RESUMO
Drugs that lower plasma apolipoprotein B (ApoB)-containing lipoproteins are central to treating advanced atherosclerosis and provide partial protection against clinical events. Previous research showed that lowering ApoB-containing lipoproteins stops plaque inflammation, but how these drugs affect the heterogeneous population of plaque cells derived from smooth muscle cells (SMCs) is unknown. SMC-derived cells are the main cellular component of atherosclerotic lesions and the source of structural components that determine the size of plaques and their propensity to rupture and trigger thrombosis, the proximate cause of heart attack and stroke. Using lineage tracing and single-cell techniques to investigate the full SMC-derived cellular compartment in progressing and regressing plaques in mice, here we show that lowering ApoB-containing lipoproteins reduces nuclear factor kappa-light-chain-enhancer of activated B cells signaling in SMC-derived fibromyocytes and chondromyocytes and leads to depletion of these abundant cell types from plaques. These results uncover an important mechanism through which cholesterol-lowering drugs can achieve plaque regression.
Assuntos
Aterosclerose , Modelos Animais de Doenças , Miócitos de Músculo Liso , Placa Aterosclerótica , Animais , Placa Aterosclerótica/patologia , Placa Aterosclerótica/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/metabolismo , Aterosclerose/patologia , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Condrócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Anticolesterolemiantes/farmacologia , Anticolesterolemiantes/uso terapêutico , Masculino , Colesterol/metabolismo , Colesterol/sangue , Camundongos , Doenças da Aorta/patologia , Doenças da Aorta/metabolismo , Análise de Célula Única , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Músculo Liso Vascular/metabolismo , NF-kappa B/metabolismoRESUMO
Acid sphingomyelinase deficiency (ASMD) or Niemann-Pick disease type A (NPA), type B (NPB) and type A/B (NPA/B), is a rare lysosomal storage disease characterized by progressive accumulation of sphingomyelin (SM) in the liver, lungs, bone marrow and, in severe cases, neurons. A disease model was established by generating liver organoids from a NPB patient carrying the p.Arg610del variant in the SMPD1 gene. Liver organoids were characterized by transcriptomic and lipidomic analysis. We observed altered lipid homeostasis in the patient-derived organoids showing the predictable increase in sphingomyelin (SM), together with cholesterol esters (CE) and triacylglycerides (TAG), and a reduction in phosphatidylcholine (PC) and cardiolipins (CL). Analysis of lysosomal gene expression pointed to 24 downregulated genes, including SMPD1, and 26 upregulated genes that reflect the lysosomal stress typical of the disease. Altered genes revealed reduced expression of enzymes that could be involved in the accumulation in the hepatocytes of sphyngoglycolipids and glycoproteins, as well as upregulated genes coding for different glycosidases and cathepsins. Lipidic and transcriptome changes support the use of hepatic organoids as ideal models for ASMD investigation.
Assuntos
Doença de Niemann-Pick Tipo A , Doenças de Niemann-Pick , Humanos , Doença de Niemann-Pick Tipo A/genética , Esfingomielinas , Fígado , Expressão GênicaRESUMO
The extraordinary plasticity of glioma cells allows them to contribute to different cellular compartments in tumor vessels, reinforcing the vascular architecture. It was recently revealed that targeting glioma-derived pericytes, which represent a big percentage of the mural cell population in aggressive tumors, increases the permeability of the vessels and improves the efficiency of chemotherapy. However, the molecular determinants of this transdifferentiation process have not been elucidated. Here we show that mutations in EGFR stimulate the capacity of glioma cells to function as pericytes in a BMX- (bone marrow and X-linked) and SOX9-dependent manner. Subsequent activation of platelet-derived growth factor receptor beta in the vessel walls of EGFR-mutant gliomas stabilized the vasculature and facilitated the recruitment of immune cells. These changes in the tumor microenvironment conferred a growth advantage to the tumors but also rendered them sensitive to pericyte-targeting molecules such as ibrutinib or sunitinib. In the absence of EGFR mutations, high-grade gliomas were enriched in blood vessels, but showed a highly disrupted blood-brain barrier due to the decreased BMX/SOX9 activation and pericyte coverage, which led to poor oxygenation, necrosis, and hypoxia. Overall, these findings identify EGFR mutations as key regulators of the glioma-to-pericyte transdifferentiation, highlighting the intricate relationship between the tumor cells and their vascular and immune milieu. Our results lay the foundations for a vascular-dependent stratification of gliomas and suggest different therapeutic vulnerabilities determined by the genetic status of EGFR. SIGNIFICANCE: This study identifies the EGFR-related mechanisms that govern the capacity of glioma cells to transdifferentiate into pericytes, regulating the vascular and immune phenotypes of the tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2142/F1.large.jpg.
Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Transdiferenciação Celular , Microambiente Celular , Glioma/irrigação sanguínea , Mutação , Pericitos/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Barreira Hematoencefálica/metabolismo , Medula Óssea , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Cromossomos Humanos X , Receptores ErbB/genética , Glioma/imunologia , Glioma/patologia , Humanos , Imunidade Celular , Isocitrato Desidrogenase/genética , Camundongos , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Piperidinas/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Transcrição SOX9 , Sunitinibe/farmacologia , Hipóxia Tumoral , Microambiente TumoralRESUMO
Despite the high frequency of EGFR and TP53 genetic alterations in gliomas, little is known about their crosstalk during tumor progression. Here, we described a mutually exclusive distribution between mutations in these two genes. We found that wild-type p53 gliomas are more aggressive than their mutant counterparts, probably because the former accumulate amplifications and/or mutations in EGFR and show a stronger activation of this receptor. In addition, we identified a series of genes associated with vesicular trafficking of EGFR in p53 wild-type gliomas. Among these genes, TMEM167A showed the strongest implication in overall survival in this group of tumors. In agreement with this observation, inhibition of TMEM167A expression impaired the subcutaneous and the intracranial growth of wild-type p53 gliomas, regardless of the presence of EGFR mutations. In the absence of p53 mutations, TMEM167A knockdown reduced the acidification of intracellular vesicles, affecting the autophagy process and impairing EGFR trafficking and signaling. This effect was mimicked by an inhibitor of the vacuolar ATPase. We propose that the increased aggressiveness of wild-type p53 gliomas might be due to the increase in growth factor signaling activity, which depends on the regulation of vesicular trafficking by TMEM167A.
RESUMO
Glioblastoma (GBM) is the most common and devastating primary brain tumor. The presence of cancer stem cells (CSCs) has been linked to their therapy resistance. Molecular and cellular components of the tumor microenvironment also play a fundamental role in the aggressiveness of these tumors. In particular, high levels of hypoxia and reactive oxygen species participate in several aspects of GBM biology. Moreover, GBM contains a large number of macrophages, which normally behave as immunosuppressive tumor-supportive cells. In fact, the presence of both, hypoxia and M2-like macrophages, correlates with malignancy and poor prognosis in gliomas. Antioxidant agents, as nutritional supplements, might have antitumor activity. Ocoxin® oral solution (OOS), in particular, has anti-inflammatory and antioxidant properties, as well as antitumor properties in several neoplasia, without known side effects. Here, we describe how OOS affects stem cell properties in certain GBMs, slowing down their tumor growth. In parallel, OOS has a direct effect on macrophage polarization in vitro and in vivo, inhibiting the protumoral features of M2 macrophages. Therefore, OOS could be a feasible candidate to be used in combination therapies during GBM treatment because it can target the highly resilient CSCs as well as their supportive immune microenvironment, without adding toxicity to conventional treatments.
Assuntos
Ácido Ascórbico/uso terapêutico , Glioblastoma/tratamento farmacológico , Macrófagos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Extratos Vegetais/uso terapêutico , Vitamina B 12/uso terapêutico , Vitamina B 6/uso terapêutico , Animais , Ácido Ascórbico/farmacologia , Ácido Fólico , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Ácido Pantotênico , Extratos Vegetais/farmacologia , Vitamina B 12/farmacologia , Vitamina B 6/farmacologia , Sulfato de ZincoRESUMO
Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling.
