RESUMO
Vascular pathology is increased in diabetes because of reactive-oxygen-species (ROS)-induced endothelial cell damage. We found that in vitro and in a streptozotocin diabetes model in vivo, metformin at diabetes-therapeutic concentrations (1-50 µM) protects tissue-intact and cultured vascular endothelial cells from hyperglycemia/ROS-induced dysfunction typified by reduced agonist-stimulated endothelium-dependent, nitric oxide-mediated vasorelaxation in response to muscarinic or proteinase-activated-receptor 2 agonists. Metformin not only attenuated hyperglycemia-induced ROS production in aorta-derived endothelial cell cultures but also prevented hyperglycemia-induced endothelial mitochondrial dysfunction (reduced oxygen consumption rate). These endothelium-protective effects of metformin were absent in orphan-nuclear-receptor Nr4a1-null murine aorta tissues in accord with our observing a direct metformin-Nr4a1 interaction. Using in silico modeling of metformin-NR4A1 interactions, Nr4a1-mutagenesis, and a transfected human embryonic kidney 293T cell functional assay for metformin-activated Nr4a1, we identified two Nr4a1 prolines, P505/P549 (mouse sequences corresponding to human P501/P546), as key residues for enabling metformin to affect mitochondrial function. Our data indicate a critical role for Nr4a1 in metformin's endothelial-protective effects observed at micromolar concentrations, which activate AMPKinase but do not affect mitochondrial complex-I or complex-III oxygen consumption rates, as does 0.5 mM metformin. Thus, therapeutic metformin concentrations requiring the expression of Nr4a1 protect the vasculature from hyperglycemia-induced dysfunction in addition to metformin's action to enhance insulin action in patients with diabetes. SIGNIFICANCE STATEMENT: Metformin improves diabetic vasodilator function, having cardioprotective effects beyond glycemic control, but its mechanism to do so is unknown. We found that metformin at therapeutic concentrations (1-50µM) prevents hyperglycemia-induced endothelial dysfunction by attenuating reactive oxygen species-induced damage, whereas high metformin (>250 µM) impairs vascular function. However, metformin's action requires the expression of the orphan nuclear receptor NR4A1/Nur77. Our data reveal a novel mechanism whereby metformin preserves diabetic vascular endothelial function, with implications for developing new metformin-related therapeutic agents.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Hiperglicemia/prevenção & controle , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Células HEK293 , Humanos , Hiperglicemia/metabolismo , Hipoglicemiantes/farmacologia , Masculino , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Estresse Oxidativo/fisiologia , Vasodilatadores/farmacologiaRESUMO
We proposed that circulating metabolites generated by the intestinal microbiota can affect vascular function. One such metabolite, indole 3-propionic acid (IPA), can activate the pregnane X receptor(PXR), a xenobiotic-activated nuclear receptor present in many tissues, including the vascular endothelium. We hypothesized that IPA could regulate vascular function by modulating PXR activity. To test this, Pxr+/+ mice were administered broad-spectrum antibiotics for 2 wk with IPA supplementation. Vascular function was evaluated by bioassay using aorta and pulmonary artery ring tissue from antibiotic-treated Pxr+/+ and Pxr-/-mice, supplemented with IPA, and using aorta tissue maintained in organ culture for 24 h in the presence of IPA. Endothelium-dependent, nitric oxide(NO)-mediated muscarinic and proteinase-activated receptor 2(PAR2)-stimulated vasodilation was assessed. Endothelial nitric oxide synthase (eNOS) abundance was evaluated in intact tissue or in aorta-derived endothelial cell cultures from Pxr+/+ and Pxr-/- mice, and vascular Pxr levels were assessed in tissues obtained from Pxr+/+ mice treated with antibiotics and supplemented with IPA. Antibiotic-treated Pxr+/+ mice exhibited enhanced agonist-induced endothelium-dependent vasodilation, which was phenocopied by tissues from either Pxr-/- or germ-free mice. IPA exposure reduced the vasodilatory responses in isolated and cultured vessels. No effects of IPA were observed for tissues obtained from Pxr-/- mice. Serum nitrate levels were increased in antibiotic-treated Pxr+/+and Pxr-/- mice. eNOS abundance was increased in aorta tissues and cultured endothelium from Pxr-/- mice. PXR stimulation reduced eNOS expression in cultured endothelial cells from Pxr+/+ but not Pxr-/- mice. The microbial metabolite IPA, via the PXR, plays a key role in regulating endothelial function. Furthermore, antibiotic treatment changes PXR-mediated vascular endothelial responsiveness by upregulating eNOS.
