RESUMEN
The Wnt signalling pathway in beta-cells has been linked to the development of type 2 diabetes. Investigating the impact of a non-canonical Wnt ligand, Wnt4, on beta-cell function we found that in INS-1 cells, Wnt4 was able to completely block Wnt3a stimulated cell growth and insulin secretion. However, despite high levels of Wnt4 protein being detected in INS-1 cells, reducing the expression of Wnt4 had no impact on cell growth or Wnt3a signalling. As such, the role of the endogenously expressed Wnt4 in beta-cells is unclear, but the data showing that Wnt4 can act as a negative regulator of canonical Wnt signalling in beta-cells suggests that this pathway could be a potential target for modulating beta-cell function.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Proteína Wnt3A/metabolismo , Proteína Wnt4/metabolismo , Animales , Línea Celular , Proliferación Celular , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Inmunohistoquímica , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Ratas , Vía de Señalización Wnt , Proteína Wnt3A/antagonistas & inhibidores , Proteína Wnt3A/farmacología , Proteína Wnt4/genética , Proteína Wnt4/farmacología , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Vascular diseases such as diabetes and hypertension cause changes to the vasculature that can lead to vessel stiffening and the loss of vasoactivity. The microstructural bases of these changes are not presently fully understood. We present a new methodology for stain-free visualization, at a microscopic scale, of the morphology of the main passive components of the walls of unfixed resistance arteries and their response to changes in transmural pressure. Human resistance arteries were dissected from subcutaneous fat biopsies, mounted on a perfusion myograph, and imaged at varying transmural pressures using a multimodal nonlinear microscope. High-resolution three-dimensional images of elastic fibers, collagen, and cell nuclei were constructed. The honeycomb structure of the elastic fibers comprising the internal elastic layer became visible at a transmural pressure of 30 mmHg. The adventitia, comprising wavy collagen fibers punctuated by straight elastic fibers, thinned under pressure as the collagen network straightened and pulled taut. Quantitative measurements of fiber orientation were made as a function of pressure. A multilayer analytical model was used to calculate the stiffness and stress in each layer. The adventitia was calculated to be up to 10 times as stiff as the media and experienced up to 8 times the stress, depending on lumen diameter. This work reveals that pressure-induced reorganization of fibrous proteins gives rise to very high local strain fields and highlights the unique mechanical roles of both fibrous networks. It thereby provides a basis for understanding the micromechanical significance of structural changes that occur with age and disease.
Asunto(s)
Adventicia/ultraestructura , Arterias/ultraestructura , Núcleo Celular/ultraestructura , Colágeno/ultraestructura , Tejido Elástico/ultraestructura , Resistencia Vascular , Adulto , Arterias/fisiología , Fenómenos Biomecánicos , Femenino , Voluntarios Sanos , Humanos , Imagenología Tridimensional , Masculino , Microscopía , Imagen Multimodal , Miografía , Presión , Grasa Subcutánea/irrigación sanguínea , Adulto JovenRESUMEN
AIMS/HYPOTHESIS: Hydrogen sulphide is a recently identified endogenous endothelium-dependent vasodilator. Animal models of diabetes have shown that low plasma H(2)S levels are associated with marked endothelial dysfunction and insulin resistance. However, human studies on H(2)S and vascular function in health and disease are lacking. METHODS: Plasma was obtained from male patients with type 2 diabetes (n = 11), overweight (n = 16) and lean (n = 11) volunteers. H(2)S levels were determined by zinc trap spectrophotometry. Anthropometric measurements (BMI/waist:hip ratio), lipid profile, systemic blood pressure, biochemical indices of diabetes (fasting glucose, insulin sensitivity, Hb(1Ac)) and microvascular function (minimum vascular resistance) were determined. RESULTS: Median plasma H(2)S levels (25th, 75th percentiles) in age-matched lean, overweight and type 2 diabetes individuals were 38.9 (29.7, 45.1) micromol/l, 22.0 (18.6, 26.7) micromol/l and 10.5 (4.8, 22.0) micromol/l, respectively. Median plasma H(2)S levels were significantly lower in patients with type 2 diabetes compared with lean (p = 0.001, Mann-Whitney) and overweight participants (p = 0.008). Median plasma H(2)S levels in overweight participants were significantly lower than in lean controls (p = 0.003). Waist circumference was an independent predictor of plasma H(2)S (R (2) = 0.423, standardised beta: -0.650, p < 0.001). This relationship was independent of diabetes, which only contributed a further 5% to the model (R (2) = 0.477). Waist circumference or other measures of adiposity (waist:hip ratio/BMI) remained independent predictors of plasma H(2)S after adjustment for systolic blood pressure, microvascular function, insulin sensitivity, glycaemic control and lipid profile. CONCLUSIONS/INTERPRETATION: Plasma H(2)S levels are reduced in overweight participants and patients with type 2 diabetes. Increasing adiposity is a major determinant of plasma H(2)S levels.
