RESUMEN
In 2001 the ACPSEM published a position paper on quality assurance in screen film mammography which was subsequently adopted as a basis for the quality assurance programs of both the Royal Australian and New Zealand College of Radiologists (RANZCR) and of BreastScreen Australia. Since then the clinical implementation of digital mammography has been realised and it has become evident that existing screen-film protocols were not appropriate to assure the required image quality needed for reliable diagnosis or to address the new dose implications resulting from digital technology. In addition, the advantages and responsibilities inherent in teleradiology are most critical in mammography and also need to be addressed. The current document is the result of a review of current overseas practice and local experience in these areas. At this time the technology of digital imaging is undergoing significant development and there is still a lack of full international consensus about some of the detailed quality control (QC) tests that should be included in quality assurance (QA) programs. This document describes the current status in digital mammography QA and recommends test procedures that may be suitable in the Australasian environment. For completeness, this document also includes a review of the QA programs required for the various types of digital biopsy units used in mammography. In the future, international harmonisation of digital quality assurance in mammography and changes in the technology may require a review of this document. Version 2.0 represented the first of these updates and key changes related to image quality evaluation, ghost image evaluation and interpretation of signal to noise ratio measurements. In Version 3.0 some significant changes, made in light of further experience gained in testing digital mammography equipment were introduced. In Version 4.0, further changes have been made, most notably digital breast tomosynthesis (DBT) testing and QC have been addressed. Some additional testing for conventional projection imaging has been added in order that sites may have the capability to undertake dose surveys to confirm compliance with diagnostic reference levels (DRLs) that may be established at the National or State level. A key recommendation is that dosimetry calculations are now to be undertaken using the methodology of Dance et al. Some minor changes to existing facility QC tests have been made to ensure the suggested procedures align with those most recently adopted by the Royal Australian and New Zealand College of Radiologists and BreastScreen Australia. Future updates of this document may be provided as deemed necessary in electronic format on the ACPSEM's website ( https://www.acpsem.org.au/whatacpsemdoes/standards-position-papers and see also http://www.ranzcr.edu.au/quality-a-safety/radiology/practice-quality-activities/mqap ).
Asunto(s)
Mamografía/normas , Garantía de la Calidad de Atención de Salud , Biopsia , Humanos , Control de CalidadRESUMEN
Xanthurenic acid (XA), an endogenous kynurenine, is a known vesicular glutamate transport (VGLUT) inhibitor and has also been proposed as an mGlu2/3 receptor agonist. Changes in these systems have been implicated in the pathophysiology of schizophrenia and other psychiatric disorders; however, little is known of how XA affects synaptic transmission. We therefore investigated the effects of XA on synaptic transmission at two hippocampal glutamatergic pathways and evaluated the ability of XA to bind to mGlu2/3 receptors. Field excitatory postsynaptic potentials (fEPSPs) were recorded from either the dentate gyrus (DG) or CA1 region of mouse hippocampal slices in vitro. Addition of XA to the bathing medium (1-10 mM) resulted in a dose-related reduction of fEPSP amplitudes (up to 52% reduction) in both hippocampal regions. In the DG, the VGLUT inhibitors Congo Red and Rose Bengal, and the mGlu2/3 agonist LY354740, also reduced fEPSPs (up to 80% reduction). The mGlu2/3 antagonist LY341495 reversed the LY354740 effect, but not the XA effect. LY354740, but not XA, also reduced DG paired-pulse depression. XA had no effect on specific binding of 1 nM [(3)H]LY341495 to membranes with human mGlu2 receptors. We conclude that XA can modulate synaptic transmission via a mechanism that may involve VGLUT inhibition rather than activation of mGlu2/3 receptors. This could be important in the pathophysiology of nervous system disorders including schizophrenia and might represent a target for developing novel pharmacological therapies.