Assuntos
Células Endoteliais/efeitos dos fármacos , Indóis/farmacologia , Receptor de Pregnano X/agonistas , Receptor de Pregnano X/fisiologia , Vasodilatação/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Células Cultivadas , Células Endoteliais/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Indóis/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota/efeitos dos fármacos , Microbiota/fisiologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Receptor de Pregnano X/genética , Vasodilatação/genéticaRESUMO
Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR2) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR2 signaling contributed to asthma symptoms via a PAR2/ß-arrestin signaling axis, identify the protease activity responsible for PAR2 signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR2/ß-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR2-/- and ß-arrestin-2-/- mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR2-/- or ß-arrestin-2-/- mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR2 signaling and induce ß-arrestin-2-/--dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR2/ß-arrestin signaling axis. Thus, ß-arrestin-biased PAR2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.
Assuntos
Inflamação/metabolismo , Receptor PAR-2/metabolismo , Serina Proteases/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestina 2/metabolismo , Alérgenos/metabolismo , Animais , Asma/metabolismo , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Serina/metabolismo , Serina Endopeptidases/metabolismoRESUMO
We propose that in the microenvironment of inflammatory tissues, including tumours, extracellular proteinases can modulate cell signalling in part by regulating proteinase-activated receptors (PARs). We have been exploring this mechanism in a variety of inflammation and tumour-related settings that include tumour-derived cultured cells from prostate and bladder cancer, as well as immune inflammatory cells that are involved in the pathology of inflammatory diseases including multiple sclerosis. Our work showed that proteinase signalling via the PARs affects prostate and bladder cancer-derived tumour cell behaviour and can regulate calcium signalling in human T-cell and macrophage-related inflammatory cells as well as in murine splenocytes. Further, we found that the tumour-derived prostate cancer cells and immune-related cells (Jurkat, THP1, mouse splenocytes) can produce PAR-regulating proteinases (including kallikreins: kallikrein-related peptidases), that can control tissue function by both a paracrine and autocrine mechanism. We suggest that this PAR-driven signalling process involving secreted microenvironment proteinases can play a key role in cancer and inflammatory diseases including multiple sclerosis.
Assuntos
Inflamação/metabolismo , Peptídeo Hidrolases/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Ativados por Proteinase/metabolismo , Microambiente Tumoral , Animais , Células Cultivadas , Humanos , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/patologiaRESUMO
Thrombin initiates human platelet aggregation by coordinately activating proteinase-activated receptors (PARs) 1 and 4. However, targeting PAR1 with an orthosteric-tethered ligand binding-site antagonist results in bleeding, possibly owing to the important role of PAR1 activation on cells other than platelets. Because of its more restricted tissue expression profile, we have therefore turned to PAR4 as an antiplatelet target. We have identified an intracellular PAR4 C-terminal motif that regulates calcium signaling and ß-arrestin interactions. By disrupting this PAR4 calcium/ß-arrestin signaling process with a novel cell-penetrating peptide, we were able to inhibit both thrombin-triggered platelet aggregation in vitro and clot consolidation in vivo. We suggest that targeting PAR4 represents an attractive alternative to blocking PAR1 for antiplatelet therapy in humans.
Assuntos
Plaquetas/metabolismo , Receptores de Trombina/química , Receptores de Trombina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Transporte Proteico/efeitos dos fármacos , Relação Estrutura-Atividade , Trombose/patologia , beta-Arrestinas/metabolismoRESUMO
Thrombin is known to signal to cells by cleaving/activating a G-protein-coupled family of proteinase-activated receptors (PARs). The signaling mechanism involves the proteolytic unmasking of an N-terminal receptor sequence that acts as a tethered receptor-activating ligand. To date, the recognized targets of thrombin cleavage and activation for signaling are PAR1 and PAR4, in which thrombin cleaves at a conserved target arginine to reveal a tethered ligand. PAR2, which like PAR1 is also cleaved at an N-terminal arginine to unmask its tethered ligand, is generally regarded as a target for trypsin but not for thrombin signaling. We now show that thrombin, at concentrations that can be achieved at sites of acute injury or in a tumor microenvironment, can directly activate PAR2 vasorelaxation and signaling, stimulating calcium and mitogen-activated protein kinase responses along with triggeringß-arrestin recruitment. Thus, PAR2 can be added alongside PAR1 and PAR4 to the targets, whereby thrombin can affect tissue function.
