RESUMO
Mutations in the gene encoding ATP-binding cassette transporter 1 ( ABC1) have been reported in Tangier disease (TD), an autosomal recessive disorder that is characterized by almost complete absence of plasma high-density lipoprotein (HDL), deposition of cholesteryl esters in the reticulo-endothelial system (RES) and aberrant cellular lipid trafficking. We demonstrate here that mice with a targeted inactivation of Abc1 display morphologic abnormalities and perturbations in their lipoprotein metabolism concordant with TD. ABC1 is expressed on the plasma membrane and the Golgi complex, mediates apo-AI associated export of cholesterol and phospholipids from the cell, and is regulated by cholesterol flux. Structural and functional abnormalities in caveolar processing and the trans-Golgi secretory pathway of cells lacking functional ABC1 indicate that lipid export processes involving vesicular budding between the Golgi and the plasma membrane are severely disturbed.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Complexo de Golgi/metabolismo , Metabolismo dos Lipídeos , Doença de Tangier/genética , Doença de Tangier/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Animais , Apoptose , Plaquetas/metabolismo , Colesterol/sangue , Colesterol/metabolismo , HDL-Colesterol/sangue , Fibroblastos/metabolismo , Genótipo , Humanos , Mucosa Intestinal/patologia , Mucosa Intestinal/ultraestrutura , Intestino Delgado/patologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Triglicerídeos/sangueRESUMO
Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31. Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22-31. We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Glicoproteínas/genética , Mutação , Doença de Tangier/genética , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Pré-Escolar , HDL-Colesterol/deficiência , HDL-Colesterol/metabolismo , Cromossomos Humanos Par 9 , Feminino , Glicoproteínas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Dados de Sequência Molecular , LinhagemRESUMO
Stored platelet concentrates (PLCs) for therapeutic purpose, develop a platelet storage lesion (PSL), characterized by impaired platelet (PLT) viability and function, platelet extracellular vesicle (PL-EV) release and profound lipidomic changes. Whereas oxidized low-density lipoprotein (oxLDL) activates PLTs and promotes atherosclerosis, effects linked to oxidized high-density lipoprotein (oxHDL) are poorly characterized. PLCs from blood donors were treated with native (nHDL) or mildly oxidized HDL (moxHDL) for 5days under blood banking conditions. Flow cytometry, nanoparticle tracking analysis (NTA), aggregometry, immunoblot analysis and mass spectrometry were carried out to analyze PL-EV and platelet exosomes (PL-EX) release, PLT aggregation, protein expression, and PLT and plasma lipid composition. In comparison to total nHDL, moxHDL significantly decreased PL-EV release by -36% after 5days of PLT storage and partially reversed agonist-induced PLT aggregation. PL-EV release positively correlated with PLT aggregation. MoxHDL improved PLT membrane lipid homeostasis through enhanced uptake of lysophospholipids and their remodeling to corresponding phospholipid species. This also appeared for sphingomyelin (SM) and d18:0/d18:1 sphingosine-1-phosphate (S1P) at the expense of ceramide (Cer) and hexosylceramide (HexCer) leading to reduced Cer/S1P ratio as PLT-viability indicator. This membrane remodeling was associated with increased content of CD36 and maturation of scavenger receptor-B1 (SR-B1) protein in secreted PL-EVs. MoxHDL, more potently than nHDL, improves PLT-membrane lipid homeostasis, partially antagonizes PL-EV release and agonist-induced PLT aggregation. Altogether, this may be the result of more efficient phospho- and sphingolipid remodeling mediated by CD36 and SR-B1 in the absence of ABCA1 on PLTs. As in vitro supplement in PLCs, moxHDL has the potential to improve PLC quality and to prolong storage.