Asunto(s)
Hipocampo/metabolismo , Quinurenina/fisiología , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular de Glutamato/antagonistas & inhibidores , Proteínas de Transporte Vesicular de Glutamato/fisiología , Xanturenatos/farmacología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transmisión Sináptica/efectos de los fármacosRESUMEN
In 2001 the ACPSEM published a position paper on quality assurance in screen film mammography which was subsequently adopted as a basis for the quality assurance programs of both the Royal Australian and New Zealand College of Radiologists (RANZCR) and of BreastScreen Australia. Since then the clinical implementation of digital mammography has been realised and it has become evident that existing screen-film protocols were not appropriate to assure the required image quality needed for reliable diagnosis or to address the new dose implications resulting from digital technology. In addition, the advantages and responsibilities inherent in teleradiology are most critical in mammography and also need to be addressed. The current document is the result of a review of current overseas practice and local experience in these areas. At this time the technology of digital imaging is undergoing significant development and there is still a lack of full international consensus about some of the detailed Quality Control tests that should be included in quality assurance (QA) programs. This document describes the current status in digital mammography QA and recommends test procedures that may be suitable in the Australasian environment. For completeness, this document also includes a review of the QA programs required for the various types of digital biopsy units used in mammography. In the future, international harmonisation of digital quality assurance in mammography and changes in the technology may require a review of this document. Accordingly, updates of this document will be provided as deemed necessary in electronic format on the ACPSEM's website (see http://www.acpsem.org.au/au/subgroup/radiology/RadiologySG_index.html).
Asunto(s)
Mamografía/instrumentación , Mamografía/normas , Garantía de la Calidad de Atención de Salud , Australia , Biopsia , Humanos , Nueva ZelandaRESUMEN
There are obvious differences in the automatic selection of kVp, anode, and filter between a digital mammography system and the equivalent film/screen system for the same thickness of PMMA absorber. To investigate the reason for these changes, a large number of images were acquired using 2, 4, 6, and 8 cm thick PMMA absorbing slabs, and various combinations of kVp, mAs, anode, and filter. The SNR and CNR were calculated using two different contrast test objects and plotted as a function of AGD. The results can be summarized as follows: 1) For any given AGD with 4, 6, and 8 cm thick PMMA absorbers, SNR(Rh/Rh) > SNR(Mo/Rh) > SNR(Mo/Mo), 2) For any given AGD: At 4 cm thickness, CNR(Mo/Mo) > CNR(Mo/Rh) > CNR(Rh/Rh), At 6 cm thickness, CNR(Rh/Rh) approximately CNR(Mo/Rh) > CNR(Mo/Mo), At 8 cm thickness, CNR(Rh/Rh) > CNR(Mo/Rh), 3) For any given absorber thickness and target/filter combination, CNR is approximately proportional to SNR, 4) For any given absorber thickness and target/filter combination, SNR (and hence CNR) is approximately proportional to (AGD)0.3 rather than the expected (AGD)(0.5), 5) CNR measured using 0.1 mm thick aluminium foil as the contrast object is more dependent on choice of kVp than using a 1 mm deep hole in a PMMA slab.
Asunto(s)
Mamografía/instrumentación , Mamografía/métodos , Intensificación de Imagen Radiográfica/instrumentación , Intensificación de Imagen Radiográfica/métodos , Interpretación de Imagen Radiográfica Asistida por Computador/métodos , Radiometría/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Fantasmas de Imagen , Dosis de Radiación , Efectividad Biológica Relativa , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The measurement of the FWHM of the slice thickness radiation dose profile of a CT scanner using a prototype low sensitivity CR imaging plate has been investigated, as an alternative to the traditional method using envelope-packed industrial film. Using a standard Agfa clinical CR system to acquire the image, the FWHM of the dose profile can be accurately measured using readily available Public Domain software. An Agfa 18 x 24 cm CR cassette gives a pixel pitch of 113.5 microm, but with interpolation, the measurement accuracy can be less than 1 pixel. For a nominal 10 mm collimation, 15 successive measurements of the FWHM using CR gave an average width of 10.00 mm with a standard deviation of 0.02 mm. This may be compared with 4 successive measurements using film and a dual exposure technique to define the optical density at half peak height, yielding an average width of 9.98 mm with a SD of 0.03 mm. This prototype NDT plate is not a commercial product, but a radiotherapy plate with a similar sensitivity is available commercially and should give similar results.
