RESUMEN

INTRODUCTION: In 2001 national health survey data, Indigenous Australians reported asthma as the second most commonly experienced health condition. This systematic review was undertaken to determine the current evidence concerning asthma in the Aboriginal and Torres Strait Islander population. METHODS: The review was conducted according to Australian National Health and Medical Research Council guidelines. Relevant databases and Internet sites were searched with selection criteria inclusive of all study designs, outcome measures and interventions that investigated asthma in Australian Indigenous participants. Studies of Level 4 evidence were graded using the READER critical appraisal tool, in the absence of an instrument validated and specifically designed for the Indigenous Australian context. RESULTS: A total of 10 descriptive studies published in scientific journals were found, which were largely cross-sectional prevalence studies with rural and remote populations and retrospective reviews of hospitalisation data. There are no published scientific papers on the prevalence of asthma in Indigenous individuals dwelling in metropolitan areas. Other papers included a review, an editorial, and a number of abstracts and letters. Three national health surveys published by the Australian Bureau of Statistics were included. Inconsistency of results regarding the prevalence of asthma was common among studies. Asthma prevalence in rural children has been described as low as 0.5% and as high as 39.4%, and in rural adults in one study as 3.3%. In remote communities, asthma prevalence has been described as 15.8% for children, and for adults between 7% and 26%. National health survey data in the last 10 years has described overall prevalence of asthma in the Indigenous population in the range 15.3%-19%. Hospitalisation data for children with asthma in the Northern Territory showed that rural Indigenous children were admitted significantly less than urban non-Indigenous children, while a Western Australian study found that admissions for asthma were 3.1 times higher in Indigenous than non-Indigenous children, and rates were higher in non-metropolitan compared with metropolitan areas. A single descriptive study of an asthma management strategy utilised with good attendance rates on a remote Torres Strait Island has been published. It is unclear whether the divergence in research findings reflects a difference in prevalence of asthma across demographic regions and among different states and territories, or results from the lack of standardisation of epidemiological research methods used. CONCLUSIONS: Previous research efforts concerning asthma in the Australian Indigenous population are insufficient, although there has been an increasing number of studies published during the past decade. All studies undertaken reside at a low level on the hierarchy of evidence scale. There remains no consensus of scientific opinion around the prevalence and aetiology of asthma in Aboriginal and Torres Strait Islander individuals. Particularly lacking are quality studies on asthma management interventions. Extensive consultation with Indigenous communities is indicated in order to determine priorities for asthma research. Following this, well-funded studies of a high methodological quality and culturally appropriate design investigating asthma in the Indigenous population should be undertaken.

RESUMEN

Two members of the GAP1 family, GAP1(IP4BP) and GAP1(m), have been shown to bind the putative second messenger Ins(1,3,4,5)P4 with high affinity and specificity, though other aspects of their behaviour suggest that in vivo, whereas GAP1(IP4BP) may function as an Ins(1,3,4,5)P4 receptor, GAP1(m) may be a receptor for the lipid second messenger PtdIns(3,4,5)P3. As a step towards clarifying their cellular roles, we describe here how we have raised and characterised antisera that are specific for the two proteins, and used these to undertake a comprehensive study of their tissue distribution. Both proteins are widely expressed, but there are several clear differences between them in the tissues that show the highest levels of expression.

Asunto(s)

Fosfatos de Inositol/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Anticuerpos/inmunología , Células COS , Femenino , Humanos , Immunoblotting , Masculino , Pruebas de Precipitina , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/inmunología , Porcinos , Distribución Tisular , Proteínas Activadoras de ras GTPasa/inmunología

RESUMEN

Ca(2+) within intracellular stores (luminal Ca(2+)) is believed to play a role in regulating Ca(2+) release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P(3))-gated Ca(2+) channel (or Ins(1,4,5)P(3) receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P(3) receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca(2+)] using K(+) as the current carrier. At a high luminal [Ca(2+)] and in the presence of optimal [Ins(1,4,5)P(3)] and cytosolic [Ca(2+)], a short burst of Ins(1,4,5)P(3) receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca(2+)] caused the channel to reactivate indefinitely. At luminal [Ca(2+)], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca(2+)] caused reactivation. We provide evidence that luminal Ca(2+) is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca(2+) overload is prevented when the energy state of the cell is compromised.

