RESUMO
Wortmannin is a fungal metabolite that so far has been shown to act as a selective inhibitor of phosphoinositide 3-kinase. It can therefore be used to investigate the convergence between two major cellular signalling systems: those involving G-protein-coupled receptors and those involving receptor tyrosine kinases. Importantly, wortmannin can enter intact cells, making whole-cell studies of the above signalling pathways possible.
Assuntos
Androstadienos/farmacologia , Fosfatidilinositóis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sondas Moleculares , Fosfatidilinositol 3-Quinases , WortmaninaRESUMO
A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Animais , Autorradiografia , Linhagem Celular , DNA/metabolismo , Temperatura Alta , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C3H , MutaçãoRESUMO
Chicken embryo fibroblasts (CEF) transformed with v-src were previously reported to revert to normal phenotype after the introduction of dominant-negative mutants of Fos or Jun, indicating that endogenous AP-1 activity is essential for the cellular transformation. The major changes in the expression levels of fos and jun family genes induced by v-src were the elevation of fra-2 and c-jun transcripts. We show here that extensive phosphorylation of the AP-1 component Fra-2 is a major qualitative change in v-src transformed CEF and that several Ser and Thr residues in a C-terminal region of Fra-2 (amino acids 266-323) are phosphorylated specifically. The induced kinase activity was detected at the position of 42 kDa by in gel kinase assay using the Fra-2 C-terminal region as a substrate, and it was identified as chicken ERK2. JNK1 and JNK2, other members of the MAP kinase family, were not significantly activated in v-src transformed CEF and Fra-2 was not a good substrate for JNKs. fra-2 promoter analysis indicated that this promoter activity is elevated in v-src transformed CEF via two AP-1 binding sites and CRE-like sequence. We propose that phosphorylation of Fra-2 by ERK2 converts it from an inefficient transcriptional activator to an active one and further that fra-2 expression is autoregulated in response to the phosphorylation status of its gene product.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Transformada , Embrião de Galinha , Ativação Enzimática , Antígeno 2 Relacionado a Fos , Genes src , Proteínas Quinases JNK Ativadas por Mitógeno , Proteína Quinase 1 Ativada por Mitógeno , Fosforilação , Fator de Transcrição AP-1/genéticaRESUMO
1. The changes in phosphorylase activity and glycogen synthetase I (active form) activity during perfusion of rat liver were studied together with their responses to added epinephrine and glucagon. 2. Phosphorylase activity of the liver from fed or fasted rats fell rapidly during perfusion, regardless of whether the perfusate was added with glucose or not. The addition of epinephrine or glucagon at the start or at 60 min of perfusion caused a prompt restoration of the initial high activity. Both glycogen breakdown and glucose liberation proceeded in parallel with the changes in phosphorylase activity. 3. The I-form of glycogen synthetase in the liver from fed rats increased rapidly when the concentration of perfusate glucose was raised to near 10 mM. This increase was promptly prevented by the addition of epinephrine or glucagon. In contrast, glycogen synthetase of the liver from fasted rats responded to neither glucose nor epinephrine (or glucagon) during perfusion. 4. Phosphorylase in the liver of fasted, adrenalectomized rats did not respond to a low concentration of epinephrine (3 - 10(-8) M) or glucagon (5 - 10(-9) M), but was increased by higher concentrations of the hormones. The treatment of adrenalectomized rats with hydrocortisone restored the response of liver phosphorylase to the low concentrations of the hormones. Thus, glucocorticoid plays a "permissive" role by increasing the affinity of liver phosphorylase to epinephrine or glucagon.
Assuntos
Epinefrina/farmacologia , Glucagon/farmacologia , Glicogênio Sintase/metabolismo , Hidrocortisona/farmacologia , Fígado/enzimologia , Fosforilases/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Jejum , Glicogênio/metabolismo , Fígado/efeitos dos fármacos , Perfusão , RatosRESUMO
1. Epinephrine-induced hyperglycemia was attenuated by the treatment of rats with pertussis vaccine, but this attenuation was abolished when endogenous insulin was suppressed by streptozotocin or anti-insulin serum. It was concluded that epinephrine-induced hyperglycemia was counterbalanced by the hypoglycemic action of insulin, the secretion of which was markedly potentiated in pertussis-sensitized rats. 2. Without epinephrine, no hypoglycemia developed in pertussis-sensitized rats despite the higher blood level of insulin. Tracer experiments with [14C,3H] glucose or [14C]bicarbaonate showed that, in pertussis-sensitized rats, more glucose was liberated into the blood from hepatic gluconeogenesis at the expense of hepatic glycogenesis, thereby accelerating the turnover of blood glucose. 3. Since this activation of hepatic glucose production was reduced by propranolol, a beta-adrenergic blocking agent, it is very likely that adrenergic beta-stimulation is, at least partly, responsible for the metabolic alterations observed in pertussis-sensitized rats.