Assuntos
Sinalização do Cálcio , Sistema de Sinalização das MAP Quinases , Receptor PAR-2/agonistas , Trombina/metabolismo , Vasodilatação , Substituição de Aminoácidos , Animais , Aorta , Arrestinas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Endotélio Vascular/fisiologia , Humanos , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Mutação , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteólise , Coelhos , Receptor PAR-2/química , Receptor PAR-2/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Vasodilatação/efeitos dos fármacos , beta-ArrestinasRESUMO
Neutrophil proteinases released at sites of inflammation can affect tissue function by either activating or disarming signal transduction mediated by proteinase-activated receptors (PARs). Because PAR1 is expressed at sites where abundant neutrophil infiltration occurs, we hypothesized that neutrophil-derived enzymes might also regulate PAR1 signaling. We report here that both neutrophil elastase and proteinase-3 cleave the human PAR1 N terminus at sites distinct from the thrombin cleavage site. This cleavage results in a disarming of thrombin-activated calcium signaling through PAR1. However, the distinct non-canonical tethered ligands unmasked by neutrophil elastase and proteinase-3, as well as synthetic peptides with sequences derived from these novel exposed tethered ligands, selectively stimulated PAR1-mediated mitogen-activated protein kinase activation. This signaling was blocked by pertussis toxin, implicating a Gαi-triggered signal pathway. We conclude that neutrophil proteinases trigger biased PAR1 signaling and we describe a novel set of tethered ligands that are distinct from the classical tethered ligand revealed by thrombin. We further demonstrate the function of this biased signaling in regulating endothelial cell barrier integrity.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Elastase de Leucócito/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Mieloblastina/metabolismo , Receptor PAR-1/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Elastase de Leucócito/genética , Mieloblastina/genética , Receptor PAR-1/genética , Trombina/genética , Trombina/metabolismoRESUMO
BACKGROUND AND PURPOSE: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. EXPERIMENTAL APPROACH: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. KEY RESULTS: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. CONCLUSION AND IMPLICATIONS: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.
Assuntos
Quimotripsina , Mucosa Intestinal , Receptor PAR-1 , Receptor PAR-2 , Animais , Humanos , Camundongos , Quimotripsina/metabolismo , Mucosa Intestinal/metabolismo , Camundongos Endogâmicos C57BL , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Transdução de SinaisRESUMO
Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1-ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.
Assuntos
Peptídeos/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Implantação do Embrião , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/química , Pichia/genética , Pichia/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise , Ratos , Receptor PAR-1/genética , Receptor PAR-2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética , Trombina/metabolismo , Tripsina/metabolismoRESUMO
(+)-JQ1, a specific chemical inhibitor of bromodomain and extraterminal (BET) family protein 4 (BRD4), has been reported to inhibit smooth muscle cell (SMC) proliferation and mouse neointima formation via BRD4 regulation and modulate endothelial nitric oxide synthase (eNOS) activity. This study aimed to investigate the effects of (+)-JQ1 on smooth muscle contractility and the underlying mechanisms. Using wire myography, we discovered that (+)-JQ1 inhibited contractile responses in mouse aortas with or without functional endothelium, reducing myosin light chain 20 (LC20) phosphorylation and relying on extracellular Ca2+. In mouse aortas lacking functional endothelium, BRD4 knockout did not alter the inhibition of contractile responses by (+)-JQ1. In primary cultured SMCs, (+)-JQ1 inhibited Ca2+ influx. In aortas with intact endothelium, (+)-JQ1 inhibition of contractile responses was reversed by NOS inhibition (L-NAME) or guanylyl cyclase inhibition (ODQ) and by blocking the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. In cultured human umbilical vein endothelial cells (HUVECs), (+)-JQ1 rapidly activated AKT and eNOS, which was reversed by PI3K or ATK inhibition. Intraperitoneal injection of (+)-JQ1 reduced mouse systolic blood pressure, an effect blocked by co-treatment with L-NAME. Interestingly, (+)-JQ1 inhibition of aortic contractility and its activation of eNOS and AKT were mimicked by the (-)-JQ1 enantiomer, which is structurally incapable of inhibiting BET bromodomains. In summary, our data suggest that (+)-JQ1 directly inhibits smooth muscle contractility and indirectly activates the PI3K/AKT/eNOS cascade in endothelial cells; however, these effects appear unrelated to BET inhibition. We conclude that (+)-JQ1 exhibits an off-target effect on vascular contractility.