Assuntos
Movimento Celular , Membrana Corioalantoide/irrigação sanguínea , Proteínas do Tecido Nervoso/metabolismo , Netrinas/metabolismo , Neuroblastoma/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Embrião de Galinha , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Netrinas/genética , Neuroblastoma/genética , Neuroblastoma/mortalidade , Neuroblastoma/secundário , Interferência de RNA , Receptores de Superfície Celular/genética , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
Alternative forms of proinsulin mRNA with differential translational capacities and unknown significance are expressed in several developing tissues and in the adult pancreas. In the chick embryo developing heart, we observed low expression of the translationally active transcript of embryonic proinsulin (Pro1B), and predominant expression of the intron 1-unspliced variant, translationally inactive. In the embryonic mouse heart, intron 1-unspliced isoform appeared after E12.5. This tight regulation is required for normal development, since overexpression of Pro1B resulted in abnormal cardiac morphogenesis in 40% of chick embryos, and was accompanied by changes in gene expression of Amhc1and Vmhc1.
Assuntos
Processamento Alternativo , Coração/embriologia , Morfogênese , Proinsulina/genética , RNA Mensageiro/genética , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Embrião de Galinha , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Proinsulina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMO
Mitochondrial Ca(2+) activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca(2+)] ([Ca(2+)](M)) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca(2+)](M) from increasing above 2-3 microM. Instead, using low-Ca(2+)-affinity aequorin probes, we have measured [Ca(2+)](M) values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca(2+)](M) to levels of 0.5-1mM was actually observed at any phosphate concentration (0-10mM) during continuous perfusion of 3.5-100 microM Ca(2+)-buffers. In spite of this high and maintained (>10 min) [Ca(2+)](M), mitochondria retained functionality and the [Ca(2+)](M) drop induced by a protonophore was fully reversible. In addition, this high [Ca(2+)](M) did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca(2+)](M). In conclusion [Ca(2+)](M) levels of 0.5-1mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Equorina/metabolismo , Animais , Arsenicais/farmacologia , Soluções Tampão , Cálcio/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Bovinos , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Perfusão , Permeabilidade/efeitos dos fármacos , Fosfatos/farmacologiaRESUMO
The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50-100 microM Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.
Assuntos
Medula Suprarrenal/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Células Cromafins/metabolismo , Vesículas Secretórias/metabolismo , Trifosfato de Adenosina/metabolismo , Medula Suprarrenal/citologia , Equorina/genética , Equorina/metabolismo , Animais , Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Catecolaminas/metabolismo , Bovinos , Compartimento Celular/efeitos dos fármacos , Compartimento Celular/fisiologia , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inositol 1,4,5-Trifosfato/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Tapsigargina/farmacologiaRESUMO
The recent availability of activators of the mitochondrial Ca(2+) uniporter allows direct testing of the influence of mitochondrial Ca(2+) uptake on the overall Ca(2+) homeostasis of the cell. We show here that activation of mitochondrial Ca(2+) uptake by 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca(2+) release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na(+)-Ca(2+) exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca(2+) uptake and release from mitochondria control the ability of local Ca(2+) microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). In addition, the ability of mitochondria to control Ca(2+) release from the ER allows them to modulate cytosolic Ca(2+) oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca(2+) oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca(2+) oscillations are exquisitely sensitive to the rates of mitochondrial Ca(2+) uptake and release, which precisely control the size of the local Ca(2+) microdomains around InsP(3)Rs and thus the ability to produce feedback activation or inhibition of Ca(2+) release.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Fibroblastos/fisiologia , Mitocôndrias/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Clonazepam/análogos & derivados , Clonazepam/farmacologia , Citosol/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Antagonistas de Estrogênios/farmacologia , Fibroblastos/efeitos dos fármacos , Células HeLa , Histamina/farmacologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/efeitos dos fármacos , Quempferóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Estimulação Química , Tiazepinas/farmacologiaRESUMO
There is increasing evidence that mitochondria play an important role in the control of cytosolic Ca2+ signaling. We show here that the main mitochondrial Ca2+-exit pathway, the mitochondrial Na+/Ca2+ exchanger, controls the pattern of cytosolic Ca2+ oscillations in non-excitable cells. In HeLa cells, the inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca2+] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na+/Ca2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca2+ release and in that way controls cytosolic Ca2+ oscillations.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Mitocôndrias/fisiologia , Trocador de Sódio e Cálcio/fisiologia , Células HeLa , HumanosRESUMO
We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.
