) of rats, compared to the vehicle (34.2 + 3.6 µg/g/tissue and 29.3 + 5.3 µg/g/tissue, respectively). NG-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg, intraperitoneal), a nitric oxide (NO) synthase inhibitor, Icatibant [HOE 140, 10 nmol/50 µl, intratracheal (i.t.)], a bradykinin B2 antagonist, and capsazepine (100 nmol/50 µl, i.t.), a TRPV1 antagonist, attenuated simvastatin-induced plasma extravasation. Simvastatin (5, 10 and 20 mg/kg) did not cause bronchoconstriction per se, but exacerbated the bronchoconstrictive response to bradykinin (30 nmol/kg, i.v.), a B2 agonist (0.7 + 0.1 ml/H2O), or capsaicin (30 nmol/kg, i.v.), a TRPV1 agonist (0.8 + 0.1 ml/H2O), compared to the vehicle (0.1 + 0.04 ml/H2O and 0.04 + 0.01 ml/H2O, respectively). The bronchoconstriction elicited by bradykinin (100 nmol/kg, i.v.) in simvastatin non-treated rats was inhibited by L-NAME. The exacerbation of bronchoconstriction induced by bradykinin or capsaicin in simvastatin-treated rats was inhibited by L-NAME, HOE 140 or capsazepine. These results suggest that treatment with simvastatin promotes the release of bradykinin, which, via B2 receptors, releases NO that can then activate the TRPV1 to promote plasma extravasation and bronchoconstriction.


Assuntos
Brônquios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Receptor B2 da Bradicinina/metabolismo , Sinvastatina/efeitos adversos , Canais de Cátion TRPV/metabolismo , Traqueia/efeitos dos fármacos , Administração Intravenosa , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Bradicinina/administração & dosagem , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/administração & dosagem , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Brônquios/metabolismo , Broncoconstrição/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Capsaicina/administração & dosagem , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Injeções Intraperitoneais , Masculino , NG-Nitroarginina Metil Éster/administração & dosagem , NG-Nitroarginina Metil Éster/farmacologia , Ratos Wistar , Sinvastatina/administração & dosagem , Canais de Cátion TRPV/antagonistas & inibidores , Traqueia/metabolismo
7.
Cells ; 10(10)2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34685589

RESUMO

Preeclampsia is one of the most frequent and severe complications of pregnancy. Symptoms of preeclampsia usually occur after 20 weeks of pregnancy and include hypertension and kidney dysfunction with proteinuria. Up to now, delivery of the infant has been the most effective and life-saving treatment to alleviate symptoms of preeclampsia because a causative treatment does not exist, which could prolong a pregnancy complicated with preeclampsia. Preeclampsia is a complex medical condition, which is attributed to a variety of different risk factors and causes. Risk factors account for insufficient placentation and impaired vasculogenesis and finally culminate in this life-threatening condition of pregnancy. Despite progress, many pathomechanisms and causes of preeclampsia are still incompletely understood. In recent years, it was found that excessive protein complex formation between G-protein-coupled receptors is a common sign of preeclampsia. Specifically, the aberrant heteromerization of two vasoactive G-protein-coupled receptors (GPCRs), the angiotensin II AT1 receptor and the bradykinin B2 receptor, is a causative factor of preeclampsia symptoms. Based on this knowledge, inhibition of abnormal GPCR protein complex formation is an experimental treatment approach of preeclampsia. This review summarizes the impact of pathological GPCR protein aggregation on symptoms of preeclampsia and delineates potential new therapeutic targets.


Assuntos
Pré-Eclâmpsia/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Feminino , Humanos , Gravidez
8.
Biomed Pharmacother ; 143: 112165, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34543986