Assuntos
Plaquetas/citologia , Coagulantes/química , Lipoproteínas HDL/química , Agregação Plaquetária , Plaquetas/metabolismo , Citometria de Fluxo , Homeostase , Humanos , Lipídeos/química , Lipoproteínas LDL/química , Lisofosfolipídeos/química , Espectrometria de Massas , Nanopartículas/química , Oxirredução , Oxigênio/química , Esfingosina/análogos & derivados , Esfingosina/químicaRESUMO
Adipophilin, a marker of lipid accumulation initially described in adipocytes, was recently shown to be induced in macrophage foam cells. We found that even freshly isolated blood monocytes express adipophilin and that the amount of adipophilin protein is variable in monocytes from different healthy individuals. However, the physiological expression of adipophilin does not correlate with the levels of free fatty acids, cholesterylesters or free cholesterol. Enzymatically modified low-density lipoprotein (E-LDL) induces rapid foam cell formation in monocytes and upregulates adipophilin mRNA and protein within 2 h of incubation. This rapid induction of adipophilin is accompanied by a significant increase of free fatty acids in monocytes incubated with E-LDL. Adipophilin facilitates the uptake of free fatty acids, and here we demonstrate that free fatty acids increase is related to the early upregulation of adipophilin expression in blood monocytes. Fatty acids are ligands for peroxisome proliferator-activated receptor-gamma (PPARgamma), and the upregulation of adipophilin mRNA by PPARgamma agonists like 15d-PGJ(2) and ciglitazone indicates that PPARgamma may mediate the induction of adipophilin expression in human blood monocytes.
Assuntos
Lipídeos/sangue , Monócitos/metabolismo , Peptídeos/metabolismo , Adulto , Sequência de Bases , Northern Blotting , Western Blotting , Primers do DNA , Citometria de Fluxo , Humanos , Lipoproteínas LDL/metabolismo , Proteínas de Membrana , Pessoa de Meia-Idade , Peptídeos/genética , Perilipina-2 , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Regulação para CimaRESUMO
CD163, also referred to as M130, a member of the scavenger receptor cysteine-rich family (SRCR) is exclusively expressed on cells of the monocyte lineage. In freshly isolated monocytes the CD14bright CD16+ monocyte subset revealed the highest expression of CD163 among all monocyte subsets. CD163 mRNA and protein expression is up-regulated during macrophage colony-stimulating factor (M-CSF)-dependent phagocytic differentiation of human blood monocytes. In contrast, monocytic cells treated with GM-CSF and interleukin-4 (IL-4) for dendritic differentiation down-regulate this antigen. CD163 expression is also suppressed by proinflammatory mediators like lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and tumor necrosis factor alpha, whereas IL-6 and the antiinflammatory cytokine interleukin-10 (IL-10) strongly up-regulate CD163 mRNA in monocytes and macrophages. The effects of the immunosuppressants dexamethasone, cyclosporin A (CA), and cortisol differ in their capacity to influence CD163 mRNA levels. Our results demonstrate that CD163 expression in monocytes/macrophages is regulated by proinflammatory and antiinflammatory mediators. This expression pattern implies a functional role of CD 163 in the antiinflammatory response of monocytes.
Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana , Monócitos/metabolismo , Receptores de Superfície Celular , Receptores de Lipoproteínas , Células Cultivadas , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Inflamação/patologia , Interferon gama/farmacologia , Interleucinas/farmacologia , Lipopolissacarídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/patologia , Monócitos/patologia , Fagocitose , Receptores Imunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The objective of the current study was to characterize the influence of high density lipoproteins (HDL) on processes related to the vascular recruitment of human monocytes, which may contribute to the anti-atherogenic properties of these lipoproteins. We show that HDL(3) and apo AI inhibit the following processes in primary human monocytes: (1) M-CSF induced cell spreading; (2) M-CSF stimulated expression of surface molecules involved in adhesion, migration, and scavenging; (3) fMLP induced chemotaxis. These processes are obviously modulated by the regulation of cellular cholesterol pools as indicated by the following findings. In Tangier monocytes with defective apo AI induced cholesterol efflux, apo AI had no influence on the spreading response. In control cells, stimulation of cholesterol efflux by p-cyclodextrin mimicked the effect of apo AI and HDL(3) on spreading and chemotaxis, whereas cholesterol loading with enzymatically modified LDL (E-LDL) showed the opposite effect. Finally, a similar inverse regulation by E-LDL and apo AI/HDL(3) was also observed in regard to the surface expression of beta(1)- and beta(2)-integrins as well as the hemoglobin/haptoglobin scavenger receptor CD163 and the Fcgamma-IIIaR CD16. CDC42 was identified as a potential downstream target linking changes in cellular cholesterol content to monocyte spreading and chemotaxis. Thus, CDC42 antisense markedly reduced spreading and, in parallel with their influence on monocyte spreading, HDL(3), apo AI and p-cyclodextrin down-regulated CDC42 expression while E-LDL had the inverse effect. The apo AI induced decrease of CDC42 protein expression was paralleled by the reduction of active GTP-bound CDC42. In summary, we provide evidence that HDL(3) and apo AI are able to inhibit processes in primary human monocytes, which are related to the recruitment of monocytes into the vessel wall and probably involve regulation of cellular cholesterol pools and CDC42 function.