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Camundongos , Humanos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Aorta/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas de Ciclo CelularRESUMO
We hypothesized that kallikrein-related peptidase 14 (KLK14) is produced by colonic tumors and can promote tumorigenesis by activating proteinase-activated receptors (PARs). We found that KLK14 is expressed in human colon adenocarcinoma cells but not in adjacent cancer-free tissue; KLK14 mRNA, present in colon cancer, leads to KLK14 protein expression and secretion; and KLK14 signals viaPAR-2 in HT-29 cells to cause (1) receptor activation/internalization, (2) increases in intracellular calcium, (3) stimulation of ERK1/2/MAP kinase phosphorylation, and (4) cell proliferation. We suggest that KLK14, acting via PAR-2, represents an autocrine/paracrine regulator of colon tumorigenesis.
Assuntos
Neoplasias do Colo/patologia , Calicreínas/metabolismo , Receptor PAR-2/metabolismo , Transdução de Sinais , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Calicreínas/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
We compared signalling pathways such as calcium transients, MAPK activation, ß-arrestin interactions and receptor internalization triggered by kallikrein-related peptidases (KLKs) 8 and 14 in human and rat proteinase-activated receptor (PAR)2-expressing human embryonic kidney (HEK) and Kirsten transformed rat kidney (KNRK) cells. Further, we analysed processing by KLK8 vs. KLK14 of synthetic human and rat PAR2-derived sequences representing the cleavage-activation domain of PAR2. Our data show that like KLK14, KLK8 can unmask a PAR2 receptor-activating sequence from a peptide precursor. However, whilst KLK8, like KLK14, can signal in rat-PAR2-expressing KNRK cells, this enzyme cannot signal via human PAR2 in HEK or KNRK cells, but rather, disarms HEK PAR1. Thus, KLK8 and KLK14 can signal differentially via the PARs to affect tissue function.
Assuntos
Calicreínas/metabolismo , Receptor PAR-2/metabolismo , Transdução de Sinais , Animais , Células HEK293 , Humanos , Transporte Proteico , RatosRESUMO
Giardia duodenalis cysteine proteases have been identified as key virulence factors and have been implicated in alterations to intestinal goblet cell activity and mucus production during Giardia infection. The present findings demonstrate a novel mechanism by which Giardia cysteine proteases modulate goblet cell activity via cleavage and activation of protease-activated receptor 2. Giardia duodenalis (assemblage A) increased MUC2 mucin gene expression in human colonic epithelial cells in a manner dependent upon both protease-activated receptor 2 activation and Giardia cysteine protease activity. Protease-activated receptor 2 cleavage within the N-terminal activation domain by Giardia proteases was confirmed using a nano-luciferase tagged recombinant protease-activated receptor 2. In keeping with these observations, the synthetic protease-activated receptor 2-activating peptide 2fLIGRLO-amide increased Muc2 gene expression in a time-dependent manner. Calcium chelation and inhibition of the ERK1/2 mitogen activated protein kinase pathway inhibited Muc2 upregulation during Giardia infection, consistent with canonical protease-activated receptor 2 signaling pathways. Giardia cysteine proteases cleaved both recombinant protease-activated receptor 1 and protease-activated receptor 2 within their extracellular activation domains with isolate-dependent efficiency that correlated with the production of cysteine protease activity. Protease-activated receptors represent a novel target for Giardia cysteine proteases, and these findings demonstrate that protease-activated receptor 2 can regulate mucin gene expression in intestinal goblet cells.
