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1.
Am J Nephrol ; 51(9): 695-704, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866949

RESUMO

BACKGROUND: Apolipoprotein L1 gene (APOL1) G1 and G2 kidney-risk variants (KRVs) cause CKD in African Americans, inducing mitochondrial dysfunction. Modifying factors are required, because a minority of individuals with APOL1 high-risk genotypes develop nephropathy. Given that APOL1 function is pH-sensitive and the pH of the kidney interstitium is <7, we hypothesized the acidic kidney interstitium may facilitate APOL1 KRV-induced mitochondrial dysfunction. METHODS: Human embryonic kidney (HEK293) cells conditionally expressing empty vector (EV), APOL1-reference G0, and G1 or G2 KRVs were incubated in media pH 6.8 or 7.4 for 4, 6, or 8 h. Genotype-specific pH effects on mitochondrial length (µm) were assessed using confocal microscopy in live cells and Fiji derivative of ImageJ software with MiNA plug-in. Lower mitochondrial length indicated fragmentation and early dysfunction. RESULTS: After 6 h doxycycline (Dox) induction in pH 6.8 media, G2-expressing cells had shorter mitochondria (6.54 ± 0.40) than cells expressing EV (7.65 ± 0.72, p = 0.02) or G0 (7.46 ± 0.31, p = 0.003). After 8 h Dox induction in pH 6.8 media, both G1- (6.21 ± 0.26) and G2-expressing cells had shorter mitochondria (6.46 ± 0.34) than cells expressing EV (7.13 ± 0.32, p = 0.002 and p = 0.008, respectively) or G0 (7.22 ± 0.45, p = 0.003 and p = 0.01, respectively). Mitochondrial length in cells incubated in pH 7.4 media were comparable after 8 h Dox induction regardless of genotype. APOL1 mRNA expression and cell viability were comparable regardless of pH or genotype after 8 h Dox induction. CONCLUSION: Acidic pH facilitates early mitochondrial dysfunction induced by APOL1 G1 and G2 KRVs in HEK293 cells. We propose that the acidic kidney interstitium may play a role in APOL1-mediated mitochondrial pathophysiology and nephropathy.


Assuntos
Apolipoproteína L1/metabolismo , Predisposição Genética para Doença , Rim/patologia , Mitocôndrias/patologia , Insuficiência Renal Crônica/genética , Negro ou Afro-Americano/genética , Apolipoproteína L1/genética , Meios de Cultura/química , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Rim/química , Rim/citologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Insuficiência Renal Crônica/patologia
2.
Nephrol Dial Transplant ; 33(11): 1960-1967, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29420808

RESUMO

Background: Viral infections can trigger chronic kidney disease (CKD) and the urine virome may inform risk. The Natural History of APOL1-Associated Nephropathy Study (NHAANS) reported that urine JC polyomavirus (JCPyV) associated with a lower risk of APOL1-associated nephropathy in African Americans. Herein, association was assessed between urine JCPyV with CKD in African Americans independent from the APOL1 genotype. Methods: Quantitative polymerase chain reaction was performed for urinary detection of JCPyV and BK polyoma virus (BKPyV) in 200 newly recruited nondiabetic African Americans. A combined analysis was performed in these individuals plus 300 NHAANS participants. Results: In the 200 new participants, urine JCPyV was present in 8.8% of CKD cases and 45.8% of nonnephropathy controls (P = 3.0 × 10-8). In those with APOL1 renal-risk genotypes, JCPyV was detected in 5.1% of cases and 40.0% of controls (P = 0.0002). In those lacking APOL1 renal-risk genotypes, JCPyV was detected in 12.2% of cases and 48.8% of controls (P = 8.5 × 10-5). BKPyV was detected in 1.3% of cases and 0.8% of controls (P = 0.77). In a combined analysis with 300 NHAANS participants (n = 500), individuals with urine JCPyV had a 63% lower risk of CKD compared with those without urine JCPyV (odds ratio 0.37; P = 4.6 × 10-6). RNA fluorescence in situ hybridization confirmed the presence of JCPyV genomic DNA and JCPyV messenger RNA (mRNA) in nondiseased kidney. Conclusions: Inverse relationships exist between JCPyV viruria and non-diabetic CKD. Future studies should determine whether renal inflammation associated with CKD is less permissive for JCPyV reactivation/replication or whether JCPyV is a marker of reduced host immune responsiveness that diminishes immune pathologic contributions to CKD.