Assuntos
Glicemia/metabolismo , Epinefrina/farmacologia , Insulina/fisiologia , Isoproterenol/farmacologia , Vacina contra Coqueluche/farmacologia , Estreptozocina/farmacologia , Animais , Bicarbonatos/metabolismo , Diabetes Mellitus/sangue , Diabetes Mellitus/induzido quimicamente , Coração/efeitos dos fármacos , Hipoglicemia/sangue , Lactatos/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Músculos/efeitos dos fármacos , Músculos/metabolismo , Miocárdio/metabolismo , Especificidade de Órgãos , RatosRESUMO
1. Epinephrine-induced increase in rat liver cyclic AMP in vivo was potentiated when the circulating insulin was suppressed by injection of anti-insulin serum or by induction of diabetes. Consequently, phosphorylase was activated, glycogen synthetase was inactivated and glycogen accumulation induced by glucose load was prevented by epinephrine in the insulin-deficient rats to a much larger extent than in normal rats. 2. Insulin lack was effective in potentiating epinephrine-induced increase in liver and muscule cyclic AMP even after the treatment of rats with theophylline; the potentiation could not be solely accounted for by the inhibition of cyclic AMP phosphodiesterase. Thus, it is likely that insulin lack enhaces epinephrine activation of adenylate cyclase. 3. Unlike epinephrine, glucagon increased liver cyclic AMP to essentially the same extent whether the rat was treated with anti-insulin serum or not. 4. Based on the difference in dose-response curves between normal and insulin-deficient rats, a possibility is discussed that there are two adenylate cylase in the liver with higher and lower affinities for epinephrine and that circulating insulin blocks the high affinity enzyme selectively.
Assuntos
AMP Cíclico/metabolismo , Epinefrina/farmacologia , Insulina/fisiologia , Fígado/metabolismo , Adenilil Ciclases/metabolismo , Tecido Adiposo/metabolismo , Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Diabetes Mellitus/induzido quimicamente , Diabetes Mellitus/metabolismo , Relação Dose-Resposta a Droga , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Anticorpos Anti-Insulina , Masculino , Músculos/metabolismo , Fosforilases/metabolismo , Ratos , Estreptozocina , Teofilina/farmacologiaRESUMO
Hepatic glycogen metabolism was studied in rats during the period of transition from the fed to fasted states. Glycogenic activity was measured in vivo based on the incorporation of [14C]glucose into liver glycogen. Its changes were almost parallel to the changes in glycogen synthase activity. Progressive accumulation of liver glycogen that occurred in the fed state was associated with a proportional increase in glycogenic activity. Within 4 h after the cessation of food intake, glycogenic activity showed a precipitous fall from the peak to its nadir without significant changes in glycogen content. Meanwhile, the glucose concentration in the portal vein decreased. Upon further development of fasting, glycogenic activity displayed a progressive regain, reciprocally as glycogen contents gradually decreased. The precipitous fall of glycogenic activity during the transition from the fed to fasted states was associated with a transient increase in plasma glucagon, and was partly overcome by the injection of anti-glucagon serum. It is concluded that the fall of portal venous concentration of glucose and secretion of glucagon act as a signal to initiate liver glycogen metabolism characteristic of the fasted or postabsorptive state.
