RESUMO
GSK3335065 is an inhibitor of kynurenine monooxygenase (KMO) being developed for the treatment of acute pancreatitis. Healthy male volunteers were administered ascending doses of GSK3335065 or matched placebo as a single intravenous bolus injection to assess safety, tolerability, pharmacokinetics and pharmacodynamics. GSK3335065 displayed an apparent volume of distribution between 20.6 L and 44.6 L, a clearance between 0.462 L/h and 0.805 L/hr and a terminal half-life between 31.3 and 34.5 hr. In the single subject who received 1.3 mg GSK3335065, changes in tryptophan pathway metabolites were observed consistent with the changes seen in preclinical species suggesting that KMO enzyme activity was partially inhibited. However, a broad complex ventricular tachycardia was observed in this subject, which was judged to be a Serious Adverse Event (SAE) and resulted in early termination of the study. While development of GSK3335065 was subsequently discontinued, significant confounding factors hinder a clear interpretation that the tachycardia was directly related to administration of the compound.
Assuntos
Cinurenina , Pancreatite , Doença Aguda , Método Duplo-Cego , Voluntários Saudáveis , Humanos , Masculino , Oxigenases de Função MistaRESUMO
Atherosclerotic artery disease is the major cause of death and an immense burden on healthcare systems worldwide. The formation of atherosclerotic plaques is promoted by high levels of low-density lipoproteins (LDL) in the blood, especially in the oxidized form. Circulating LDL is taken up by conventional and non-classical endothelial cell receptors and deposited in the vessel wall. The exact mechanism of LDL interaction with vascular endothelial cells is not fully understood. Moreover, it appears to depend on the type and location of the vessel affected and the receptor involved. Here, we analyze how native LDL (nLDL) and oxidized LDL (oxLDL) modulate the expression of their receptors-classical LDLR and alternative LOX-1-in endothelial cells derived from human umbilical artery (HUAECs), used as an example of a medium-sized vessel, which is typically affected by atherosclerosis. Exposure of HUAECs to nLDL resulted in moderate nLDL uptake and gradual increase in LDLR, but not LOX-1, expression over 24 h. Conversely, exposure of HUAECs to oxLDL, led to significant accumulation of oxLDL and rapid induction of LOX-1, but not LDLR, within 7 h. These activation processes were associated with phosphorylation of protein kinases ERK1/2 and p38, followed by activation of the transcription factor AP-1 and its binding to the promoters of the respective receptor genes. Both nLDL-induced LDLR mRNA expression and oxLDL-induced LOX-1 mRNA expression were abolished by blocking ERK1/2, p-38 or AP-1. In addition, oxLDL, but not nLDL, was capable of inducing LOX-1 through the NF-κB-controlled pathway. These observations indicate that in arterial endothelial cells nLDL and oxLDL signal mainly via LDLR and LOX-1 receptors, respectively, and engage ERK1/2 and p38 kinases, and AP-1, as well as NF-κB transcription factors to exert feed-forward regulation and increase the expression of these receptors, which may perpetuate endothelial dysfunction in atherosclerosis.
Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Lipoproteínas LDL/farmacologia , Receptores de LDL/metabolismo , Receptores Depuradores Classe E/metabolismo , Artérias Umbilicais/citologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Oxirredução , Regiões Promotoras Genéticas/genética , Receptores de LDL/genética , Receptores Depuradores Classe E/genética , Fator de Transcrição AP-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A model-informed drug discovery and development strategy played a key role in the novel glucose-responsive insulin MK-2640's early clinical development strategy and supported a novel clinical trial paradigm to assess glucose responsiveness. The development and application of in silico modeling approaches by leveraging substantial published clinical insulin pharmacokinetic-pharmacodynamic (PKPD) data and emerging preclinical and clinical data enabled rapid quantitative decision making. Learnings can be applied to define PKPD properties of novel insulins that could become therapeutically meaningful for diabetic patients.