Assuntos
Apolipoproteína L1/genética , Negro ou Afro-Americano/genética , Infecções por Polyomavirus/virologia , Insuficiência Renal Crônica/prevenção & controle , Infecções Tumorais por Vírus/virologia , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Vírus JC/genética , Vírus JC/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Infecções por Polyomavirus/etnologia , Infecções por Polyomavirus/urina , Insuficiência Renal Crônica/etnologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/virologia , Infecções Tumorais por Vírus/etnologia
3.
J Am Soc Nephrol ; 28(4): 1093-1105, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27821631

RESUMO

APOL1 G1 and G2 variants facilitate kidney disease in blacks. To elucidate the pathways whereby these variants contribute to disease pathogenesis, we established HEK293 cell lines stably expressing doxycycline-inducible (Tet-on) reference APOL1 G0 or the G1 and G2 renal-risk variants, and used Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics analyses involved mitochondrial function; results from immunoblotting, immunofluorescence, and functional assays validated these findings. Overexpression of APOL1 by doxycycline induction in HEK293 Tet-on G1 and G2 cells led to impaired mitochondrial function, with markedly reduced maximum respiration rate, reserve respiration capacity, and mitochondrial membrane potential. Impaired mitochondrial function occurred before intracellular potassium depletion or reduced cell viability occurred. Analysis of global gene expression profiles in nondiseased primary proximal tubule cells from black patients revealed that the nicotinate phosphoribosyltransferase gene, responsible for NAD biosynthesis, was among the top downregulated transcripts in cells with two APOL1 renal-risk variants compared with those without renal-risk variants; nicotinate phosphoribosyltransferase also displayed gene expression patterns linked to mitochondrial dysfunction in HEK293 Tet-on APOL1 cell pathway analyses. These results suggest a pivotal role for mitochondrial dysfunction in APOL1-associated kidney disease.


Assuntos
Apolipoproteínas/genética , Nefropatias/genética , Lipoproteínas HDL/genética , Doenças Mitocondriais/genética , Apolipoproteína L1 , População Negra , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco
4.
J Lipid Res ; 57(1): 120-30, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26586272

RESUMO

APOL1 gene renal-risk variants are associated with nephropathy and CVD in African Americans; however, little is known about the circulating APOL1 variant proteins which reportedly bind to HDL. We examined whether APOL1 G1 and G2 renal-risk variant serum concentrations or lipoprotein distributions differed from nonrisk G0 APOL1 in African Americans without nephropathy. Serum APOL1 protein concentrations were similar regardless of APOL1 genotype. In addition, serum APOL1 protein was bound to protein complexes in two nonoverlapping peaks, herein referred to as APOL1 complex A (12.2 nm diameter) and complex B (20.0 nm diameter). Neither of these protein complexes associated with HDL or LDL. Proteomic analysis revealed that complex A was composed of APOA1, haptoglobin-related protein (HPR), and complement C3, whereas complex B contained APOA1, HPR, IgM, and fibronectin. Serum HPR was less abundant on complex B in individuals with G1 and G2 renal-risk variant genotypes, relative to G0 (P = 0.0002-0.037). These circulating complexes may play roles in HDL metabolism and susceptibility to CVD.


Assuntos
Apolipoproteínas/sangue , Negro ou Afro-Americano , Lipoproteínas HDL/sangue , Adulto , Apolipoproteína L1 , Apolipoproteínas/genética , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Humanos , Nefropatias/sangue , Nefropatias/genética , Lipoproteínas HDL/genética , Masculino , Pessoa de Meia-Idade , Proteômica , Fatores de Risco
5.
J Am Soc Nephrol ; 26(2): 339-48, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25012173

RESUMO

Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.


Assuntos
Apolipoproteínas/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Lipoproteínas HDL/metabolismo , Células Mesangiais/metabolismo , RNA Mensageiro/metabolismo , Apolipoproteína L1 , Biópsia , Linhagem Celular , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Técnicas In Vitro , Rim/patologia , Rim/cirurgia , Glomérulos Renais/patologia , Túbulos Renais Proximais/patologia , Células Mesangiais/patologia , Microscopia de Fluorescência , Nefrectomia , Podócitos/metabolismo , Podócitos/patologia
6.
Arterioscler Thromb Vasc Biol ; 33(4): 752-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23329133

RESUMO

OBJECTIVE: Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. APPROACH AND RESULTS: Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. CONCLUSIONS: Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.


