RESUMO
The p66ShcA protein controls cellular responses to oxidative stress, senescence, and apoptosis. Here, we test the hypothesis that aging phenotype(s) commonly associated with the broad category of chronic kidney disease are accelerated in diabetic kidneys and linked to the p66ShcA locus. At the organ level, tissue stem cells antagonize senescent phenotypes by replacing old dysfunctional cells. Using established methods, we isolated a highly purified population of stem cell antigen-1-positive mesenchymal stem cells (Sca-1+ MSCs) from kidneys of wild-type (WT) and p66 knockout (p66 KO) mice. Cells were plated in culture medium containing normal glucose (NG) or high glucose (HG). Reactive oxygen species (ROS) metabolism was substantially increased in WT MSCs in HG medium in association with increased cell death by apoptosis and acquisition of the senescent phenotype. DNA microarray analysis detected striking differences in the expression profiles of WT and p66 KO-MSCs in HG medium. Unexpectedly, the analysis for p66 KO-MSCs revealed upregulation of Wnt genes implicated in self-renewal and differentiation. To test the in vivo consequences of constitutive p66 expression in diabetic kidneys, we crossed the Akita diabetic mouse with the p66KO mouse. Homozygous mutation at the p66 locus delays or prevents aging phenotype(s) in the kidney that may be precursors to diabetic nephropathy.
Assuntos
Envelhecimento/metabolismo , Nefropatias Diabéticas/metabolismo , Rim/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Fatores Etários , Envelhecimento/genética , Envelhecimento/patologia , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Senescência Celular , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Glucose/metabolismo , Rim/patologia , Células-Tronco Mesenquimais/patologia , Camundongos Knockout , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/deficiência , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Nicho de Células-Tronco , Via de Sinalização WntRESUMO
We have developed a state-of-the-art apparatus for laser-based spin- and angle-resolved photoemission spectroscopy with micrometer spatial resolution (µ-SARPES). This equipment is realized by the combination of a high-resolution photoelectron spectrometer, a 6 eV laser with high photon flux that is focused down to a few micrometers, a high-precision sample stage control system, and a double very-low-energy-electron-diffraction spin detector. The setup achieves an energy resolution of 1.5 (5.5) meV without (with) the spin detection mode, compatible with a spatial resolution better than 10 µm. This enables us to probe both spatially-resolved electronic structures and vector information of spin polarization in three dimensions. The performance of µ-SARPES apparatus is demonstrated by presenting ARPES and SARPES results from topological insulators and Au photolithography patterns on a Si (001) substrate.
RESUMO
We study ultrafast population dynamics in the topological surface state of Sb[Formula: see text]Te[Formula: see text] in two-dimensional momentum space with time- and angle-resolved two-photon photoemission spectroscopy. Linearly polarized mid-infrared pump pulses are used to permit a direct optical excitation across the Dirac point. We show that this resonant excitation is strongly enhanced within the Dirac cone along three of the six [Formula: see text]-[Formula: see text] directions and results in a macroscopic photocurrent when the plane of incidence is aligned along a [Formula: see text]-[Formula: see text] direction. Our experimental approach makes it possible to disentangle the decay of transiently excited population and photocurent by elastic and inelastic electron scattering within the full Dirac cone in unprecedented detail. This is utilized to show that doping of Sb[Formula: see text]Te[Formula: see text] by vanadium atoms strongly enhances inelastic electron scattering to lower energies, but only scarcely affects elastic scattering around the Dirac cone.
RESUMO
The GATA-6 transcription factor is reported to be expressed in vascular myocytes. Because glomerular mesangial cells (GMCs) and vascular myocytes have similar properties, we examined whether GATA-6 was expressed in cultured GMCs and whether overexpression of GATA-6 induced cell cycle arrest in GMCs, using a recombinant adenovirus that expresses GATA-6 (Ad GATA-6). GATA-6 expression in GMCs was downregulated when quiescent GMCs were stimulated by serum to reenter the cell cycle. [(3)H]thymidine uptake was inhibited in GMCs infected with Ad GATA-6 in a dose- and time-dependent manner. The expression of cyclin A protein was decreased and that of the cyclin-dependent kinase inhibitor p21(cip1) was increased in GMCs infected with Ad GATA-6. Although the expression of p21(cip1) transcripts did not change remarkably, p21(cip1) protein was stabilized in GMCs infected with Ad GATA-6, suggesting a post-transcriptional regulation of p21(cip1) expression. Northern blot analysis showed that expression of the cyclin A transcript was decreased in Ad GATA-6-infected cells, whereas this decrease of cyclin A was not observed in GMCs derived from p21(cip1) null mice. Our results demonstrate that GATA-6 is endogenously expressed in GMCs and that overexpression of GATA-6 can induce cell cycle arrest. Our results also show that GATA-6-induced cell cycle arrest is associated with inhibition of cyclin A expression and p21(cip1) upregulation. Finally, our results indicate that the GATA-6-induced suppression of cyclin A expression depends on the presence of p21(cip1).