Asunto(s)
Análisis de Falla de Equipo/instrumentación , Análisis de Falla de Equipo/métodos , Garantía de la Calidad de Atención de Salud/métodos , Protección Radiológica/instrumentación , Servicio de Radiología en Hospital , Radiometría/instrumentación , Tomografía Computarizada por Rayos X/instrumentación , Australia , Diseño de Equipo , Dosis de Radiación , Protección Radiológica/métodos , Radiometría/métodosRESUMEN
The stimulation of glucose transport is one of the early cellular responses to growth factors and is essential for cell proliferation, yet the molecular processes that underlie this response are poorly defined. The aim of this study was to characterize the role of the low-molecular-mass G-proteins, Ras and Rho, and their downstream targets, Raf protein kinase and phosphatidylinositol 3-kinase, in the regulation of glucose transport in Xenopus oocytes by two distinct growth-factor receptors: the insulin-like growth factor I (IGF-I) tyrosine kinase receptor and the heterotrimeric G-protein-coupled lysophosphatidic acid (LPA) receptor. Microinjection of a neutralizing anti-Ras antibody partially blocked IGF-I-stimulated deoxyglucose uptake but was without effect on LPA-stimulated deoxyglucose uptake. In contrast, microinjection of the C3 coenzyme of botulinum toxin, which selectively ADP-ribosylates and inactivates Rho, inhibited LPA-stimulated, but not IGF-I-stimulated, deoxyglucose uptake. Similarly, LPA- but not IGF-I-stimulated deoxyglucose uptake was attenuated in oocytes expressing a dominant negative rho construct. Cells expressing a dominant negative mutant of Raf protein kinase exhibited markedly reduced sensitivity to both LPA and IGF-I, consistent with a role for endogenous Raf in glucose uptake by both growth factors. Furthermore, expression of a constitutively activated form of raf-1 resulted in a growth-factor-independent increase in deoxyglucose uptake. Measurements of phosphatidylinositol 3-kinase activity in microinjected cells support the hypothesis that the IGF-I receptor stimulates glucose transport by a Ras-dependent activation of phosphatidylinositol 3-kinase, whereas the G-protein-coupled LPA receptor controls this response by a pathway that involves Rho-dependent activation of a distinct phosphatidylinositol 3-kinase. Thus we provide evidence for clear differences in the signalling pathways that control glucose transport by G-protein-coupled and tyrosine kinase growth-factor receptors. Furthermore this is the first demonstration that active Rho is involved in the signalling pathways that regulate glucose uptake in response to some growth factors.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Glucosa/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Lisofosfolípidos/farmacología , Oocitos/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal , Animales , Transporte Biológico , Activación Enzimática , Femenino , Oocitos/metabolismo , Fosfatidilinositol 3-Quinasas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Xenopus laevisAsunto(s)
Proteínas de Unión al GTP/metabolismo , Oocitos/fisiología , Proteínas ras/metabolismo , Animales , Transporte Biológico , Desoxiglucosa/metabolismo , Femenino , Factor I del Crecimiento Similar a la Insulina/farmacología , Lisofosfolípidos/farmacología , Oocitos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Xenopus laevisRESUMEN
The exposure of 3T3-L1 fibroblasts to growth factors results in a 2-to-3-fold increase in 2-deoxyglucose transport and a approximately 50% to 80% increase in cell-surface transferrin receptor levels. We sought to determine the role of phosphatidylinositol-3'-kinase and p70 ribosomal S6 kinase in these stimulations, using selective inhibitors of these enzymes. Both basal and growth factor-stimulated deoxyglucose transport are blocked by wortmannin, but with different IC50 values (65 nM vs. 15 nM, respectively), suggesting a functional difference between these two states. This is accompanied by the accumulation of glucose transporters in intracellular locations. Both basal and growth factor-stimulated cell-surface transferrin receptor levels are downregulated by wortmannin, but with identical IC50 values (approximately 15 nM). These two proteins are known to recycle between an intracellular site and the plasma membrane in these cells, thus implying a functional role for phosphatidylinositol-3'-kinase in membrane recycling. In an effort to determine whether the effect of wortmannin was selective for the protein component of this recycling, we examined fluid-phase endocytosis of radiolabeled mannitol. Wortmannin was without effect on the fluid phase accumulation of mannitol, suggesting that the effects on membrane traffic are limited to the protein component of recycling membranes. Rapamycin, an inhibitor of p70 ribosomal S6 kinase, was without effect on any of these parameters, but both rapamycin and wortmannin inhibit growth factor-stimulated p70 ribosomal S6 kinase activity. These data support an important role for phosphatidylinositol-3'-kinase, but not p70 ribosomal S6 kinase, in the regulation of membrane protein traffic. We suggest that this enzyme may be involved in sorting of membrane proteins during trafficking.