Assuntos
Glicemia/efeitos dos fármacos , Hipoglicemiantes/administração & dosagem , Insulina/análogos & derivados , Modelos Biológicos , Animais , Simulação por Computador , Tomada de Decisões , Diabetes Mellitus/tratamento farmacológico , Desenvolvimento de Medicamentos , Descoberta de Drogas/métodos , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Insulina/administração & dosagem , Insulina/farmacocinética , Insulina/farmacologiaRESUMO
Aldosterone synthesis is primarily regulated by angiotensin II and potassium ions. In addition, endothelial cell-secreted factors have been shown to regulate mineralocorticoid release. We analyzed the pathways that mediate endothelial cell-factor-induced aldosterone release from adrenocortical cells, NCI-H295R using endothelial cell-conditioned medium (ECM). The cAMP antagonist Rp-cAMP caused a 44% decrease in the ECM-induced aldosterone release but inhibition of cAMP-dependent PKA had no effect on aldosterone release. Interestingly, inhibition of cAMP-regulated guanine nucleotide exchange factor Epac with brefeldin-A decreased the ECM-induced aldosterone release by 45%. Similarly, inhibition of p38 MAP-kinase; PI-3-kinase and PKB significantly reduced the ECM-induced aldosterone release whereas inhibition of ERK1/2 and PKC did not decrease aldosterone release. These results provide evidence for the existence of a cAMP-dependent but PKA-independent pathway in mediating the ECM-induced aldosterone release and the significant influence of more than one signaling mechanism.
Assuntos
Aldosterona/metabolismo , Meios de Cultivo Condicionados/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/química , Células Endoteliais/metabolismo , Transdução de Sinais/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Células Endoteliais/citologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The major source for dehydroepiandrosterone (DHEA) and its sulphate compound DHEA-S is the inner zone of the adrenal cortex, which is in direct contact to adrenomedullary chromaffin cells. Due to their close proximity, direct interactions of DHEA and DHEA-S with chromaffin cells during adrenal gland development and throughout the whole life span are hypothesized. A possible direct effect of DHEA-S and the cellular and molecular mechanisms of DHEA-S action on chromaffin cells remain unresolved. Therefore, in this study, we aimed at clarifying DHEA-S effects and mechanisms of action on rat chromaffin PC12 cells. DHEA-S (10(-6)mol/l) inhibited nerve growth factor (NGF, 20ng/ml)-induced cell proliferation by 66% (n=4, p<0.001). In NGF-stimulated cells, neuronal differentiation was inhibited by DHEA-S, as demonstrated by a 22% reduction (n=3; p<0.05) of neuronal differentiation marker expression, synaptosome-associated protein of 25kDa (SNAP-25), and a 59% (n=6; p<0.001) decrease in neurite outgrowth. Moreover, DHEA-S stimulated expression of endocrine marker chromogranin A (CgA) by 31% (n=4; p<0.05 vs. control) and catecholamine release from NGF-treated PC12 cells by 229% (n=3-5; p<0.001), indicating a DHEA-S-induced shift towards neuroendocrine differentiation. On a molecular level, DHEA-S diminished NGF-induced ERK1/2 phosphorylation. Taken together, DHEA-S inhibited NGF-induced proliferation and neuronal differentiation and shifted cells towards a more endocrine phenotype. Interference of DHEA-S with NGF-stimulated ERK1/2 activation might be involved in this effect. Our study provides support for the notion that adrenocortical-derived DHEA-S impacts adrenomedullary chromaffin cells during development and differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Sulfato de Desidroepiandrosterona/farmacologia , Células PC12 , Animais , Cromogranina A/metabolismo , Dopamina/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Células PC12/efeitos dos fármacos , Células PC12/fisiologia , Ratos , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
The goal of this investigation was to examine clinical translation of glucose responsiveness of MK-2640, which is a novel insulin saccharide conjugate that can bind the insulin receptor or mannose receptor C type 1 (MRC1), the latter dependent upon glucose concentration. In a rising dose study in 36 healthy adults under euglycemic clamp conditions, rising exposures revealed saturation of MK-2640 clearance, likely due to saturation of clearance by MRC1. Potency of MK-2640 was ~25-fold reduced relative to regular human insulin. In a randomized, 2-period crossover trial in 16 subjects with type 1 diabetes mellitus to evaluate glucose-responsiveness of i.v. administered MK-2640, we were unable to demonstrate a glucose-dependent change in MK-2640 clearance, although a significant glucose-dependent augmentation of glucose infusion rate was observed. These pharmacokinetic (PK) and pharmacodynamic (PD) data provide crucial insights into next steps for developing an insulin saccharide conjugate as a clinically effective glucose-responsive insulin analog.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Insulina/análogos & derivados , Administração Intravenosa , Adolescente , Adulto , Antígenos CD/efeitos dos fármacos , Estudos Cross-Over , Diabetes Mellitus Tipo 1/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Técnica Clamp de Glucose , Humanos , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/farmacocinética , Insulina/efeitos adversos , Insulina/farmacocinética , Insulina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Receptor de Insulina/efeitos dos fármacos , Adulto JovemRESUMO
