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1.
Biochem Biophys Res Commun ; 608: 156-162, 2022 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-35398613

RESUMO

Calpains are cysteine proteases activated in response to intracellular calcium signaling. Activated calpains regulate various cellular functions by degrading substrate molecules in a site-specific manner. Although most calpains are localized in the cytosol, we previously reported that calpain-5 exists in the mitochondria. The mitochondrial calpain-5 is activated during endoplasmic reticulum (ER) stress. However, the substrate of calpain-5, as well as the physiological significance of calpain-5 activation, has not yet been elucidated. In the present study, we treated HeLa cells with A23187, tunicamycin, or hydrogen peroxide to induce intracellular calcium increase, resulting in cell death. The cells treated with A23187 or tunicamycin exhibited the activation of calpain-5 and truncation of caspase-4. The truncation of caspase-4 was inhibited by the repression of calpain-5 expression with the appropriate siRNA. Additionally, both calpain-5 and caspase-4 were observed in the mitochondria. Our study is the first to demonstrate that the activation of mitochondrial calpain-5 triggers the truncation of caspase-4, suggesting that mitochondrial calpain-5 regulates the downstream pathway of caspase-4, including cell death and the inflammatory cascade. The results of the present study provide new insights into ER-stress-related diseases such as Alzheimer's disease and cancer. These perspectives allow us to propose new therapeutic strategies such as the development of inhibitors or activators of calpain-5, which may be useful in the development of treatment for ER-stress-related diseases.


Assuntos
Calpaína , Caspases Iniciadoras , Estresse do Retículo Endoplasmático , Mitocôndrias , Apoptose , Calcimicina , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Calpaína/metabolismo , Caspases Iniciadoras/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Tunicamicina/farmacologia
2.
Int J Mol Sci ; 23(3)2022 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-35163382

RESUMO

Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca2+ concentration and is expressed on smooth muscle cells (SMCs). It is implicated in the myogenic constriction of cerebral arteries. We hypothesized that TRPM4 has a general role in intracellular Ca2+ signal amplification in a wide range of blood vessels. TRPM4 function was tested with the TRPM4 antagonist 9-phenanthrol and the TRPM4 activator A23187 on the cardiovascular responses of the rat, in vivo and in isolated basilar, mesenteric, and skeletal muscle arteries. TRPM4 inhibition by 9-phenanthrol resulted in hypotension and a decreased heart rate in the rat. TRPM4 inhibition completely antagonized myogenic tone development and norepinephrine-evoked vasoconstriction, and depolarization (high extracellular KCl concentration) evoked vasoconstriction in a wide range of peripheral arteries. Vasorelaxation caused by TRPM4 inhibition was accompanied by a significant decrease in intracellular Ca2+ concentration, suggesting an inhibition of Ca2+ signal amplification. Immunohistochemistry confirmed TRPM4 expression in the smooth muscle cells of the peripheral arteries. Finally, TRPM4 activation by the Ca2+ ionophore A23187 was competitively inhibited by 9-phenanthrol. In summary, TRPM4 was identified as an essential Ca2+-amplifying channel in peripheral arteries, contributing to both myogenic tone and agonist responses. These results suggest an important role for TRPM4 in the circulation. The modulation of TRPM4 activity may be a therapeutic target for hypertension. Furthermore, the Ca2+ ionophore A23187 was identified as the first high-affinity (nanomolar) direct activator of TRPM4, acting on the 9-phenanthrol binding site.


Assuntos
Sinalização do Cálcio , Canais de Cátion TRPM/metabolismo , Vasoconstrição , Administração Intravenosa , Animais , Artérias/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Calcimicina/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Ionóforos/farmacologia , Masculino , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/irrigação sanguínea , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Norepinefrina/farmacologia , Fenantrenos/administração & dosagem , Fenantrenos/farmacologia , Cloreto de Potássio/farmacologia , Ratos Wistar , Canais de Cátion TRPM/agonistas , Vasoconstrição/efeitos dos fármacos
3.
Biochim Biophys Acta Biomembr ; 1864(5): 183883, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35181295