Assuntos
Glicemia/metabolismo , Jejum , Glicogênio/metabolismo , Fígado/metabolismo , Animais , Relação Dose-Resposta a Droga , Glucagon/farmacologia , Glicogênio Sintase/metabolismo , Insulina/farmacologia , Fígado/enzimologia , Masculino , Fosforilases/metabolismo , Ratos , Fatores de TempoRESUMO
Insulin/dexamethasone/methylisobutylxanthine (hormones/IBMX) induce 3T3-L1 fibroblasts to differentiate into adipocytes. Our previous study suggested that pertussis toxin (IAP)-sensitive GTP-binding protein(s) (G-protein) is involved in the process of differentiation by hormones/IBMX, accompanied by c-fos induction. Northern blotting indicated that among the IAP-sensitive G-proteins, the levels of Gi2 alpha, Go alpha, and Gi3 alpha mRNA were decreased, increased and unchanged, respectively. Gi1 alpha was undetectable and IAP attenuated the decrease in Gi2 alpha mRNA level but did not affect the change in Go alpha mRNA level during the adipocyte differentiation. These results indicate that IAP-sensitive Gi2 alpha mRNA level is decreased during adipocyte differentiation. A combination of phosphatidylinositol-specific phospholipase C (PI-PLC) and IBMX induced c-fos expression in 3T3-L1 fibroblasts similar to that induced with hormones/IBMX. c-fos induced by both stimulators was also diminished by anti-inositolglycan antibody or anti-PI-PLC antiserum. Insulin stimulated the release of inositolproteoglycan and diacylglycerol from 3T3-L1 fibroblasts, which was suppressed by IAP treatment. These findings suggested that one of the pathways of adipocyte differentiation induced by hormones/IBMX occurs via the inositolglycan-specific PI-PLC cascade coupled to IAP-sensitive G-protein(s). Both activation of glycerophosphate dehydrogenase and stimulation of insulin-dependent 2-deoxyglucose uptake induced by hormones/IBMX were enhanced in protein kinase C-depleted cells exposed to phorbol 12-myristate 13-acetate (PMA), and attenuated in IAP-treated cells. The level of a 32P-labeled 52 kDa protein in plasma membrane fractions immunoprecipitated by anti-PI-PLC antiserum was increased by PMA stimulation, abolished in PMA-treated cells, and increased in IAP-treated cells. These findings suggest that protein kinase C phosphorylates PI-PLC, resulting in a decrease in PI-PLC activity related to the signal transduction pathway of adipocyte differentiation of 3T3-L1 fibroblasts.
Assuntos
Adipócitos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Proteína Quinase C/metabolismo , Células 3T3 , Animais , Diferenciação Celular , Diglicerídeos/metabolismo , Regulação para Baixo , Proteínas de Ligação ao GTP/genética , Insulina/farmacologia , Camundongos , Toxina Pertussis , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , RNA Mensageiro/análise , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Cellular cyclic AMP formation in response to prostaglandin (PG) E1 was markedly potentiated by the chemoattractant formyl-Met-Leu-Phe (fMLP) in guinea pig neutrophils. This potentiation by fMLP was abolished by prior treatment of the cells with pertussis toxin, but not by the prevention of an fMLP-induced intracellular Ca2+ increase in the cells, indicating the direct involvement of the inhibitory GTP-binding protein (Gj), but not Ca2+, in the fMLP-induced potentiation of cyclic AMP formation. Cyclic AMP formation in the neutrophils was also unique in response to forskolin; the diterpene inhibited cyclic AMP formation stimulated by PGE1 plus fMLP at low concentrations, but it slightly stimulated the basal and fMLP-induced cyclic AMP formation at high concentrations. Such a forskolin-induced inhibition was also observed in the adenylyl cyclase of the cell membranes and detergent extract therefrom only when the cyclase was activated by GTP or its nonhydrolyzable analogue (GTP gamma S). The forskolin-inhibitable activity could be affinity-purified from the GTP gamma S-treated cell membranes with a forskolin-agarose column. The cyclase appeared to be purified as a complex with the GTP gamma S-bound alpha subunit of the stimulatory GTP-binding protein (Gs alpha), but not with the beta gamma subunits, as judged from immunoblot analysis with specific antisera. The GTP gamma S-bound Gs alpha-stimulated cyclase activity was further enhanced by beta gamma, and this enhancement was again inhibited by forskolin. These results suggest that the GTP-bound Gs alpha produced by PGE1 receptor stimulation and the beta gamma subunits released from Gj by fMLP receptor stimulation were acting synergistically in the cyclic AMP formation of intact neutrophils.