Assuntos
Apolipoproteína A-I/metabolismo , HDL-Colesterol/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Apolipoproteína A-I/genética , Western Blotting , Células Cultivadas , HDL-Colesterol/genética , Citometria de Fluxo , Expressão Gênica , Humanos , Valores de Referência , Sensibilidade e Especificidade , Doença de Tangier/metabolismo , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
The object of these experiments was to investigate whether noradrenaline is the signal neurotransmitter between the sympathetic nervous system and rat thymocytes. Using immunocytochemistry, evidence was obtained that the rat thymus (thymic capsule, subcapsular region and connective tissue septa) is innervated by noradrenergic varicose axons terminals (tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunostained nerve fibres). This innervation is mainly associated with the vasculature and separately from vessels along the thymic tissue septa it branches into the thymic parenchyma. Using electron microscopy, classical synapses between thymocytes and neuronal elements were not observed. The neurochemical study revealed that these nerve terminals are able to take up, store and release noradrenaline upon axonal stimulation in a [Ca2+]o-dependent manner. The release was tetrodotoxin (1 microM)-sensitive, and reserpine pretreatment prevented axonal stimulation to release noradrenaline, indicating vesicular origin of noradrenaline. In addition, it was found that the release of noradrenaline was subjected to negative feedback modulation via presynaptic alpha 2-adrenoreceptors. Using a patch-clamp technique, electrophysiological evidence was obtained showing that noradrenaline inhibits in a concentration-dependent manner outward voltage-dependent potassium (k+) current recorded from isolated thymocytes. Since noradrenergic varicose axon terminals enter the parenchyma thymocytes and the boutons are not in close apposition to their target cells, noradrenaline released from these terminals diffuses away from release site to reach its targets, thymocytes, and to exert its inhibitory effect on voltage-dependent K+ -current. Since K+ channels are believed to be involved in T cell proliferation and differentiation, the modulation of K+ channel gating by noradrenaline released in response to axonal activity suggests that signals from blood-born or locally released hormones and cytokines. In this respect, noradrenaline released from non-synaptic neuronal varicosities and exerting its effect within the radius of diffusion may serve as a chemical link between the sympathetic nervous system and thymocytes and may have physiological and pathological importance in the thymus during stress and inflammatory/immune responses.
Assuntos
Norepinefrina/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Sistema Nervoso Simpático/fisiologia , Timo/inervação , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Sistema Cromafim/citologia , Sistema Cromafim/metabolismo , Estimulação Elétrica , Eletrofisiologia , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Norepinefrina/metabolismo , Sistema Nervoso Parassimpático/metabolismo , Sistema Nervoso Parassimpático/ultraestrutura , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/ultraestrutura , Timo/citologia , Timo/fisiologiaRESUMO
Histamine, a principal mediator in various physiological and pathological cell functions is synthesized from L-histidine exclusively by histidine decarboxylase, an enzyme, which is expressed in many tissues of mammalian organism. Histamine plays a role in various cellular functions, including cell differentiation. The aim of this study was to determine the presence and to characterize the role of the endogenously produced histamine during in vitro dendritic cell (DC) differentiation induced by interleukin-4 (IL-4) and granulocyte-monocyte colony stimulating factor (GM-CSF). The changes in intracellular histamine content, biosynthesis and gene expression of histidine decarboxylase were investigated during this process. One also studied how histamine receptor antagonists