RESUMO
While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor 2 (Fgfr2) is necessary for kidney and ureter mesenchymal development. Our objective was to determine the role of Fgfr2 in bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in bladder mesenchyme (Fgfr2BM-/-). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblotting, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with controls, embryonic (E) day 16.5 (E16.5) Fgfr2BM-/- bladders have thin muscle layers with reduced α-smooth muscle actin levels and thickened lamina propria with increased collagen expression that intrudes into muscle. From postnatal (P) day 1 (P1) to P30, Fgfr2BM-/- bladders demonstrate progressive muscle loss and increased collagen expression. Postnatal Fgfr2BM-/- bladder sheets exhibit decreased contractility and increased passive stretch tension compared with controls. In vivo cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2BM-/- bladders compared with controls. Mechanistically, while Shh expression appears normal, mRNA and protein readouts of hedgehog activity are increased in E16.5 Fgfr2BM-/- bladders compared with controls. Moreover, E16.5Fgfr2BM-/- bladders exhibit higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, than controls. Fgfr2 is critical for bladder mesenchyme patterning by virtue of its role in modulation of hedgehog signaling.
Assuntos
Padronização Corporal , Mesoderma/metabolismo , Músculo Liso/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Bexiga Urinária/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Apoptose , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Colágeno/genética , Colágeno/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Idade Gestacional , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Masculino , Camundongos Knockout , Contração Muscular , Músculo Liso/embriologia , Músculo Liso/fisiopatologia , Miócitos de Músculo Liso , Fenótipo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/efeitos dos fármacos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Bexiga Urinária/embriologia , Bexiga Urinária/fisiopatologia , UrodinâmicaRESUMO
While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor (Fgfr)2 is necessary for kidney and ureter mesenchymal development. The objective of the present study was to determine the role of Fgfr2 in the bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in the bladder mesenchyme (Fgfr2(BM-/-)). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblot analysis, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with control bladders, embryonic day 16.5 (E16.5) Fgfr2(BM-/-) bladders had thin muscle layers with less α-smooth muscle actin and thickened lamina propria with increased collagen type Ia and IIIa that intruded into the muscle. The reciprocal changes in mutant layer thicknesses appeared partly due to a cell fate switch. From postnatal days 1 to 30, Fgfr2(BM-/-) bladders demonstrated progressive muscle loss and increased collagen expression. Postnatal Fgfr2(BM-/-) bladder sheets exhibited decreased agonist-mediated contractility and increased passive stretch tension versus control bladder sheets. Cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2(BM-/-) versus control bladders. Mechanistically, whereas Shh expression appeared normal, mRNA and protein readouts of hedgehog activity were increased in E16.5 Fgfr2(BM-/-) versus control bladders. Moreover, E16.5 Fgfr2(BM-/-) bladders exhibited higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, compared with control bladders. In conclusion, loss of Fgfr2 in the bladder mesenchyme leads to abnormal bladder morphology and decreased compliance and contractility.
Assuntos
Padronização Corporal , Mesoderma/metabolismo , Músculo Liso/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Bexiga Urinária/metabolismo , Animais , Apoptose , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Complacência (Medida de Distensibilidade) , Fibrose , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Idade Gestacional , Proteínas Hedgehog/metabolismo , Imunoglobulina G/metabolismo , Masculino , Mesoderma/anormalidades , Camundongos Knockout , Contração Muscular , Músculo Liso/anormalidades , Músculo Liso/fisiopatologia , Tamanho do Órgão , Fenótipo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Bexiga Urinária/anormalidades , Bexiga Urinária/fisiopatologia , UrodinâmicaRESUMO
During the period of aging of spontaneously hypertensive rats (SHR) between 6 and 13 weeks the systolic arterial pressure increased from 131+/-2 up to 176+/-3 mm Hg while in the control group of WKY rats it reached 122+/-2 mmHg. The hypertension was combined with myocardial hypertrophy -- the relative weight of SHR heart was 24% higher. The contractile myocardial function of the isolated isovolumic heart of SHR group did not differ from WKY group in a wide range of coronary perfusion rates. During oxidative stress induced by 40-min intracoronary introduction of H(2)O(2) function of hypertrophied SHR hearts fell significantly deeper. This coincided with decreased myocardial activity of superoxide dismutase and glutathione peroxidase by 29-30%, and increased catalase activity by 18%. The rate of generation of active forms of oxygen (hydroxyl radicals HO(.-)) in mitochondria from SHR hearts was higher as compared with WKY. Thus, the development of hypertension was combined with decreased antioxidant protection of the myocardium. The addition of ubiquinone to drinking water (approximately 10 mg/kg/day) for 6 weeks did not affect arterial pressure level, but was associated with two times lesser degree of myocardial hypertrophy. The hearts of SHR that received ubiquinone differed from those not treated with ubiquinone by increased maximal level of myocardial contractile function, and by improved myocardial relaxability and distensibility. After administration of H(2)O(2), myocardial function of SHR was kept on higher level. That was combined with less myocardial oedema, better preservation of antioxidant enzymes and reduced rate of succinate-dependent generation of superoxide radicals in mitochondria from hearts of ubiquinone treated SHR. The results have shown, that administration of ubiquinone to rats with hereditary hypertension reduces degree of myocardial hypertrophy, improves functional properties of the myocardium, promotes effective protection of antioxidant enzymes and increases the resistance of the cardiac muscle to oxidative stress.