Assuntos
Artérias Cerebrais/metabolismo , Circulação Cerebrovascular , Células Endoteliais/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Canais de Potássio/metabolismo , Vasodilatação , Animais , Benzopiranos/farmacologia , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Artérias Cerebrais/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Imidazóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/agonistas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
7.
JCI Insight ; 8(10)2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-37129980

RESUMO

Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-ß (Aß) release, offering a mechanistic link between type 2 diabetes and Alzheimer's disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits Kir6.2/KCNJ11 and SUR1/ABCC8 were expressed on excitatory and inhibitory neurons in the human brain, and cortical expression of KCNJ11 and ABCC8 changed with AD pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aß pathology in a potentially novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (i.e., amyloid precursor protein [APP]/PS1 Kir6.2-/- mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemia-dependent increases in interstitial fluid levels of Aß, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a potentially new role for Kir6.2-KATP channels in AD and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aß pathology in patients with diabetes or prediabetes.


Assuntos
Doença de Alzheimer , Diabetes Mellitus Tipo 2 , Hiperglicemia , Humanos , Camundongos , Animais , Canais KATP/metabolismo , Doença de Alzheimer/patologia , Diabetes Mellitus Tipo 2/complicações , Glucose , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo
8.
Am J Physiol Heart Circ Physiol ; 300(6): H2080-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21421821

RESUMO

Insulin resistance (IR) impairs cerebrovascular responses to several stimuli in Zucker obese (ZO) rats. However, cerebral artery responses after subarachnoid hemorrhage (SAH) have not been described in IR. We hypothesized that IR worsens vascular reactions after a mild SAH. Hemolyzed blood (300 µl) or saline was infused (10 µl/min) into the cisterna magna of 11-13-wk-old ZO (n = 25) and Zucker lean (ZL) rats (n = 25). One day later, dilator responses of the basilar artery (BA) and its side branch (BA-Br) to acetylcholine (ACh, 10(-6) M), cromakalim (10(-7) M, 10(-6) M), and sodium nitroprusside (10(-7) M) were recorded with intravital videomicroscopy. The baseline diameter of the BA was increased both in the ZO and ZL rats 24 h after the hemolysate injection. Saline-injected ZO animals showed reduced dilation to ACh (BA = 9 ± 3 vs. 22 ± 4%; and BA-Br = 23 ± 5 vs. 37 ± 7%) compared with ZL rats. Hemolysate injection blunted the response to ACh in both the ZO (BA = 4 ± 2%; and BA-Br = 12 ± 3%) and ZL (BA = 7 ± 2%; and BA-Br = 11 ± 3%) rats. Cromakalim (10(-6) M)-induced dilation was significantly reduced in the hemolysate-injected ZO animals compared with the saline control (BA = 13 ± 3 vs. 26 ± 5%; and BA-Br = 28 ± 8 vs. 44 ± 9%) and in the hemolysate-injected ZL rats compared with their saline control (BA = 24 ± 4 vs. 32 ± 4%; but not BA-Br = 39 ± 6 vs. 59 ± 9%). No significant difference in sodium nitroprusside reactivity was observed. Western blot analysis of the BA showed a lower baseline level of neuronal nitric oxide synthase expression and an enhanced cyclooxygenase-2 level in the hemolysate-injected ZO animals. In summary, cerebrovascular reactivity to both endothelium-dependent and -independent stimuli is severely compromised by SAH in IR animals.