Asunto(s)

Adenosina Trifosfato/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Citosol/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Adenosina Trifosfato/farmacología , Animales , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Cerebelo , Citosol/efectos de los fármacos , Conductividad Eléctrica , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Activación del Canal Iónico/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Potasio/metabolismo , Proteolípidos/efectos de los fármacos , Proteolípidos/metabolismo , Ratas

RESUMEN

To try to further define the mechanism of action of the putative second messenger inositol 1,3,4,5-tetrakisphosphate (InsP4), we have studied its effects in permeabilized cells expressing different levels of inositol trisphosphate receptor (InsP3R) types I and III and of the GTPase-activating protein GAP1IP4BP. During the growth curve of human HL-60 cells and mouse T15 cells there was an increase in these proteins, which was further increased by differentiation (HL-60) and, marginally, by transformation (T15). T15 cells entering the stationary phase showed much lower concentrations of these proteins and expression was below detection in apoptotic HL-60 cells. Rasp21 showed a different pattern of expression. The ratios of InsP3R subtypes seem to affect the dose-response curve for inositol 2,4,5-trisphosphate Ins(2,4,5)P3. In permeabilized T15 cells the curve was approximately 5-fold to the right of that obtained using HL-60 cells. However, permeabilized untreated and differentiated HL-60 cells and T15 cells all showed a comparable synergistic effect of InsP4 on Ca2+ release stimulated by a concentration of Ins(2,4,5)P3, releasing approximately 20% of the Ins(1,4,5)P3 sensitive Ca2+ pool. The data indicate that under these conditions InsP4 is acting independently of cell type, of the ratio of inositol trisphosphate receptor subtypes, and of the concentration of GAP1IP4BP.

Asunto(s)

Canales de Calcio/metabolismo , Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Diferenciación Celular , Permeabilidad de la Membrana Celular , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica , Fibroblastos/metabolismo , Fibroblastos/patología , Células HL-60/efectos de los fármacos , Células HL-60/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/farmacología , Ratones , NADPH Oxidasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Tretinoina/farmacología

RESUMEN

We have previously shown that addition of Ins(1,3,4,5)P4 to permeabilized L1210 cells increases the amount of Ca2+ mobilized by a submaximal concentration of Ins(2,4,5)P3, and we suggested that, in doing this, Ins(1,3,4,5)P4 is not working via an InsP3 receptor but indirectly via an InsP4 receptor [Loomis-Husselbee, Cullen, Dreikhausen, Irvine and Dawson (1996) Biochem. J. 314, 811-816]. Here we have investigated whether this effect might be mediated by GAP1(IP4BP), recently identified as a putative receptor for Ins(1,3, 4,5)P4. GAP1(IP4BP) is a protein that interacts with one or more monomeric G-proteins, so we sought evidence for involvement of monomeric G-proteins in the effects of Ins(1,3,4,5)P4 in permeabilized L1210 cells. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) enhanced the effect of Ins(1,3,4,5)P4 on Ins(2,4, 5)P3-stimulated Ca2+ mobilization, but had no effect on the action of Ins(2,4,5)P3 alone. A specific enhancement of only the action of Ins(1,3,4,5)P4 was also seen with GTP[S]-loaded R-Ras or Rap1a (two G-proteins known to interact with GAP1(IP4BP)), whereas H-Ras was inactive at similar concentrations. Guanosine 5'-[beta-thio]diphosphate (GDP[S]) did not alter the action of either Ins(2,4,5)P3 or Ins(1,3,4,5)P4. Finally, the addition of exogenous GAP1(IP4BP), purified from platelets, markedly enhanced the effect of Ins(1,3,4,5)P4, and again, the amount of Ca2+ mobilized by Ins(2,4,5)P3 alone was unaltered. We conclude that the increase in Ins(2,4,5)P3-stimulated Ca2+ mobilization by Ins(1,3,4, 5)P4 may be mediated by GAP1(IP4BP) or a closely related protein (such as GAP1(m)), and if so, the action of the GAP1 is not solely to regulate GTP loading of a G-protein, but rather it acts with a G-protein to cause its effect.