RESUMO

Cells are dynamic systems with complex mechanical properties, regulated by the presence of different species of proteins capable to assemble (and disassemble) into filamentous forms as required by different cells functions. Giant unilamellar vesicles (GUVs) of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) are systems frequently used as a simplified model of cells because they offer the possibility of assaying separately different stimuli, which is no possible in living cells. Here we present a study of the effect of acting protein on mechanical properties of GUVs, when the protein is inside the vesicles in either monomeric G-actin or filamentous F-actin. For this, rabbit skeletal muscle G-actin is introduced inside GUVs by the electroformation method. Protein polymerization inside the GUVs is promoted by adding to the solution MgCl2 and the ion carrier A23187 to allow the transport of Mg+2 ions into the GUVs. To determine how the presence of actin changes the mechanical properties of GUVs, the vesicles are deformed by the application of an AC electric field in both cases with G-actin and with polymerized F-actin. The changes in shape of the vesicles are characterized by optical microscopy and from them the bending stiffness of the membrane are determined. It is found that G-actin has no appreciable effect on the bending stiffness of DMPC GUVs, but the polymerized actin makes the vesicles more rigid and therefore more resistant to deformations. This result is supported by evidence that actin filaments tend to accumulate near the membrane.


Assuntos
Actinas/química , Dimiristoilfosfatidilcolina/química , Eletricidade , Lipossomas Unilamelares/química , Citoesqueleto de Actina/química , Actinas/metabolismo , Animais , Calcimicina/química , Cloreto de Magnésio/química , Cloreto de Magnésio/metabolismo , Microscopia , Músculo Esquelético/metabolismo , Coelhos , Tensão Superficial , Lipossomas Unilamelares/metabolismo , Viscosidade
4.
J Thromb Haemost ; 20(5): 1223-1235, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35146910

RESUMO

BACKGROUND: Platelets contain a high amount of potentially active A subunit dimer of coagulation factor XIII (cellular FXIII; cFXIII). It is of cytoplasmic localization, not secreted, but becomes translocated to the surface of platelets activated by convulxin and thrombin (CVX+Thr). OBJECTIVE: To explore the difference in cFXIII translocation between receptor mediated and non-receptor mediated platelet activation and if translocation can also be detected on platelet-derived microparticles. Our aim was also to shed some light on the mechanism of cFXIII translocation. METHODS: Gel-filtered platelets were activated by CVX+Thr or Ca2+ -ionophore (calcimycin). The translocation of cFXIII and phosphatidylserine (PS) to the surface of activated platelets and platelet-derived microparticles was investigated by flow cytometry, immunofluorescence, and immune electron microscopy. Fluo-4-AM fluorescence was used for the measurement of intracellular Ca2+ concentration. RESULTS: Receptor mediated activation by CVX+Thr exposed cFXIII to the surface of more than 60% of platelets. Electron microscopy revealed microparticles with preserved membrane structure and microparticles devoid of labeling for membrane glycoprotein CD41a. cFXIII was observed on both types of microparticles but was more abundant in the absence of CD41a. Rhosin, a RhoA inhibitor, significantly decreased cFXIII translocation. Non-receptor mediated activation of platelets by calcimycin elevated intracellular Ca2+ concentration, induced the translocation of PS to the surface of platelets and microparticles, but failed to expose cFXIII. CONCLUSIONS: The elevation of intracellular Ca2+ concentration is sufficient for the translocation of PS from the internal layer of the membrane, while the translocation of cFXIII from the platelet cytoplasm requires additional receptor mediated mechanism(s).


Assuntos
Micropartículas Derivadas de Células , Fator XIII , Plaquetas , Calcimicina/farmacologia , Proteínas de Transporte , Humanos , Fosfatidilserinas , Ativação Plaquetária , Trombina/farmacologia
5.
Plant Cell Rep ; 41(4): 1043-1057, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35190883

RESUMO

KEY MESSAGE: After cryopreservation, the Ca2+ content increased, which affected the intracellular ROS content, then participated in the occurrence of programmed cell death in pollen. Programmed cell death (PCD) is one of the reasons for the decline in pollen viability after cryopreservation. However, the role of calcium ions (Ca2+) in PCD during pollen cryopreservation has not been revealed in the existing studies. In this study, Paeonia lactiflora 'Fen Yu Nu' pollen was used as the research material for investigating the effects of Ca2+ changes on PCD indices and reactive oxygen species (ROS) during pollen cryopreservation. The results showed that after cryopreservation, with the decrease of pollen viability, the Ca2+ content significantly increased. The regulation of Ca2+ content had a significant effect on PCD indices, which showed that the Ca2+ carrier A23187 accelerated the decrease of mitochondrial membrane potential level and increased the activity of caspase-3-like and caspase-9-like proteases and the apoptosis rate. The expression levels of partial pro-PCD genes were upregulated, the anti-PCD gene BI-1 was downregulated, and the addition of Ca2+-chelating agent EGTA had the opposite effect. The addition of the Ca2+ carrier A23187 after cryopreservation significantly increased the ROS content of pollen, the addition of the Ca2+-chelating agent EGTA had the opposite effect, and Ca2+ regulators also had significant effects on the contents of ROS production and clearance-related substances. Ca2+ affected intracellular ROS content by acting on the ROS production and clearance system during the cryopreservation of pollen and is thus involved in the occurrence of PCD.