and a histamine synthesis blocker influence the expression of differentiation antigens on the DC during in vitro maturation. During in vitro differentiation parallel culture incubations were performed by adding H1 receptor antagonist triprolidine, H2 receptor antagonist tiotidine, the tamoxifene derivate DPPE which blocks the intracellular binding of histamine, and an irreversible blocker of histidine decarboxylase, alpha-fluoromethyl histamine (alpha-FMH). The results show simultaneous increase in both histidine decarboxylase level and histamine content during differentiation of elutriated monocytes toward DC. Both blockade of de novo histamine production (by alpha-FMH) and inhibition of histamine binding (by H1 and H2 receptor antagonists, triprolidine and tiotidine, respectively) markedly decreased CD40 expression and that of CD45 from the 3rd day of treatment. DPPE by disturbing intracellular interaction of histamine with cytochrome P-450 moieties was able to decrease the expression of CD45, CD86, HLA-DR, CD33, CD40 and CD11c. Based on the data it is suggested that endogenous histamine is actively synthesized during cytokine-induced in vitro DC differentiation. The functional relevance and autocrine and paracrine action of endogenously produced histamine is supported by the data showing that inhibition of histamine synthesis by HDC, blocking of histamine binding by both 'extracellular' histamine receptors (by specific antagonists, triprolidine and tiotidine) and 'intracellular' antagonists (DPPE) disturb the differentiation of DC. This conclusion is supported by the fact, that by the inhibition of histamine acting in an autocrine/paracrine way, the expression pattern of differentiation markers on DC is markedly changed.
Assuntos
Células Dendríticas/citologia , Histamina/biossíntese , Antígenos CD/biossíntese , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Cimetidina/análogos & derivados , Cimetidina/farmacologia , Células Dendríticas/metabolismo , Antagonistas dos Receptores Histamínicos H1/farmacologia , Antagonistas dos Receptores H2 da Histamina/farmacologia , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Humanos , Triprolidina/farmacologiaRESUMO
The effect of supramaximal electric field stimulation on [3H]dopamine (DA) release by rat adrenal capsule-glomerulosa preparations was studied using a micro-volume perfusion system. When the tissues were preloaded with [3H]DA, a considerable amount of [3H]DA and [3H]noradrenaline (NA) were released in response to field stimuli. Reserpinization, calcium removal or tetrodotoxin blocking of Na+ influx all completely inhibited the stimulation-evoked release of DA/NA, indicating that the radioactivity released is of neuronal and vesicular origin. In the adrenal cortex, a substantial proportion of tyrosine hydroxylase and dopamine-beta-hydroxylase immunoreactive nerve fibres and varicosities were observed around the zona glomerulosa. DA-containing nerves were not seen in the adrenal cortex; however, the same immunocytochemical procedures clearly demonstrated dopaminergic nerve cells and fibres in the substantia nigra and the striatum respectively, and cells of the adrenal medulla. Like the NA release from noradrenergic varicosities in the zona glomerulosa, the DA release from noradrenergic endings is not subject to negative feedback modulation through DA2 receptors since apomorphine, a DA2-receptor agonist, and sulpiride, a selective DA2-receptor antagonist, failed to affect the release. After in-vivo i.v. administration of [3H]DA, the glomerulosa content of DA and NA and the in-vitro release of [3H]DA and [3H]NA of zona glomerulosa both increased, indicating that the local varicose axon terminals were able to accumulate DA from the circulation, convert it into NA and release it in response to neural activity. This local arrangement of noradrenergic axon terminals, able to take up DA from the circulation and release it or convert it into NA, provides the possibility of a fine tuning of local circulation and aldosterone synthesis in the zona glomerulosa.
