RESUMO
Membrane microviscosity, phospholipid composition, and turnover were measured in cultured vascular smooth muscle cells isolated from mesenteric arteries of stroke-prone spontaneously hypertensive and age-matched, normotensive Wistar-Kyoto rats. Membrane microviscosity, measured with fluorescence polarization, revealed greater microviscosity (lower fluidity) of the membranes isolated from smooth muscle cells from hypertensive as compared with those isolated from normotensive rats (p less than 0.01). Preincubation of membranes from hypertensive rats with 5 mM calcium reduced membrane microviscosity in "core" and in "surface" regions of the bilayer toward values observed in Wistar-Kyoto rats. Phospholipid composition did not differ between intact aortas and cultured mesenteric cells or between those tissues obtained from normotensive and from hypertensive rats. The total lipid-associated radioactivity was significantly lower in cells from stroke-prone spontaneously hypertensive rats than in those from Wistar-Kyoto controls (p less than 0.01). Phosphatidylcholine incorporated 70% and phosphatidylinositol 16% of total lipid-associated radioactivity, with no difference between cells from hypertensive and normotensive animals. Turnover of phosphatidylethanolamine was greater in cells from Wistar-Kyoto rats (p = 0.02), whereas turnover of phosphatidylserine was greater in cells from stroke-prone spontaneously hypertensive rats (p = 0.04). The greater microviscosity of the lipid bilayer in hypertension is a generalized defect of the matrix in which the transport proteins function. We hypothesize that this defect is responsible for the multiple abnormalities of membrane transport systems that have been described in genetic hypertension.
Assuntos
Membrana Celular/química , Hipertensão/genética , Lipídeos de Membrana/análise , Animais , Aorta , Plaquetas/química , Cálcio/farmacologia , Membrana Celular/ultraestrutura , Células Cultivadas , Hipertensão/patologia , Lipídeos de Membrana/metabolismo , Artérias Mesentéricas , Músculo Liso Vascular/metabolismo , Ácidos Fosfatídicos/análise , Fosfatidilcolinas/análise , Fosfatidiletanolaminas/análise , Fosfatidilinositóis/análise , Fosfatidilserinas/análise , Polarografia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Esfingomielinas/análise , Viscosidade/efeitos dos fármacosRESUMO
In unmodified synaptosomal brain membranes the presence of NaCl inhibited the binding to mu receptors of the tritiated opioid agonists etorphine, Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, and sufentanil by 53, 43, and 37%, respectively, and increased that of the antagonist [3H]naltrexone by 54%. On the other hand, in membranes whose microviscosity was increased by incorporation of cholesteryl hemisuccinate (CHS) the effects of sodium on opioid agonist and antagonist binding were abolished and strongly reduced, respectively. Furthermore, in the modified membranes the ability of sodium to protect the opioid receptor from inactivation by the sulfhydryl-reactive agent N-ethylmaleimide (NEM) was diminished. In CHS-treated membranes whose elevated microviscosity was reduced by the incorporation of oleic acid, the effectiveness of sodium in modulating opioid binding and attenuating receptor inactivation by NEM was restored. The results implicate membrane microviscosity in the mechanism by which sodium modulates the conversion between agonist- and antagonist-favoring states of mu opioid receptor.
Assuntos
Encéfalo/metabolismo , Encefalinas/metabolismo , Etorfina/metabolismo , Naltrexona/metabolismo , Receptores Opioides mu/metabolismo , Sufentanil/metabolismo , Animais , Ésteres do Colesterol/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina , Masculino , Fluidez de Membrana , Membranas/metabolismo , Ácido Oleico , Ácidos Oleicos/farmacologia , Ratos , Ratos Sprague-Dawley , Cloreto de Sódio/farmacologia , ViscosidadeRESUMO
In synaptosomal membranes from rat and monkey brain cortex, the addition of petroselenic (18:1, cis-delta 6) acid, oleic (18:1, cis-delta 9) acid, and vaccenic (18:1, cis-delta 11) acid or their corresponding methyl esters at 0.5 mumol/mg of membrane protein caused a similar 7-10% decrease in the microviscosity of the membrane core, whereas at the membrane surface the microviscosity was reduced 5-7% by the fatty acids but only 1% by their methyl esters. Concomitantly, the fatty acids, but not the methyl esters, inhibited the specific binding of the tritiated mu-, delta-, and kappa-opioids Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE), and U69,593, respectively. As shown with oleic acid, the sensitivity of opioid receptor binding toward inhibition by fatty acids was in the order delta greater than mu much greater than kappa, whereby the binding of [3H]DPDPE was abolished, but significant inhibition of [3H]U69,593 binding, determined in membranes from monkey brain, required membrane modification with a twofold higher fatty acid concentration. Except for the unchanged KD of [3H]U69,593, the inhibition by oleic acid involved both the Bmax and affinity of opioid binding. Cholesteryl hemisuccinate (0.5-3 mumol/mg of protein), added to membranes previously modified by fatty acids, reversed the fluidization caused by the latter compounds and restored inhibited mu-, delta-, and kappa-opioid binding toward control values. In particular, the Bmax of [3H]-DPDPE binding completely recovered after being undetectable.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Benzenoacetamidas , Encéfalo/metabolismo , Antagonistas de Entorpecentes , Sinaptossomos/metabolismo , Viscosidade , Animais , Ésteres do Colesterol/farmacologia , Ésteres/farmacologia , Ácidos Graxos Monoinsaturados/farmacologia , Fluidez de Membrana , Membranas/metabolismo , Pirrolidinas/metabolismo , Propriedades de SuperfícieRESUMO