Assuntos
Circulação Cerebrovascular/fisiologia , Resistência à Insulina/fisiologia , Obesidade/fisiopatologia , Hemorragia Subaracnóidea/fisiopatologia , Vasoespasmo Intracraniano/fisiopatologia , Acetilcolina/farmacologia , Animais , Circulação Cerebrovascular/efeitos dos fármacos , Cromakalim/farmacologia , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Nitroprussiato/farmacologia , Obesidade/metabolismo , Ratos , Ratos Zucker , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologia
9.
Kidney360 ; 1(12): 1353-1362, 2020 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-35372896

RESUMO

Background: Kidney risk variants (KRVs) in the APOL1 gene are associated with mitochondrial dysfunction. However, the molecular spectrum of metabolites affected by the G1 and G2 KRVs, and the downstream mitochondrial pathways they affect, remain unknown. Methods: We performed a metabolomics analysis using HEK293 Tet-on cells conditionally expressing APOL1 G0, G1, and G2 KRVs to determine the patterns of metabolites and pathways potentially involved in nephropathy. The Welch two-sample t test, matched-pairs t test, and two-way repeated measures ANOVA were used to identify differential metabolites. Random forest, a supervised classification algorithm that uses an ensemble of decision trees, and the mean-decrease-accuracy metric were applied to prioritize top metabolites. Results: Alterations in the tricarboxylic acid cycle, increased fatty acid oxidation, and compromised redox homeostasis were the major pathways affected by overexpression of APOL1 KRVs. Conclusions: Impairment of mitochondrial membrane respiratory chain complex I appeared to account for critical metabolic consequences of APOL1 KRVs. This finding supports depletion of the mitochondrial membrane potential, as has been reported.


Assuntos
Apolipoproteína L1 , Predisposição Genética para Doença , Apolipoproteína L1/genética , Células HEK293 , Humanos , Metabolômica , Mitocôndrias/genética
10.
Kidney Int Rep ; 5(6): 891-904, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32518871

RESUMO

INTRODUCTION: APOL1 G1 and G2 nephropathy-risk variants cause mitochondrial dysfunction and contribute to kidney disease. Analyses were performed to determine the genetic regulation of APOL1 and elucidate potential mechanisms in APOL1-nephropathy. METHODS: A global gene expression analysis was performed in human primary renal tubule cell lines derived from 50 African American individuals. Follow-up gene knock out, cell-based rescue, and microscopy experiments were performed. RESULTS: APOL1 genotypes did not alter APOL1 expression levels in the global gene expression analysis. Expression quantitative trait locus (eQTL) analysis in polyinosinic-polycytidylic acid (poly IC)-stimulated renal tubule cells revealed that single nucleotide polymorphism (SNP) rs513349 adjacent to BAK1 was a trans eQTL for APOL1 and a cis eQTL for BAK1; APOL1 and BAK1 were co-expressed in cells. BAK1 knockout in a human podocyte cell line resulted in diminished APOL1 protein, supporting a pivotal effect for BAK1 on APOL1 expression. Because BAK1 is involved in mitochondrial dynamics, mitochondrial morphology was examined in primary renal tubule cells and HEK293 Tet-on cells of various APOL1 genotypes. Mitochondria in APOL1 wild-type (G0G0) tubule cells maintained elongated morphology when stimulated by low-dose poly IC, whereas those with G1G1, G2G2, and G1G2 genotypes appeared to fragment. HEK293 Tet-on cells overexpressing APOL1 G0, G1, and G2 were created; G0 cells appeared to promote mitochondrial fusion, whereas G1 and G2 induced mitochondrial fission. The mitochondrial dynamic regulator Mdivi-1 significantly preserved cell viability and mitochondrial cristae structure and reversed mitochondrial fission induced by overexpression of G1 and G2. CONCLUSION: Results suggest the mitochondrial fusion/fission pathway may be a therapeutic target in APOL1-nephropathy.

11.
Microcirculation ; 16(7): 629-39, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19657965

RESUMO

OBJECTIVE: N-methyl-d-aspartate (NMDA) is a powerful cerebrovascular dilator in vivo. Cortical spreading depression (CSD) has recently been shown to contribute to the pial arteriolar dilation in mice. Our main aim was to examine the participation of CSD in the overall cerebrovascular response to NMDA in the rat. METHODS: Anesthetized Wistar rats (eight weeks old) were equipped with a closed cranial window to allow topical application of NMDA (10(-5)-10(-3) M) to the parietal cortex. Cortical blood flow (CoBF) under and outside the cranial window was simultaneously monitored by using a two-channel laser-Doppler flowmeter. CSDs were detected by recording the changes in the cortical DC potential. RESULTS: Concentrations of 10(-4) and 10(-3) M of NMDA evoked single CSDs associated with rapid, transient hyperemia, followed by a sustained, but reduced, increase in CoBF. The latency and magnitude of the CoBF responses were dose dependent. The higher dose resulted in shorter latency (100+/-5* vs. 146+/-11 seconds, *P<0.05; mean+/-standard error of the mean) and larger overall flow response (77+/-12* vs. 28+/-3% from baseline) under, but not outside, the cranial window. CONCLUSIONS: NMDA elicits dose-dependent increases in CoBF that are composed of CSD-dependent and -independent components in rats.