Assuntos
Axônios/metabolismo , Dopamina/metabolismo , Fenômenos Fisiológicos do Sistema Nervoso , Norepinefrina/metabolismo , Zona Glomerulosa/metabolismo , Córtex Suprarrenal/inervação , Animais , Estimulação Elétrica , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-DawleyRESUMO
The effect of supramaximal electric field stimulation on [3H]noradrenaline (NA) release and hormone production by rat adrenal capsule-glomerulosa preparations was studied using a microvolume perfusion system. A substantial proportion (about 20%) of nerve endings (varicosities) were observed close to zona glomerulosa cells, and about half of them appeared to be catecholaminergic, as judged by the chromaffin reaction of the synaptic vesicles studied at electron microscopic level. In tissue, preloaded with [3H]NA, the release of NA in response to electrical stimulation was frequency-dependent. Reserpinization, calcium removal or inhibition of Na+ influx by tetrodotoxin completely blocked NA release by field stimulation, indicating that the release resulted from axonal activity and is of vesicular origin. Neither the alpha 2-adrenoceptor agonist xylazine nor the muscarine-receptor agonist oxotremorine affected the stimulation-evoked release of [3H]NA, suggesting that, in contrast with other neurones present in the central nervous system or in the peripheral autonomic nervous system but like those in the median eminence, these axon terminals contained few presynaptic modulatory receptors. The NA (10.20 +/- 1.79 (S.E.M.) micrograms/g, n = 9), adrenaline (24.38 +/- 5.50 micrograms/g, n = 9) and dopamine (0.35 +/- 0.09 micrograms/g, n = 6) contents of the preparations were high, as determined by high-performance liquid chromatography. Our observations that the release and content of NA is high, and that a substantial proportion of catecholaminergic axon terminals lie in close proximity to zona glomerulosa cells (median value of the distance 300 nm) or to smooth muscle cells of the vessels, suggest that NA released from local adrenergic neurones without being presynaptically modulated may play an important role in fine-tuning both steroid production and/or blood flow through the gland, itself a powerful modulator of the adrenocortical response. This local modulating effect of NA may be especially significant when sympathetic activity is enhanced.
Assuntos
Corticosteroides/metabolismo , Fibras Adrenérgicas/metabolismo , Norepinefrina/fisiologia , Zona Glomerulosa/metabolismo , Aldosterona/metabolismo , Animais , Axônios/fisiologia , Corticosterona/metabolismo , Estimulação Elétrica , Masculino , Microscopia Eletrônica , Ratos , Ratos Sprague-Dawley , Reserpina/farmacologia , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/ultraestruturaRESUMO
Both [3H]noradrenaline ([3H]NA) and ATP were released in response to supramaximal electric field stimulation in superfused rat adrenal capsule-glomerulosa preparations. The voltage-dependent potassium channel blocker 4-aminopyridine enhanced, while the ATP-sensitive potassium channel blocker glibenclamide failed to affect the stimulation-evoked release of [3H]NA. The selective alpha 2-adrenoceptor antagonist CH-38083 enhanced the evoked release of [3H]NA while the P2 receptor agonist ATP and alpha, beta-methylene-ATP failed to affect it. Neither the adenosine A1 receptor agonist N6-cyclopentyl-adenosine (CPA) nor the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) influenced the stimulation-evoked [3H]NA release. The data showed that ATP was released from capsule-glomerulosa preparations in response to field stimulation together with but independently from [3H]NA, and that the local noradrenergic varicose axon terminals are not equipped with purinoceptors sensitive to ATP and/or adenosine. High concentrations of ATP also stimulated steroid hormone secretion in vitro, and thus may have a physiological role in this tissue. The presence of ecto-Ca(2+)-ATPases, enzymes able to terminate the effect of ATP, was demonstrated around the nerve profiles at the border of the capsule and zona glomerulosa tissue.