Asunto(s)
Adiposidad/fisiología , Diabetes Mellitus Tipo 2/sangre , Sulfuro de Hidrógeno/sangre , Obesidad/sangre , Sobrepeso/sangre , Adulto , Anciano , Presión Sanguínea , Diabetes Mellitus Tipo 2/fisiopatología , Humanos , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Obesidad/fisiopatología , Sobrepeso/fisiopatología , Análisis de Regresión , Estadísticas no Paramétricas , Circunferencia de la CinturaRESUMEN
Human endothelial cells (ECs) are heterogeneous, although little is known regarding regional variations in their regulation of vascular tone. This study compares activation of the key enzyme phospholipase D (PLD) by the vasoconstrictors angiotensin II (AII) and lysophosphatidylcholine (lysoPC), and the vasodilator insulin, in primary human microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs). PLD activity was measured by [(3)H]phosphatidylethanol production in cells labeled with [(3)H]myristic acid. AII maximally activated PLD in both cell types at 1 nmol/L. AII also significantly activated PLD at 100 pmol/L in HUVECS but not in HMVECs. LysoPC dose dependently activated PLD in both cell types, although HUVECs were more sensitive to the agonist; being significantly activated by 10 micromol/L lysoPC and displaying an approximately sevenfold greater PLD activity with 20 micromol/L lysoPC compared to HMVECs. Insulin significantly increased PLD activity in both cell types with maximum activation at 1 nmol/L. Again differential sensitivity was observed; 10 nmol/L insulin significantly stimulated PLD in HUVECs but not HMVECs. Differential sensitivity of PLD activation in human endothelial cells from different vascular beds in response to vasoactive agents was observed, with the HUVECs displaying greater sensitivity to vasoconstricting agents than HMVECs.
Asunto(s)
Angiotensina II/farmacología , Endotelio Vascular/enzimología , Activación Enzimática/efectos de los fármacos , Insulina/farmacología , Lecitinas/farmacología , Microcirculación/fisiología , Fosfolipasa D/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Cinética , Microcirculación/efectos de los fármacos , Venas Umbilicales/citología , Venas Umbilicales/fisiologíaRESUMEN
AIMS/HYPOTHESIS: Insulin is known to stimulate endothelial nitric oxide synthesis, although much remains unknown about the intracellular mechanisms involved. This study aims to examine, in human endothelial cells, the specific contribution of heterotrimeric Gi proteins and extracellular signal-regulated protein kinases 1/2 (ERK1/2) in insulin signalling upstream of nitric-oxide-dependent cyclic GMP production. METHODS: Human umbilical vein endothelial cells were treated with 1 nmol/l insulin in the presence or absence of inhibitors of tyrosine kinases (erbstatin), Gi proteins (pertussis toxin) or ERK1/2 (PD098059 or U0126), and nitric oxide production was examined by quantification of intracellular cyclic GMP. Activation/phosphorylation of ERK1/2 by insulin was examined by immunoblotting with specific antibodies, and direct association of the insulin receptor with Gi proteins was examined by immunoprecipitation. RESULTS: Treatment of cells with a physiological concentration of insulin (1 nmol/l) for 5 min increased nitric-oxide-dependent cyclic GMP accumulation by 3.3-fold, and this was significantly inhibited by erbstatin. Insulin-stimulated cyclic GMP production was significantly reduced by pertussis toxin and by the inhibitors of ERK1/2, PD098059 and U0126. Immunoblotting indicated that insulin stimulated the phosphorylation of ERK1/2 after 5 min and 1 h, and that this was completely abolished by pertussis toxin, but insensitive to the nitric oxide synthase inhibitor L-NAME. No direct interaction of the insulin receptor beta with Gialpha2 could be demonstrated by immunoprecipitation. CONCLUSIONS/INTERPRETATION: This study demonstrates, for the first time, that nitric oxide production induced by physiologically relevant concentrations of insulin, is mediated by the post-receptor activation of a pertussis-sensitive GTP-binding protein and subsequent downstream activation of the ERK1/2 cascade.