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclina A/metabolismo , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mesângio Glomerular/metabolismo , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Animais , Proteínas Sanguíneas/farmacologia , Northern Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ciclina A/genética , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Fator de Transcrição GATA6 , Expressão Gênica/efeitos dos fármacos , Mesângio Glomerular/citologia , Mesângio Glomerular/efeitos dos fármacos , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Timidina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , Regulação para CimaRESUMO
BACKGROUND: Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. Levels of AM are markedly increased in the fetoplacental circulation during pregnancy, although its function there remains unknown. To clarify the physiological functions of AM, we chose a gene-targeting strategy in mice. METHODS AND RESULTS: Targeted null mutation of the AM gene is lethal in utero: the mortality rate among AM(-/-) embryos was >80% at E13.5. The most apparent abnormality in surviving AM(-/-) embryos at E13.5 to E14.0 was severe hemorrhage, readily observable under the skin and in visceral organs. Hemorrhage was not detectable at E12.5 to E13.0, although the yolk sac lacked well-developed vessels. Electron microscopic examination showed endothelial cells to be partially detached from the basement structure at E12.5 in vitelline vessels and hepatic capillaries, which allowed efflux of protoerythrocytes through the disrupted barrier. The basement membrane was not clearly recognizable in the aorta and cervical artery, and the endothelial cells stood out from the wall of the lumen, only partially adhering to the basement structure. AM(+/-) mice survived to adulthood but exhibited elevated blood pressures with diminished nitric oxide production. CONCLUSIONS: AM is indispensable for the vascular morphogenesis during embryonic development and for postnatal regulation of blood pressure by stimulating nitric oxide production.
Assuntos
Vasos Sanguíneos/anormalidades , Anormalidades Cardiovasculares/patologia , Hipertensão/patologia , Peptídeos/deficiência , Adrenomedulina , Animais , Vasos Sanguíneos/patologia , Vasos Sanguíneos/ultraestrutura , Perda do Embrião/etiologia , Perda do Embrião/patologia , Endotélio Vascular/embriologia , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Feminino , Marcação de Genes , Genes Letais , Genótipo , Hemodinâmica/genética , Hemorragia/embriologia , Hemorragia/genética , Hemorragia/patologia , Heterozigoto , Homozigoto , Hipertensão/genética , Hipertensão/fisiopatologia , Endogamia , Bombas de Infusão , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Peptídeos/administração & dosagem , Peptídeos/genética , Fenótipo , Proteínas Recombinantes/administração & dosagem , Membrana Vitelina/irrigação sanguínea , Membrana Vitelina/embriologia , Membrana Vitelina/patologiaRESUMO
The vasopressin receptor subtype that causes nitric oxide (NO) release remains controversial. To elucidate this receptor-ligand interaction, we examined the effects of vasopressin receptor antagonists on vasopressin-induced release of NO from isolated perfused rat kidneys by using a sensitive chemiluminescence assay. Vasopressin increased renal perfusion pressure and NO signals in the perfusate in a dose-dependent manner. N omega-Monomethyl-L-arginine abolished this increase in NO release; however, a similar increase in renal perfusion pressure induced by prostaglandin F2 alpha was not associated with the increase in NO release. OPC-21268, a V1 receptor antagonist, significantly reduced the vasopressin-evoked renal vasoconstriction and NO release, whereas OPC-31260, a V2 receptor antagonist, had no effects. Moreover, desmopressin, a selective V2 receptor agonist, did not increase the NO signal. NO release by vasopressin was markedly attenuated in deoxycorticosterone acetate (DOCA)-salt hypertensive rat kidneys compared with control kidneys (10(-10) mol/L vasopressin: +0.8 +/- 0.3 versus +6.9 +/- 1.4 fmol/min per gram kidney, DOCA versus control; P < .001). Histochemical analysis for renal NO synthase revealed a substantial attenuation of the staining of endothelial NO synthase in DOCA-salt rats. These results directly demonstrate that vasopressin stimulates NO release via the endothelial V1 receptor in the rat kidney.