Assuntos
Adenilil Ciclases/metabolismo , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Neutrófilos/metabolismo , Receptores Imunológicos/fisiologia , Receptores de Peptídeos/fisiologia , Alprostadil/fisiologia , Animais , Cálcio/metabolismo , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Colforsina/farmacologia , Ativação Enzimática , Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/metabolismo , Cobaias , Substâncias Macromoleculares , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Receptores de Formil Peptídeo , Transdução de Sinais , Fosfolipases Tipo C/metabolismoRESUMO
Chemical modification of amino groups in the molecule of islet-activating protein (IAP), pertussis toxin, resulted in differential modification of biological activities of the toxin estimated in vivo with rats. Acetamidination of epsilon-amino groups of 50% (or more) of lysine residues in the IAP molecule totally abolished the lymphocytosis-promoting activity, but exerted no effects on the epinephrine-hyperglycemia inhibitory activity, of the toxin. Both activities were abolished by acylation of 50% or more of the amino groups probably due to the destruction of the toxin's quarternary structure. In contrast, the subunit assembly of IAP was maintained after exhaustive acetamidination of its lysine residues. The ADP-ribosyltransferase (or NAD-glycohydrolase) activity of the A-promoter (the biggest subunit) of IAP, which is responsible for the principal action of the toxin, enhancing insulin secretory responses and thereby inhibiting epinephrine hyperglycemia, was not affected by acetamindination of lysine residues. Thus, the A-protomer moiety of IAP is not directly involved in, but the amino groups of lysine residues in other subunits are selectively essential for, the development of the toxin-induced lymphocytosis.
Assuntos
Toxinas Bacterianas/toxicidade , Linfocitose/induzido quimicamente , Acilação , Aminoácidos/análise , Animais , Toxinas Bacterianas/isolamento & purificação , Glicemia/metabolismo , Epinefrina/farmacologia , Hiperglicemia/metabolismo , Indicadores e Reagentes , Linfócitos/efeitos dos fármacos , Masculino , Toxina Pertussis , Ratos , Ratos Endogâmicos , Relação Estrutura-Atividade , Fatores de Virulência de BordetellaRESUMO
Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein composed of an A-protomer and a B-oligomer. There seem to be at least two molecular mechanisms by which IAP exerts its various effects in vivo and in vitro. On the one hand, some of the effects were not significantly affected by acetamidination of the epsilon-amino groups of the lysine residues in the molecule. These include the activities in vitro (1) catalyzing ADP-ribosylation of one of the membrane proteins directly, (2) enhancing membrane adenylate cyclase activity in C6 cells, (3) reversing receptor-mediated inhibition of insulin or glycerol release from pancreatic islets or adipocytes, respectively, and the activities in vivo (4) inhibiting epinephrine-induced hyperglycemia, (5) potentiating glucose-induced hyperinsulinemia, (6) reducing hypertension and increasing the heart rate in genetically hypertensive rats. These activities are concluded to develop as a result of ADP-ribosylation catalyzed by the A-protomer which is rendered accessible to its intramembrane substrate thanks to the associated B-oligomer moiety. Thus, neither the enzymic activity of the A-protomer nor the transporting activity of the B-oligomer needs free amino groups of the lysine residues in the IAP molecule. On the other hand, additional effects of IAP, such as (1) mitogenic, (2) lymphocytosis-promoting, (3) histamine-sensitizing, (4) adjuvant and (5) vascular permeability increasing, were markedly suppressed by acetamidination of the intrapeptide lysine residues. The free epsilon-amino group of lysine would play an indispensable role in the firm (or divalent) attachment of the B-oligomer of IAP to the cell surface that is responsible for development of these activities.