Assuntos
Apoptose , Pólen , Calcimicina/metabolismo , Calcimicina/farmacologia , Quelantes/farmacologia , Criopreservação/métodos , Ácido Egtázico/metabolismo , Ácido Egtázico/farmacologia , Pólen/genética , Espécies Reativas de Oxigênio/metabolismo
6.
Pflugers Arch ; 474(5): 541-551, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35157133

RESUMO

The higher permeability of the venules in jejunal microcirculation to albumin contributes to the increased mesenteric lymph formation. Recently, we demonstrated that water intake induced serotonin release from enterochromaffin cells in rat jejunum, serotonin of which circulated through the portal vein into blood circulation and then increased the mesenteric lymph formation. The mode of action of serotonin remains unclear. Therefore, we aimed to clarify the mechanisms involved in the regulation of the jejunal lymph formation with permeant albumin in in vivo rat experiments. We investigated the effects of intravenous administration of serotonin or water intake on the jejunal-originated lymph volume and the concentration of albumin in the lymph in the presence or absence of L-NAME. The effects of intravenous administration of L-NAME, nicardipine, A23187, and ML-7 on the lymph formation with permeant albumin were also evaluated. Serotonin or water intake significantly increased the mesenteric lymph volume with permeant albumin in the jejunal microcirculation. The serotonin- and water intake-mediated responses were significantly reduced by the pretreatment with intravenous administration of L-NAME. Intravenous administration of L-NAME itself also decreased significantly the jejunal lymph formation. Administration of A23187 and ML-7 significantly reduced the jejunal lymph formation with permeant albumin. In contrast, administration of nicardipine significantly increased the lymph formation. In conclusion, portal venous blood flow- or serotonin-mediated NO release from venular endothelial cells plays physiologically key roles in the lymph formation in rat jejunum via the extrusion of calcium ions and inactivation of MLCK in endothelial cells.


Assuntos
Jejuno , Serotonina , Albuminas , Animais , Calcimicina/farmacologia , Células Endoteliais , NG-Nitroarginina Metil Éster/farmacologia , Nicardipino/farmacologia , Ratos , Serotonina/farmacologia
7.
J Thromb Haemost ; 20(4): 989-995, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35034417

RESUMO

BACKGROUND: During thrombosis, procoagulant platelets expose phosphatidylserine (PS), which enhances local thrombin generation. Reducing platelet PS exposure could be a novel anti-thrombotic approach. PS is confined to the inner leaflet of the plasma membrane in unstimulated platelets by ATP-dependent "flippase" activity. Ca2+ ionophores trigger all platelets to expose a high level of PS by activating a scramblase protein and inactivating the flippase. Although R5421 was previously shown to reduce Ca2+ ionophore-induced PS exposure, its mechanism of action is unknown. OBJECTIVES: To determine the mechanism by which R5421 reduces platelet PS exposure. METHODS: Washed human platelets were stimulated with the Ca2+ ionophore, A23187, to induce procoagulant platelet formation while bypassing proximal receptor signalling. Platelets PS exposure was detected using annexin V or lactadherin in flow cytometry. NBD (7-nitro-2-1,3-benzoxadiazol-4-yl)-PS was used to assess scramblase and flippase activity. Thrombin generation was monitored using a fluorogenic substrate. RESULTS AND CONCLUSIONS: R5421 reduced the extent of A23187-stimulated platelet PS exposure, as demonstrated with annexin V or lactadherin binding. R5421 also maintained flippase activity in procoagulant platelets. Although R5421 appeared to inhibit scramblase activity in procoagulant platelets, it did not once the flippase had been inhibited, demonstrating that scramblase activity is not directly inhibited. Furthermore, R5421 inhibited the contribution of A23187-stimulated platelets to thrombin generation. Together these data demonstrate that R5421 reduces the extent of PS exposure in procoagulant platelets by maintaining flippase activity. Maintaining flippase activity in procoagulant platelets is a novel and effective approach to reducing thrombin generation.