Assuntos
Cisteína Proteases , Giardia lamblia , Mucinas , Receptor PAR-2 , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Expressão Gênica , Giardia lamblia/enzimologia , Giardia lamblia/genética , Células Caliciformes/metabolismo , Humanos , Mucinas/metabolismo , Receptor PAR-2/genética , Receptor PAR-2/metabolismoRESUMO
Hypocapnia and hypercapnia constrict and relax airway smooth muscle, respectively, through pH- and calcium (Ca(2+))-mediated mechanisms. In this study we explore a potential role for the airway epithelium in these responses to carbon dioxide (CO(2)). Contractile and relaxant responses of isolated rat bronchial rings were measured under hypocapnic, eucapnic, and hypercapnic conditions. Substance P was added to methacholine precontracted bronchial rings with and without epithelium. The role of Ca(2+) was assessed using Ca(2+)-free solutions and a Ca(2+) channel blocker, nifedipine. The effects of pH were assessed in solutions with HEPES buffer. Hypocapnic challenge increased the organ bath's pH and increased bronchial smooth muscle resting tension. This effect was abolished with HEPES buffer and partially inhibited by nifedipine. Hypocapnic conditions suppressed substance P-induced epithelium-dependent relaxation, whereas hypercapnia augmented the response. The epithelial hypocapnic effect was pH dependent, whereas the hypercapnic effect was pH independent. CO(2) had no effect on the epithelial independent smooth muscle agonists methacholine and isoproterenol. In conclusion our data indicate that, in addition to the effects of pH and Ca(2+), CO(2) affects airway smooth muscle by a pH-independent, epithelium-mediated mechanism. These findings could potentially lead to new treatments for asthma involving CO(2)-sensing receptors in the airways.
Assuntos
Brônquios/efeitos dos fármacos , Dióxido de Carbono/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Substância P/farmacologia , Animais , Brônquios/metabolismo , Brônquios/fisiologia , Cálcio/metabolismo , Sinergismo Farmacológico , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/fisiologia , Concentração de Íons de Hidrogênio , Hipercapnia/metabolismo , Hipercapnia/fisiopatologia , Hipocapnia/metabolismo , Hipocapnia/fisiopatologia , Isoproterenol/farmacologia , Masculino , Cloreto de Metacolina/farmacologia , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Nifedipino/farmacologia , Ratos , Ratos Sprague-Dawley , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismoRESUMO
[This corrects the article DOI: 10.3389/fimmu.2020.629726.].
RESUMO
In the present study, we report the synthesis and biological evaluation of a series of new non-peptide PAR(1) mimetic receptor antagonists, based on conformational analysis of the S(42)FLLR(46) tethered ligand (TL) sequence of PAR(1). These compounds incorporate the key pharmacophore groups in the TL sequence, guanidyl, amino and phenyl, which are essential for triggering receptor activity. Compounds 5 and 15 (50-100 microM) inhibited both TFLLR-amide (10 microM) and thrombin-mediated (0.5 and 1 U/ml; 5 and 10 microM) calcium signaling in a cultured human HEK cell assay.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Mimetismo Molecular , Receptor PAR-1/química , Receptores de Trombina/antagonistas & inibidores , Trombina/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Amidas/farmacologia , Arginina/análogos & derivados , Arginina/química , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Cicloexanos/síntese química , Cicloexanos/química , Cicloexanos/farmacologia , Dipeptídeos/síntese química , Dipeptídeos/química , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/química , Guanidinas/síntese química , Guanidinas/química , Guanidinas/farmacologia , Humanos , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Ligantes , Oligopeptídeos/antagonistas & inibidores , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Receptor PAR-1/agonistas , Relação Estrutura-Atividade , Trombina/metabolismoRESUMO
Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators. Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel. Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2. Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.