RESUMO

The injury of endothelial cells is one of the initiating factors in restenosis after endovascular treatment. Human urinary kallidinogenase (HUK) is a tissue kallikrein which is used for ischemia-reperfusion injury treatment. Studies have shown that HUK may be a potential therapeutic agent to prevent stenosis after vascular injury, however, the precise mechanisms have not been fully established. This study is to investigate whether HUK can protect endothelial cells after balloon injury or H2O2-induced endothelial cell damage through the proline-rich tyrosine kinase 2 (Pyk2)/mitochondrial calcium uniporter (MCU) pathway. Intimal hyperplasia, a decrease of pinocytotic vesicles and cell apoptosis were found in the common carotid artery balloon injury and H2O2-induced endothelial cell damage, Pyk2/MCU was also up-regulated in such pathological process. HUK could prevent these injuries partially via the bradykinin B2 receptor by inhibiting Pyk2/MCU pathway, which prevented the mitochondrial damage, maintained calcium balance, and eventually inhibited cell apoptosis. Furthermore, MCU expression was not markedly increased if Pyk2 was suppressed by shRNA technique in the H2O2 treatment group, and cell viability was significantly better than H2O2-treated only. In short, our results indicate that the Pyk2/MCU pathway is involved in endothelial injury induced by balloon injury or H2O2-induced endothelial cell damage. HUK plays an protective role by inhibiting the Pyk2/MCU pathway in the endothelial injury.


Assuntos
Canais de Cálcio/metabolismo , Lesões das Artérias Carótidas/tratamento farmacológico , Artéria Carótida Primitiva/efeitos dos fármacos , Quinase 2 de Adesão Focal/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Calicreínas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Canais de Cálcio/genética , Lesões das Artérias Carótidas/enzimologia , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva/enzimologia , Artéria Carótida Primitiva/ultraestrutura , Células Cultivadas , Modelos Animais de Doenças , Quinase 2 de Adesão Focal/genética , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Peróxido de Hidrogênio/toxicidade , Calicreínas/urina , Masculino , Neointima , Ratos Sprague-Dawley , Receptor B2 da Bradicinina/metabolismo , Transdução de Sinais
9.
J Diabetes Res ; 2021: 6626627, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34557552

RESUMO

BACKGROUND: Circulating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown. METHODS: Cord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated ß-galactosidase (SA-ß-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot. RESULTS: B2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK. CONCLUSION: BK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.


Assuntos
Bradicinina/farmacologia , Senescência Celular/efeitos dos fármacos , Células Progenitoras Endoteliais/efeitos dos fármacos , Receptor B2 da Bradicinina/metabolismo , Estudos de Casos e Controles , Células Cultivadas , DNA Mitocondrial/genética , Diabetes Gestacional , Dinoprosta/análogos & derivados , Dinoprosta/sangue , Células Progenitoras Endoteliais/citologia , Feminino , Sangue Fetal , Dosagem de Genes , Glucose/farmacologia , Humanos , Recém-Nascido , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo III/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Telomerase , Telômero
10.
Peptides ; 146: 170646, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34500007

RESUMO

Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg9-BK, an antagonist of B1. BK induced the stall of megalin in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.


Assuntos
Albuminas/metabolismo , Bradicinina/farmacologia , Endocitose/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Túbulos Renais Proximais/metabolismo , Células LLC-PK1 , Proteína Quinase C/metabolismo , Receptor B2 da Bradicinina/metabolismo , Suínos
11.
Cells ; 10(8)2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34440682

RESUMO

The kallikrein-kinin system (KKS) contributes to retinal inflammation and neovascularization, notably in diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD). Bradykinin type 1 (B1R) and type 2 (B2R) receptors are G-protein-coupled receptors that sense and mediate the effects of kinins. While B2R is constitutively expressed and regulates a plethora of physiological processes, B1R is almost undetectable under physiological conditions and contributes to pathological inflammation. Several KKS components (kininogens, tissue and plasma kallikreins, and kinin receptors) are overexpressed in human and animal models of retinal diseases, and their inhibition, particularly B1R, reduces inflammation and pathological neovascularization. In this review, we provide an overview of the KKS with emphasis on kinin receptors in the healthy retina and their detrimental roles in DR and AMD. We highlight the crosstalk between the KKS and the renin-angiotensin system (RAS), which is known to be detrimental in ocular pathologies. Targeting the KKS, particularly the B1R, is a promising therapy in retinal diseases, and B1R may represent an effector of the detrimental effects of RAS (Ang II-AT1R).