Assuntos
Trombina , Trombose , Anexina A5 , Plaquetas/metabolismo , Calcimicina/farmacologia , Humanos , Ionóforos/efeitos adversos , Ionóforos/metabolismo , Fosfatidilserinas/metabolismo , Trombina/metabolismo , Trombose/metabolismo
8.
Andrology ; 10(2): 367-376, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34542939

RESUMO

BACKGROUND: Polyphenylene carboxymethylene (PPCM) sodium salt is a promising multipurpose technology for prevention of both sexually transmitted infections (STIs) and pregnancy. In preclinical studies, PPCM has demonstrated significant (1) antimicrobial activity against several important viral and bacterial pathogens and (2) contraceptive activity associated with premature acrosome loss. OBJECTIVE: To further evaluate a vaginal antimicrobial compound as a contraceptive agent in preclinical studies utilizing a repurposed hyaluronan binding assay (HBA). MATERIALS AND METHODS: Semen samples containing either neat semen or washed spermatozoa were treated with increasing concentrations of PPCM or calcium ionophore A23187 (positive control). Sperm inactivation was measured by two methods: (1) double acrosome staining (AS), and (2) a hyaluronan binding assay (HBA® ). Percentage of inactivated sperm was compared between untreated control sperm and those treated with PPCM or A23187. RESULTS: PPCM had a significant (p < 0.05) and dose-dependent effect on sperm inactivation in both assays, with HBA detecting a higher proportion of inactivated sperm than AS. PPCM did not affect sperm motility and exhibited equivalent responses in the neat and washed samples. DISCUSSION: Both HBA and AS confirmed that spermatozoa were rapidly inactivated at PPCM concentrations likely present in the vagina under actual use conditions and in a time-frame comparable to in vivo migration of spermatozoa out of seminal plasma into cervical mucus. CONCLUSION: PPCM vaginal gel may provide contraceptive protection as well as help with STI prevention. HBA may be a sensitive and much needed biomarker for sperm activity in future contraceptive development.


Assuntos
Acrossomo/efeitos dos fármacos , Anticoncepcionais/farmacologia , Polímeros/farmacologia , Espermatozoides/efeitos dos fármacos , Cremes, Espumas e Géis Vaginais/farmacologia , Calcimicina/farmacologia , Feminino , Humanos , Ácido Hialurônico , Masculino , Gravidez , Sêmen/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos
9.
Parasitol Res ; 121(4): 1169-1177, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34386856

RESUMO

Neospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca++ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca++ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions.


Assuntos
Coccidiose , Neospora , Parasitos , Toxoplasma , Animais , Calcimicina , Cálcio , Bovinos , Coccidiose/parasitologia , Coccidiose/veterinária , Células Endoteliais , Ionóforos/farmacologia
10.
Platelets ; 33(4): 562-569, 2022 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34348059

RESUMO

Experiments were undertaken to identify the nature of a previously identified inhibitor of PAF-induced platelet aggregation (PA) in human saliva. Human saliva fractionated by preparative thin layer chromatography (TLC) yielded a fraction that co-migrated with fatty acids (FAs) and inhibited PAF-induced aggregation of platelets. Synthetic FAs tested for their capacities to inhibit 0.1 nM PAF-induced PA showed that only the cis-unsaturated compounds were inhibitory with activities of some of the polyunsaturated FAs (PUFA) reaching almost 100% at 20 µM. Eicosapentanoic acid (EPA) and 8,11,14-eicosatrienoic acid also deaggregated the PAF-induced aggregates. With the exception of oleic acid (OLA), cis-monounsaturated FAs, and elaidic acid, the trans isomer of OLA, were poor inhibitors. In a direct comparison with other platelet agonists, ADP, thrombin, and ionophore A23187, the active saliva fraction and selected individual FAs inhibited, to greater or lesser extent, PA induced by each of the agonists. EPA, OLA, linoleic acid (LNA), and the active saliva fraction were potent inhibitors of ADP-induced PA, EPA completely inhibited thrombin-induced PA and the saliva fraction showed only weak - moderate inhibitory activity to both thrombin- and ionophore A23187-induced PA. Other reports of endogenous PAF inhibitors in mammalian tissues are compared to the present results. PAF can trigger and amplify inflammatory cascades suggesting a possible modulation role for cis-unsaturated FAs in some diseases.


Assuntos
Fator de Ativação de Plaquetas , Agregação Plaquetária , Difosfato de Adenosina/farmacologia , Animais , Plaquetas , Calcimicina/análise , Calcimicina/farmacologia , Ácidos Graxos/análise , Ácidos Graxos/farmacologia , Humanos , Ionóforos/análise , Ionóforos/farmacologia , Mamíferos , Fator de Ativação de Plaquetas/análise , Fator de Ativação de Plaquetas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Saliva/química , Trombina/farmacologia
11.
Int Immunopharmacol ; 101(Pt A): 108319, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34740079