Assuntos
Arginina Vasopressina/farmacologia , Rim/metabolismo , Óxido Nítrico/metabolismo , Receptores de Vasopressinas/classificação , Fármacos Renais/farmacologia , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Arginina Vasopressina/metabolismo , Benzazepinas/farmacologia , Desoxicorticosterona , Dinoprosta/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Rim/efeitos dos fármacos , Masculino , Perfusão , Piperidinas/farmacologia , Quinolonas/farmacologia , Ratos , Ratos Wistar , Receptores de Vasopressinas/metabolismo , Fármacos Renais/metabolismo , ômega-N-Metilarginina/farmacologiaRESUMO
To examine whether endothelial dysfunction in hypertension is reversible or not, we studied the effects of imidapril, an angiotensin-converting enzyme inhibitor, on nitric oxide release in stroke-prone spontaneously hypertensive rats (SHR) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. After a 4-week treatment with imidapril (1 or 10 mg/d SC) or vehicle, acetylcholine-induced vasodilation and nitric oxide release in the isolated kidneys were determined. Nitric oxide release was measured by a chemiluminescense assay. Imidapril lowered blood pressure in stroke-prone SHR in a dose-dependent manner. Untreated stroke-prone SHR exhibited significantly attenuated responses to acetylcholine (10(-8) mol/L) of both renal perfusion pressure (stroke-prone SHE 42 +/- 4% versus Wistar-Kyoto rats [WKY] 58 +/- 4% [mean +/- SE], P < .01) and nitric oxide release (stroke-prone SHR +7.6 +/- 2.1 versus WKY +29.7 +/- 9.7 fmol/min per gram of kidney wt, P < .01). Imidapril at 10 mg/d significantly increased acetylcholine-induced renal vasodilation and nitric oxide release in stroke-prone SHR (renal perfusion pressure, 56 +/- 3%; nitric oxide release, +27.1 +/- 6.4 fmol/min per gram of kidney wt; both P < .01 versus stroke-prone SHR treated with vehicle). On the other hand, imidapril neither decreased blood pressure nor changed nitric oxide release induced by acetylcholine in DOCA-salt hypertensive rats. Staining for endothelial nitric oxide synthase and brain nitric oxide synthase was clearly detected in the kidneys of both stroke-prone SHR and WKY, whereas staining intensity was weaker in DOCA-salt hypertensive rats. Inducible nitric oxide synthase immunoreactivity was barely noticeable in any type of rat. Thus, imidapril restored endothelial damage by pressure-dependent mechanisms. Most of the nitric oxide detected in the perfusate seemed to be derived from constitutive nitric oxide synthase.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Hipertensão/metabolismo , Imidazóis/administração & dosagem , Imidazolidinas , Rim/metabolismo , Óxido Nítrico/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Bombas de Infusão Implantáveis , Rim/fisiopatologia , Ratos , Ratos Endogâmicos SHRRESUMO
We previously reported that adrenomedullin (AM), a potent vasodilator peptide discovered in pheochromocytoma cells, stimulates nitric oxide (NO) release in the rat kidney. To further investigate whether the NO-cGMP pathway is involved in the mechanisms of AM-induced vasodilation, we examined the effects of E-4021, a cGMP-specific phosphodiesterase inhibitor, on AM-induced vasorelaxation in aortic rings and perfused kidneys isolated from Wistar rats. We also measured NO release from the kidneys using a chemiluminescence assay. AM (10(-10) to 10(-7) mol/L) relaxed the aorta precontracted with phenylephrine in a dose-dependent manner. Denudation of endothelium (E) attenuated the vasodilatory action of AM (10(-7) mol/L AM: intact (E+) -25.7+/-5.2% versus denuded (E-) -7. 8+/-0.6%, P<0.05). On the other hand, pretreatment with 10(-8) mol/L E-4021 augmented AM-induced vasorelaxation in the intact aorta (-49. 0+/-7.9%, P<0.05) but not in the denuded one. E-4021 also enhanced acetylcholine (ACh)-induced vasorelaxation in the rat intact aorta (10(-7) mol/L ACh -36.6+/-8.4% versus 10(-8) mol/L E-4021+10(-7) mol/L ACh -62.7+/-3.1%, P<0.05). In perfused kidneys, AM-induced vasorelaxation was also augmented by preincubation with E-4021 (10(-9) mol/L AM -15.4+/-0.6% versus 10(-8) mol/L E-4021+10(-9) mol/L AM -23.6+/-1.2%, P<0.01). AM significantly increased NO release from rat kidneys (DeltaNO: +11.3+/-0.8 fmol. min-1. g-1 kidney at 10(-9) mol/L AM), which was not affected by E-4021. E-4021 enhanced ACh-induced vasorelaxation (10(-9) mol/L ACh -9.7+/-1.7% versus 10(-8) mol/L E-4021+10(-9) mol/L ACh -18.8+/-2.9%, P<0.01) but did not affect ACh-induced NO release from the kidneys. In the aorta and the kidney, 10(-4) mol/L of NG-nitro-L-arginine methyl ester, an NO synthase inhibitor, and 10(-5) mol/L of methylene blue, a guanylate cyclase inhibitor, reduced the vasodilatory effect of AM. These results suggest that the NO-cGMP pathway is involved in the mechanism of AM-induced vasorelaxation, at least in the rat aorta and kidney.