Assuntos
Toxinas Bacterianas/farmacologia , Adenosina Difosfato Ribose/metabolismo , Toxina Adenilato Ciclase , Animais , Toxinas Bacterianas/isolamento & purificação , Pressão Sanguínea/efeitos dos fármacos , Fenômenos Químicos , Química , Histamina/toxicidade , Imidoésteres , Indicadores e Reagentes , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Linfocitose/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos , Mitógenos , Toxina Pertussis , Coelhos , Ratos , Ratos Endogâmicos SHR , Relação Estrutura-Atividade , Fatores de Virulência de BordetellaRESUMO
Prostaglandin (PG) E2 binding protein, a putative PGE2 receptor, was purified 26-fold with 0.4% recovery from canine renal outer medullary membranes solubilized with 12% digitonin with the sequential use of a Superose 12, Wheat Germ Agglutinin (WGA) Affigel 10, DEAE-5PW and Ampholine column chromatographies. The final preparation retained the binding activity specific for PGE2, but lost most of the sensitivity to guanosine-5'-(gamma-thio)triphosphate (GTP gamma S). An antibody against alpha subunit of the inhibitory guanine nucleotide-binding protein (alpha Gi)1 and alpha Gi2 or that against common sequences of alpha subunit of guanine nucleotide-binding proteins (alpha G(common)) reacted at 41 kDa protein in the sample of each step of purification, but failed to do so in the final preparation. An antibody against alpha Gi3 or alpha Go had no effect. In fact, peaks of the binding activity and immunoreactivity for alpha Gi1,2 were chromatographically separated by isoelectric focusing. Moreover, antibodies against alpha G(common) or alpha Gi1,2, but not that against alpha Gi3 and alpha Go, precipitated PGE2 binding activity in the active fractions of WGA-Affigel 10 column chromatography. These results suggest that the PGE2 receptor is an acidic glycoprotein and that Gi1 or Gi2 is physically associated with the PGE2 receptor and dissociates from the receptor protein during purification procedures.
Assuntos
Dinoprostona/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Medula Renal/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Western Blotting , Cromatografia Líquida , Cães , Eletroforese em Gel de Poliacrilamida , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Focalização Isoelétrica , Testes de Precipitina , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/isolamento & purificação , Receptores de Prostaglandina ERESUMO
We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.
Assuntos
Citosol/química , Proteínas de Ligação ao GTP/metabolismo , Mastócitos/química , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , Difosfato de Adenosina/metabolismo , Animais , Western Blotting , Membrana Celular/química , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/efeitos dos fármacos , Proteínas de Ligação ao GTP/isolamento & purificação , Cinética , Sarcoma de Mastócitos , Camundongos , Células Tumorais CultivadasRESUMO
cDNA clones encoding three types of Gi alpha, the alpha subunit of GTP-binding protein (Gi1 alpha, Gi2 alpha, and Gi3 alpha), were isolated from a cDNA library of the guinea-pig lung. Nucleotide sequence analysis revealed a high degree of homology with other mammalian Gi alpha cDNAs. By RNA blot analysis, the expression pattern of Gi1 alpha was more tissue-specific than those of other types of Gi alphas in the guinea-pig tissues examined. While Gi2 alpha and Gi3 alpha mRNAs were ubiquitously expressed in all tissues examined, Gi1 alpha mRNA was mainly expressed in the brain, lung and kidney. These results suggest that each Gi alpha protein may have a different role.
Assuntos
Proteínas de Ligação ao GTP/biossíntese , Pulmão/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação ao GTP/análise , Cobaias , Dados de Sequência Molecular , RNA Mensageiro/análise , Alinhamento de SequênciaRESUMO
The mechanism of G protein beta gamma subunit (G beta gamma)-induced activation of the muscarinic K+ channel (KACh) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. G beta gamma and GTP-gamma S-bound alpha subunits (G alpha *'s) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg2+, G beta gamma activated the KACh channel in a concentration-dependent fashion. 10 nM G beta gamma almost fully activated the channel in 132 of 134 patches (98.5%). The G beta gamma-induced maximal channel activity was equivalent to or sometimes larger than the GTP-gamma S-induced one. Half-maximal activation occurred at approximately 6 nM G beta gamma. Detergent (CHAPS) and boiled G beta gamma preparation could not activate the KACh channel. G beta gamma suspended by Lubrol PX instead of CHAPS also activated the channel. Even when G beta gamma was pretreated in Mg(2+)-free EDTA internal solution containing GDP analogues (24-48 h) to inactivate possibly contaminating G i alpha *'s, the G beta gamma activated the channel. Furthermore, G beta gamma preincubated with excessive GDP-bound G o alpha did not activate the channel. These results indicate that G beta gamma itself, but neither the detergent CHAPS nor contaminating G i alpha *, activates the KACh channel. Three different