RESUMO

The weaponry possessed by Mycobacterium tuberculosis (M. tb) in the form of immunodominant antigens hijack the host immune system to give a survival advantage to this intracellular fiend, but the mechanism of this control is not entirely known. Since we have previously reported the mechanism of autophagy inhibition by early secreted antigenic target 6 kDa (ESAT-6) through microRNA (miR)-30a-3p in Calcimycin treated differentiated THP-1 (dTHP-1) cells, the present study was undertaken to deduce the effect of miR-30a on the immunomodulatory profile of ESAT-6 treated cells and the mechanism involved thereof, if any. Initially, the effect of recombinant ESAT-6 (rESAT-6) on the immunomodulatory profile in Calcimycin-treated phorbol 12-myristate 13-acetate (PMA) dTHP-1 cells was checked. Later, transfection studies using miR-30a-3p inhibitor or -5p mimic highlighted the contrary roles of different arms of the same miRNA in regulating IL-18 response by ESAT-6 in dTHP-1 cells after Calcimycin treatment. By using either IL-18 neutralizing antibody or inhibitors of phosphoinositide 3-kinase (PI3K)/NF-κB/phagosome-lysosome fusion in the miRNA-30a transfected background, IL-18 mediated signaling and intracellular killing of mycobacteria was reversed in the presence of ESAT-6. Overall, the results of this study conclusively prove the contrary roles of miR-30a-3p and miR-30a-5p in regulating IL-18 signaling by ESAT-6 in dTHP-1 cells upon Calcimycin treatment that affected phagosome-lysosome fusion and intracellular survival of mycobacteria.


Assuntos
Antibacterianos/farmacologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Calcimicina/farmacologia , Interleucina-18/metabolismo , Lisossomos/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Western Blotting , Linhagem Celular , Citometria de Fluxo , Humanos , Lisossomos/metabolismo , MicroRNAs/metabolismo , Microscopia Confocal , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tuberculose/imunologia , Tuberculose/metabolismo
12.
J Assist Reprod Genet ; 38(12): 3125-3133, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34642877

RESUMO

PURPOSE: To evaluate whether ionophore application at the oocyte stage changes the morphokinetics of the associated embryos in cases of artificial oocyte activation. METHODS: In a prospective sibling oocyte approach, 78 ICSI patients with suspected fertilization problems had half of their MII-oocytes treated with a ready-to-use ionophore (calcimycin) immediately following ICSI (study group). Untreated ICSI eggs served as the control group. Primary analyses focused on morphokinetic behavior and the presence of irregular cleavages. The rates of fertilization, utilization, pregnancy, and live birth rate were also evaluated. RESULTS: Ionophore-treated oocytes showed a significantly earlier formation of pronuclei (t2PNa) and a better synchronized third cell cycle (s3) (P < .05). The rate of irregular cleavage was unaffected (P > .05). Ionophore treatment significantly improved the overall rates of fertilization (P < .01) and blastocyst utilization (P < .05). CONCLUSION: Ionophore application does not negatively affect cleavage timing nor is it associated with irregular cleavage.


Assuntos
Ionóforos/farmacologia , Oócitos/efeitos dos fármacos , Adulto , Coeficiente de Natalidade , Blastocisto/efeitos dos fármacos , Calcimicina/farmacologia , Transferência Embrionária/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização In Vitro/métodos , Humanos , Nascido Vivo , Masculino , Gravidez , Taxa de Gravidez , Estudos Prospectivos , Estudos Retrospectivos , Injeções de Esperma Intracitoplásmicas/métodos
13.
Int J Mol Sci ; 22(20)2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34681651

RESUMO

Mast cells play a very important role in skin allergy and inflammation, including atopic dermatitis and psoriasis. In the past, it was found that neferine has anti-inflammatory and anti-aging effects on the skin, but its effect on mast cells has not yet been studied in detail. In this study, we used mast cells (RBL-2H3 cells) and mouse models to study the anti-allergic and inflammatory effects of neferine. First, we found that neferine inhibits the degranulation of mast cells and the expression of cytokines. In addition, we observed that when mast cells were stimulated by A23187/phorbol 12-myristate-13-acetate (PMA), the elevation of intracellular calcium was inhibited by neferine. The phosphorylation of the MAPK/NF-κB pathway is also reduced by pretreatment of neferine. The results of in vivo studies show that neferine can improve the appearance of dermatitis and mast cell infiltration caused by dinitrochlorobenzene (DNCB). Moreover, the expressions of barrier proteins in the skin are also restored. Finally, it was found that neferine can reduce the scratching behavior caused by compound 48/80. Taken together, our results indicate that neferine is a very good anti-allergic and anti-inflammatory natural product. Its effect on mast cells contributes to its pharmacological mechanism.