The adrenal androgen dehydroepiandrosterone (DHEA) is produced in the inner zone of the adrenal cortex, which is in direct contact to adrenal medullary cells. Due to their close anatomical proximity and tightly intermingled cell borders, a direct interaction of adrenal cortex and medulla has been postulated. In humans congenital adrenal hyperplasia due to 21-hydroxylase deficiency results in androgen excess accompanied by severe adrenomedullary dysplasia and chromaffin cell dysfunction. Therefore, to define the mechanisms of DHEA action on chromaffin cell function, we investigated its effect on cell survival and differentiation processes on a molecular level in the chromaffin cell line PC12. DHEA lessened the positive effect of NGF on cell survival and neuronal differentiation. Nerve growth factor (NGF)-mediated induction of a neuronal phenotype was inhibited by DHEA as indicated by reduced neurite outgrowth and decreased expression of neuronal marker proteins such as synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein-2. We examined whether DHEA may stimulate the cells toward a neuroendocrine phenotype. DHEA significantly elevated catecholamine release from unstimulated PC12 cells in the presence but not absence of NGF. Accordingly, DHEA enhanced the expression of the neuroendocrine marker protein chromogranin A. Next, we explored the possible molecular mechanisms of DHEA and NGF interaction. We demonstrate that NGF-induced ERK1/2 phosphorylation was reduced by DHEA. In summary, our data show that DHEA influences cell survival and differentiation processes in PC12 cells, possibly by interacting with the ERK1/2 MAPK pathway. DHEA drives NGF-stimulated cells toward a neuroendocrine phenotype, suggesting that the interaction of intraadrenal steroids and growth factors is required for the maintenance of an intact adrenal medulla.
Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cromafins/efeitos dos fármacos , Desidroepiandrosterona/farmacologia , Fator de Crescimento Neural/farmacologia , Tumores Neuroendócrinos/patologia , Feocromocitoma/patologia , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cromafins/metabolismo , Células Cromafins/patologia , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neuritos/efeitos dos fármacos , Tumores Neuroendócrinos/metabolismo , Células PC12 , Fenótipo , Feocromocitoma/metabolismo , Ratos , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Adrenocortical androgens, including dehydroepiandrosterone (DHEA), are produced in the inner zone of the adrenal cortex that is in direct contact with the neural crest-derived catecholamine-producing chromaffin cells. DHEA has recently been identified as a crucial regulator of neuronal stem cell proliferation. Thus, DHEA might play a hitherto unknown role in intra-adrenal tissue formation. In the present study, we examined the influence of DHEA on nerve growth factor (NGF)-mediated survival in serum-deprived PC12 cells and analyzed the influence of DHEA on NGF-induced ERK1/2 mitogen-activated protein (MAP) kinase activation by enzyme-linked immunosorbent assay (ELISA). Cell survival promoted by NGF in serum-deprived PC12 cells and neurite outgrowth was reduced by DHEA, pointing toward a role of DHEA in the differentiation process of chromaffin cells. Furthermore, NGF-induced ERK 1/2 activation was significantly inhibited by DHEA. Hence, we speculate that DHEA might influence NGF-mediated chromaffin differentiation processes using the ERK1/2 MAP kinase pathway during adrenal tissue development.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Desidroepiandrosterona/farmacologia , Fator de Crescimento Neural/farmacologia , Animais , Meios de Cultura Livres de Soro , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células PC12 , RatosRESUMO
The steroid hormone aldosterone is important for salt and water homeostasis as well as for pathological tissue modifications in the cardiovascular system and the kidney. The mechanisms of action include a classical genomic pathway, but physiological relevant nongenotropic effects have also been described. Unlike for estrogens or progesterone, the mechanisms for these nongenotropic effects are not well understood, although pharmacological studies suggest a role for the mineralocorticoid receptor (MR). Here we investigated whether the MR contributes to nongenotropic effects. After transfection with human MR, aldosterone induced a rapid and dose-dependent phosphorylation of ERK1/2 and c-Jun NH2-terminal kinase (JNK) 1/2 kinases in Chinese hamster ovary or human embryonic kidney cells, which was reduced by the MR-antagonist spironolactone and involved cSrc kinase as well as the epidermal growth factor receptor. In primary human aortic endothelial cells, similar results were obtained for ERK1/2 and JNK1/2. Inhibition of MAPK kinase (MEK) kinase but not of protein kinase C prevented the rapid action of aldosterone and also reduced aldosterone-induced transactivation, most probably due to impaired nuclear-cytoplasmic shuttling of MR. Cytosolic Ca2+ was increased by aldosterone in mock- and in human MR-transfected cells to the same extend due to Ca2+ influx, whereas dexamethasone had virtually no effect. Spironolactone did not prevent the Ca2+ response. We conclude that some nongenotropic effects of aldosterone are MR dependent and others are MR independent (e.g. Ca2+), indicating a higher degree of complexity of rapid aldosterone signaling. According to this model, we have to distinguish three aldosterone signaling pathways: 1) genomic via MR, 2) nongenotropic via MR, and 3) nongenotropic MR independent.