Asunto(s)

Calcio/metabolismo , Proteínas de Unión al GTP/fisiología , Fosfatos de Inositol/farmacología , Proteínas/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Proteínas Activadoras de GTPasa , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Ratones , Tapsigargina/farmacología , Tionucleótidos/metabolismo , Células Tumorales Cultivadas , Proteínas Activadoras de ras GTPasa , Proteínas ras/fisiología

RESUMEN

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a 'bell-shaped Ca2+ dependence'. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the 'tip-dip' technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 microM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 microM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free -Ca2+- to 4 microM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 microM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.

Asunto(s)

Canales de Calcio/fisiología , Calcio/fisiología , Activación del Canal Iónico , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Membrana Celular/fisiología , Sistema Libre de Células , Cerebelo/fisiología , Quelantes/farmacología , Citosol/fisiología , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Métodos , Ratas

RESUMEN

The intracellular receptor for inositol 1,4,5-trisphosphate (IP3) is responsible for generation and control of very complex Ca2+ signals. New experimental approaches to studying the kinetics of the IP3 receptor are now beginning to give some insight into the mechanisms behind its rather bizarre properties.

Asunto(s)

Canales de Calcio/fisiología , Calcio/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Transducción de Señal/fisiología , Animales , Canales de Calcio/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Modelos Moleculares , Receptores Citoplasmáticos y Nucleares/metabolismo

Asunto(s)

Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Proteínas Activadoras de GTPasa , Homeostasis , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas/química , Receptores Citoplasmáticos y Nucleares/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

The Met protein is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional growth factor with mitogenic, motogenic and morphogenic properties. A morphologically altered variant of the MDCK cell line, MDCK-1, spontaneously exhibits a number of features associated with a partial HGF/SF-Met induced phenotype (less adhesive colonies in culture, enhanced invasion and motility, nascent tubule formation), but paradoxically does not respond to HGF/SF treatment. Although the overall cell surface expression and distribution of Met were found to be similar in parental MDCK cells and the MDCK-1 cell line, p145met autophosphorylation (+/ HGF/SF) was significantly reduced in MDCK-1 cells in vitro and in vivo when compared with parental MDCK cells. In contrast, EGF induced cell proliferation and EGF receptor autophosphorylation to similar levels in both cell lines. The basal levels of protein tyrosine phosphorylation were higher in MDCK-1 cells when compared with parental MDCK cells, including that of two prominent proteins with molecular masses of approximately 185 kDa and 220 kDa. Moreover, both p185 and p220 are present and tyrosine phosphorylated in Met immunoprecipitates from MDCK-1 cells (+/-HGF/SF), but not parental MDCK cells. In addition, Met immunocomplexes from MDCK-1 cells exhibited an approximately 3-fold increased tyrosine kinase activity in vitro when compared with MDCK cells, correlating with the higher basal levels of total phosphotyrosine. Treatment of MDCK-1 cells with the tyrosine kinase inhibitor herbimycin A reverted the cell phenotype to a more MDCK-like morphology in culture, with a concomitant reduction in the tyrosine phosphorylation predominantly of p220. Taken together these data suggest that aberrations in Met activity and associated signalling render MDCK-1 cells insensitive to HGF/SF, and may also mediate alterations in MDCK-1 cell behaviour.

Asunto(s)

Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Benzoquinonas , Línea Celular , Movimiento Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Perros , Resistencia a Medicamentos , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Lactamas Macrocíclicas , Fenotipo , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-met , Quinonas/farmacología , Rifabutina/análogos & derivados , Transducción de Señal , Tirosina/metabolismo

RESUMEN

The solubilized partially purified Ins(1,4,5)P3-sensitive Ca2+ channel from rat cerebellum has been reconstituted into planar lipid bilayer membranes by the 'tip-dip' method [Ehrlich (1992) Methods Enzymol. 207, 463-471] allowing low noise current records. Single-channel events have been recorded. In the presence of 10 microM Ins(1,4,5)P3, 50 microM ATP, and 0.2 microM Ca2+ the Ins(1,4,5)P3 receptor channel opens to a conductance level of 53 pS. In the presence of 100 microM thimerosal (TMS), a sulphydryl-oxidizing agent, three subconductance levels (60 pS, 80 pS and 120 pS) were observed. More than one population of mean open times was found, both in the absence and presence of TMS, although TMS affected the length of the open time by decreasing the short opening significantly from 4.05 ms to 2.78 ms and increasing the longer open time from 27.8 ms to 94.8 ms. The results indicate that TMS enhances Ins(1,4,5)P3-induced Ca2+ release by both altering the open times of the channel significantly and causing a shift to higher subconductance levels.