Asunto(s)
Androstadienos/farmacología , Desoxiglucosa/metabolismo , Endocitosis/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Transferrina/metabolismo , Células 3T3 , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Transportador de Glucosa de Tipo 1 , Ratones , Proteínas de Transporte de Monosacáridos/metabolismo , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Polienos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas , Sirolimus , WortmaninaRESUMEN
Lysophosphatidic acid (LPA) stimulated the transport of deoxyglucose into oocytes isolated from Xenopus laevis. This stimulation was accounted for entirely by an increase in the Vmax for transport. Various LPAs with different acyl groups in the sn-1 position and phosphatidic acid stimulated deoxyglucose (deGlc) transport in these cells with a rank order potency of 1-oleoyl-LPA > 1-palmitoyl-LPA > phosphatidic acid = 1-stearoyl-LPA > 1-myristoyl-LPA. The phosphatidylinositol 3'-kinase inhibitor LY294002 completely blocked LPA-stimulated deoxyglucose uptake (IC50 approximately 2 microM). In marked contrast, wortmannin, which can completely block both insulin-like growth factor-I (IGF-I)-stimulated deGlc uptake in oocytes and phosphatidylinositol 3'-kinase activation at concentrations as low as 20 nM [Gould, Jess, Andrews, Herbst, Plevin and Gibbs (1994) J. Biol. Chem. 269, 26622-26625], was a relatively poor inhibitor of LPA-stimulated deGlc transport, even at concentrations as high as 100 nM. We further show that LPA stimulates phosphatidylinositol 3'-kinase activity(s) that can phosphorylate both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate, and that this stimulation is inhibited by LY294002 but is relatively insensitive to wortmannin, again in marked contrast to IGF-I-stimulated phosphatidylinositol 3'-kinase activity. Antibodies against the p85 regulatory subunit of phosphatidylinositol 3'-kinase or antiphosphotyrosine antibodies immunoprecipitated IGF-I-stimulated but not LPA-stimulated phosphatidylinositol 3'-kinase activity. We conclude that LPA stimulates glucose uptake in Xenopus oocytes by a mechanism that may involve activation of a form of phosphatidylinositol 3'-kinase that is distinguished from other isoforms by its resistance to wortmannin and by its substrate specificity. Since the LPA-activated form of phosphatidylinositol 3'-kinase is pharmacologically and immunologically distinct from that which is involved in IGF-I-stimulated glucose transport in these cells, we suggest that distinct isoforms of this enzyme are able to function with the same biological effect, at least in the regulation of sugar transport.
Asunto(s)
Desoxiglucosa/metabolismo , Glucosa/metabolismo , Lisofosfolípidos/farmacología , Oocitos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Androstadienos/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Cromonas/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Femenino , Cinética , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas , Relación Estructura-Actividad , Wortmanina , Xenopus laevisRESUMEN
PURPOSE: To assess the accuracy of three-dimensional CT angiography (CTA) in the detection and characterization of intracranial aneurysms and to help determine its role as a screening test for aneurysms in the asymptomatic population and as an adjunct to angiography in subarachnoid hemorrhages and in the follow-up of untreated aneurysms. METHODS: In a blinded, prospective study, the 3-D CTA studies in 80 patients with symptomatic aneurysms were analyzed for the presence and morphology of aneurysms. Angiography or surgery acted as the control. RESULTS: Ninety-four aneurysms were found in 63 patients. Negative findings at angiography were noted in 17. Sensitivity and specificity of 3-D CTA for all aneurysms, all patients, and aneurysms 5 mm or smaller were 90.4% and 50%, 98.4% and 82.4%, and 78.8% and 51.9%, respectively. CONCLUSION: Three-dimensional CTA may have a role in noninvasive screening for asymptomatic aneurysms in the general population, but caution is advocated when data obtained from symptomatic patients are extrapolated to the asymptomatic population who harbor smaller aneurysms. Also, 3-D CTA may be useful as an adjunct to angiography in the characterization of berry aneurysms and in the follow-up of untreated aneurysms.