Assuntos
Aldosterona/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transdução de Sinais , Aldosterona/farmacologia , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Citosol/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Homeostase , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores de Mineralocorticoides/genéticaRESUMO
UNLABELLED: Mineralocorticoid Receptor (MR) activation is involved in blood pressure regulation and the pathogenesis of cardiovascular diseases, such as cardiac fibrosis, vascular inflammation and arterial aging. Recent investigations suggest a role for MR activation in metabolic dysregulation. OBJECTIVE: To test the effect of MR blockade on basal and postprandial glucose and lipid levels after a meal high in fat and glucose in healthy males. SUBJECTS AND METHODS: A prospective, self-controlled study was performed in 13 healthy adult males aged 18-45years. Blood was drawn before, 2h, and 4h after a high fat/high glucose meal (50g fat, 75g glucose), followed by low-dose eplerenone treatment (50mg daily) for 14days. Subjects returned for a second high fat/high glucose meal after the medication period. Basal and postprandial blood glucose and lipid levels were compared before and after eplerenone treatment. RESULTS: Eplerenone treatment affected neither basal nor postprandial glucose and lipid levels in our study population. CONCLUSION: Our results suggest that low-dose, non-blood pressure-affecting, MR blockade does not alter postprandial lipid and glucose homeostasis in healthy adult subjects.
Assuntos
Glucose/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Receptores de Mineralocorticoides/metabolismo , Espironolactona/análogos & derivados , Adolescente , Adulto , Glicemia/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Colesterol/sangue , Eplerenona , Humanos , Insulina/sangue , Resistência à Insulina , Masculino , Pessoa de Meia-Idade , Período Pós-Prandial/efeitos dos fármacos , Estudos Prospectivos , Análise de Regressão , Espironolactona/farmacologia , Adulto JovemRESUMO
OBJECTIVE: To test the hypothesis that aliskiren improves the metabolic phenotype in a genetic mouse model of the metabolic syndrome (the caveolin-1 (cav-1) knock out (KO) mouse). MATERIALS/METHODS: Eleven-week-old cav-1 KO and genetically matched wild-type (WT) mice were randomized to three treatment groups: placebo (n=8/group), amlodipine (6 mg/kg/day, n=18/ group), and aliskiren (50 mg/kg/day, n=18/ group). After three weeks of treatment, all treatment groups were assessed for several measures of insulin resistance (fasting insulin and glucose, HOMA-IR, and the response to an intraperitoneal glucose tolerance test (ipGTT)) as well as for triglyceride levels and the blood pressure response to treatment. RESULTS: Treatment with aliskiren did not affect the ipGTT response but significantly lowered the HOMA-IR and insulin levels in cav-1 KO mice. However, treatment with amlodipine significantly degraded the ipGTT response, as well as the HOMA-IR and insulin levels in the cav-1 KO mice. Aliskiren also significantly lowered triglyceride levels in the cav-1 KO but not in the WT mice. Moreover, aliskiren treatment had a significantly greater effect on blood pressure readings in the cav-1 KO vs. WT mice, and was marginally more effective than amlodipine. CONCLUSIONS: Our results support the hypothesis that aliskiren reduces insulin resistance as indicated by improved HOMA-IR in cav-1 KO mice whereas amlodipine treatment resulted in changes consistent with increased insulin resistance. In addition, aliskiren was substantially more effective in lowering blood pressure in the cav-1 KO mouse model than in WT mice and marginally more effective than amlodipine.