Assuntos
Trifosfato de Adenosina/metabolismo , Glândulas Suprarrenais/fisiologia , ATPases Transportadoras de Cálcio/metabolismo , Estimulação Elétrica , Norepinefrina/metabolismo , 4-Aminopiridina/farmacologia , Trifosfato de Adenosina/farmacologia , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Berberina/análogos & derivados , Berberina/farmacologia , ATPases Transportadoras de Cálcio/análise , Glibureto/farmacologia , Histocitoquímica , Masculino , Microscopia Eletrônica , Técnicas de Cultura de Órgãos , Bloqueadores dos Canais de Potássio , Ratos , Ratos Wistar , Tetrodotoxina/farmacologia , TrítioRESUMO
Using histochemical and immunocytochemical methods, cholinergic nerve fibres were demonstrated in the rat adrenal cortex, primarily in the capsule and zona glomerulosa, and in the medulla. Some terminated among the glomerulosa cells or around blood vessels. Occasional fibres were also seen in the fasciculata, ending in islets of chromaffin tissue without ramifications on cortical cells. To clarify the role of cholinergic innervation, a microvolume perifusion system was used to study steroid production by the rat adrenal capsule-glomerulosa. Acetylcholine (ACh) itself had no reproducible effects; however, since variable amounts of endogenous ACh were present, the actions of antagonists were also studied. The M1 muscarinic receptor antagonist pirenzepine (10 and 100 microM) stimulated aldosterone secretion. This stimulation was abolished by co-incubation with carbachol, the M1 agonist McN A-343 and by atropine. We found that the action of pirenzepine was blocked by nifedipine (Ca2+ channel blocker), suggesting that pirenzepine (through release of endogenous ACh) provides an acute stimulus by enhancing Ca2+ inflow. Hemicholine, a choline uptake blocker, reduced the stimulatory effect of pirenzepine on steroid secretion, confirming that stimulation was of neural origin. Neither the non-selective muscarinic receptor antagonist atropine, the selective M1-M3 muscarinic receptor antagonist 4-DAMP, nor the selective M2 muscarinic receptor antagonist methoctramine influenced aldosterone output. Receptor-binding studies revealed the existence of M3 receptors in capsule-glomerulosa homogenates. We conclude that pirenzepine acts on presynaptic M1 autoreceptors to increase spontaneous ACh release from varicose axon terminals that lie in close proximity to the glomerulosa cells. In turn ACh may thus stimulate steroidogenesis acutely through M3 receptors. These results support the concept of a direct cholinergic influence on zona glomerulosa function in the rat.
Assuntos
Acetilcolina/análise , Fibras Colinérgicas/fisiologia , Zona Glomerulosa/inervação , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Acetilcolina/farmacologia , Acetilcolina/fisiologia , Aldosterona/metabolismo , Animais , Atropina/farmacologia , Carbacol/farmacologia , Colina O-Acetiltransferase/análise , Histocitoquímica , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Técnicas de Cultura de Órgãos , Pirenzepina/farmacologia , Ratos , Ratos Sprague-Dawley , Zona Glomerulosa/química , Zona Glomerulosa/efeitos dos fármacosRESUMO
The coexpression analysis of the 55-kDa lipopolysaccharide receptor (CD14) and the Fc gamma-receptor III (CD16) reveals a broad heterogeneity of blood monocytes which in our previous work could be divided into five subpopulations based on correlated differences in expression of the pan-myeloid antigen CD33 and the adhesion antigens CD11a, CD11b and CD56. An even larger complexity of myeloid cells with antigen presenting capacity in peripheral blood is suggested by the description of small populations of immature and mature precursors of dendritic cells which rapidly develop potent costimulatory activity and a dendritic morphology in in vitro culture. The identity of the subsets of cells which have been described based on heterogeneous analytical approaches, however, remains unclear. The goal of this study, therefore, was the correlated analysis of monocyte subpopulations and dendritic cell precursors in a quantitative whole blood assay. This was achieved based on simultaneous expression analysis of the monocyte markers CD14 and CD16 with antigens such as CD33, HLA-DR, the integrin CD11c, and the interleukin-3 receptor alpha-chain (CD123) which in absence of lineage-related antigens have been used for description of dendritic cell precursors. The selected marker panel revealed identity of cells previously described as CD33bright CD14dim dendritic cell precursors with CD11c+lin-HLA-DR+ cells. Dendritic cell precursors considered to be less mature which have been alternatively described as CD33+ CD14dim CD16- cells or CD123hi dendritic cell precursors, however, were shown to differ in phenotype from each other with regard to expression density of CD33 and expression of CD14. In summary, our study revealed a complex heterogeneity of monocytes and dendritic cell precursors in peripheral blood and indicates that a direct comparison of the analytical approaches of different authors is needed to further clarify the ontogeny of human monocytes and dendritic cells.