Naturally occurring human insulin receptor mutants Ser1200 and Thr1134, and a site-directed mutant Arg1030 overexpressed in Chinese hamster ovary (CHO) cells, bind insulin with affinities identical to wildtype receptors but are apparently kinase deficient. Cells expressing the Ser1200 receptor exhibit insulin stimulation of glycogen synthesis similar to these bearing the wildtype receptor, but fail to mediate insulin-responsive DNA synthesis. In contrast, the Thr1134 and Arg1030 mutants exhibit no response to insulin. The activity of Mitogen Activated Protein (MAP) kinase in cells transfected with wildtype receptor is more responsive to insulin than that detected in untransfected parental cells, while cells bearing any of the mutant receptors are less responsive than the parental cells. These differences in the stimulation of MAP kinase activity are paralleled by differences in insulin-dependent phosphorylation of the enzyme. These results suggest that the p42 MAP kinase is not universally required for the metabolic effects of insulin.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Insulina/farmacologia , Mutação , Receptor de Insulina/biossíntese , Animais , Células CHO , Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Fosforilação , Receptor de Insulina/genética , Proteínas Recombinantes/biossíntese , Tirosina/metabolismoRESUMO
The Eph/Eck subfamily of receptor protein tyrosine kinases is currently the largest subfamily of receptor protein tyrosine kinases with a dozen members (Van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. Rev. Cell Biol. 10, 251-337). Using the cytoplasmic domain of Eck as bait in a yeast two-hybrid screen of mouse embryonic and T-cell cDNA libraries, it was discovered that the p85 subunit of phosphatidylinositol 3-kinase bound Eck. Further, using glutathione S-transferase fusion proteins, it was found that the C-terminal src homology 2 domain of the p85 subunit specifically interacted with Eck. Additionally, Eck coimmunoprecipitated with p85 in ligand activated cells confirming their interaction in vivo. In keeping with the above observations, activation of Eck by its ligand, B61, increased phosphatidylinositol 3-kinase activity. This is the first description of a signal transduction pathway initiated by any member of the Eph/Eck family.
Assuntos
Proteínas de Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Clonagem Molecular , Primers do DNA , DNA Complementar/metabolismo , Embrião de Mamíferos , Ativação Enzimática , Expressão Gênica , Biblioteca Gênica , Glutationa Transferase/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Músculo Liso Vascular/enzimologia , Fosfatidilinositol 3-Quinases , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Ratos , Receptor EphA2 , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Linfócitos T/enzimologia , Transcrição GênicaRESUMO
Although the insulin-dependent hydrolysis of glycosyl-phosphatidylinositol (GPI) may play an important role in insulin action, an absolute requirement for this glycolipid has not been demonstrated. Human K562 cells were mutated to produce a cell line (IA) incapable of the earliest step in PI glycosylation, the formation of PI-GlcNAc. Another cell line (IVD) was deficient in the deacetylation of PI-GlcNAc to form PI-GlcN and subsequent mannosylated species. Each line was transfected with wild-type human insulin receptors. Similar insulin-stimulated receptor autophosphorylation was observed in all three lines, along with a nearly identical increase in the association of phosphorylated insulin receptor substrate 1 with endogenous PI 3-kinase. Both normal and GPI-defective lines also displayed a similar 2- to 3-fold increase in phosphorylation of the Shc protein and its association with growth factor receptor-bound protein 2 in response to insulin. In contrast to these results, striking differences were noted in insulin-stimulated glycogen synthesis. In normal cells, glycogen synthesis was significantly increased by insulin, whereas no insulin stimulation was observed in GPI-deficient IA cells, and only a trace of stimulation was detected in IVD cells. These results indicate that tyrosine phosphorylation produced by insulin is not dependent on GPI synthesis, and this effect is not sufficient to elicit at least some of the metabolic effects of the hormone. In contrast, GPI synthesis is required for the stimulation of glycogen synthesis by insulin in these cells. These findings support the existence of divergent pathways in the action of insulin.