Asunto(s)

Canales de Calcio/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Activación del Canal Iónico , Receptores Citoplasmáticos y Nucleares/metabolismo , Reactivos de Sulfhidrilo/farmacología , Timerosal/farmacología , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Cerebelo/química , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Membrana Dobles de Lípidos , Ratas

RESUMEN

We have previously found that for permeabilized L1210 cells, low micromolar concentrations of Ins(1,3,4,5)P4 added prior to Ins(2,4,5)P3 enhance the effects of suboptimal concentrations of Ins(2,4,5)P3 in causing Ca2+ release from InsP3-sensitive Ca2+ stores [Cullen, Irvine and Dawson (1990) Biochem J. 271, 549-553]. If this was due either to some conversion of added Ins(1,3,4,5)P4 into Ins(1,4,5)P3 by the 3-phosphatase, or to Ins(1,3,4,5)P4 acting as a weak (or partial) agonist on the InsP3 receptor it would be expected that,in the presence of thimerosal to sensitize the InsP3 receptor, the dose-response curve to Ins(1,3,4,5)P4 would be left-shifted by the same extent as that of Ins(1,4,5)P3. This was found not to be the case; the dose-response curve to Ins(1,3,4,5)P4 was not shifted at all by thimerosal. Furthermore, L-Ins(1,3,4,5)P4, which can displace radiolabelled D-Ins(1,3,4,5)P4 but not D-Ins(1,4,5)P3 from their respective high-affinity binding sites, mimicked the effects of D-Ins(1,3,4,5)P4 in enhancing the slow phase of Ins(2,4,5)P3-stimulated Ca2+ release. Ins(1,3,4,5)P4 caused an increase in magnitude of the slow phase of InsP3-stimulated Ca2+ release leaving the magnitude of the fast phase unaltered, in contrast to increasing Ins(2,4,5)P3 concentrations which increased the size of both phases. In addition, Ins(1,3,4,5)P4 decreased the rate constant for the slow phase of Ca2+ release. These findings point strongly to the conclusion that InsP4 is not working directly via the InsP3 receptor but indirectly via an InsP4 receptor.

Asunto(s)

Canales de Calcio/metabolismo , Calcio/metabolismo , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/farmacología , Leucemia L1210/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Sitios de Unión , Canales de Calcio/efectos de los fármacos , Electroporación , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Ratones , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Timerosal/farmacología , Células Tumorales Cultivadas

RESUMEN

The modulating effect of calcium cell signalling agonists on tissue growth was studied in a rabbit lens cell line (NN1003A). Calcium mobilisation was measured after Fura-2 incorporation and growth assayed either by direct Coulter counting or [3H]-thymidine incorporation. Transient increases in cytoplasmic calcium were elicited by rabbit serum, histamine, ATP and PDGF. Thapsigargin induced a prolonged increase and all of the above agonists failed to elicit a response after thapsigargin. Rabbit serum and PDGF both increased cell growth in a concentration-dependent manner. While histamine and ATP had little effect in serum-free medium, they reduced serum-stimulated growth. Acetylcholine and FGF did not produce a marked rise in cytoplasmic calcium and neither did they modulate growth. Both thapsigargin and caffeine greatly inhibited growth. These findings indicate that, in lens cells, agonists that mobilise calcium, whether by acting through G-protein or tyrosine kinase receptors, also modulate lens cell growth. Agents such as thapsigargin and caffeine that inactivate the same calcium store also inhibit growth.

Asunto(s)

Calcio/metabolismo , Cristalino/citología , Acetilcolina/farmacología , Adenosina Trifosfato/farmacología , Animales , Cafeína/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular/citología , Línea Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Factores de Crecimiento de Fibroblastos/farmacología , Fura-2 , Inhibidores de Crecimiento/farmacología , Cristalino/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Conejos , Terpenos/farmacología , Tapsigargina

RESUMEN

The rate of unidirectional efflux of 45Ca from rat liver microsomal vesicles loaded with 45Ca and then treated with thapsigargin is not inhibited by increased [Ca2+] in the external medium, although the net efflux rate is substantially inhibited. We have used this property to measure the electrochemical gradient of Ca2+ from the inside to the outside of the vesicles at a series of Ca2+ loadings, by measuring the external [Ca2+]free at which there is zero net efflux. At a loading of 7.9 +/- 0.6 nmol/mg of microsomal protein, the apparent internal [Ca2+]free is 21 +/- 1.6 microM. As the loading is increased, the internal [Ca2+]free increases linearly up to a value of 47 +/- 3.6 microM at a loading of 21.6 +/- 1.6 nmol/mg. Using a similar technique, the value for [Ca2+]free in the endoplasmic reticulum of permeabilized L1210 cells was found to be 12.5 microM.