Assuntos
Córtex Cerebral/irrigação sanguínea , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Hiperemia/induzido quimicamente , N-Metilaspartato/farmacologia , Animais , Circulação Cerebrovascular , Relação Dose-Resposta a Droga , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional
12.
J Cereb Blood Flow Metab ; 28(6): 1090-103, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18212794

RESUMO

Previously, we have shown that the selective mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener BMS-191095 (BMS) induces neuronal preconditioning (PC); however, the exact mechanism of BMS-induced neuroprotection remains unclear. In this study, we have identified key components of the cascade resulting in delayed neuronal PC with BMS using isolated rat brain mitochondria and primary cultures of rat cortical neurons. BMS depolarized isolated mitochondria without an increase in reactive oxygen species (ROS) generation and induced rapid phosphorylation of Akt and glycogen synthase kinase-3beta. Long-term (3 days) treatment of neurons with BMS resulted in sustained mitochondrial depolarization, decreased basal ROS generation, and elevated ATP levels. This treatment also elicited almost complete protection against glutamate excitotoxicity, which could be abolished using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, but not with the superoxide dismutase (SOD) mimetic M40401. Long-term BMS treatment induced a PI3K-dependent increase in the expression and activity of catalase without affecting manganese SOD and copper/zinc-dependent SOD. Finally, the catalase inhibitor 3-aminotriazole dose-dependently antagonized the neuroprotective effect of BMS-induced PC. In summary, BMS depolarizes mitochondria without ROS generation, activates the PI3K-Akt pathway, improves ATP content, and increases catalase expression. These mechanisms appear to play important roles in the neuroprotective effect of BMS.


Assuntos
Benzopiranos/farmacologia , Imidazóis/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Canais de Potássio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Citosol/efeitos dos fármacos , Citosol/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Homeostase/efeitos dos fármacos , Indóis/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Técnicas de Cultura de Tecidos
13.
J Neurochem ; 105(4): 1115-28, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18182041

RESUMO

1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619), a potent activator of the large conductance Ca2+ activated potassium (BK(Ca)) channel, has been demonstrated to induce preconditioning (PC) in the heart. The aim of our study was to test the delayed PC effect of NS1619 in rat cortical neuronal cultures against oxygen-glucose deprivation, H2O2, or glutamate excitotoxicity. We also investigated its actions on reactive oxygen species (ROS) generation, and on mitochondrial and plasma membrane potentials. Furthermore, we tested the activation of the phosphoinositide 3-kinase (PI3K) signaling pathway, and the effect of NS1619 on caspase-3/7. NS1619 dose-dependently protected the cells against the toxic insults, and the protection was completely blocked by a superoxide dismutase mimetic and a PI3K antagonist, but not by BK(Ca) channel inhibitors. Application of NS1619 increased ROS generation, depolarized isolated mitochondria, hyperpolarized the neuronal cell membrane, and activated the PI3K signaling cascade. However, only the effect on the cell membrane potential was antagonized by BK(Ca) channel blockers. NS1619 inhibited the activation of capase-3/7. In summary, NS1619 is a potent inducer of delayed neuronal PC. However, the neuroprotective effect seems to be independent of cell membrane and mitochondrial BK(Ca) channels. Rather it is the consequence of ROS generation, activation of the PI3K pathway, and inhibition of caspase activation.