Assuntos
Antialérgicos/farmacologia , Anti-Inflamatórios/farmacologia , Benzilisoquinolinas/farmacologia , Mastócitos/efeitos dos fármacos , Animais , Antialérgicos/uso terapêutico , Benzilisoquinolinas/uso terapêutico , Calcimicina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/patologia , Dinitroclorobenzeno/farmacologia , Modelos Animais de Doenças , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
14.
Life Sci ; 285: 119939, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34506836

RESUMO

AIMS: Nitric oxide synthases (NOSs) are key enzymes regulating vascular function. Previously, we reported that ß-adrenergic (ß-AR) overstimulation, a common feature of cardiovascular diseases, did not impair endothelium-dependent vasodilation, although it resulted in endothelial NOS (eNOS) uncoupling and reduced NO bioavailability. In addition to NO, neuronal NOS (nNOS) produces H2O2, which contributes to vasodilation. However, there is limited information regarding vascular ß-AR signaling and nNOS. In the present study, we assessed the possible role of nNOS-derived H2O2 and caveolins on endothelial vasodilation function following ß-AR overstimulation. MAIN METHODS: Male C57BL/6 wild-type and nNOS knockout mice (nNOS-/-) were treated with the ß-AR agonist isoproterenol (ISO, 15 mg·kg-1·day-1, s.c.) or vehicle (VHE) for seven days. Relaxation responses of aortic rings were evaluated using wire myograph and H2O2 by Amplex Red. KEY FINDINGS: Acetylcholine- or calcium ionophore A23187-induced endothelium-dependent relaxation was similar in aortic rings from VHE and ISO. However, this relaxation was significantly reduced in aortas from ISO compared to VHE when (1) caveolae were disrupted, (2) nNOS was pharmacologically inhibited or genetically suppressed and (3) H2O2 was scavenged. NOS-derived H2O2 production was higher in the aortas of ISO mice than in those of VHE mice. Aortas from ISO-treated mice showed increased expression of caveolin-1, nNOS and catalase, while caveolin-3 expression did not change. SIGNIFICANCE: The results suggest a role of caveolin-1 and the nNOS/H2O2 vasodilatory pathway in endothelium-dependent relaxation following ß-AR overstimulation and reinforce the protective role of nNOS in cardiovascular diseases associated with high adrenergic tone.


Assuntos
Caveolina 1/fisiologia , Óxido Nítrico Sintase Tipo I/fisiologia , Receptores Adrenérgicos alfa/metabolismo , Vasodilatação/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Caveolina 1/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Peróxido de Hidrogênio/metabolismo , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo I/genética , Vasodilatação/efeitos dos fármacos , Vasodilatação/genética
15.
Front Endocrinol (Lausanne) ; 12: 692082, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335469

RESUMO

Calcium is a crucial factor in regulating the biological behavior of cells. The imbalance of calcium homeostasis in cytoplasm will cause abnormal behavior of cells and the occurrence of diseases. In intracytoplasmic sperm injection (ICSI) cycle, the dysfunction of oocyte activation caused by insufficient release of Ca2+ from endoplasmic reticulum is one of the main reasons for repeated fertilization failure. Calcium ionophore (A23187) is a highly selective calcium ionophore, which can form stable complex with Ca2+ and pass through the cell membrane at will, effectively increasing intracellular Ca2+ levels. It has been reported that calcium ionophore (A23187) can activate oocytes and obtain normal embryos. However, there are few studies on unfertilized oocytes after calcium ionophore (A23187) rescue activation in ICSI cycle. The purpose of this study was to analyze the effects of calcium ionophore (A23187) rescue activation on the activation of unfertilized oocytes, embryonic development potential, embryonic development timing and chromosomal aneuploidy, and to compare and analyze the clinical data of patients with calcium ionophore (A23187) activation in clinical application. The results showed that a certain proportion of high-quality blastocysts with normal karyotype could be obtained after calcium ionophore (A23187) rescue activation of unfertilized oocytes, and it did not have a significant effect on the timing of embryo development. In clinical practice, direct activation with calcium ionophore (A23187) after ICSI was better than rescue activation the next day. In conclusions, the studies on the effectiveness and safety of calcium ionophore (A23187) rescue activation for oocytes with ICSI fertilization failure can enable some patients to obtain usable, high-quality embryos during the first ICSI cycle.