Assuntos
Cininas/metabolismo , Degeneração Macular/patologia , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Retina/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Humanos , Sistema Calicreína-Cinina , Degeneração Macular/metabolismo , Neovascularização Patológica , Sistema Renina-Angiotensina , Retina/patologia
12.
Clin Sci (Lond) ; 135(9): 1145-1163, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33899912

RESUMO

Compound 21 (C21), a selective agonist of angiotensin II type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of the present study was to assess the effect of C21 on thoracic aorta endothelial function in a model of diet-induced obesity (DIO) and to elucidate the potential cross-talk among AT2R, Mas receptor (MasR) and/or bradykinin type 2 receptor (B2R) in this response. Five-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1 mg/kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (HECs; EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels. Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favored the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine (ACh) due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways. In conclusion, C21 favors the interaction among AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Imidazóis/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Receptor B2 da Bradicinina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sulfonamidas/uso terapêutico , Tiofenos/uso terapêutico , Doenças Vasculares/prevenção & controle , Animais , Aorta Torácica/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dieta Hiperlipídica , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais da Veia Umbilical Humana , Humanos , Imidazóis/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/complicações , Obesidade/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Cross-Talk , Receptor Tipo 2 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo
13.
Skin Pharmacol Physiol ; 34(3): 162-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33794540

RESUMO

Bradykinin increases skin blood flow via a cGMP mechanism but its role in sweating in vivo is unclear. There is a current need to translate cell culture and nonhuman paw pad studies into in vivo human preparations to test for therapeutic viability for disorders affecting sweat glands. Protocol 1: physiological sweating was induced in 10 healthy subjects via perfusing warm (46-48°C) water through a tube-lined suit while bradykinin type 2 receptor (B2R) antagonist (HOE-140; 40 µM) and only the vehicle (lactated Ringer's) were perfused intradermally via microdialysis. Heat stress increased sweat rate (HOE-140 = +0.79 ± 0.12 and vehicle = +0.64 ± 0.10 mg/cm2/min), but no differences were noted with B2R antagonism. Protocol 2: pharmacological sweating was induced in 6 healthy subjects via intradermally perfusing pilocarpine (1.67 mg/mL) followed by the same B2R antagonist approach. Pilocarpine increased sweating (HOE-140 = +0.38 ± 0.16 and vehicle = +0.32 ± 0.12 mg/cm2/min); again no differences were observed with B2R antagonism. Last, 5 additional subjects were recruited for various control experiments which identified that a functional dose of HOE-140 was utilized and it was not sudorific during normothermic conditions. These data indicate B2R antagonists do not modulate physiologically or pharmacologically induced eccrine secretion volumes. Thus, B2R agonist/antagonist development as a potential therapeutic target for hypo- and hyperhidrosis appears unwarranted.


Assuntos
Antagonistas de Receptor B2 da Bradicinina/farmacologia , Bradicinina/análogos & derivados , Sudorese/efeitos dos fármacos , Bradicinina/farmacologia , Resposta ao Choque Térmico/efeitos dos fármacos , Resposta ao Choque Térmico/fisiologia , Humanos , Pilocarpina/farmacologia , Receptor B2 da Bradicinina/metabolismo , Pele/metabolismo , Sudorese/fisiologia
14.
Aging (Albany NY) ; 13(5): 7499-7516, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33686021

RESUMO

Bradykinin receptor B2 (BDKRB2) has been reported as an oncogene in several malignancies. In glioma, the role of BDKRB2 remains unknown. This study aimed at investigating its clinical significance and biological function in glioma at the transcriptional level. We selected 301 glioma patients with microarray data from CGGA database and 697 with RNAseq data from TCGA database. Transcriptome and clinical data of 998 samples were analyzed. Statistical analysis and figure generating were performed with R language. BDKRB2 expression showed a positive correlation with the WHO grade of glioma. BDKRB2 was increased in IDH wildtype and mesenchymal subtype of glioma. Gene ontology analysis demonstrated that BDKRB2 was profoundly associated with extracellular matrix organization in glioma. GSEA analysis revealed that BDKRB2 was particularly correlated with epithelial-to-mesenchymal transition (EMT). GSVA analysis showed that BDKRB2 was significantly paralleled with several EMT signaling pathways, including PI3K/AKT, hypoxia, and TGF-ß. Moreover, BDKRB2 expression was significantly correlated with key biomarkers of EMT, especially with N-cadherin, snail, slug, vimentin, TWIST1, and TWIST2. Finally, higher BDKRB2 indicated significantly shorter survival for glioma patients. In conclusion, BDKRB2 was associated with more aggressive phenotypes of gliomas. Furthermore, BDKRB2 was involved in the EMT process and could serve as an independent prognosticator in glioma.