Asunto(s)

Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Hígado/metabolismo , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Permeabilidad de la Membrana Celular , Digitonina , Retículo Endoplásmico/efectos de los fármacos , Cinética , Leucemia L1210/metabolismo , Leucemia L1210/patología , Masculino , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Ratas , Terpenos/farmacología , Tapsigargina , Células Tumorales Cultivadas

RESUMEN

Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) is produced rapidly from inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in stimulated cells. Despite extensive experimentation, no clearly defined cellular function has yet been described for this inositol phosphate. Binding sites specific for Ins(1,3,4,5)P4 have been identified in several tissues, and we have purified one such protein to homogeneity. Its high affinity for Ins(1,3,4,5)P4, and its exquisite specificity for this isomeric configuration, suggest it may be an Ins(1,3,4,5)P4 receptor. Here we report the cloning and characterization of this protein as a GTPase-activating protein, specifically a member of the GAP1 family. In vitro it shows GAP activity against both Rap and Ras, but only the Ras GAP activity is inhibited by phospholipids and is specifically stimulated by Ins(1,3,4,5)P4.

Asunto(s)

Fosfatos de Inositol/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa , Humanos , Proteínas de Transporte de Membrana/química , Datos de Secuencia Molecular , Fosfolípidos/metabolismo , Unión Proteica , Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rap , Proteínas Activadoras de ras GTPasa , Proteínas ras/metabolismo

RESUMEN

The role of the ras oncogene in the signalling pathway triggered by platelet-derived growth factor BB (PDGF-BB) has been investigated in a cell line which normally differentiates into myotubes. Following the activation of the N-ras oncogene, however, the cells proliferate and form foci. PDGF-BB stimulated the phosphorylation of tyrosine in several cellular proteins of molecular weight 185, 160, 94, 54, 44, 42 kDa and furthermore Ca2+ was released from internal stores. Activation of the N-ras gene by treatment of cells with dexamethasone (DEX) inhibited these responses to PDGF-BB. On the other hand, both ras-induced and -non induced cells responded to bradykinin (BK), foetal calf serum (FCS) and ionomycin (ION) by releasing Ca2+ from intracellular stores. The inhibition of the response to PDGF-BB in ras-activated cells has been further investigated. The binding of [125I]-PDGF-BB to its receptors was low and western blotting showed a low level of PDGF-BB receptor protein. This was in marked contrast to the receptor number seen in cells grown in growth medium or fusion promoting medium. These results indicate that cells transformed with the N-ras oncogene fail to respond to platelet-derived growth factor and exhibit a very low level of PDGF receptors. This suggests a role for the ras oncogene in the earliest steps of the signalling pathway.

Asunto(s)

Calcio/metabolismo , Expresión Génica , Genes ras , Músculo Esquelético/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Animales , Becaplermina , Bradiquinina/farmacología , Línea Celular , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ionomicina/farmacología , Cinética , Ratones , Peso Molecular , Músculo Esquelético/efectos de los fármacos , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosforilación , Fosfotirosina , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Tirosina/análogos & derivados , Tirosina/análisis

RESUMEN

We have studied the effects of CoA and palmitoyl-CoA on Ca2+ movements and GTP-dependent vesicle fusion in rat liver microsomes. (1) Inhibition of membrane fusion by CoA depends on esterification of CoA to long-chain acyl-CoA using endogenous non-esterified fatty acids. (2) Binding of long-chain acyl-CoA to microsomal membranes is inhibited by BSA, which also relieves inhibition of membrane fusion. (3) Under conditions where acyl-CoA binding is inhibited, CoA causes increased Ca2+ accumulation, apparently by decreasing the Ca2+ leak rate. (4) Conversely, palmitoyl-CoA, in the presence of BSA, causes Ca2+ efflux. (5) The decrease in Ca(2+)-permeability caused by CoA does not depend on the presence of ATP or GTP, and is irreversible in the short term. (6) Using 14C-labelled CoA we show that CoA derivatives can be formed from endogenous components of microsomal membranes in the absence of ATP. (7) The results are interpreted in terms of a Ca(2+)-permeability which is controlled by CoA and/or long-chain acyl-CoA esters.