Assuntos
Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Oócitos/efeitos dos fármacos , Aneuploidia , Blastocisto/efeitos dos fármacos , Cromossomos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Humanos , Injeções de Esperma Intracitoplásmicas
16.
Artigo em Inglês | MEDLINE | ID: mdl-34444489

RESUMO

Streptomyces chartreusis NRRL 3882 produces the polyether ionophore calcimycin and a variety of analogs, which originate from the same biosynthetic gene cluster. The role of calcimycin and its analogs for the producer is unknown, but calcimycin has strong antibacterial activity. Feeding experiments were performed in chemically defined medium systematically supplemented with proteinogenic amino acids to analyze their individual effects on calcimycin synthesis. In the culture supernatants, in addition to known calcimycin analogs, eight so far unknown analogs were detected using LC-MS/MS. Under most conditions cezomycin was the compound produced in highest amounts. The highest production of calcimycin was detected upon feeding with glutamine. Supplementation of the medium with glutamic acid resulted in a decrease in calcimycin production, and supplementation of other amino acids such as tryptophan, lysine, and valine resulted in the decrease in the synthesis of calcimycin and of the known intermediates of the biosynthetic pathway. We demonstrated that the production of calcimycin and its analogs is strongly dependent on amino acid supply. Utilization of amino acids as precursors and as nitrogen sources seem to critically influence calcimycin synthesis. Even amino acids not serving as direct precursors resulted in a different product profile regarding the stoichiometry of calcimycin analogs. Only slight changes in cultivation conditions can lead to major changes in the metabolic output, which highlights the hidden potential of biosynthetic gene clusters. We emphasize the need to further study the extent of this potential to understand the ecological role of metabolite diversity originating from single biosynthetic gene clusters.


Assuntos
Aminoácidos , Espectrometria de Massas em Tandem , Calcimicina , Cromatografia Líquida , Streptomyces
17.
Eur J Pharmacol ; 910: 174448, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34454926

RESUMO

Reduced skin blood flow has been reported in neuropathic pain patients as well as various peripheral neuropathic pain model animals. We have previously shown that vasodilators, which improves reduced skin blood flow, correlatively alleviate neuropathic pain in chronic constriction injury (CCI) mice, a model of neuropathic pain from peripheral nerve injury. Here, we sought to elucidate the mechanism underlying the reduced skin blood flow in CCI rats. The skin blood flow of the ipsilateral plantar arteries was significantly reduced compared to that of the contralateral ones 4 weeks after loose ligation of the sciatic nerve. The contraction induced by noradrenaline, serotonin, and U46619, a thromboxane receptor agonist, in the isolated ipsilateral plantar arteries was significantly enhanced compared to that in the contralateral ones. KB-R7943, a Na+/Ca2+ exchanger (NCX) inhibitor, shifted the concentration-response curves of noradrenaline to the left in the contralateral arteries but had no effect on the ipsilateral side. There was no significant difference in concentration-response curves of noradrenaline between the ipsilateral and contralateral arteries in the presence of KB-R7943. Amiloride, a non-specific inhibitor of Na+ channels and transporters, comparably shifted concentration-response curves of noradrenaline to the left in both the contralateral and ipsilateral arteries. One hundred nM of noradrenaline induced intracellular Ca2+ elevation in the ipsilateral arteries, which was significantly larger than that induced by 300-nM noradrenaline in the contralateral arteries. These results suggest that reduced peripheral blood flow after nerve injury is due to Na+-dependent inactivation of NCX in the ipsilateral plantar arteries.


Assuntos
Circulação Sanguínea/efeitos dos fármacos , Neuralgia/metabolismo , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Vasodilatadores/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Amilorida/farmacologia , Animais , Artérias/efeitos dos fármacos , Compostos de Boro/farmacologia , Calcimicina/farmacologia , Cálcio/metabolismo , Ionóforos de Cálcio/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Contração Muscular/efeitos dos fármacos , Nifedipino/farmacologia , Norepinefrina/farmacologia , Ouabaína/farmacologia , Ratos Wistar , Serotonina/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Vasoconstritores/farmacologia
18.
Mol Biol Cell ; 32(18): 1724-1736, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34081532

RESUMO

The vascular system is precisely regulated to adjust blood flow to organismal demand, thereby guaranteeing adequate perfusion under varying physiological conditions. Mechanical forces, such as cyclic circumferential stretch, are among the critical stimuli that dynamically adjust vessel distribution and diameter, but the precise mechanisms of adaptation to changing forces are unclear. We find that endothelial monolayers respond to cyclic stretch by transient remodeling of the vascular endothelial cadherin-based adherens junctions and the associated actomyosin cytoskeleton. Time-resolved proteomic profiling reveals that this remodeling is driven by calcium influx through the mechanosensitive Piezo1 channel, triggering Rho activation to increase actomyosin contraction. As the mechanical stimulus persists, calcium signaling is attenuated through transient down-regulation of Piezo1 protein. At the same time, filamins are phosphorylated to increase monolayer stiffness, allowing mechanoadaptation to restore junctional integrity despite continuing exposure to stretch. Collectively, this study identifies a biphasic response to cyclic stretch, consisting of an initial calcium-driven junctional mechanoresponse, followed by mechanoadaptation facilitated by monolayer stiffening.