Assuntos
Glioma/mortalidade , Receptor B2 da Bradicinina/metabolismo , Adulto , Biomarcadores Tumorais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Perfilação da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Transdução de Sinais , Análise de Sobrevida , Análise Serial de Tecidos
16.
Clin Exp Hypertens ; 43(5): 408-415, 2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-33687297

RESUMO

OBJECTIVE: To demonstrate that the kallikrein-kinin system (KKS) is upstream of angiogenic signaling pathway, and to determine the role of the kinin B1 and B2 receptors in myocardial angiogenesis induced by exercise training. METHODS: Forty Wistar rats were randomly assigned to an exercise control (EC) group, a B1 receptor antagonist (B1Ant) group, a B2 receptor antagonist (B2Ant) group, and a double receptor antagonist ((B1+ B2)Ant) group. A myocardial infarction model was employed. Animals in all groups received 30 min of exercise training for 4 weeks. The expression of VEGF and eNOS, capillary supply, and apoptosis rate were evaluated. RESULTS: The mRNA and protein expression of VEGF and eNOS showed similar trends in all groups, and were lowest in the (B1+ B2) Ant group, and highest in the EC group. Levels of VEGF and eNOS mRNA were significantly lower in the B1Ant group than in the B2Ant group (p< .001 and p< .05, respectively). VEGF and eNOS protein in the B1Ant group was also significantly lower (p< .01 and p< .05, respectively) than in the B2Ant group. The capillary numbers in the (B1+ B2) Ant group were significantly lower than in the EC group (395.8 ± 105 vs. 1127.9 ± 192.98, respectively). The apoptosis rate of cardiomyocytes was highest in the (B1+ B2) Ant group. CONCLUSION: KKS may act as an upstream signal transduction pathway for angiogenic factors in myocardial angiogenesis. The B1 and B2 receptors exert additive effects, and the B1 receptor has the most prominent role in mediating KKS-induced myocardial angiogenesis.


Assuntos
Miocárdio/metabolismo , Neovascularização Fisiológica , Condicionamento Físico Animal , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Animais , Capilares/metabolismo , Cininas/metabolismo , Masculino , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
Eur J Med Chem ; 210: 112948, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33139111

RESUMO

Βradykinin stimulation of B2 receptor is known to activate the oncogenic ERK pathway and overexpression of bradykinin receptors B1 and B2 has been reported to occur in glioma, colorectal and cervical cancers. B1R and B2R antagonists have been shown to reverse tumor proliferation and invasion. Paradoxically, B1R and B2R agonism has also been reported to elicit antiproliferative benefits. In order to complement the data accumulated to date with the natural substrate bradykinin and peptidic B2R antagonists, we decided to examine for the first time the response elicited by B2R stimulation in breast cancer lines with a non-peptidic small molecule B2R agonist. We synthesized and assessed the highly selective and potent B2R partial agonist FR-190997 in MCF-7 and MDA-MBA-231 breast cancer lines and found it possessed significant antiproliferative activity (IC50 2.14 and 0.08 µΜ, respectively). The modular nature of FR-190997 allowed us to conduct a focused SAR study and discover compound 10 which exhibits subnanomolar antiproliferative activity (IC 50 0.06 nΜ) in the TNBC MDA-MBA-231 cell line. This performance surpasses, in most cases by several orders of magnitude, those of established anticancer agents and FDA-approved breast cancer drugs. In line with the established literature we suggest that this remarkable activity precipitates from a dual mode of action involving agonist-induced receptor internalization/degradation combined with sequestration of functional intracellular B2 receptors and inhibition of the associated endosomal signaling. The latter mode may be realized by appropriate ligands regardless of B2R agonist/antagonist designation which only relates to membrane residing GCPRs. Under this prism the controversy over the antiproliferative effects of B2 agonists and antagonists is potentially neutralized.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proliferação de Células/efeitos dos fármacos , Quinolinas/química , Quinolinas/farmacologia , Receptor B2 da Bradicinina/agonistas , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Receptor B2 da Bradicinina/metabolismo
18.
Cells ; 9(12)2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33321704