Assuntos
Benzimidazóis/farmacologia , Precondicionamento Isquêmico/métodos , Neurônios/fisiologia , Canais de Potássio Cálcio-Ativados/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Feminino , Masculino , Neurônios/efeitos dos fármacos , Gravidez , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
14.
J Cereb Blood Flow Metab ; 27(7): 1318-26, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17213862

RESUMO

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays a significant role in postischemic brain injury. We assessed the contribution of PARP activation to the blood-brain barrier (BBB) disruption and edema formation after ischemia-reperfusion. In male Wistar rats, global cerebral ischemia was achieved by occluding the carotid arteries and lowering arterial blood pressure for 20 mins. The animals were treated with saline or with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N, N-dimethylacetamide.HCl (PJ34); (10 mg/kg, i.v.) before ischemia. After 40 mins, 24, and 48 h of reperfusion, the permeability of the cortical BBB was determined after Evans Blue (EB) and Na-fluorescein (NaF) administration. The water content of the brain was also measured. The permeability of the BBB for EB increased after ischemia-reperfusion compared with the nonischemic animals after 24 and 48 h reperfusion but PARP inhibition attenuated this increase at 48 h (nonischemic: 170+/-9, saline: 760+/-95, PJ34: 472+/-61 ng/mg tissue). The extravasation of NaF showed similar changes and PJ34 post-treatment attenuated the permeability increase even at 24 h. PARP inhibition decreased the brain edema seen at 48 h. Because PARP has proinflammatory properties, the neutrophil infiltration of the cortex was determined, which showed lower values after PJ34 treatment. Furthermore, PJ34 treatment decreased the loss of the tight junction protein occludin at 24 and 48 h. The inhibition of PARP activity accompanied by reduced post-ischemic BBB disturbance and decreased edema formation suggests a significant role of this enzyme in the development of cerebral vascular malfunction


Assuntos
Barreira Hematoencefálica/patologia , Ativação Enzimática , Hipóxia-Isquemia Encefálica/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Traumatismo por Reperfusão/enzimologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiopatologia , Western Blotting , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/enzimologia , Edema Encefálico/etiologia , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Hipóxia-Isquemia Encefálica/complicações , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Ocludina , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Ratos , Ratos Wistar , Traumatismo por Reperfusão/complicações , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Life Sci ; 80(7): 633-7, 2007 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-17113604

RESUMO

Previous studies raised the possibility that nitric oxide synthase is present in heart mitochondria (mtNOS) and the existence of such an enzyme became generally accepted. However, original experimental evidence is rather scarce and positive identification of the enzyme is lacking. We aimed to detect an NOS protein in human and mouse heart mitochondria and to measure the level of NO released from the organelles. Western blotting with 7 different anti-NOS antibodies failed to detect a NOS-like protein in mitochondria. Immunoprecipitation or substrate-affinity purification of the samples concentrated NOS in control preparations but not in mitochondria. Release of NO from live respiring human mitochondria was below 2 ppb after 45 min of incubation. In a bioassay system, mitochondrial suspension failed to cause vasodilation of human mammary artery segments. These results indicate that mitochondria do not produce physiologically relevant quantities of NO in the heart and are unlikely to have any physiological importance as NO donors, nor do they contain a recognizable mtNOS enzyme.


Assuntos
Mitocôndrias Cardíacas/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Animais , Western Blotting , Humanos , Camundongos , Óxido Nítrico/análise , Óxido Nítrico Sintase/análise
16.
J Appl Physiol (1985) ; 100(4): 1392-9, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16322371

RESUMO

Prostaglandins produced in cerebral endothelial cells (CECs) are the final signal transduction mediators from the periphery to the brain during fever response. However, prostaglandins are organic anions at physiological pH, and they enter cells poorly using simple diffusion. Several transporters have been described that specifically transport prostaglandins across cell membranes. We examined the expression of the two principal prostaglandin carriers, prostaglandin transporter (PGT), and multidrug resistance-associated protein 4 (MRP4) in cells of the blood-brain barrier and in choroid epithelial cells in vitro as well as in vivo in rat brain in control conditions and after lipopolysaccharide (LPS) challenge. We detected PGT in primary cultures of rat CECs, astrocytes, pericytes, and choroid epithelial cells. LPS stimulation had no effect on the expression level of PGT in these cells; however, after LPS stimulation the polarized, dominantly luminal, expression pattern of PGT significantly changed. MRP4 is also expressed in CECs, and its level was not influenced by LPS treatment. In rat brain, PGT was highly expressed in the supraoptic and paraventricular nuclei of the hypothalamus, in the ependymal cell layer of the third ventricle, and in the choroid plexus. LPS treatment increased the expression of PGT in the supraoptic and paraventricular nuclei. Our results suggest that PGT and MRP4 likely play a role in transporting prostaglandins through the blood-brain and blood-cerebrospinal fluid barriers and may be involved in the maintenance of prostaglandin homeostasis in the brain and in the initiation of fever response.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Animais , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Células Cultivadas , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Ânions Orgânicos/genética , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Prostaglandinas/metabolismo , RNA Mensageiro , Ratos , Ratos Wistar , Núcleo Supraóptico/efeitos dos fármacos , Núcleo Supraóptico/metabolismo , Fatores de Tempo
17.
Diabetes ; 53(5): 1352-9, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15111506