Asunto(s)
Angiografía Cerebral , Aneurisma Intracraneal/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Adulto , Anciano , Femenino , Humanos , Aneurisma Intracraneal/cirugía , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
The uptake of 2-deoxyglucose into KB cells was stimulated about 2-fold by interleukin-1 (IL1), anisomycin or insulin-like growth factor-1 (IGF1). Stimulation by IL1 and anisomycin was prevented by SB 203580, a specific inhibitor of the mitogen-activated protein (MAP) kinase homologue termed 're-activating kinase' [RK; also known as p38, p40 and CSBP (cytokine synthesis anti-inflammatory-drug-binding protein)], but was unaffected by PD 98059, a specific inhibitor of the activation of the classical MAP kinase pathway. In contrast, the stimulation of 2-deoxyglucose uptake by IGF1 was blocked by PD 98059 and unaffected by SB 203580. Consistent with these observations, IL1 and anisomycin were potent activators of MAP kinase-activated protein (MAPKAP) kinase-2, a physiological substrate of RK, whereas IGF1 was only a very weak activator of MAPKAP kinase-2. Conversely, IGF1 was a stronger activator of p42 MAP kinase than IL1 or anisomycin. These results imply that the activation of distinct MAP kinase pathways is required for the stimulation of glucose transport by IL1/anisomycin and IGF1 in KB cells, and suggest that the combined use of SB 203580 and PD 98059 is a powerful new approach to explore the roles of different MAP kinase cascades in cell regulation.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Desoxiglucosa/farmacocinética , Factor I del Crecimiento Similar a la Insulina/farmacología , Interleucina-1/farmacología , Proteínas Quinasas Activadas por Mitógenos , Anisomicina/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Células KB , Proteínas de Transporte de Monosacáridos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Estimulación Química , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
A phosphatidic-acid-hydrolysing phospholipase A2 was purified from rat brain and characterized. This phospholipase A2 was purified by sequential cation, hydrophobic, heparin and gel-filtration chromatography. The purified protein had a mass of approximately 58 kDa as assayed by SDS/PAGE, had a pH optimum of 6.0, and was Ca(2+)-independent. This enzyme was apparently phosphatidic-acid-selective and had little measurable catalytic activity when phosphatidylcholine, phosphatidylethanolamine or diacylglycerol was used as substrate. On the basis of its physical and catalytic properties, we conclude that this phospholipase A2 is unique from those previously purified, and we speculate that it may be important for the production of the bioactive lipid lysophosphatidic acid.
Asunto(s)
Encéfalo/enzimología , Ácidos Fosfatidicos/metabolismo , Fosfolipasas A/aislamiento & purificación , Animales , Calcio/farmacología , Catálisis , Cromatografía , Concentración de Iones de Hidrógeno , Hidrólisis , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Ratas , Especificidad por SustratoRESUMEN
The phospholipase A2 (PLA2) inhibitors, quinacrine, p-bromophenacyl bromide, ONO-RS-082, aristolochic acid and chloracysine blocked the priming effect of LHRH, but not acute LHRH-induced gonadotrophin release measured in anterior pituitary pieces in pro-oestrous rats in vitro. These results suggest that the intracellular mechanisms underlying LHRH priming are distinct from those which mediate LH release in the present circumstances in that they involve PLA2. Furthermore, neither LHRH-induced LH release from preprimed tissue nor Ca(2+)-induced LH release were attenuated by quinacrine, indicating that this inhibitor does not interfere with the general Ca(2+)-dependent secretory apparatus of the gonadotroph and that the critical period for its action is in the induction of priming. LHRH induced the release of [3H]arachidonic acid ([3H]AA) from [3H]AA-prelabelled anterior pituitary tissue from pro-oestrous rats; a response which was sensitive to inhibitors of PLA2, of protein kinase C (PKC) and of protein synthesis. Activation of PKC also resulted in [3H]AA release which was inhibited with exactly the same pharmacological profile as the response to LHRH. Both gonadotrophin secretion and [3H]AA release responses to LHRH and to phorbol ester varied in parallel during the oestrous cycle and in ovariectomized/oestradiol-17 beta-replaced animals, as did their sensitivity to quinacrine and the protein synthesis inhibitor cycloheximide. These results indicate that LHRH priming is dependent on a hormonally regulated cascade involving a distinct form of PKC acting through a protein synthesis-dependent step to release AA by means of PLA2 activity. The priming effect was mimicked (at least in part) by conditioning preincubation with AA, confirming the functional relevance of this signalling cascade. Results using standard inhibitors of lipoxygenase/epoxygenase pathways were equivocal as to whether these pathways were critically involved, whilst cyclo-oxygenase inhibitors were completely without effect. The steps downstream from AA (and its possible metabolites) by which stimulus-secretion coupling is up-regulated in priming remain to be clarified.