Asunto(s)
GMP Cíclico/metabolismo , Endotelio Vascular/fisiología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Insulina/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Óxido Nítrico/fisiología , Venas Umbilicales/fisiología , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Humanos , Hidroquinonas/farmacología , Toxina del Pertussis/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Embarazo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacosRESUMEN
AIMS: Nitric oxide (NO) is an important regulator of cardiovascular homeostasis. Lysophosphatidylcholine (lyso-PC), a major constituent of oxidized low density lipoproteins (oxLDL), has been reported to impair nitric oxide-dependent vasodilatation. This study investigated the possible mechanism of the lyso-PC effect on insulin-stimulated NO-dependent of cyclic guanosine 3',5'-monophosphate (cGMP) generation in human endothelial cells. METHODS: The intracellular concentration of cGMP in cultured human umbilical vein endothelial cells (HUVECs) was used to estimate NO production. The levels of endothelial nitric oxide synthase (eNOS) protein expression were assessed by Western blotting analyses. RESULTS: Both insulin, at physiological concentration, and lyso-PC stimulated rapid and prolonged intracellular of cGMP production, and together induced a marked synergistic response (for short-term stimulation: 1185 +/- 285.9% over control level (100%) compared with insulin and lyso-PC alone (384.8 +/- 67.4% and 357 +/- 205%, respectively; P < 0.001), for long-term stimulation: 3495 +/- 1377%, compared with insulin and lyso-PC alone (663 +/- 131% and 487 +/- 250%, P = 0.002)). Stimulated levels of cGMP accumulation were completely abrogated by NOS inhibitor, indicating NO involvement in the effects of insulin and lyso-PC. Stimulated NO synthesis was not associated with altered eNOS protein expression. Cell subfractionation studies demonstrate that insulin and lyso-PC each alone induced translocation of eNOS from the membrane to the cytosolic compartment and together caused a synergistic translocation. CONCLUSIONS: The presented data suggest that insulin and lyso-PC synergistically upregulate endothelial NO production via eNOS translocation from the membrane fraction to the cytosol. This study raises the possibility that an interplay between various factors accompanying diabetes can lead to endothelial NO overproduction or desensitization of NO-dependent responses. Appropriate rather than necessarily high levels of nitric oxide is the determinant of vascular health.
Asunto(s)
GMP Cíclico/biosíntesis , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Insulina/farmacología , Lisofosfatidilcolinas/farmacología , Óxido Nítrico/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , GMP Cíclico/análisis , Citosol/enzimología , Sinergismo Farmacológico , Humanos , Immunoblotting/métodos , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Estadísticas no Paramétricas , Estimulación QuímicaRESUMEN
Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [gamma-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCbeta (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 degrees C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Isoenzimas/metabolismo , Neutrófilos/metabolismo , Fosfatidilinositoles/metabolismo , Fosfolipasas de Tipo C/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Activación Enzimática , Humanos , Lípidos , Neutrófilos/ultraestructura , Ácidos Fosfatidicos/metabolismo , Fosfolipasa C betaRESUMEN
Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads to loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils. PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity. C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF. To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the Gi family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for Gi2/Gi3 protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of Gi proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Fosfolipasa D/metabolismo , Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Transporte Biológico , Células Cultivadas , Citosol/enzimología , Activación Enzimática , Humanos , Neutrófilos/enzimología , Toxina del Pertussis , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Formil Péptido , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Phospholipase D (PLD) is responsible for the hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. Human neutrophils contain PLD activity which is regulated by the small GTPases, ADP-ribosylation factor (ARF) and Rho proteins. In this study we have examined the subcellular localization of the ARF-regulated PLD activity in non-activated neutrophils and cells 'primed' with N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe). We report that PLD activity is localized at the secretory vesicles in control cells and is mobilized to the plasma membrane upon stimulation with fMetLeuPhe. We conclude that the ARF-regulated PLD activity is translocated to the plasma membrane by secretory vesicles upon stimulation of neutrophils with fMetLeuPhe in inflammatory/priming doses. We propose that this relocalization of PLD is important for the subsequent events occurring during neutrophil activation.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Fosfolipasa D/metabolismo , Factores de Ribosilacion-ADP , Fraccionamiento Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Electroforesis/métodos , Humanos , Activación Neutrófila , Neutrófilos/inmunología , Neutrófilos/metabolismoRESUMEN
Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3) play an essential role in the regulation of neutrophil functions by the chemoattractant formylmethionyl-leucylphenylalanine (FMLP). Here we show that permeabilization of human neutrophils leads to loss of cytosolic components, including PI3Kgamma, and causes the loss of FMLP-dependent production of PIP3. FMLP-sensitive synthesis of PIP3 could be restored by reconstitution of permeabilized neutrophils with recombinant PI3Kgamma. Admixture of recombinant phosphatidylinositol transfer protein (PITP) to the reconstitution cocktail produced a further increase of PIP3 synthesis, whereas pertussis toxin suppressed the FMLP-dependent production of PIP3. We conclude that FMLP-sensitive PIP3 formation in human neutrophils involves the FMLP receptor, heterotrimeric G-proteins of the Gi type, PI3Kgamma and PITP.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Bacterianas , Permeabilidad de la Membrana Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Immunoblotting , Toxina del Pertussis , Fosfatidilinositol 3-Quinasas , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferencia de Fosfolípidos , Proteínas Recombinantes/metabolismo , Transducción de Señal , Estreptolisinas/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
ADP-ribosylation factor (ARF), a small GTPase required for vesicle formation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [14C]acetate for 72 h and, after disruption, fractionated on a linear sucrose gradient. ARF1-regulated PLD activity in each fraction was assessed by measurement of phosphatidylethanol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and plasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-dependent PLD activity in endomembranes and plasma membrane, and also identified an additional activity in the cytosol. In formyl-Met-Leu-Phe-stimulated cells, PLD activity as assessed by phosphatidylethanol formation was also associated with both the plasma membrane and endomembranes. Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), the distributions of inositol lipids and the kinases responsible for lipid phosphorylation were examined. PIP2 was highly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 synthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membrane as the major site of PIP2 production. However, endomembranes possessed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that:(1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments to function as a cofactor for ARF-regulated PLD, and (3) in intact cells, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Glicerofosfolípidos , Fosfolipasa D/metabolismo , 1-Fosfatidilinositol 4-Quinasa , Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Acetatos/metabolismo , Biomarcadores/análisis , Fraccionamiento Celular , Membrana Celular/química , Células Cultivadas , Centrifugación por Gradiente de Densidad , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Aparato de Golgi/metabolismo , Guanosina Trifosfato/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neomicina/farmacología , Ácidos Fosfatidicos/análisis , Ácidos Fosfatidicos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilinositoles/análisis , Fosfatidilinositoles/metabolismo , Fosfolípidos/análisis , Fosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Human neutrophil PLD activity stimulated with GTP-gamma-S was reconstituted with recombinant ARF1 in cytosol-depleted cells. PMA-pretreatment of intact cells greatly enhanced the subsequent reconstitution of the ARF1-regulated PLD activity. This enhancement was only observed provided that the intact cells were pretreated with PMA, suggesting the stable recruitment of a cytosolic component, presumably protein kinase C, to the membranes. rARF1-reconstituted PLD activity was not dependent on MgATP, but could be considerably enhanced by MgATP. Maximal effects of MgATP were seen at 1 mM. This enhancement by MgATP could not be attributed to protein kinase C. Neomycin was found to inhibit ARF1-regulated PLD activity suggesting the requirement for polyphosphoinositides. We conclude: (i) that many of the observed effects of PMA may be dependent on the presence of the small GTP-binding protein, ARF, and (ii) polyphosphoinositides are required for ARF1-stimulated PLD activity.
Asunto(s)
Adenosina Trifosfato/farmacología , Proteínas de Unión al GTP/metabolismo , Neutrófilos/enzimología , Fosfolipasa D/sangre , Acetato de Tetradecanoilforbol/farmacología , Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Citosol/química , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Fosfatos de Fosfatidilinositol/fisiología , Proteínas Recombinantes/metabolismoRESUMEN
The transbilayer distribution of phospholipids in plasma membrane vesicles derived from BHK cells by treatment with iodoacetamide or fluoride and merocyanine 540 has been examined by exposing the vesicles to bee venom phospholipase A2 (PLA2) or to Bacillus cereus sphingomyelinase. The results show that almost all of the phosphatidylserine (PS) is on the inner lipid leaflet and most of the sphingomyelin is on the outer lipid leaflet. In contrast, about 50% of the phosphatidylcholine (PC) and 30-40% of the phosphatidylethanolamine (PE) is rapidly degraded by PLA2 and thus appears to be present on the surface of the vesicles. The pools of PC and PE which are accessible only slowly to PLA2 are degraded with halftimes of about 5 h and 2 h, respectively, and it is suggested that this rate reflects the rate of transbilayer migration of these lipids. We conclude that the profound energy depletion caused by treatment with iodoacetamide or fluoride does not alter the asymmetric distribution of PS across the plasma membrane but does have a marked effect on the transbilayer distribution of PE. Residual cells after treatment with fluoride and MC540 were also exposed to PLA2. The results were broadly in agreement with those obtained with vesicles, suggesting that the vesicles were representative of the BHK cell plasma membrane in terms of phospholipid asymmetry. Fluoride or MC540 added separately caused little vesicle release but did lead to significant loss of phospholipid asymmetry. When centrifuged on a sucrose density gradient, vesicles were separated into two major fractions accounting for about two thirds and about 20%, respectively, of total phospholipid but no significant differences were seen in the transbilayer phospholipid asymmetry of the two fractions.