Assuntos
Aorta/fisiologia , GMP Cíclico/fisiologia , Óxido Nítrico/fisiologia , Peptídeos/farmacologia , Transdução de Sinais/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologia , Adrenomedulina , Animais , Aorta/efeitos dos fármacos , Rim/fisiologia , Masculino , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
The mechanisms for the vascular actions of vasodilatory beta-blockers remain undetermined. For some kinds of beta-blockers, the involvement of nitric oxide (NO) has been suggested. We studied the effects of vasodilatory beta-blockers on renal perfusion pressure (RPP) and NO release in the rat kidney. Infusion of bopindolol, celiprolol, and nebivolol caused a dose-dependent reduction in RPP and an increase in NO release (RPP: bopindolol 10(-6) mol/L, -23+/-2%; celiprolol 10(-4) mol/L, -27+/-2%; nebivolol 10(-5) mol/L, -35+/-3%; NO: bopindolol 10(-6) mol/L, +33+/-2; celiprolol 10(-4) mol/L, +41+/-2; nebivolol 10(-5) mol/L, +45+/-5 fmol. min-1. g kidney-1, mean+/-SEM). Metergoline (10(-6) mol/L), a 5-hydroxytryptamine (5-HT)1/2 antagonist, or NAN-190 (10(-6) mol/L), a 5-HT1A antagonist, almost completely abolished the vasorelaxation and NO release caused by bopindolol, celiprolol, and nebivolol. However, neither propranolol nor bisoprolol decreased RPP. Celiprolol and nebivolol caused vasodilation in the rat thoracic aorta, and it was markedly reduced by endothelial denudation, Nomega-nitro-L-arginine methyl ester (10(-4) mol/L), or NAN-190 (10(-6) mol/L). In deoxycorticosterone acetate-salt hypertensive rats, 4-week administration of celiprolol (50 mg. kg-1. d-1 IV) restored the responses regarding RPP and NO release to acetylcholine. These results suggest that several beta-blockers exert their vasodilatory action through the 5-HT1A receptor/NO pathway and that treatment with these beta-blockers may protect against endothelial injury in hypertension.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Circulação Renal/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Benzopiranos/farmacologia , Celiprolol/farmacologia , Desoxicorticosterona , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Etanolaminas/farmacologia , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Técnicas In Vitro , Rim/fisiologia , Rim/fisiopatologia , Masculino , Metergolina/farmacologia , Músculo Liso Vascular/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Nebivolol , Óxido Nítrico Sintase Tipo III , Fenilefrina/farmacologia , Pindolol/análogos & derivados , Pindolol/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Quinazolinas/farmacologia , Ratos , Ratos Wistar , Circulação Renal/fisiologia , Antagonistas da Serotonina/farmacologia , Vasoconstrição/efeitos dos fármacosRESUMO
Adrenomedullin, which was discovered as a vasodilating peptide, has been reported to be produced in various organs, in which adrenomedullin regulates not only vascular tone but also cell proliferation and differentiation in an autocrine/paracrine manner. We evaluated the effect of adrenomedullin on endothelial cell apoptosis. Human umbilical vein endothelial cells underwent apoptosis when cultured in serum-free medium. Treatment with adrenomedullin reduced the number of cells with pyknotic nuclei (Hoechst 33258 staining) and inhibited cell death (dimethylthiazol-diphenyltetrazolium bromide assay) in a dose-dependent manner. The administration of adrenomedullin did not alter the expression levels of Bcl-2 family proteins. Experiments with analogs of cAMP or a cAMP-elevating agonist demonstrated that elevation of the intracellular cAMP concentration does not mediate the antiapoptotic effect of adrenomedullin. The coadministration of N-nitro-L-arginine methyl ester (2 mmol/L), an inhibitor of nitric oxide synthase, abrogated the effect of adrenomedullin. Lower doses of sodium nitroprusside (1 to 10 micromol/L), a nitric oxide donor, mimicked the antiapoptotic effect of adrenomedullin. The antiapoptotic effect of sodium nitroprusside was not attenuated by the inhibition of soluble guanylyl cyclase with 1 micromol/L oxadiazolo-quinoxalin-1-one nor could apoptosis be inhibited by the incubation of human umbilical vein endothelial cells with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analog. These results indicate that adrenomedullin and nitric oxide inhibit endothelial cell apoptosis via a cGMP-independent mechanism.
Assuntos
Apoptose/efeitos dos fármacos , GMP Cíclico/fisiologia , Endotélio Vascular/efeitos dos fármacos , Óxido Nítrico/fisiologia , Peptídeos/farmacologia , Adrenomedulina , Sobrevivência Celular , Células Cultivadas , AMP Cíclico/fisiologia , Endotélio Vascular/citologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/análiseRESUMO
To elucidate the effect of aging and blood pressure on glucose tolerance, we examined the subjects who received 75g OGTT annually for 10 years. They were classified into 2 groups by blood pressure, and into 4 groups by age. The study group consisted of 34 cases (mean 62 years old), including 11 hypertensive (HT) and 23 normotensive (NT) control. All cases had their body weight fluctuate in less than 4 kgs. After blood pressure was measured, blood was drawn for determination of plasma glucose and insulin concentrations in each subject before 75g OGTT and 30, 60, 90, 120 minutes afterward. sigma BS and sigma IRI were defined as the sum of 5 points in BS and IRI, respectively. Insulinogenic index (I.I) was defined as deltaIRI/deltaBS in 30 min. sigma IRI/sigma BS and I.I of each year were calculated serially. They were compared between HT and NT group, and among 4 groups of age; 40s, 50s, 60s and 70s. sigma IRI/sigma BS was significantly decreased in both HT and NT groups (0.52 +/- 0.05 (mean +/- SE) to 0.29 +/- 0.04; p <0.0001 vs. 0.42 +/- 0.05 to 0.23 +/- 0.02; p < 0.0001, respectively). But significant difference between the 2 groups was not observed. No significant difference of sigma IRI/sigma BS among 4 groups of age was found. There were significant differences between I.I of the first year and that of several years, but they did not correlate linearly. These data indicate that the effect of age on the diminution of sigma IRI/sigma BS was significant for 10 years follow-up of the same subjects in both HT and NT groups. But there were no significant differences in classified groups by blood pressure and age.