Assuntos
Hipertensão/tratamento farmacológico , Contração Miocárdica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ubiquinona/uso terapêutico , Animais , Pressão Sanguínea/efeitos dos fármacos , Catalase/metabolismo , Modelos Animais de Doenças , Seguimentos , Glutationa Peroxidase/metabolismo , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Contração Miocárdica/fisiologia , Miocárdio/enzimologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Superóxido Dismutase/metabolismoRESUMO
Using EPR spectroscopy it was established that Fe ions released from ferritin under the action of glutathione and superoxide took part in the formation of dinitrosyl complexes of iron with glutathione (DNIC). The reaction between O2-. and NO resulted in the formation of peroxynitrite, which oxidized glutathione to the thiyl radical. In these conditions, DNIC did not inhibit the formation of thiyl radicals but effectively slowed down the oxidative destruction of beta-carotene by peroxynitrite and free radicals of lipids. In the presence of glutathione, the inversion of the antioxidant properties of DNIC into prooxidant ones took place. S-nitrosoglutathione prevented this inversion and suppressed the free-radical oxidation of beta-carotene induced by ferritin. It was proposed that the equilibrium between S-nitrosoglutathione, DNIC, "free Fe" ions and ferritin may determine the balance between prooxidant and antioxidant processes in living organisms.
Assuntos
Ferritinas/química , Óxidos de Nitrogênio/síntese química , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Oxigênio/química , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Glutationa/química , Hidrazinas/química , Ferro/química , Lipídeos/química , Óxido Nítrico/química , Óxidos de Nitrogênio/química , Oxirredução , Ácido Peroxinitroso/química , S-Nitrosoglutationa/química , Detecção de Spin , Superóxidos/química , beta Caroteno/químicaRESUMO
The effect of iron dinitrosyl complexes, S-nitrosoglutathione, and glutathione on free radical oxidation of rat heart mitochondria induced by tert-butyl hydroperoxide and metmyoglobin or their combination with ferritin was studied. It was shown that iron dinitrosyl complexes or the combination of S-nitrosoglutathione and glutathione inhibited most effectively the peroxidation of mitochondrial membranes. It was found that ferritin stimulated the prooxidant action of metmyoglobin. Using EPR spectroscopy, it was established that, in conditions of O2*- generation, the destruction of iron dinitrosyl complexes took place. Iron dinitrosyl complexes also inhibited the formation of thiyl radicals, which appeared during O2*- generation in the system containing glutathione and S-nitrosoglutathione. It is essential that the formation of iron dinitrosyl complexes in this reaction system took place with the involvement of ferritin. It was proposed that the prooxidant action of ferritin and myoglobin could be inverted to the antioxidant one.
Assuntos
Ferritinas/metabolismo , Peroxidação de Lipídeos , Mitocôndrias Cardíacas/metabolismo , Mioglobina/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Glutationa/farmacologia , Técnicas In Vitro , Ferro/farmacologia , Óxidos de Nitrogênio/farmacologia , Ratos , Ratos Endogâmicos WKY , S-Nitrosoglutationa/farmacologia , Superóxidos/metabolismoRESUMO
This study examined the origin of spontaneous activity in neonatal and adult rat bladders and the effect of stretch and muscarinic agonists and antagonists on spontaneous activity. Rats were anesthetized and their bladders were excised, cannulated, and loaded with voltage- and Ca(2+)-sensitive dyes. Intracellular Ca(2+) and membrane potential transients were mapped using photodiode arrays in whole bladders, bladder sheets, or cross-section preparations at 37 degrees C. Intravesical pressure was recorded from whole bladders. In neonatal bladders and sheets, spontaneous Ca(2+) and electrical signals arose at a site near the dome and spread in a coordinated manner throughout the bladder with different dome-to-neck conduction velocities (Ca(2+): 3.7 +/- 0.4 mm/s; membrane potential: 46.2 +/- 3.1 mm/s). In whole bladders, optical signals were associated with spontaneous contractions (10-20 cmH(2)O). By contrast, in adult bladders spontaneous Ca(2+) and electrical activity was uncoordinated, originating at multiple sites and was associated with smaller (2-5 cmH(2)O) contractions. Spontaneous contractions and optical signals were insensitive to tetrodotoxin (2 muM) but were blocked by nifedipine (10 muM). Stretch or low carbachol concentrations (50 nM) applied to neonatal whole bladders enhanced the amplitude (to 20-35 cmH(2)O) of spontaneous activity, which was blocked by atropine. Bladder cross sections revealed that Ca(2+) and membrane potential transients produced by stretch or carbachol began near the urothelial-suburothelial interface and then spread to the detrusor. In conclusion, spontaneous activity in neonatal bladders, unlike activity in adult bladders, is highly organized, originating in the urothelium-suburothelium near the dome. Activity is enhanced by stretch or carbachol and this enhancement is blocked by atropine. It is hypothesized that acetylcholine is released from the urothelium during bladder filling to enhance spontaneous activity.