Asunto(s)
Membrana Celular/química , Fosfolípidos/química , Animales , Línea Celular , Cricetinae , Yodoacetamida , Riñón , Membrana Dobles de Lípidos/química , Fosfolipasas A/farmacología , Fosfolipasas A2 , Pirimidinonas , Esfingomielina Fosfodiesterasa/farmacologíaRESUMEN
Sphingomyelin, which has been degraded at the BHK cell surface by exogenous sphingomyelinase, is converted back into sphingomyelin with kinetics similar to those of plasma membrane recycling. Resynthesis of sphingomyelin under these conditions proceeds at a rate about 4-fold higher than normal biosynthesis of sphingomyelin. Neither resynthesis of sphingomyelin nor its return to the surface is inhibited by brefeldin A (BFA), which is a potent blocker of vesicular transport through the Golgi but has no effect on plasma membrane recycling. However, resynthesis of plasma membrane sphingomyelin is greatly decreased in cells undergoing mitosis or energy depletion, where endocytosis is inhibited. We conclude that the main site of surface sphingomyelin synthesis in BHK cells could be in recycling endosomes and not in the Golgi apparatus as proposed previously. We also suggest a model pathway by which cholesterol may reach the plasma membrane via recycling endosomes.
Asunto(s)
Membrana Celular/metabolismo , Esfingomielinas/biosíntesis , Animales , Transporte Biológico , Células Cultivadas , Colesterol/metabolismo , Cricetinae , Endocitosis , Aparato de Golgi/metabolismo , CinéticaRESUMEN
Treatment of BHK or HL60 cell lines with merocyanine 540 in the presence of the sulphydryl blocker iodoacetamide caused budding of the cell surface to release vesicles about 50-100 nm in diameter which accounted for up to 25% of the total surface membrane lipid. Smaller amounts of vesicular material were released in the presence of fluoride and merocyanine 540. The vesicles had a membrane lipid composition which was characteristic of other purified plasma membranes, with large amounts of sphingomyelin, phosphatidylserine and cholesterol and low proportions of phosphatidylinositol, phosphatidylcholine, triacylglycerol and cholesterol ester. This procedure for the isolation of vesicles should be a general method for the purification of plasma membrane components from a wide range of different cell types.
Asunto(s)
Membrana Celular/efectos de los fármacos , Yodoacetamida/farmacología , Lípidos de la Membrana/aislamiento & purificación , Pirimidinonas/farmacología , Animales , Línea Celular/efectos de los fármacos , Línea Celular/ultraestructura , Membrana Celular/química , Colesterol/análisis , Cricetinae , Relación Dosis-Respuesta a Droga , Humanos , Lípidos de la Membrana/análisis , Mesocricetus , Fosfolípidos/análisis , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/ultraestructuraRESUMEN
An analysis was made of the rate, extent, and reversibility of the morphological transitions which were induced in human erythrocytes after loading with 150 microM or 1 mM Ca2+. The rate and extent of proteolytic cleavage of cytoskeletal proteins were monitored simultaneously, particularly those of the ankyrins and band 4.1, and were found not to reflect the rate of shape change. These observations were made when intact cells were incubated either in a buffer which supported glycolysis or in a simple isotonic Tris buffer without glucose. The composition of the buffer affected the initial morphology of the cells, the rate of morphological transition, the rate of proteolysis of cytoskeletal proteins, and the extent and kinetics of the reversal of morphology from the echinocyte to discocyte after removal of the ionophore A23187 and Ca2+. The morphology of cells transformed to spheroechinocytes by loading metabolically depleted cells for 15 min with 1 mM Ca2+, and which retained 50% band 2.1, was reversed in the presence of substrates for ATP synthesis to that of a mixture of 60% stage 1 echinocytes plus 25% discocytes, suggesting that ankyrin may not be essential for the maintenance of a disc-like morphology. Echinocytes which were depleted of greater than 50% band 4.1 were unable to undergo the transition back to discs.