Assuntos
Envelhecimento , Pressão Sanguínea , Intolerância à Glucose , Hipertensão/fisiopatologia , Insulina/sangue , Idoso , Humanos , Hipertensão/sangue , Pessoa de Meia-IdadeRESUMO
Ischemic acute renal failure is associated with vascular endothelial dysfunction. We examined whether vasodilatory antihypertensive agents would improve endothelial function in rats with ischemia/reperfusion renal injury. Rat kidneys were isolated and perfused after clipping of the bilateral renal arteries for 45 min and reperfusion for 24 h, and renal perfusion pressure and nitric oxide concentration in the venous effluent (chemiluminescence assay) were monitored. Preischemic administration of celiprolol (a beta-blocker; 100 mg/kg p.o.), benidipine (a calcium channel blocker; 1 mg/kg p.o.), or imidapril (an angiotensin converting-enzyme inhibitor; 3 mg/kg p.o.) restored endothelial function in rats subjected to acute renal ischemia (deltarenal perfusion pressure [10(-8) M acetylcholine]: sham -42+/-3%, ischemia -31+/-1%, ischemia +celiprolol -39+/-1%*, ischemia+benidipine -38+/-2%*, ischemia+imidapril -42+/-2%*; *p<0.05 vs. ischemia). Serum urea nitrogen and creatinine levels were also lower in the treated groups. Furthermore, ischemia-induced decreases in the response to acetylcholine and renal excretory function were smaller in SHR than in deoxycorticosterone-salt hypertensive rats, in which endothelial damage was marked. These results suggest that preischemic endothelial function may influence the degree of ischemic renal injury. Calcium channel blockers, converting-enzyme inhibitors, and endothelial NO synthase-activating beta-blockers had beneficial effects on renovascular endothelial dysfunction due to ischemia.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Celiprolol/farmacologia , Endotélio Vascular/metabolismo , Imidazolidinas , Traumatismo por Reperfusão/tratamento farmacológico , Vasodilatadores/farmacologia , Acetilcolina/farmacologia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Nitrogênio da Ureia Sanguínea , Peso Corporal , Bloqueadores dos Canais de Cálcio/farmacologia , Creatinina/sangue , Desoxicorticosterona , Di-Hidropiridinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Imidazóis/farmacologia , Masculino , Óxido Nítrico/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/metabolismoRESUMO
Acetylcholine (ACh)-induced vasodilation is mainly due to endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF). To explore the mechanisms underlying attenuated endothelium-dependent vasodilation in hypertensive arteries, we measured the EDNO released from isolated kidneys of spontaneously hypertensive rats (SHR) using a sensitive chemiluminescence assay system of NO. ACh-induced renal vasodilation was significantly smaller in SHR than in the normotensive control, Wistar-Kyoto rats (WKY). However, ACh-induced NO release did not differ between SHR and WKY (10(-7) M: SHR +37 +/- 2 [SE] vs. WKY +32 +/- 4 fmol/min/g kidney). Perfusion with a 20 mEq/L high-K+ buffer, which is reported to inhibit action of EDHF, significantly reduced ACh-induced vasorelaxation in WKY but not in SHR, resulting in identical renal perfusion pressure in SHR and wKY under these conditions. These results indicate that attenuated ACh-induced vasorelaxation in the SHR kidney may be attributed to a decrease in EDHF rather than that in EDNO.