RESUMO

Insulin resistance (IR) impairs vascular function in the peripheral and coronary circulations, but its effects on cerebral arteries are virtually unexplored. We examined the vascular responses of the basilar artery (BA) and its side branches through a cranial window in Zucker lean (ZL) and IR Zucker obese (ZO) rats. Nitric oxide (NO) and K+ channel-mediated dilator responses, elicited by acetylcholine, iloprost, cromakalim, and elevated [K+], were greatly diminished in the ZO rats compared with ZL rats. In contrast, sodium nitroprusside induced similar relaxations in the two experimental groups. Expressions of the K+ channel pore-forming subunits were not affected by IR, while endothelial NO synthase was upregulated in the ZO arteries compared with ZL arteries. Protein kinase C (PKC) activity and production of superoxide anion were increased in the cerebral arteries of ZO rats, and pretreatment with superoxide dismutase restored all examined dilator responses. In contrast, application of PKC inhibitors improved only receptor-linked NO-mediated relaxation, but not K+ channel-dependent responses. Thus, IR induces in ZO rats cerebrovascular dysfunction, which is mediated by oxidative stress and partly by PKC activation. The revealed impairment of NO and K+ channel-dependent dilator responses may be responsible for the increased risk of cerebrovascular events and neurodegenerative disorders in IR.


Assuntos
Circulação Cerebrovascular , Obesidade/fisiopatologia , Estresse Oxidativo , Proteína Quinase C/metabolismo , Animais , Ativação Enzimática , Resistência à Insulina , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Obesidade/metabolismo , Canais de Potássio/metabolismo , Isoformas de Proteínas/metabolismo , Ratos , Ratos Zucker , Espécies Reativas de Oxigênio/metabolismo , Vasodilatação
18.
Brain Res ; 1039(1-2): 84-9, 2005 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-15781049

RESUMO

Cortical spreading depression (CSD) has been documented to confer ischemic tolerance on brain. Although nitric oxide (NO) is a crucial mediator in preconditioning under certain circumstances, the role of NO in CSD-induced neuroprotection is unclear. We examined the effect of L-NAME, an inhibitor of NO synthase, on CSD-induced tolerance against transient focal cerebral ischemia. A solution of 0.5 M KCl was applied for 2 h on the right hemisphere to induce CSD. Animals received either vehicle or L-NAME (4 mg/kg, iv) 30 min before CSD. Temporary occlusion (120 min) of the right middle cerebral artery was induced 4 days after preconditioning and the infarct volume was measured. Additionally, ERK 1/2 activation and cyclooxygenase-2 (COX-2) expression in the cerebral cortex were examined by Western blotting analysis immediately after cessation of CSD, or at 1, 2, 4, 8, and 24 h after CSD. CSD reduced infarct volume from 275 +/- 15 mm3 (mean +/- SEM) in the non-CSD group to 155 +/- 14 mm3 in the CSD group (P < 0.05). L-NAME abolished this protection (281 +/- 14 mm3; P < 0.05 vs. CSD group). Elevated ERK activation and COX-2 expression were observed immediately after or 8 h after preconditioning, respectively. Those responses are significantly augmented by L-NAME (3-fold for ERK and 4-fold for COX-2). These results suggest a crucial role of NO in the establishment of preconditioning with CSD.