kinds of G i alpha * at 10 pM-10 nM could weakly activate the KACh channel. However, they were effective only in 40 of 124 patches (32.2%) and their maximal channel activation was approximately 20% of that induced by GTP-gamma S or G beta gamma. Thus, G i alpha * activation of the KACh channel may not be significant. On the other hand, G i alpha *'s effectively activated the ATP-sensitive K+ channel (KATP) in the ventricular cell membrane when the KATP channel was maintained phosphorylated by the internal solution containing 100 microM Mg.ATP. G beta gamma inhibited adenosine or mACh receptor-mediated, intracellular GTP-induced activation of the KATP channel. G i alpha *'s also activated the phosphorylated KATP channel in the atrial cell membrane, but did not affect the background KACh channel. G beta gamma subsequently applied to the same patch caused prominent KACh channel activation. The above results may indicate two distinct regulatory systems of cardiac K+ channels by PT-sensitive G proteins: G i alpha activation of the KATP channel and G beta gamma activation of the KACh channel.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/fisiologia , Átrios do Coração/citologia , Parassimpatomiméticos/farmacologia , Canais de Potássio/fisiologia , Animais , Função Atrial , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Relação Dose-Resposta a Droga , Guanosina Trifosfato , Cobaias , Átrios do Coração/ultraestrutura , Ventrículos do Coração/citologia , Ventrículos do Coração/ultraestrutura , Toxina Pertussis , Canais de Potássio/efeitos dos fármacos , Fatores de Tempo , Tripsina/farmacologia , Função Ventricular , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.
Assuntos
Agonistas Adrenérgicos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Miocárdio/metabolismo , Canais de Potássio/metabolismo , Receptores Muscarínicos/metabolismo , Animais , Eletrodos , Etilmaleimida/farmacologia , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cobaias , Átrios do Coração/citologia , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Miocárdio/citologia , Toxina Pertussis , Canais de Potássio/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
alpha 1-Adrenergic receptor-mediated responses are overwhelming in adult rat hepatocytes. Inversely, beta-responses are predominant over alpha 1-responses in the hepatocytes that have been cultured at a low cell density (10(4) cells/cm2) for 24 h. The insulin-EGF-induced DNA synthesis in the beta-response-dominant hepatocytes was doubled by beta-agonists or cAMP-generating agents added far behind (16-20 h) the addition of insulin/EGF; i.e., immediately before the entry into the S-phase of the cell cycle. Agonists of alpha 1-adrenergic or other Ca2+, mobilising receptors added to the alpha 1-response-dominant hepatocytes increased DNA synthesis only if they were added within 1-2 h after the addition of insulin/EGF, at the early stage of G1-phase. Agonists of "non-dominant" receptors were rather antagonistic to agonists of "dominant" receptors. Thus, agonists of alpha 1-adrenergic (and other Ca2+ mobilising) receptors and agonists of beta-adrenergic (and other cAMP-generating) receptors acted as comitogens in their own particular manners in the presence of growth factors in hepatocytes in which the respective receptor functions were dominant.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Fígado/efeitos dos fármacos , Mitose/efeitos dos fármacos , Antagonistas Adrenérgicos alfa/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Contagem de Células/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Células Cultivadas , Fígado/citologia , Fígado/metabolismo , Mitógenos/farmacologia , Ratos , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/fisiologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The membrane fraction prepared from rat brain was incubated with 0.5 mM N-ethylmaleimide (NEM) for 10 min. 3H-labelled agonist binding to muscarinic, A1-adenosine, opiate and alpha 2-adrenergic receptors was markedly inhibited by this NEM treatment of membranes, which interfered with the subsequent ADP-ribosylation of endogenous G-proteins by pertussis toxin. This indicated that the toxin target cysteine residues of the G-protein were modified by NEM. The NEM-induced inhibition of agonist bindings was mostly reversed by reconstitution of the alpha-subunits of purified Gi or Go into the membranes. The NEM-induced inhibition, together with the reversal by the G alpha reconstitution, was due to changes in the relative number of high- to low-affinity receptors solely without change in the total (high- plus low-affinity) receptor number. Thus, in NEM-treated membranes endogenous G-proteins become uncoupled from receptors, which were coupled to either Gi alpha or Go alpha. Reconstitution of NEM pre-treated membranes showed that Go acted in preference to Gi in interaction with muscarinic receptors and vice versa in interaction with three other types of receptor. The possible involvement of Go in mediating phospholipase C activation and Gi in mediating adenylate cyclase inhibition is discussed.