Asunto(s)

Acilcoenzima A/farmacología , Calcio/metabolismo , Coenzima A/farmacología , Microsomas Hepáticos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Membrana Celular/metabolismo , Masculino , Ratas

RESUMEN

The effect of thapsigargin on the activation by partial proteolysis of the plasma membrane Ca(2+)-ATPase was studied in intact human erythrocyte membranes and in the purified enzyme. The enzyme was maximally activated in the absence of thapsigargin within 1 min of exposure to trypsin. However, in the presence of thapsigargin maximal activation was achieved only after 5 min trypsin digestion. Thapsigargin did not alter the pattern of proteolysis as revealed by SDS-PAGE of the tryptic fragments, although it slowed down the rate of appearance of the fragments. Thapsigargin also enhanced the activation of the enzyme by calmodulin. These findings suggest that, although thapsigargin at low concentrations has no effect on the catalytic activity of the Ca(2+)-ATPase in vitro in the absence of calmodulin, it could interfere with its regulation in vivo.

Asunto(s)

ATPasas Transportadoras de Calcio/sangre , Membrana Eritrocítica/enzimología , Terpenos/farmacología , Sitios de Unión , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Calmodulina/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/metabolismo , Humanos , Técnicas In Vitro , Cinética , Terpenos/metabolismo , Tapsigargina , Tripsina/farmacología

RESUMEN

The specificity of the inositol 1,3,4,5-tetrakisphosphate binding protein purified from porcine platelets [Cullen et al. (1995) Biochem. J. 305, 139-143] was examined using all the isomers of myo-inositol tetrakisphosphate. From the relative potencies of these compounds it appears that phosphorylation of the 1, 3 and 5 positions is essential for high affinity binding, that there is some tolerance of phosphorylation of the 6-hydroxyl, but none of a phosphate in the 2-position, and that phosphorylation of the 4-hydroxyl has very little influence. The binding of Ins(1,3,4,5)P4 was not appreciably altered by physiological Mg2+ concentrations, and the pH dependence of binding under physiological conditions showed a decline from pH 5.5 to pH 9.0.

Asunto(s)

Plaquetas/metabolismo , Fosfatos de Inositol/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Unión Proteica , Receptores Citoplasmáticos y Nucleares/aislamiento & purificación , Porcinos

RESUMEN

A novel Ins(1,3,4,5)P4-binding protein has been purified to apparent homogeneity from solubilized membranes derived from pig platelets. It has a high affinity for Ins(1,3,4,5)P4 (Kd 6.3 +/- 0.4 nM), a Bmax of 2.5-6.0 nmol/mg of protein, and a high specificity for Ins(1,3,4,5)P4 [Kd values for Ins(1,3,4,5,6)P5, InsP6, GroPtdIns(3,4,5)P3, Ins(1,4,5)P3, Ins(3,4,5,6)P4 and L-Ins(1,3,4,5)P4 of 85.0 +/- 4.1 nM, 800.0 +/- 20.2 nM, 65.6 +/- 2.6 nM, > 10 microM, 793.3 +/- 55.6 nM and 81.0 +/- 5.9 nM respectively]. The protein has an apparent molecular mass of 104 kDa, suggesting that this peripheral tissue protein may be different from Ins(1,3,4,5)P4 binding proteins previously isolated from neuronal tissues.

Asunto(s)

Plaquetas/química , Plaquetas/ultraestructura , Receptores Citoplasmáticos y Nucleares/aislamiento & purificación , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Sitios de Unión , Plaquetas/metabolismo , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/aislamiento & purificación , Proteínas Sanguíneas/metabolismo , Membrana Celular/química , Membrana Celular/ultraestructura , Fosfatos de Inositol/sangre , Cinética , Unión Proteica , Receptores Citoplasmáticos y Nucleares/análisis , Solubilidad , Porcinos

Asunto(s)

ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Calcio/metabolismo , Terpenos/historia , Animales , Historia del Siglo XX , Humanos , Fosfatidilinositoles/metabolismo , Terpenos/farmacología , Tapsigargina