RESUMO

Neurotrophin nerve growth factor (NGF) has been demonstrated to upregulate the gene expression of bradykinin receptor 2 (B2R) on sensory neurons, thus facilitating nociceptive signals. The aim of the present study is to investigate the involvement of B2R in the NGF mechanism of action in nonsensory neurons in vitro by using rat mixed cortical primary cultures (CNs) and mouse hippocampal slices, and in vivo in Alzheimer's disease (AD) transgenic mice (5xFAD) chronically treated with NGF. A significant NGF-mediated upregulation of B2R was demonstrated by microarray, Western blot, and immunofluorescence analysis in CNs, indicating microglial cells as the target of this modulation. The B2R involvement in the NGF mechanism of action was also demonstrated by using a selective B2R antagonist which was able to reverse the neuroprotective effect of NGF in CNs, as revealed by viability assay, and the NGF-induced long-term potentiation (LTP) in hippocampal slices. To confirm in vitro observations, B2R upregulation was observed in 5xFAD mouse brain following chronic intranasal NGF treatment. This study demonstrates for the first time that B2R is a key element in the neuroprotective activity and synaptic plasticity mediated by NGF in brain cells.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Fator de Crescimento Neural/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Administração Intranasal , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fator de Crescimento Neural/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Cultura Primária de Células , Ratos , Regulação para Cima
19.
PLoS One ; 15(12): e0243326, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33270804

RESUMO

Bradykinin-related peptides (BRPs) family is one of the most significant myotropic peptide families derived from frog skin secretions. Here, a novel BRP callitide was isolated and identified from the red-eyed leaf frog, Agalychnis callidryas, with atypical primary structure FRPAILVRPK-NH2. The mature peptide was cleaved N-terminally at a classic propeptide convertase cleavage site (-KR-) and at the C-terminus an unusual -GKGKGK sequence was removed using the first G residue as an amide donor for the C-terminally-located K residue. Thereafter, the synthetic replicates of callitide were assessed the myotropic activity and showed a significant contraction of balder, with the 0.63 nM EC50 value, more potent than most discovered myotropic peptides. The binding mode was further speculated by molecular docking and stimulation. The result indicated that the C-terminal of callitide might selectively bind to bradykinin receptor B2 (BKRB2). Further investigation of the callitide needs to be done in the future to be exploited as potential future drug leads.


Assuntos
Proteínas de Anfíbios/química , Anuros/genética , Simulação de Acoplamento Molecular , Contração Muscular/efeitos dos fármacos , Receptor B2 da Bradicinina , Pele/química , Bexiga Urinária/metabolismo , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Proteínas de Anfíbios/farmacologia , Animais , Anuros/metabolismo , Feminino , Ratos , Ratos Wistar , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/química , Receptor B2 da Bradicinina/metabolismo , Pele/metabolismo
20.
Cells ; 9(10)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003441

RESUMO

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that Gq-coupled receptors and constitutively active Gαq/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical Gq/phospholipase C (PLC) signaling. Here, we further characterized Gq/11 regulation of SphK1. SphK1 translocation by the M3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCß3, but accelerated by wild-type PLCß3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M3 receptor-stimulated inositol phosphate production, suggesting competition at Gαq. Embryonic fibroblasts from Gαq/11 double-deficient mice were used to show that amino acids W263 and T257 of Gαq, which interact directly with PLCß3 and p63RhoGEF, were important for bradykinin B2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gαq binding motif, ALXXPI, in PLCß and p63RhoGEF. After M3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCß/PLCδ(PH) expression are important for regulation of SphK1 by Gq/11.


Assuntos
Membrana Celular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Membrana Celular/genética , Cromatografia Líquida de Alta Pressão , Fibroblastos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Receptor B2 da Bradicinina/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/genética , Esfingosina/metabolismo , Espectrometria de Massas em Tandem , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
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