Assuntos
Rim/irrigação sanguínea , Óxido Nítrico/fisiologia , Vasodilatação/fisiologia , Acetilcolina/administração & dosagem , Animais , Arginina/administração & dosagem , Arginina/análogos & derivados , Medições Luminescentes , Óxido Nítrico/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Vasodilatação/efeitos dos fármacos , ômega-N-MetilargininaRESUMO
BACKGROUND: Nitric oxide (NO) plays an important role in renal hemodynamics and function. Although production of NO in the glomeruli has been found to be increased in animal models of glomerulonephritis, it remains unclear whether its endogenous production is enhanced in patients with chronic glomerulonephritis (CGN). SUBJECTS AND METHODS: We measured NO output in exhaled air as an indicator of its local production in the lungs and plasma and urinary nitrite plus nitrate (NO2-/NO3-) levels as indicators of its production in the whole body in 21 patients with CGN in 31 healthy controls. RESULTS: The patients exhaled higher concentrations of NO (29.5 +/- 1.4 vs. 18.7 +/- 1.0 parts per billion (ppb), mean +/- SEM, p < 0.0001) and exhaled NO output was also higher than in controls (166.6 +/- 6.8 vs. 95.5 +/- 5.6 nl/min/m2, p < 0.0001). Plasma NO2-/NO3- concentrations were also significantly greater in the patients than in the controls (81.6 +/- 7.2 vs. 41.1 +/- 4.3 micromol/l, p < 0.001). In patients with CGN, exhaled NO output correlated negatively with creatinine clearance (r = -0.62, p < 0.05). Oral administration of prednisolone (60 mg/day) for two weeks did not significantly affect the exhaled NO output in the patients (160 +/- 7 vs. 200 +/- 30 nl/min/m2, p = NS) despite a decrease in urinary protein excretion (12.0 +/- 2.9 vs. 1.4 +/- 0.6 g/day, p < 0.01). CONCLUSION: These findings suggested that endogenous NO production is increased in patients with CGN. Increased endogenous NO production may play some pathophysiological role in these patients.
Assuntos
Sequestradores de Radicais Livres/análise , Glomerulonefrite/metabolismo , Óxido Nítrico/análise , Respiração , Administração Oral , Análise de Variância , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Estudos de Casos e Controles , Doença Crônica , Creatinina/sangue , Feminino , Sequestradores de Radicais Livres/metabolismo , Glomerulonefrite/sangue , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/urina , Humanos , Glomérulos Renais/metabolismo , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Nitratos/sangue , Nitratos/urina , Óxido Nítrico/biossíntese , Nitritos/sangue , Nitritos/urina , Prednisolona/administração & dosagem , Prednisolona/uso terapêutico , Proteinúria/tratamento farmacológico , Proteinúria/urina , EspirometriaRESUMO
The role of nitric oxide (NO) produced by NO synthase 1 (NOS1) in the renal vasculature remains undetermined. In the present study, we investigated the influence of systemic inhibition of NOS1 by intravenous administration of N(omega)-propyl-L-arginine (L-NPA; 1 mg. kg(-1). h(-1)) and N(5)-(1-imino-3-butenyl)-L-ornithine (v-NIO; 1 mg. kg(-1). h(-1)), highly selective NOS1 inhibitors, on renal cortical and medullary blood flow and interstitial NO concentration in Sprague-Dawley rats. Arterial blood pressure was significantly decreased by administration of both NOS1-selective inhibitors (-11 +/- 1 mmHg with L-NPA and -7 +/- 1 mmHg with v-NIO; n = 9/group). Laser-Doppler flowmetry experiments demonstrated that blood flow in the renal cortex and medulla was not significantly altered following administration of either NOS1-selective inhibitor. In contrast, the renal interstitial level of NO assessed by an in vivo microdialysis oxyhemoglobin-trapping technique was significantly decreased in both the renal cortex (by 36-42%) and medulla (by 32-40%) following administration of L-NPA (n = 8) or v-NIO (n = 8). Subsequent infusion of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg. kg(-1). h(-1)) to rats pretreated with either of the NOS1-selective inhibitors significantly increased mean arterial pressure by 38-45 mmHg and significantly decreased cortical (25-29%) and medullary (37-43%) blood flow. In addition, L-NAME further decreased NO in the renal cortex (73-77%) and medulla (62-71%). To determine if a 40% decrease in NO could alter renal blood flow, a lower dose of L-NAME (5 mg. kg(-1). h(-1); n = 8) was administered to a separate group of rats. The low dose of L-NAME reduced interstitial NO (cortex 39%, medulla 38%) and significantly decreased blood flow (cortex 23-24%, medulla 31-33%). These results suggest that NOS1 does not regulate basal blood flow in the renal cortex or medulla, despite the observation that a considerable portion of NO in the renal interstitial space appears to be produced by NOS1.