Assuntos
Toxina Adenilato Ciclase , Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Toxina Pertussis , Receptores de Superfície Celular/metabolismo , Fatores de Virulência de Bordetella/metabolismo , Adenosina Difosfato Ribose/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Encefalinas/metabolismo , Etilmaleimida/farmacologia , Técnicas In Vitro , Ratos , Receptores de Superfície Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
To develop an AIDS vaccine for human use as well as a suitable animal model for AIDS research, we constructed a series of HIV-1/SIVmac chimeric viruses (SHIVs). We successfully generated a SHIV (designated as NM-3rN) having the HIV-1 env gene, which enabled the evaluation of the efficacy of HIV-1 Env-targeted vaccines in macaque monkeys instead of chimpanzees. Two NM-3rN derivatives (NM-3 and NM-3n) induced long-term anti-virus immunities without manifesting the disease. The monkeys vaccinated with NM-3 or NM-3n became resistant to a challenge inoculation with NM-3rN. Serum from a monkey vaccinated with NM-3 neutralized not only the parental HIV-1 (NL432), but also an antigenically different HIV-1 (MN). In vivo experiments confirmed the heterologous protection against an SHIV having the HIV-1 (MN) env. In addition to specific immunity including neutralizing antibodies and cytotoxic T lymphocyte activity, nonspecific immunity such as natural killer activity is associated with this protection. These data suggest that the live vaccine has the ability to protect individuals against various types of HIVs. These SHIVs should contribute to the development of future anti-HIV-1 live vaccines in humans.
Assuntos
Vacinas contra a AIDS , HIV-1/imunologia , Vírus Reordenados/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas contra a AIDS/toxicidade , Animais , Anticorpos Antivirais/biossíntese , Feminino , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Produtos do Gene nef/imunologia , Produtos do Gene vpr/imunologia , Genes env , Genes gag , Genes nef , Genes vpr , Anticorpos Anti-HIV/biossíntese , HIV-1/genética , Humanos , Células Matadoras Naturais/imunologia , Macaca fascicularis , Masculino , Testes de Neutralização , Vírus Reordenados/genética , Vírus da Imunodeficiência Símia/genética , Linfócitos T Citotóxicos/imunologia , Vacinação , Vacinas Atenuadas , Viremia/etiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Human polymorphonuclear leukocytes (PMN) express receptors for complement (C) C3b and C3bi termed CR1 and CR3, respectively. The addition of PMA or fMLP to PMN enhances the capacity of these receptors to promote binding of C3b- and C3bi-coated erythrocytes. fMLP-dependent increase of the binding of these ligand-coated erythrocytes was completely abolished by prior exposure of the PMN to pertussis toxin (IAP). GTP-binding protein (Gi alpha) was ADP-ribosylated and dysfunctional by this treatment. On the other hand, PMA-dependent binding of these ligands, as well as control binding, was inhibited only slightly, if at all, by the IAP treatment. The levels of C receptor expression on cell surface were determined by flow cytometry using monoclonal antibody against CR1 and those against the alpha and beta chains of CR3 (CR3 is composed of alpha and beta chain). Upon exposure of PMN to the chemotactic factor or PMA, or upon incubation of the cells at 37 degrees C, the surface expression of CR1 and CR3 alpha was increased. IAP also blocked an fMLP-induced increase of CR1 and CR3 alpha, but did not block the temperature- or PMA-dependent increase of these receptors. Opsonized zymosan (SOZ), another ligand for CR3, also led to an increase of both CR1 and CR3 alpha. Neither PMA nor SOZ brought about an increase of the surface expression of CR3 beta, but fMLP caused a slight increase of CR3 beta in an IAP-sensitive manner. Based on the IAP-sensitivity of the receptor expression, therefore, it appears that at least two separate mechanisms are operative in the control of C receptors. In addition, the alpha and beta chains of CR3 are regulated independently. The present data offer evidence suggesting that C receptor functions are in part regulated through a GTP-binding protein via modulation of their surface expression.