Assuntos
Amidas/farmacologia , Anti-Hipertensivos/farmacologia , Fumaratos/farmacologia , Resistência à Insulina/fisiologia , Síndrome Metabólica/tratamento farmacológico , Renina/antagonistas & inibidores , Animais , Glicemia/metabolismo , Pressão Sanguínea/fisiologia , Caveolina 1/deficiência , Modelos Animais de Doenças , Teste de Tolerância a Glucose , Insulina/sangue , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Distribuição Aleatória , Renina/metabolismo , Triglicerídeos/sangueRESUMO
How interactions of an individual's genetic background and environmental factors, such as dietary salt intake, result in age-associated blood pressure elevation is largely unknown. Lysine-specific demethylase-1 (LSD1) is a histone demethylase that mediates epigenetic regulation and modification of gene transcription. We have shown previously that hypertensive African-American minor allele carriers of the LSD1 single nucleotide polymorphism (rs587168) display blood pressure salt sensitivity. Our goal was to further examine the effects of LSD1 genotype variants on interactions between dietary salt intake, age, and blood pressure. We found that LSD1 single nucleotide polymorphism (rs7548692) predisposes to increasing salt sensitivity during aging in normotensive Caucasian subjects. Using a LSD1 heterozygous knockout mouse model, we compared blood pressure values on low (0.02 % Na(+)) vs. high (1.6 % Na(+)) salt intake. Our results demonstrate significantly increased blood pressure salt sensitivity in LSD1-deficient compared to wild-type animals with age, confirming our findings of salt sensitivity in humans. Elevated blood pressure in LSD1(+/-) mice is associated with total plasma volume expansion and altered renal Na(+) excretion. In summary, our human and animal studies demonstrate that LSD1 is a genetic factor that interacts with dietary salt intake modifying age-associated blood pressure increases and salt sensitivity through alteration of renal Na(+) handling.
Assuntos
Envelhecimento/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Histona Desmetilases/metabolismo , Hipertensão/fisiopatologia , Oxirredutases N-Desmetilantes/metabolismo , Sódio na Dieta/administração & dosagem , Adulto , Idoso , Envelhecimento/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Hipertensão/etiologia , Hipertensão/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Sódio na Dieta/efeitos adversos , Adulto JovemRESUMO
OBJECTIVE: Both aldosterone and cortisol can activate the mineralocorticoid receptor (MR). Polymorphisms in the MR gene have been inconsistently shown to be associated with risk of hypertension and aldosterone and cortisol levels. The purpose of this project was to investigate the association of MR gene variants with serum aldosterone and a previously identified hypertension subgroup with higher urinary free cortisol (UFC) levels (high-mode UFC) in a rigorously phenotyped Caucasian hypertensive cohort. MATERIALS AND METHODS: A haplotype-based tagging single nucleotide polymorphism (htSNP) association study was conducted in the HyperPATH cohort of 570 hypertensive Caucasian subjects on a salt-controlled diet. Haplotypes generated from 74 htSNP representing the common genetic variations of the entire MR gene were analyzed by comparing high- vs. normal-mode UFC groups and the association with serum aldosterone levels. RESULTS: Of the observed 20 haplotype blocks, there were three main linkage disequilibrium (LD) regions with high recombination rates between adjacent regions. Overlaying gene structure on this LD map revealed that block 1-8 corresponded to exon 5-9 [ligand binding domain (LBD)], blocks 9-18 to exon 3-4 [DNA binding domain (DBD)], and block 19-20 to exon 1-2 (N-terminal domain). Haplotype association results showed that DBD-aligned LD blocks were associated with high-mode UFC status (global P values, 0.0004 to 0.05). The LBD-aligned LD blocks showed significant associations with serum aldosterone levels. CONCLUSIONS: These findings imply that there may be differential functional importance of the DBD and LBD of the MR in the regulation of glucocorticoid and aldosterone levels in hypertensive subjects.