Assuntos
Agonistas Muscarínicos/farmacologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Bexiga Urinária/fisiologia , Animais , Animais Recém-Nascidos , Atropina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Diacetil/análogos & derivados , Diacetil/farmacologia , Estimulação Elétrica , Eletrofisiologia , Pressão Hidrostática , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Estimulação Física , Ratos , Suramina/farmacologia , Tetrodotoxina/farmacologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/inervação , Urotélio/efeitos dos fármacos , Urotélio/fisiologiaRESUMO
Changes in myocardium were studied during oxidative stress induced by infusion of hydrogen peroxide in the coronary vessels of isolated rat heart. Moderate concentrations of H2O2 increased the heart rate but decreased the contractile force, whereas higher concentrations of H2O2 decreased both parameters and increased the end diastolic pressure. The effect of H2O2 was stable, cumulative, and was associated with disturbance in respiration of mitochondria, increased production of ROS in them, and decrease in activities of antioxidant enzymes in the myocardium. Changes in the antioxidant status of the myocardium induced by long-term addition of coenzyme Q(10) into food was accompanied by decrease in the negative inotropic effect of H2O2, whereas the levels of superoxide dismutase and glutathione peroxidase after oxidative stress were virtually unchanged. The activities of these enzymes displayed a high positive correlation with the cardiac function. The findings suggest that coenzyme Q(10) should increase resistance of the myocardium to oxidative stress not only by a direct antioxidant mechanism but also indirectly, due to increased protection of antioxidant enzymes.
Assuntos
Antioxidantes/metabolismo , Antioxidantes/farmacologia , Miocárdio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , Animais , Coenzimas , Peróxido de Hidrogênio , Contração Miocárdica/efeitos dos fármacos , Miocárdio/enzimologia , Oxidantes , Estresse Oxidativo/fisiologia , Ratos , Ratos WistarRESUMO
It is shown that dinitrosyl-iron complexes (DNIC) with glutathione can reduce oxoferrylmyoglobin forming on interaction of tert-butyl hydroperoxide and metmyoglobin. A rapid decrease in the DNIC concentration was observed under the conditions of production of tert-butyl free radicals; however, destruction of DNIC in the presence of oxoferrylmyoglobin alone was negligible. It is demonstrated that DNIC reduces oxoferrylmyoglobin more than an order more efficiently than S-nitrosoglutathione and glutathione. DNIC also inhibits formation of the thiyl radicals of glutathione in a medium containing metmyoglobin and tert-butyl hydroperoxide. A mechanism of the antioxidant action of DNIC based on regeneration of the nitrosyl complexes from the products of their interaction with oxoferrylheme is proposed.
Assuntos
Ferro/metabolismo , Metamioglobina/metabolismo , Óxidos de Nitrogênio/metabolismo , Antioxidantes/metabolismo , Óxidos N-Cíclicos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/metabolismo , Glutationa/metabolismo , Óxido Nítrico , S-Nitrosoglutationa/metabolismo , terc-Butil Hidroperóxido/metabolismoRESUMO
Ubiquinone Q(10) (coenzyme Q) is an important component of the mitochondrial electron transport chain and an antioxidant. The purpose of this work was to find out whether an increase in the level of coenzyme Q in the heart changes its maximal working capacity and resistance to oxidative stress. Male Wistar rats were treated with coenzyme Q (10 mg/kg body weight per day) for six weeks, and this increased its content in the myocardium by 63%. The myocardial content of malonic dialdehyde and activities of key antioxidant enzymes were unchanged, except nearly 2.5-fold decrease in the activity of superoxide dismutase. The maximal working capacity of the isolated isovolumic heart did not change, but under conditions of oxidative stress induced by 45-min infusion of hydrogen peroxide (70 micro M) into coronary vessels the contractile function of these hearts decreased significantly more slowly. This was associated with less pronounced lesions in the ultrastructure of cardiomyocytes and lesser disorders in the oxidative metabolism of mitochondria that suggested increased antioxidant protection of the myocardium.