Asunto(s)
Ácidos Aristolóquicos , Hormona Liberadora de Gonadotropina/metabolismo , Fosfolipasas A/fisiología , Adenohipófisis/metabolismo , Proestro/metabolismo , Acetofenonas/farmacología , Aminobenzoatos/farmacología , Animales , Ácido Araquidónico/metabolismo , Calcio/farmacología , Clorobenzoatos , Cinamatos/farmacología , Cicloheximida/farmacología , Femenino , Hormona Liberadora de Gonadotropina/farmacología , Técnicas In Vitro , Hormona Luteinizante/metabolismo , Fenantrenos/farmacología , Fenotiazinas/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Quinacrina/farmacología , Ratas , Ratas Wistar , ortoaminobenzoatosRESUMEN
A specific binding site for 1-[3H]stearoyl-lysophosphatidic acid (stearoyl-LPA) was identified and characterized in membranes prepared from rat brain and Swiss 3T3 fibroblasts. Specific binding of [3H]LPA to these sites was protein dependent, was saturable, reached equilibrium in 15 min, and was displacable by the addition of excess unlabeled LPA. Scatchard analysis of saturation binding experiments indicated that these sites had affinities of 2.0 +/- 0.5 nM and 5.4 +/- 2.6 nM and densities of 19 +/- 3 fmol/micrograms of protein and 38 +/- 6 fmol/micrograms of protein in rat brain and 3T3 cell membranes, respectively. Various LPAs, with different acyl groups in the sn-1-position, competed with [3H]LPA for these binding sites, with a rank order of potency of 1-oleoyl-LPA > 1-stearoyl-LPA = 1-palmitoyl-LPA > 1-myristoyl-LPA. Phosphatidic acid also bound to these sites, but with lower affinity than any LPA tested. Neither lysophosphatidylcholine, lysophosphatidylethanolamine, nor any free fatty acid competed with [3H]LPA for these binding sites. Binding of [3H]LPA to these sites was regulated by nonhydrolyzable guanine nucleotides in both rat brain and 3T3 cell membranes. Furthermore, in 3T3 cells, these sites were regulated by cell density. It was subsequently determined that LPA induced a transient increase in intracellular Ca2+ levels in 3T3 cells. The concentrations required for this response, as well as the rank order of potency of the various LPAs and phosphatidic acid, correlated with the affinity of these compounds for the [3H]LPA binding site. These results suggest that the specific, high affinity, binding sites for [3H]LPA are G protein-coupled receptors.