Assuntos
Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Circulação Renal/fisiologia , Anestesia , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Córtex Renal/irrigação sanguínea , Córtex Renal/enzimologia , Medula Renal/irrigação sanguínea , Medula Renal/enzimologia , Fluxometria por Laser-Doppler , Masculino , Microdiálise , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I , Nitroarginina/farmacologia , Ratos , Ratos Sprague-Dawley , Cloreto de Sódio/farmacologiaRESUMO
Vascular endothelial cells are unique in that they exit from the cell cycle when they come into contact with each other. Although the phenomenon is called "contact inhibition," little is known about the cellular mechanisms involved. Here we show that the phosphatase inhibitor sodium orthovanadate (SOV) induced the reentry of contact-inhibited human umbilical vascular endothelial cells (HUVECs) into the cell cycle and that reentry was associated with activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K)/Akt pathways. SOV stimulated [(3)H]thymidine uptake of contact-inhibited HUVECs in a time- and dose-dependent manner. SOV-induced increase in [(3)H]thymidine uptake was significantly inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 and by the PI 3-K inhibitor LY294002. SOV also stimulated the expression of cyclin D1, cyclin E, and cyclin A, and the activity of CDK2 kinase, whereas it decreased the expression of p27(kip1). In marked contrast, growth media alone did not induce these changes. Furthermore, these SOV-induced changes were abolished by pretreatment with PD98059 and LY294002. SOV stimulated phosphorylation of ERK and Akt in contact-inhibited HUVECs, while growth media alone did not. This phosphorylation was associated with inhibition of phosphatase activity in the cells. Finally, overexpression of high cell density-enhanced protein tyrosine phosphatase 1 inhibited c-fos and cyclin A promoter activity. Taken together, our results suggest that in contact-inhibited HUVECs, increased phosphatase activity suppressed the ERK and PI 3-K/Akt pathways, resulting in exit from the cell cycle by down-regulation of cyclin D1, cyclin E, and cyclin A and by up-regulation of p27(kip1).
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclo Celular , Endotélio Vascular/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Animais , Bovinos , Comunicação Celular , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Junções Intercelulares , Morfolinas/farmacologia , Transdução de SinaisRESUMO
Although it is well established that endothelin-1 (ET-1) has not only vasoconstrictive effects but also mitogenic effects, which seem to be implicated in vascular remodeling, little is known about the molecular mechanisms by which ET-1 induces cell-cycle progression. In this study, we examined the effects of ET-1 on the cell-cycle regulatory machinery, including cyclins, cyclin-dependent kinase (cdk), and cdk inhibitors in NIH3T3 cells. ET-1 increased cyclin D1 protein (5.1+/-1.9-fold increase, 8 hours after stimulation, P<0.05), cdk4 kinase activity (2.8+/-0. 5-fold increase, 12 hours after stimulation, P<0.01), and cdk2 kinase activity (2.1+/-0.4-fold increase, 16 hours after stimulation, P<0.05) in a time- and dose-dependent manner. ET-1-induced increase in cyclin D1 protein, and cdk4 kinase activity was not significantly inhibited by an inhibitor of the mitogen-activated protein kinase kinase 1/2, PD98059, nor by the protein kinase C inhibitor calphostin C, whereas ET-1-induced upregulation of cyclin D1 protein and cdk4 kinase activity was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. In contrast, ET-1-induced activation of cdk2 kinase was significantly inhibited by PD98059, calphostin C, and LY294002. ET-1 increased 3H-thymidine uptake in a time-dependent fashion (0 hours, 4216+/-264 cpm per well; 8 hours, 5025+/-197 cpm per well; 16 hours, 9239+/-79 cpm per well, P<0.001 versus 0 hours). ET-1-induced increase in 3H-thymidine uptake was significantly inhibited by PD98059, calphostin C, and LY294002. These results suggest that ET-1-induced cell-cycle progression is, at least in part, mediated by the extracellular signal-regulated kinase, protein kinase C, and phosphatidylinositol 3-kinase and that those pathways may be involved in the progression of the cell cycle at distinct points.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Ciclo Celular/fisiologia , Endotelina-1/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Fosfatidilinositol 3-Quinases/fisiologia , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas , Células 3T3 , Animais , Western Blotting , Ciclo Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Inibidores Enzimáticos/farmacologia , Genes ras , MAP Quinase Quinase 1 , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Fase S/efeitos dos fármacos , Fase S/fisiologia , Timidina/metabolismoRESUMO
OBJECTIVE: To determine if nitric oxide output in exhaled air is increased in patients with liver cirrhosis. DESIGN: Cross-sectional study. SETTING: A university hospital. PATIENTS: 50 patients with liver cirrhosis, 6 patients with chronic hepatitis, and 15 healthy controls. MEASUREMENTS: Nitric oxide in exhaled air was measured using a chemiluminescence analyzer. Cardiac index was determined using echocardiography. RESULTS: Patients with decompensated liver cirrhosis had higher levels of exhaled nitric oxide output (Child C patients, 190 +/- 11 nL/min per m2 body surface area) than controls (97 +/- 8 nL/min per m2 body surface area; P < 0.001), whereas patients with compensated liver cirrhosis or chronic hepatitis had levels of nitric oxide output similar to those found in controls. Cardiac index was greater in patients with liver cirrhosis (Child C patients, 4.3 +/- 0.3 L/min per m2 body surface area) than in controls (2.9 +/- 0.2 L/min per m2 body surface area; P < 0.001). Cardiac index correlated with nitric oxide levels (r = 0.621; P < 0.001). CONCLUSIONS: Increased nitric oxide output in exhaled air is associated with systemic circulatory disturbances in patients with liver cirrhosis.