Assuntos
Cálcio/metabolismo , Catecolaminas/metabolismo , Neurônios/metabolismo , Vesículas Secretórias/metabolismo , Equorina/genética , Equorina/metabolismo , Animais , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células PC12 , Transporte Proteico , Proteínas R-SNARE , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Ca(2+) uptake by mitochondria is a key element in the control of cellular Ca(2+) homeostasis and Ca(2+)-dependent phenomena. It has been known for many years that this Ca(2+) uptake is mediated by the mitochondrial Ca(2+) uniporter, a specific Ca(2+) channel of the inner mitochondrial membrane. We have shown previously that this channel is strongly activated by a series of natural phytoestrogenic flavonoids. We show here that several agonists and antagonists of estrogen receptors (ERs) also modulate the activity of the uniporter. The specific alpha-ER agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was the strongest activator, increasing the rate of mitochondrial Ca(2+) uptake in permeabilized HeLa cells by 10-fold at 2 microM. Consistently, PPT largely increased the histamine-induced mitochondrial [Ca(2+)] peak and reduced the cytosolic one. Diethylstilbestrol and 17-beta-estradiol (but not 17-alpha-estradiol) were active at pharmacological concentrations while the beta-estrogen-receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was little effective. The ER modulators tamoxifen and 4-hydroxy-tamoxifen inhibited mitochondrial Ca(2+) uptake (IC(50) 2.5+/-1.5 and 2.5+/-1.4 microM, mean+/-s.d., respectively) both in the presence and in the absence of PPT, but raloxifene and the pure estrogen antagonist ICI 182,780 produced no effect. Activation by PPT was immediate and inhibition by tamoxifen or 4-hydroxy-tamoxifen required only 5 min to reach maximum. Tamoxifen did not modify mitochondrial membrane potential and PPT induced a slow mitochondrial depolarization at higher concentrations than those required to activate mitochondrial Ca(2+) uptake. These results suggest that some kind of ER or related protein located in mitochondria controls the activity of the Ca(2+) uniporter by a nongenomic mechanism. This novel mechanism of action of estrogen agonists and antagonists can provide a new interpretation for several previously reported effects of these compounds.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Mitocôndrias/metabolismo , Fenóis/farmacologia , Pirazóis/farmacologia , Receptores de Estrogênio/agonistas , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Equorina/biossíntese , Equorina/genética , Cálcio/análise , Linhagem Celular Tumoral , Dietilestilbestrol/farmacologia , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Estrogênios não Esteroides/farmacologia , Feminino , Células HeLa , Humanos , Substâncias Luminescentes , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Compostos Organometálicos , Receptores de Estrogênio/antagonistas & inibidores , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Tamoxifeno/farmacologia , TransfecçãoRESUMO
During cell activation, mitochondria play an important role in Ca2+ homoeostasis due to the presence of a fast and specific Ca2+ channel in its inner membrane, the mitochondrial Ca2+ uniporter. This channel allows mitochondria to buffer local cytosolic [Ca2+] changes and controls the intramitochondrial Ca2+ levels, thus modulating a variety of phenomena from respiratory rate to apoptosis. We have described recently that SB202190, an inhibitor of p38 MAPK (mitogen-activated protein kinase), strongly activated the uniporter. We show in the present study that a series of natural plant flavonoids, widely distributed in foods, produced also a strong stimulation of the mitochondrial Ca2+ uniporter. This effect was of the same magnitude as that induced by SB202190 (an approx. 20-fold increase in the mitochondrial Ca2+ uptake rate), developed without measurable delay and was rapidly reversible. In intact cells, the mitochondrial Ca2+ peak induced by histamine was also largely increased by the flavonoids. Stimulation of the uniporter by either flavonoids or SB202190 did not require ATP, suggesting a direct effect on the uniporter or an associated protein which is not mediated by protein phosphorylation. The most active compound, kaempferol, increased the rate of mitochondrial Ca2+ uptake by 85+/-15% (mean+/-S.E.M., n=4) and the histamine-induced mitochondrial Ca2+ peak by 139+/-19% (mean+/-S.E.M., n=5) at a concentration of 1 microM. Given that flavonoids can reach this concentration range in plasma after ingestion of flavonoid-rich food, these compounds could be modulating the uniporter under physiological conditions.