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Receptores de Superficie Celular/química , Receptores Acoplados a Proteínas G , Células 3T3 , Animales , Encéfalo/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/metabolismo , Receptores del Ácido Lisofosfatídico , Distribución TisularRESUMEN
Lysophosphatidic acid (LPA) is an important constituent of serum and shares its mitogenic activity. Serum induction of several genes is regulated, at least in part, by sequences related to the c-fos serum response element (SRE). A Rat-2 fibroblast cell line containing the beta-galactosidase reporter gene under SRE control was treated with LPA. Lysophosphatidic acid induced a time- and dose-dependent increase in beta-galactosidase activity. After 5 hours of treatment with 1-oleoyl-LPA a 3-fold increase in beta-galactosidase activity was observed. In contrast, endogenous alkaline phosphatase activity did not change in parallel with the beta-galactosidase activity indicating that the induction was specific. Various LPAs with different acyl groups in the sn-1 position induced beta-galactosidase activity with a rank order potency of 1-oleoyl-LPA > 1-palmitoyl-LPA > or = 1-myristoyl-LPA > 1-stearoyl-LPA. Phosphatidic acid was approximately equal to 1-stearoyl-LPA. Neither the calcium ionophore (A23187) nor 12-O-tetradecanoylphorbol 13-acetate, induced beta-galactosidase activity. These data suggest that LPA may exert some of its effects by regulation of SRE controlled genes.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Genes fos , Lisofosfolípidos/farmacología , Regiones Promotoras Genéticas , Animales , Sangre , Calcimicina/farmacología , Línea Celular , Técnicas In Vitro , ARN Mensajero/genética , Ratas , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
A MAP kinase activity assay was developed to determine whether the LHRH receptor could activate this enzyme (particularly during LHRH priming). In anterior pituitary tissue from prooestrous rats LHRH caused concentration-dependent activation of MAP kinase after 5-10 min and continued for up to 60 min of incubation. The magnitude of this response correlated with that of LHRH priming on various days of the oestrous cycle but not with the magnitude of 1st hour (unprimed) LHRH-induced LH release. The response to LHRH was mimicked by a phorbol ester but not by ionomycin and was blocked with high potency by GF 109203X but not by H7 (in a similar manner to the PKC species that mediates LHRH priming). Neither the tyrosine kinase inhibitor lavendustin A nor the protein synthesis inhibitor cycloheximide blocked LHRH-induced MAP kinase activation. The possible functional significance of MAP kinase activation in gonadotrophs is considered with respect to LHRH priming.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Hormona Liberadora de Gonadotropina/farmacología , Adenohipófisis/enzimología , Animales , Relación Dosis-Respuesta a Droga , Activación Enzimática , Femenino , Hormona Luteinizante/metabolismo , Ovariectomía , Forbol 12,13-Dibutirato/farmacología , Adenohipófisis/efectos de los fármacos , Adenohipófisis/metabolismo , Proestro/metabolismo , Ratas , Ratas WistarRESUMEN
Minor abnormalities in sensory perception are common in elderly people but the significance of these findings is uncertain. In order to define the most relevant clinical tests for the diagnosis of significant neuropathy in the elderly diabetic patient, quantified perception of vibration, temperature, pain, and light touch was assessed in 200 (100 hospitalized, 100 community) consecutive non-diabetic elderly subjects without apparent neurological disease, using an established scoring system. The changes in sensory perception were similar in the two groups and data were pooled for further analysis. Progressive loss of peripheral sensation was apparent with increasing age (neuropathy deficit score vs age: r = .04, p < 0.0001). Loss of vibration perception was particularly marked; deficit scores for vibration were significantly lower in the < 70 years age group than in all the older age groups (vibration score: < 70 years vs 80-84 years mean (95% CI) 0.89 (0.54) vs 3.02 (0.6), p < 0.0001). In contrast, perception of light touch and pain was relatively preserved in old age. Assessment of vibration perception is of limited value in elderly people since the distinction between 'normal ageing' and significant neuropathy is unclear. Perception of light touch and pain are likely to be the most reliable clinical indicators of significant neuropathy in an elderly diabetic population.