Assuntos
Citoesqueleto de Actina/metabolismo , Actomiosina , Antígenos CD/metabolismo , Caderinas/metabolismo , Sinalização do Cálcio , Mecanotransdução Celular , Actomiosina/metabolismo , Junções Aderentes/fisiologia , Antígenos CD/genética , Fenômenos Biomecânicos , Caderinas/genética , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Citocalasina D/farmacologia , Filaminas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Mapas de Interação de Proteínas , Quinases Ativadas por p21/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
19.
Biomed Pharmacother ; 141: 111835, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34146852

RESUMO

Thymic stromal lymphopoietin (TSLP) produced by mast cells is involved in allergic inflammation pathogenesis. Chloroquine (CQ) is known to be an anti-malarial drug; however, additional protective functions of CQ have been discovered. This study aims to clarify an anti-inflammatory effect of CQ through modulating TSLP levels using an in vitro model of phorbol myristate acetate (PMA) + A23187-activated human mast cell line (HMC-1) and an in vivo model of PMA-irritated ear edema. CQ treatment reduced the production and mRNA expression levels of TSLP in activated HMC-1 cells. CQ down-regulated caspase-1 (CASP1), MAPKs, and NF-κB levels enhanced by stimulation with PMA + A23187. Moreover, ear thickness in ear edema was suppressed following CQ treatment. CQ decreased CASP1 and NF-κB levels in the ear tissue. TSLP levels in the ear tissue and serum were reduced following CQ treatment. Collectively, the above findings elucidate that CQ inhibits the pro-inflammatory mechanisms of TSLP via the down-regulation of distinct intracellular signaling cascade in mast cells. Therefore, CQ may have protective roles against TSLP-mediated inflammatory disorders.


Assuntos
Caspase 1/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Cloroquina/farmacologia , Citocinas/biossíntese , Mastócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Estromais/metabolismo , Timo/metabolismo , Animais , Calcimicina/farmacologia , Linhagem Celular , Otopatias/tratamento farmacológico , Edema/tratamento farmacológico , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Estromais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Timo/efeitos dos fármacos
20.
Andrology ; 9(5): 1631-1651, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33998170

RESUMO

BACKGROUND: Exposure to the calcium ionophore A23187 may present a "universal" sperm treatment for IVF, as it bypasses capacitation pathways. However, success in utilizing A23187 is variable, especially in equine spermatozoa. Notably, albumin is used during A23187 treatment but paradoxically is thought to suppress A23187 action. Essentially no critical data are available on the effects of A23187 and albumin concentrations, ratios, or addition protocols on changes in intracellular calcium ([Ca]i ) in any cell type. OBJECTIVE: To determine factors that affect the action of A23187 on [Ca]i in equine and murine spermatozoa. METHODS: Spermatozoa were loaded with Fluo-4 and changes in fluorescence after A23187 treatment were measured under various conditions using a microplate reader. RESULTS: Concentrations of bovine serum albumin (BSA) and A23187, type of BSA, makeup of A23187 stock solutions (i.e., 1° stock (DMSO) or 2° stock made with medium, water or DMSO), order of addition of spermatozoa and A23187, incubation of media before sperm addition, species of spermatozoa, and time of addition of BSA all affected [Ca]i in response to A23187 treatment. In equine spermatozoa already exposed to 10 µM A23187, addition of BSA to 33 mg/ml to "quench" the A23187 did not affect [Ca]i . When this concentration of BSA was added to spermatozoa exposed to 1 µM A23187, [Ca]i in murine spermatozoa returned to baseline, however, equine spermatozoa continued to exhibit increased [Ca]i . Addition of BSA to 33 mg/ml to media containing 1 µM A23187, prior to addition of spermatozoa, completely inhibited change in [Ca]i in both murine and equine spermatozoa. CONCLUSION: These results represent some of the first critical data on the effects of albumin and other procedural factors on A23187-induced changes in [Ca]i in any cell type. Our findings help to explain the variability in reported response of spermatozoa to A23187 among species and among laboratories.


Assuntos
Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Cálcio/metabolismo , Soroalbumina Bovina/metabolismo , Espermatozoides/efeitos dos fármacos , Animais , Cavalos , Masculino , Camundongos
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