Assuntos
Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Ataque Isquêmico Transitório/enzimologia , Precondicionamento Isquêmico , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/fisiologia , Animais , Encéfalo/irrigação sanguínea , Ciclo-Oxigenase 2 , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Infarto da Artéria Cerebral Média/enzimologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Degeneração Neural/enzimologia , Degeneração Neural/prevenção & controle , Óxido Nítrico Sintase/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Wistar
19.
Life Sci ; 77(18): 2262-72, 2005 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-15958270

RESUMO

Insulin resistance (IR) impairs endothelium-mediated vasodilation in cerebral arteries as well as K+ channel function in vascular smooth muscle. Peripheral arteries also show an impaired endothelium-dependent vasodilation in IR and concomitantly show an enhanced contractile response to endothelin-1 (ET-1). However, the contractile responses of the cerebral arteries in IR have not been examined systematically. This study examined the contractile responses of pressurized isolated middle cerebral arteries (MCAs) in fructose-fed IR and control rats. IR MCAs showed no difference in pressure-mediated (80 mmHg) vasoconstriction compared to controls, either in time to develop spontaneous tone (control: 61+/-3 min, n=30; IR: 63+/-2 min, n=26) or in the degree of that tone (control: 60 min: 33+/-2%, n=22 vs. IR 60 min: 34+/-3%, n=17). MCAs treated with ET-1 (10(-8.5) M) constrict similarly in control (53+/-3%, n=14) and IR (53+/-3%, n=14) arteries. Constrictor responses to U46619 (10(-6) M) are also similar in control (48+/-9%, n=8) and IR (42+/-5%, n=6) MCAs as are responses to extraluminal uridine 5'-triphosphate (UTP; 10(-4.5) M) (control: 35+/-7%, n=11 vs. IR: 38+/-3%, n=10). These findings demonstrate that constrictor responses remain intact in IR despite a selective impairment of dilator responses and endothelial and vascular smooth muscle K+ channel function in cerebral arteries. Thus, it appears that the increased susceptibility to cerebrovascular abnormalities associated with IR and diabetes (including cerebral ischemia, stroke, vertebrobasilar transient ischemic attacks) is not due to an enhanced vasoreactivity to constrictor agents.


Assuntos
Artérias Cerebrais/fisiologia , Resistência à Insulina/fisiologia , Vasoconstrição/fisiologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Pressão Sanguínea , Western Blotting , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Endotelina/metabolismo , Fatores de Tempo , Uridina Trifosfato/farmacologia , Vasoconstrição/efeitos dos fármacos
20.
Stroke ; 35(4): 964-9, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14976323

RESUMO

BACKGROUND AND PURPOSE: Insulin resistance (IR) increases the risk of stroke in humans. One possible underlying factor is cerebrovascular dysfunction resulting from altered K(+) channel function. Thus, the goal of this study was to examine K+ channel-mediated relaxation in IR cerebral arteries. METHODS: Experiments were performed on pressurized isolated middle cerebral arteries (MCAs) from fructose-fed IR and control rats. RESULTS: Dilator responses to iloprost, which are BK(Ca) channel mediated, were reduced in the IR compared with control arteries (19+/-2% versus 33+/-2% at 10(-6) mol/L). Similarly, relaxation to the K(ATP) opener pinacidil was diminished in the IR MCAs (17+/-2%) compared with controls (38+/-2% at 10(-5) mol/L). IR also reduced the K(ATP) channel-dependent component in calcitonin gene-related peptide-induced dilation; however, the magnitude of the relaxation remained unchanged in IR because of a nonspecified K+ channel-mediated compensatory mechanism. In contrast, K(ir) channel-mediated relaxation elicited by increases in extracellular [K+] (4 to 12 mmol/L) was similar in the control and IR arteries. Blockade of the K(ir) and K(v) channels with Ba2+ and 4-aminopyridine, respectively, constricted the MCAs in both experimental groups with no significant difference. Pretreatment of arteries with superoxide dismutase (200 U/mL) plus catalase (150 U/mL) restored the dilatory responses to iloprost and pinacidil in the IR arteries. Immunoblots showed that the expressions of the pore-forming subunits of the examined K+ channels are not altered by IR. CONCLUSIONS: IR induces a type-specific K+ channel dysfunction mediated by reactive oxygen species. The alteration of K(ATP) and BK(Ca) channel-dependent vascular responses may be responsible for the increased risk of cerebrovascular events in IR.


Assuntos
Resistência à Insulina/fisiologia , Artéria Cerebral Média/fisiopatologia , Canais de Potássio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Vasodilatação , Animais , Técnicas de Cultura , Iloprosta/farmacologia , Masculino , Artéria Cerebral Média/efeitos dos fármacos , Pinacidil/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
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