Assuntos
Glucocorticoides/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Mineralocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Adulto , Aldosterona/sangue , DNA/genética , Éxons/genética , Feminino , Genótipo , Haplótipos , Humanos , Hidrocortisona/urina , Desequilíbrio de Ligação/genética , Masculino , Polimorfismo de Nucleotídeo Único , Potássio na Dieta/farmacologia , Sódio na Dieta/farmacologia , População BrancaRESUMO
An accumulation of milk fat globule EGF-8 protein (MFG-E8) occurs within the context of arterial wall inflammatory remodeling during aging, hypertension, diabetes mellitus, or atherosclerosis. MFG-E8 induces VSMC invasion, but whether it affects VSMC proliferation, a salient feature of arterial inflammation, is unknown. Here, we show that in the rat arterial wall in vivo, PCNA and Ki67, markers of cell cycle activation, increase with age between 8 and 30 months. In fresh and early passage VSMC isolated from old aortae, an increase in CDK4 and PCNA, an increase in the acceleration of cell cycle S and G2 phases, decrease in the G1/G0 phase, and an increase in PDGF and its receptors confer elevated proliferative capacity, compared to young VSMC. Increased coexpression and physical interaction of MFG-E8 and integrin αvß5 occur with aging in both the rat aortic wall in vivo and in VSMC in vitro. In young VSMC in vitro, MFG-E8 added exogenously, or overexpressed endogenously, triggers phosphorylation of ERK1/2, augmented levels of PCNA and CDK4, increased BrdU incorporation, and promotes proliferation, via αvß5 integrins. MFG-E8 silencing, or its receptor inhibition, or the blockade of ERK1/2 phosphorylation in these cells reduces PCNA and CDK4 levels and decelerates the cell cycle S phase, conferring a reduction in proliferative capacity. Collectively, these results indicate that MFG-E8 in a dose-dependent manner coordinates the expression of cell cycle molecules and facilitates VSMC proliferation via integrin/ERK1/2 signaling. Thus, an increase in MFG-E8 signaling is a mechanism of the age-associated increase in aortic VSMC proliferation.
Assuntos
Antígenos de Superfície/metabolismo , Integrinas/metabolismo , Proteínas do Leite/metabolismo , Músculo Liso Vascular/citologia , Fatores Etários , Animais , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Processos de Crescimento Celular/fisiologia , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Masculino , Proteínas do Leite/biossíntese , Proteínas do Leite/genética , Músculo Liso Vascular/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Ratos Sprague-DawleyRESUMO
The purpose of the present article is to provide an overview of plasma membrane steroid hormone receptors and their implications in nongenomic signaling. We especially focus on recent evidence supporting the notion of a possible membrane-associated aldosterone receptor, whether this receptor is different from the classic nuclear receptor, and the possible implications of such a receptor for nongenomic and genomic aldosterone effects in physiological and pathophysiological processes.
Assuntos
Receptores de Superfície Celular/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transdução de Sinais , Aldosterona/metabolismo , Animais , Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Humanos , Modelos BiológicosRESUMO
Dehydroepiandrosterone sulfate is classically seen as an inactive reservoir for the production of dehydroepiandrosterone. Steroid sulfatase is the enzyme that catalyzes the hydrolysis of dehydroepiandrosterone sulfate to dehydroepiandrosterone, which can then be further metabolized to other steroid hormones. Recent studies, however, indicate that dehydroepiandrosterone sulfate can mediate biological effects without being converted to dehydroepiandrosterone. This study aims to evaluate whether dehydroepiandrosterone sulfate itself influences the differentiation of PC-12 cells or if its desulfation to dehydroepiandrosterone is required. dehydroepiandrosterone and dehydroepiandrosterone sulfate both influence the differentiation of chromaffin PC-12 cells. Blocking steroid sulfatase activity and thereby the conversion of dehydroepiandrosterone sulfate to dehydroepiandrosterone by the enzyme blocker estrone sulfamate showed that the effect of dehydroepiandrosterone sulfate is independent of its conversion to dehydroepiandrosterone. Dehydroepiandrosterone sulfate, similar to dehydroepiandrosterone, reduced nerve growth factor-induced neurite outgrowth of PC-12 cells and the expression of synaptosomal-associated membrane protein of 25 kDa, increased the expression of chromogranin A and significantly increased dopamine release of PC-12 cells. In addition, dehydroepiandrosterone sulfate, dehydroepiandrosterone and membrane impermeable dehydroepiandrosterone-BSA all significantly reduced NGF-induced MAPK ERK1/2 signaling after 5 min. In summary, this study provides evidence that dehydroepiandrosterone sulfate, independent of its conversion to dehydroepiandrosterone, directs PC-12 cells' differentiation to a neuroendocrine direction. Furthermore, employing membrane-impermeable dehydroepiandrosterone-BSA indicates the involvement of plasma-membrane bound receptors.