Assuntos
Glicogênio/biossíntese , Glicosilfosfatidilinositóis/biossíntese , Insulina/farmacologia , Leucemia Eritroblástica Aguda/metabolismo , Receptor de Insulina/fisiologia , Linhagem Celular , Citometria de Fluxo , Glucose/metabolismo , Humanos , Cinética , Mutagênese , Fosforilação , Fosfotirosina , Receptor de Insulina/biossíntese , Receptor de Insulina/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais Cultivadas , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
Activation of the tyrosine kinase activity of the insulin receptor by autophosphorylation leads to phosphorylation of cellular substrates on tyrosine. Thus far, the best characterized is the insulin receptor substrate (IRS) 1, which has been proposed to serve as a docking protein for other molecules involved in signal transduction. A number of other proteins that become phosphorylated in response to insulin have been identified, some of which are reported to be tissue-specific. A 60 kDa phosphoprotein has been detected in adipocytes after insulin stimulation [Lavan and Lienhard (1993) J. Biol. Chem. 268, 5921-5928]. We have identified a protein of similar molecular mass in rat hepatoma cells transfected with the human insulin receptor. The 60 kDa protein in hepatoma cells is tyrosine-phosphorylated in response to insulin in a dose-dependent manner, with maximal phosphorylation occurring at 50 nM insulin. Although the dose-response of p60 phosphorylation mirrors that of IRS-1, the time course is slightly slower, with maximal phosphorylation observed 5 min after addition of insulin. Like the adipocyte protein, the 60 kDa protein detected in liver cells binds to the SH2 domain of the p85 regulatory subunit of phosphatidylinositol 3-kinase, but not to other SH2 domains. Binding of p60 to p85 is similar to the interaction between p85 and IRS-1 in that a tyrosine-phosphorylated peptide containing the YVXM motif can inhibit the association. The presence of this 60 kDa tyrosine-phosphorylated protein in adipocytes and hepatoma cells suggests that it represents another important intermediate in the insulin-receptor signal-transduction pathway.
Assuntos
Insulina/farmacologia , Fígado/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptor de Insulina/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Neoplasias Hepáticas Experimentais/metabolismo , Dados de Sequência Molecular , Peso Molecular , Peptídeos/química , Fosfatidilinositol 3-Quinases , Fosfoproteínas/química , Fosfotirosina , Ligação Proteica , Ratos , Transdução de Sinais , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The precise mechanism by which insulin regulates glucose metabolism is not fully understood. However, it is known that insulin activates two enzymes, phosphatidylinositol 3'-kinase (PI 3'-K) and mitogen-activated protein kinase (MAPK), which may be involved in stimulating the metabolic effects of insulin. The role of these enzymes in glucose metabolism was examined by comparing the effects of insulin, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in 3T3-L1 adipocytes. Treatment of the cells with PDGF or EGF for 5 min increased the MAPK activity 3-5-fold, while insulin treatment produced a 2.5-fold increase. The MAPK activity remained elevated for 1 h after either PDGF or insulin treatment. PDGF and insulin, but not EGF, caused a transient increase in the amount PI 3'-K activity coprecipitated with tyrosine phosphorylated proteins. Although PDGF and insulin caused a similar increase in the activities of these two enzymes, only insulin caused substantial increases in glucose utilization. Insulin increased the transport of glucose and the synthesis of lipid 4- and 17-fold, respectively, while PDGF did not affect these processes significantly. Glycogen synthesis was increased 15-fold in response to insulin and only 3-fold in response to PDGF. Thus, the activation of MAPK and PI 3'-K are not sufficient for the complete stimulation of glucose transport, lipid synthesis, or glycogen synthesis by hormones in 3T3-L1 adipocytes, suggesting a requirement for other signaling mechanisms that may be uniquely responsive to insulin.
Assuntos
Adipócitos/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Glucose/metabolismo , Glicogênio/biossíntese , Substâncias de Crescimento/farmacologia , Insulina/farmacologia , Lipídeos/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Células 3T3 , Adipócitos/efeitos dos fármacos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Radioisótopos de Carbono , Desoxiglucose/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Cinética , Camundongos , Fosfatidilinositol 3-Quinases , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fatores de TempoRESUMO
Insulin stimulates the activity of mitogen-activated protein kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual specificity kinase that phosphorylates MAPK on threonine and tyrosine. The potential role of MAPK activation in insulin action was investigated with the specific MEK inhibitor PD98059. Insulin stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6 myotubes (1.4-fold) was completely abolished by pretreatment of cells with the MEK inhibitor, as was the phosphorylation of MAPK and pp90Rsk, and the transcriptional activation of c-fos. Insulin receptor autophosphorylation on tyrosine residues and activation of phosphatidylinositol 3'-kinase were unaffected. Pretreatment of cells with PD98059 had no effect on basal and insulin-stimulated glucose uptake, lipogenesis, and glycogen synthesis. Glycogen synthase activity in extracts from 3T3-L1 adipocytes and L6 myotubes was increased 3-fold and 1.7-fold, respectively, by insulin. Pretreatment with 10 microM PD98059 was without effect. Similarly, the 2-fold activation of protein phosphatase 1 by insulin was insensitive to PD98059. These results indicate that stimulation of the MAPK pathway by insulin is not required for many of the metabolic activities of the hormone in cultured fat and muscle cells.