Asunto(s)
Anciano , Examen Neurológico , Neuronas Aferentes/fisiología , Enfermedades del Sistema Nervioso Periférico/diagnóstico , Factores de Edad , Anciano de 80 o más Años , Neuropatías Diabéticas/diagnóstico , Neuropatías Diabéticas/fisiopatología , Femenino , Humanos , Pacientes Internos , Masculino , Persona de Mediana Edad , Pacientes Ambulatorios , Percepción , Enfermedades del Sistema Nervioso Periférico/epidemiología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Prevalencia , VibraciónRESUMEN
The protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu) induced the release of both luteinizing hormone (LH) and growth hormone (GH) from proestrous rat anterior pituitary pieces in vitro. Phorbol 12,13-dibutyrate-induced LH, but not GH release was readily inhibited by the phospholipase A2 (PLA2) inhibitors, quinacrine, aristolochic acid, ONO-RS-082 and chloracysine. Furthermore, PDBu induced release of [3H]arachidonic acid ([3H]AA) from pre-labelled anterior pituitary tissue that was prevented in the presence of quinacrine, aristolochic acid and ONO-RS-082 but not the diglyceride lipase inhibitor RHC 80267. The effect of PDBu was completely inhibited by staurosporine and the selective PKC inhibitor Ro 31-8220 but only partially by low concentrations of H7; consistent with the involvement of both H7-sensitive and H7-resistant forms of PKC in the activation of PLA2 by PDBu. The protein synthesis inhibitor cycloheximide inhibited the release of both [3H]AA and LH that had been induced by PDBu, whereas LH release induced by the PLA2 activator mellitin was cycloheximide-insensitive. These results suggest that PKC activators may induce LH but not GH release from anterior pituitary tissue by a mechanism involving activation of a PLA2, brought about by a process which is reliant on protein synthesis.
Asunto(s)
Ácidos Aristolóquicos , Hormona del Crecimiento/metabolismo , Hormona Luteinizante/metabolismo , Forbol 12,13-Dibutirato/farmacología , Fosfolipasas A/fisiología , Adenohipófisis/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Alcaloides/farmacología , Aminobenzoatos/farmacología , Animales , Ácido Araquidónico/metabolismo , Clorobenzoatos , Cinamatos/farmacología , Ciclohexanonas/farmacología , Cicloheximida/farmacología , Activación Enzimática/efectos de los fármacos , Femenino , Indoles/farmacología , Ionomicina/farmacología , Isoquinolinas/farmacología , Meliteno/farmacología , Fenantrenos/farmacología , Fenotiazinas/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Piperazinas/farmacología , Adenohipófisis/efectos de los fármacos , Adenohipófisis/enzimología , Proestro , Proteína Quinasa C/metabolismo , Quinacrina/farmacología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Estaurosporina , ortoaminobenzoatosRESUMEN
We examined the role of voltage-activated, L-type, Ca2+ channels in phorbol ester-induced luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue. The L-type Ca2+ channel inhibitor, nimodipine (NMD), inhibited phorbol 12,13-dibutyrate (PDBu)-induced GH release but had no significant effect on LH release. The L-type Ca2+ channel activator BAY K 8644 had no effect on PDBu-induced GH release but potentiated PDBu-induced LH release. In contrast, 60 mM K(+)-induced LH and GH release were inhibited by NMD, whereas BAY K 8644 had no effect. When PDBu and either K+ or BAY K 8644 were used together, they acted synergistically to evoke levels of LH release greater than addition of release caused by each secretagogue alone. However, the release of GH was additive with PDBu and either K+, BAY K 8644. The protein kinase C (PKC) inhibitor staurosporine inhibited both PDBu-induced LH release and GH release. A structurally different PKC inhibitor, H7, significantly inhibited PDBu-induced LH release but had no effect on PDBu-induced GH release. Both staurosporine and H7 inhibited LH release induced by PDBu and BAY K 8644 together. In contrast, although staurosporine inhibited GH release induced by PDBu and BAY K 8644, H7 significantly potentiated this response. A difference in the action of these two inhibitors was also apparent on K(+)-induced hormone release where staurosporine partially blocked K(+)-induced LH and GH release but H7 had no effect on the release of either hormone. Data obtained in 45Ca2+ influx experiments further suggested that a staurosporine-sensitive, but H7-resistant, PKC-like kinase may tonically maintain L-channels in a voltage-sensitive state, as down-regulation of PKC in dispersed anterior pituitary cells by long term PDBu treatment caused a significant reduction in K(+)-induced 45Ca2+ influx. We conclude that phorbol ester-induced GH release, but not LH release, is a result of L-type Ca2+ channel activation which may occur by means of alterations in the channel itself to increase its responsiveness to a given depolarisation.