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Sulfato de Desidroepiandrosterona/farmacologia , Animais , Biomarcadores/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cromogranina A/genética , Cromogranina A/metabolismo , Desidroepiandrosterona/análogos & derivados , Desidroepiandrosterona/farmacologia , Dopamina/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Sistemas Neurossecretores/efeitos dos fármacos , Sistemas Neurossecretores/metabolismo , Células PC12 , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soroalbumina Bovina/farmacologia , Esteril-Sulfatase/genética , Esteril-Sulfatase/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de TempoRESUMO
Arterial aging is a predominant risk factor for the onset of cardiovascular diseases, such as hypertension, myocardial infarction, or stroke. Aging is associated with intravascular renin-angiotensin system activation, increased vascular stiffness, intima-media thickening, and a proinflammatory phenotype. Little is known about the influence of aldosterone on arterial aging. Hence, we hypothesized that aldosterone and mineralocorticoid receptor (MR) activation might contribute to and possibly accelerate the arterial aging process. We demonstrate increased MR expression in whole aortae and early passage aortic vascular smooth muscle cells from aged (30 months) compared with adult (8 months) F344XBN rats. Sensitivity to aldosterone-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activity is increased in aged cells. MR blockade and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase inhibition prevent age-associated increases of transforming growth factor-beta, intercellular adhesion molecule 1, and procollagen 1. Aldosterone increases expression of proinflammatory marker proteins, shifting the phenotype of adult vascular smooth muscle cells toward the proinflammatory phenotype of aged rats. Epidermal growth factor receptor expression is increased with age and by aldosterone, and inhibition of epidermal growth factor receptor tyrosine kinase decreases age-associated proinflammatory marker expression. Our data support the hypothesis that increased constitutive MR signaling may promote and amplify age-associated inflammation that accompanies arterial aging through increased angiotensin II-stimulated expression of MR and enhanced sensitivity to aldosterone-mediated extracellular signal-regulated kinase 1/2 activation, likely related to increased epidermal growth factor receptor expression.
Assuntos
Receptores ErbB/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/fisiologia , Receptores de Mineralocorticoides/metabolismo , Envelhecimento/fisiologia , Análise de Variância , Animais , Aorta Torácica/citologia , Western Blotting , Células Cultivadas , Receptores ErbB/genética , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Modelos Animais , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Fenótipo , Probabilidade , Distribuição Aleatória , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Receptores de Mineralocorticoides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Modification of low-density lipoprotein (LDL) and abnormal aldosterone and cortisol metabolism have been implicated in the pathogenesis of type 2 diabetes (DM2) and diabetic vascular disease. Since LDL serves as a major cholesterol source for adrenal steroidogenesis, we investigated whether LDL modification in prediabetic and diabetic subjects influences adrenocortical aldosterone and cortisol release. LDL was isolated from 30 subjects with normal glucose tolerance (NGT-LDL), 30 subjects with impaired glucose tolerance (IGT-LDL), and 26 patients with DM2 (DM2-LDL). Oxidation and glycoxidation characteristics of LDL apolipoprotein B100 of each individual was assessed by gas chromatography-mass spectrometry analysis. Human adrenocortical cells (NCI-H295R) were incubated for 24 h with 100 microg/ml LDL and after removal of supernatants stimulated for a further 24 h with angiotensin II (AngII). In supernatants, aldosterone and cortisol secretion was measured. IGT-LDL and DM2-LDL were substantially more modified than NGT-LDL. Each of the five measured oxidation/glycoxidation markers was significantly positively associated with glycemic control, measured as HbA(1c). LDL from all subjects stimulated both the basal and AngII-induced aldosterone and cortisol release from adrenocortical cells. However, hormone secretion was significantly inversely related to the degree of LDL oxidation/glycoxidation. We conclude that LDL modifications in IGT and DM2 subjects may have significant clinical benefits by counteracting prediabetic and diabetic overactivity of the renin-angiotensin-aldosterone system and enhanced cortisol generation.