Assuntos
Antígenos CD/imunologia , Linhagem da Célula/imunologia , Células Dendríticas/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Monócitos/citologia , Células Dendríticas/imunologia , Citometria de Fluxo , Humanos , Imunofenotipagem , Monócitos/imunologiaRESUMO
Lipid droplets, the storage places of cholesterol in adrenocortical cells, exhibit a relatively uniform appearance studied by the electron microscope but they are heterogeneous in respect of their optical polarizing properties. Optical birefringency was studied in cryosections of normal and hyperfunctioning adrenal cortex by a polarizing microscope, equipped with a cold/hot stage working in the temperature range from -40 to 40 degrees C. The majority of lipid droplets in normal adrenal cortex were optically anisotropic in each cortical zone at room temperature (22 degrees C) indicating a long-range molecular order of the lipid components. The lipids of the zona glomerulosa, in the cases of Conn's and Bartter's syndromes, became anisotropic when the temperature was lowered below ambient. The birefringency of the lipids of the zona fasciculata in the case of Cushing's disease was observed at temperatures below -10 degrees C indicating ordered packing of the components of lipid droplets at this temperature. Thus the lipids were more fluid in the hyperfunctioning, hormone-producing cells--this may represent an optimal precondition for their mobilization and processing by the hydrolyzing enzyme system. The changes in fluidity of the intracellular lipids can be attributed to different functional states in the adrenal cortex. Study of the thermotropic phase transitions of the lipid droplets by polarizing microscopy may be a useful additional method for the diagnosis of some adrenocortical diseases.
Assuntos
Córtex Suprarrenal/patologia , Hiperfunção Adrenocortical/metabolismo , Colesterol/metabolismo , Metabolismo dos Lipídeos , Córtex Suprarrenal/metabolismo , Hiperfunção Adrenocortical/patologia , Hiperfunção Adrenocortical/cirurgia , Adulto , Birrefringência , Feminino , Congelamento , Humanos , Masculino , Microscopia de Polarização , Pessoa de Meia-Idade , Especificidade de Órgãos , Valores de Referência , TermodinâmicaRESUMO
Joining peptide 1-18 (JP 1-18), added alone in concentrations of 10(-13)-10(-7) M to collagenase-dispersed human adrenocortical cells, did not affect the basal production of corticosterone, cortisol, aldosterone, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS). JP 1-18 potentiated the ACTH-stimulated production of steroids. When administered in combination with histamine (10(-8)-10(-3) M), JP 1-18 (10(-8) or 10(-10) M), enhanced the synthesis of DHEA and DHEAS. JP 1-18, together with histamine, may play a role in the regulation of DHEA and DHEAS production.
Assuntos
Córtex Suprarrenal/metabolismo , Desidroepiandrosterona/metabolismo , Histamina/farmacologia , Fragmentos de Peptídeos/farmacologia , Pró-Opiomelanocortina/farmacologia , Córtex Suprarrenal/citologia , Córtex Suprarrenal/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Sequência de Aminoácidos , Sulfato de Desidroepiandrosterona/metabolismo , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Dados de Sequência MolecularRESUMO
Classic Refsum disease (RD) is a rare, autosomal recessively-inherited disorder of peroxisome metabolism due to a defect in the initial step in the alpha oxidation of phytanic acid (PA), a C16 saturated fatty acid with four methyl side groups, which accumulates in plasma and lipid enriched tissues (please see van den Brink and Wanders, Cell Mol Life Sci 63:1752-1765, 2006). It has been proposed that the disease complex in RD is in part due to the high affinity of phytanic acid for retinoid X receptors and peroxisome proliferator-activated receptors. Structurally, epidermal hyperplasia, increased numbers of cornified cell layers, presence of cells with lipid droplets in stratum basale and reduction of granular layer to a single layer have been reported by Blanchet-Bardon et al. (The ichthyoses, SP Medical & Scientific Books, New York, pp 65-69, 1978). However, lamellar body (LB) density and secretion were reportedly normal. We recently examined biopsies from four unrelated patients, using both OsO4 and RuO4 post-fixation to evaluate the barrier lipid structural organization. Although lamellar body density appeared normal, individual organelles often had distorted shape, or had non-lamellar domains interspersed with lamellar structures. Some of the organelles seemed to lack lamellar contents altogether, showing instead uniformly electron-dense contents. In addition, we also observed mitochondrial abnormalities in the nucleated epidermis. Stratum granulosum-stratum corneum junctions also showed co-existence of non-lamellar and lamellar domains, indicative of lipid phase separation. Also, partial detachment or complete absence of corneocyte lipid envelopes (CLE) was seen in the stratum corneum of all RD patients. In conclusion, abnormal LB contents, resulting in defective lamellar bilayers, as well as reduced CLEs, likely lead to impaired barrier function in RD.