Assuntos
Artrite Psoriásica/imunologia , Sinalização do Cálcio/imunologia , Macrófagos/imunologia , Receptor PAR-2/imunologia , Triptases/imunologia , Artrite Psoriásica/patologia , Feminino , Humanos , Macrófagos/patologia , MasculinoRESUMO
The expression of proteinase-activated receptor (PAR)(2) in human hepatocellular carcinoma (HCC) was established by reverse transcription-polymerase chain reaction, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures and HCC tumor tissue. Stimulation of HCC cells with trypsin and the PAR(2)-selective activating peptide, 2-furoyl-LIGRLO-NH(2), increased cell invasion across Matrigel. Both effects were blocked by a PAR(2)-selective pepducin antagonist peptide (pal-PAR(2)) and by PAR(2) silencing with specific small interfering RNA (siRNA). PAR(2)-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by downregulation of Met with specific siRNA. The involvement of Met in PAR(2)-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 mitogen-activated protein Kinases was found to be critical for the PAR(2)-Met receptor tyrosine kinase-invasive signaling axis in HCC cells. Our study establishes an important link between the PAR(2) and Met receptor tyrosine kinase signaling in promoting HCC cell invasion.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas/fisiologia , Receptor PAR-2/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Ativação Transcricional , Sequência de Aminoácidos , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Dados de Sequência Molecular , Invasividade Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-met , Espécies Reativas de Oxigênio/metabolismo , Receptor PAR-2/análiseRESUMO
This overview deals with mechanisms whereby hyperglycemia-induced oxidative stress compromises vascular endothelial function and provides a background for a recently published study illustrating the beneficial impact of endothelial sodium-glucose cotransporter 2 (SGLT2) inhibitors in attenuating hyperglycemia-induced vascular dysfunction in vitro. The data provide new insight that can possibly lead to improved drug therapy for people with type 2 diabetes. The working hypotheses that underpinned the experiments performed are provided, along with the findings of the study. For the causes of hyperglycemia-induced vascular endothelial dysfunction, the findings point to the key roles of: 1) functional endothelial SGLT2; 2) oxidative stress-induced signalling pathways including mammalian sarcoma virus kinase, the EGF receptor-kinase and protein kinase C; and 3) mitochondrial dysfunction triggered by hyperglycemia was mitigated by an SGLT2 inhibitor in the hyperglycemic mouse aorta vascular organ cultures. The overview sums up the approaches implicated by the study that can potentially counteract the detrimental impact of hyperglycemia on vascular function in people with diabetes, including the clinical use of SGLT2 inhibitors for those with type 2 diabetes already being treated, for example, with metformin, along with dietary supplementation with broccoli-derived sulforaphane and tetrahydrobiopterin. The caveats associated with the study for extending the findings from mice to humans are summarized, pointing to the need to validate the work using vascular tissues from humans. Suggestions for future clinical studies are made, including the assessment of the impact of the therapeutic strategies proposed on measurements of blood flow in subjects with diabetes.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Hiperglicemia/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Transportador 2 de Glucose-Sódio/química , Biomarcadores/análise , Biopterinas/análogos & derivados , Biopterinas/uso terapêutico , Glicemia/análise , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/metabolismo , Angiopatias Diabéticas/epidemiologia , Angiopatias Diabéticas/metabolismo , Endotélio Vascular/patologia , Humanos , Incidência , Isotiocianatos/uso terapêutico , Prognóstico , SulfóxidosRESUMO
The proteinase-activated receptor-2 (PAR2)-activating peptide with an N-terminal furoyl group modification, 2-furoyl-LIGRLO-NH2 (2fLI), was derivatized via its free ornithine amino group to yield [3H]propionyl-2fLI and Alexa Fluor 594-2fLI that were used as receptor probes for ligand binding assays and receptor visualization both for cultured cells in vitro and for colonic epithelial cells in vivo. The binding of the radiolabeled and fluorescent PAR2 probes was shown to be present in PAR2-transfected Kirsten normal rat kidney cells, but not in vector-alone-transfected cells, and was abolished by pretreatment of cells with saturating concentrations of receptor-selective PAR2 peptide agonists such as SLIGRL-NH2 and the parent agonist 2fLI but not by reverse-sequence peptides such as 2-furoyl-OLRGIL-NH2 that cannot activate PAR2. The relative orders of potencies for a series of PAR2 peptide agonists to compete for the binding of [3H]propionyl-2fLI (2fLI >> SLIGRL-NH2 approximately= trans-cinnamoyl-LIGRLO-NH2 > SLIGKV-NH2 > SLIGKT-NH2) mirrored qualitatively their relative potencies for PAR2-mediated calcium signaling in the same cells or for vasorelaxation in a rat aorta vascular assay. In the vascular assay, the potency of Alexa Fluor 594-2fLI was the same as 2fLI. We conclude that ornithine-derivatized 2fLI peptides are conveniently synthesized PAR2 probes that will be of value for future studies of receptor binding and visualization.