Assuntos
Testes Respiratórios , Cirrose Hepática/metabolismo , Óxido Nítrico/metabolismo , Idoso , Débito Cardíaco , Doença Crônica , Estudos Transversais , Hemodinâmica , Hepatite Viral Humana/metabolismo , Humanos , Cirrose Hepática/fisiopatologia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/biossínteseRESUMO
Nitric oxide exerts multiple effects on renal function. It remains unclear whether endogenous nitric oxide production is increased or decreased in patients with chronic renal failure. To evaluate endogenous nitric oxide production in these patients we studied exhaled nitric oxide output by an ozone chemiluminescence method and plasma NO2(-)/NO3(-) levels by the Griess method in 40 patients with end-stage chronic renal failure who underwent regular continuous ambulatory peritoneal dialysis (n=30) or haemodialysis (n=10), and in 28 healthy subjects. Patients with chronic renal failure had a higher exhaled nitric oxide concentration [39+/-3 versus 19+/-1 parts per billion, (mean+/-S.E.M.), P<0.0001], a greater nitric oxide output (177+/-11 versus 96+/-7 nl.min-1.m-2, P<0.001) and a higher plasma NO2(-)/NO3(-) concentration (96+/-14 versus 33+/-4 micromol, P<0.01) than controls. These values did not differ between patients on haemodialysis and those on continuous ambulatory peritoneal dialysis. Patients with chronic renal failure had significantly higher plasma concentrations of both interleukin-1beta and interferon-gamma than controls. The exhaled nitric oxide output did not correlate with plasma NO2(-)/NO3(-) or with peritoneal dialysate NO2(-)/NO3(-), but plasma NO2(-)/NO3(-) correlated with dialysate NO2(-)/NO3(-) in patients who underwent continuous ambulatory peritoneal dialysis (r=0.77, P<0.01). Haemodialysis for 4 h acutely decreased plasma NO2(-)/NO3(-) (92+/-17 versus 50+/-8 micromol, P<0.05) and cGMP concentration (16.5+/-4.3 versus 5.1+/-1. 7 pmol/ml, P<0.01), but did not decrease exhaled nitric oxide output. The increase in exhaled nitric oxide with the simultaneous increase in circulating cytokines suggests that nitric oxide synthase seems to be induced significantly in patients with chronic renal failure. Increased endogenous nitric oxide production may have a pathophysiological role in patients with uraemia.
Assuntos
Falência Renal Crônica/metabolismo , Óxido Nítrico/metabolismo , Análise de Variância , Fator Natriurético Atrial/sangue , Testes Respiratórios , Distribuição de Qui-Quadrado , GMP Cíclico/sangue , Feminino , Humanos , Interferon gama/sangue , Interleucina-1/sangue , Medições Luminescentes , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/sangue , Nitratos/sangue , Óxido Nítrico/análise , Óxido Nítrico/sangue , Nitritos/sangue , Diálise Peritoneal Ambulatorial Contínua , Diálise Renal , Fatores de TempoRESUMO
BACKGROUND: Adrenomedullin (AM) is a newly discovered peptide that has a potent vasorelaxant activity. To investigate its potential roles in hypoxia-induced renal injury, we examined whether AM production in the kidney increased under hypoxic conditions. METHODS: The AM transcript levels in Madin-Darby canine kidney (MDCK) cells, rat vascular smooth muscle cells (VSMCs), and rat mesangial cells were assessed by Northern blot analyses under normoxic and hypoxic conditions. The AM peptide in culture media was measured by radioimmunoassay. The effects of hypoxia on accumulation of cAMP in VSMCs were also examined. The stability of AM transcripts under normoxic and hypoxic conditions was compared in the presence of actinomycin D. The effects of hypoxia on AM promoter activity was assessed by transient transfection assays using the AM promoter subcloned upstream of luciferase gene. RESULTS: The expression of AM transcripts increased significantly in MDCK cells, rat VSMCs, and rat mesangial cells under hypoxic conditions without changes in the stability of AM transcripts; however, the AM promoter activity under hypoxic was not elevated significantly. The accumulation of AM peptide in culture media also increased significantly under hypoxic conditions in MDCK cells (2.2 +/- 0.1 fmol/10(5) cells in normoxia vs. 3.5 +/- 0.3 fmol/10(5) cells in hypoxia, 6 hr after hypoxia induction, P < 0.001), and in rat VSMCs (5.5 +/- 0.3 fmol/10(5) cells in normoxia vs. 7.8 +/- 0.4 fmol/10(5) cells in hypoxia, 8 hr after hypoxia induction, P < 0.01). Under hypoxic conditions, cAMP levels in rat VSMCs increased significantly compared with those under normoxic conditions (13.3 +/- 1.4 pmol/well vs. 4.6 +/- 0.4 pmol/well, P < 0.01). CONCLUSIONS: Renal parenchymal cells as well as renal vessels may produce AM under hypoxic conditions.