Assuntos
Gotículas Lipídicas/ultraestrutura , Doença de Refsum/patologia , Pele/ultraestrutura , Idoso , Biópsia , Feminino , Humanos , Metabolismo dos Lipídeos/genética , Microscopia Eletrônica , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Mutação/genética , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Doença de Refsum/diagnóstico , Doença de Refsum/genética , Pele/metabolismoAssuntos
Arteriopatias Oclusivas/diagnóstico por imagem , Perna (Membro)/irrigação sanguínea , Intensificação de Imagem Radiográfica , Obstrução da Artéria Renal/diagnóstico por imagem , Idoso , Angiografia , Arteriopatias Oclusivas/complicações , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obstrução da Artéria Renal/complicaçõesRESUMO
Chronic lymphocytic leukemia (CLL) is a malignancy of mature B-lymphocytes that manifests in a variety of clinical courses. The accumulation of CLL-cells is primarily caused by defective apoptosis; however, a higher proliferative capacity has also been found to correlate with poorer prognostic factors. Proliferating CLL-cells are confined to specialized structures called pseudofollicles, which contain CLL-cells, T-lymphocytes, and stromal cells. We established an in vitro model for pseudofollicles to characterize the behavior of CLL-cells in relation to clinical courses with different outcomes. Only CLL-cells from progressive clinical cases were inducible to proliferate by a combination of soluble CD40L/IL-2/IL-10 in co-culture with stromal cells. Proliferating CLL-cells showed a higher and more extensive expression of antigens, which are important in T-B-cell interactions such as CD40, MHC II, and adhesion molecules. IL-4 increased interferon regulatory factor-4 expression and induced a specific immunophenotype, which may imply plasmacytic differentiation. Furthermore, it was shown that co-cultured stromal cells protected CLL-cells from apoptosis. CLL-cells from clinically indolent cases had a far worse survival rate in medium than the cells from poor prognostic cases. Thus, we can assume that not only a different resistance to apoptosis, but also proliferation contributes to the progression of CLL resulting in bone marrow failure with thrombocytopenia and anemia.
Assuntos
Anemia/patologia , Apoptose/fisiologia , Medula Óssea/patologia , Leucemia Linfocítica Crônica de Células B/patologia , Trombocitopenia/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Ligante de CD40/farmacologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Meios de Cultura/farmacologia , Feminino , Humanos , Imunofenotipagem , Interleucina-10/farmacologia , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Células Estromais/citologiaRESUMO
AOX1, a member of the cytosolic molybdenum hydroxylase family, has been identified by us earlier as an ABCA1-interacting protein. AOX1 is well-described as xenobiotic metabolizing enzyme, which upon oxidation of acetaldehyde and retinaldehyde to acetic acid and retinoic acid generates reactive oxygen species. Here we show that knock-down of AOX1 in HepG2 by small interfering RNA significantly reduced ABCA1-dependent lipid efflux and enhanced phagocytic uptake of microspheres similar to ABCA1 deficiency, without affecting ABCA1 mRNA and protein levels. ABCA1 and AOX1 are coexpressed in human hepatocytes, kidney proximal tubular epithelial cells, Leydig, and adrenocortical cells. Expression of ABCA1 and AOX1 was investigated by immunohistochemistry in liver tissue arrays. A strong AOX1 expression was found in normal liver, and in cirrhosis. In contrast, hepatocellular carcinomas showed either a complete loss or reduced expression of AOX1. Significant correlations were found between reduced AOX1 expression and tumor stage, or metastatic or regional lymph node states. Deregulation was also observed for ABCA1 expression but to a lesser extent. Our findings show that the interaction of ABCA1 with AOX1 modulates ABCA1-linked cellular functions such as lipid efflux and phagocytosis in hepatocytes, and the reduced expression of AOX1 in malignant transformed hepatocytes supports the differentiation dependent upregulation of AOX1.