Assuntos
Córtex Suprarrenal/metabolismo , Aldosterona/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hidrocortisona/metabolismo , Lipoproteínas LDL/metabolismo , Estado Pré-Diabético/metabolismo , Adulto , Linhagem Celular , Diabetes Mellitus Tipo 2/sangue , Feminino , Teste de Tolerância a Glucose , Glicosilação , Humanos , Lipoproteínas LDL/sangue , Masculino , Oxirredução , Estado Pré-Diabético/sangueRESUMO
D-Serine selectively causes necrosis of S(3) segments of proximal tubules in rats. This leads to aminoaciduria and glucosuria. Coinjection of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) prevents the tubulopathy. D-serine is selectively reabsorbed in S(3), thereby gaining access to peroxisomal D-amino acid oxidase (D-AAO). D-AAO-mediated metabolism produces reactive oxygen species. We determined the fractional excretion of amino acids and glucose in rats after intraperitoneal injection of d-serine alone or together with reduced glutathione (GSH) or AIB. Both compounds prevented the hyperaminoaciduria. We measured GSH concentrations in renal tissue before (control) and after D-serine injection and found that GSH levels decreased to approximately 30% of control. This decrease was prevented when equimolar GSH was coinjected with D-serine. To find out why AIB protected the tubule from D-serine toxicity, we microinfused D-[(14)C]serine or [(14)C]AIB (0.36 mmol/l) together with [(3)H]inulin in late proximal tubules in vivo and measured the radioactivity in the final urine. Fractional reabsorption of D-[(14)C]serine and [(14)C]AIB amounted to 55 and 70%, respectively, and 80 mmol/l of AIB or D-serine mutually prevented reabsorption to a great extent. D-AAO activity measured in vitro (using D-serine as substrate) was not influenced by a 10-fold higher AIB concentration. We conclude from these results that 1) D-AAO-mediated d-serine metabolism lowers renal GSH concentrations and thereby provokes tubular damage because reduction of reactive oxygen species by GSH is diminished and 2) AIB prevents d-serine-induced tubulopathy by inhibition of D-serine uptake in S(3) segments rather than by interfering with intracellular D-AAO-mediated D-serine metabolism.
Assuntos
Ácidos Aminoisobutíricos/uso terapêutico , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Serina/toxicidade , Aminoácidos/sangue , Animais , D-Aminoácido Oxidase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Glucose/metabolismo , Glutationa/metabolismo , Glicosúria/induzido quimicamente , Peróxido de Hidrogênio/metabolismo , Injeções Intraperitoneais , Insulina , Rim/efeitos dos fármacos , Rim/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Alça do Néfron/efeitos dos fármacos , Alça do Néfron/metabolismo , Masculino , Oxirredução , Ratos , Ratos Wistar , Serina/administração & dosagem , Serina/metabolismoRESUMO
Aldosterone plays a key role in cardiovascular and renal injury. The underlying mechanisms are not completely understood. Because the epidermal growth factor receptor (EGFR) is involved in the development of fibrosis and vascular dysfunction, upregulation of EGFR expression by aldosterone-bound mineralocorticoid receptor (MR) is an attractive hypothesis. We investigated the effect of aldosterone on EGFR expression in the aorta of adrenalectomized rats and in human aorta smooth muscle cells (HAoSMC) in primary culture. Aldosterone, but not dexamethasone, stimulated EGFR expression in vivo in the aorta as well as in HAoSMC. EGFR degradation was not affected. Aldosterone-induced EGFR expression in HAoSMC was dose dependent and prevented by spironolactone. Furthermore, incubation of HAoSMC with aldosterone led to enhanced EGF-induced ERK1/2 phosphorylation and an EGFR-dependent increase in media fibronectin. EGFR promoter reporter gene assay as well as chromatin immunoprecipitation data indicate that MR interacts with the EGFR promoter. With deletion constructs we gained evidence that this interaction takes place between the hMR and the EGFR promoter regions 316-163 (stronger activation site, EC50 approximately 1.0 nM) and 163-1 (weaker activation site, EC50 approximately 0.7 nM), which do not comprise canonical glucocorticoid response elements and are not activated by the human glucocorticoid receptor. The interactions require in part the NH2-terminal domains of MR. ELISA-based transcription factor DNA binding assay with in vitro synthesized hMR suggest direct binding to region 163-1. Our results indicate that aldosterone leads to enhanced EGFR expression via an interaction with the EGFR promoter, which is MR specific and could contribute to the aldosterone-induced increase in fibronectin abundance.