RESUMO
Sodium butyrate, an erythroid differentiation inducer and a histone deacetylase inhibitor, increases G alpha(i2) levels in differentiating K562 cells. Here we show that sodium butyrate induces G alpha(i2) gene transcription via sequences at -50/-36 and -92/-85 in the G alpha(i2) gene promoter. Both sequences contain core sequence motif for Sp1 binding; electrophoretic mobility shift as well as supershift assays confirmed binding to Sp1. Transcription from the G alpha(i2) gene promoter was also activated by two other histone deacetylase inhibitors, trichostatin A and Helminthsporium carbonium toxin (HC toxin), which also induce erythroblastic differentiation in K562 cells. However, hydroxyurea, a potent erythroid differentiation inducer in these cells, did not activate transcription from this gene promoter, indicating that promoter activation is inducer-specific. Mutations within the Sp1 sites at -50/-36 and -92/-85 in the G alpha(i2) gene promoter substantially decreased transcriptional activation by sodium butyrate, trichostatin A, or HC toxin. Transfection with constitutively activated ERKs indicated that this promoter can be activated through the MEK-ERK signal transduction pathway. Inhibition of the MEK-ERK pathway with U0126 or reduction in the expression of endogenous ERK with an antisense oligonucleotide to ERK significantly inhibited sodium butyrate- and HC toxin-induced transcription but had no effect on trichostatin A-induced transcription. Inhibition of the JNK and p38 MAPKs, using selective inhibitors, had no effect on sodium butyrate-induced transcription. In cells in which sodium butyrate induction of promoter activation had been inhibited by various concentrations of U0126, constitutively activated ERK2 reversed this inhibition. These results show that the MEK-ERK signal transduction pathway is important in butyrate signaling, which eventually converges in the cell nucleus.
Assuntos
Butiratos/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/genética , MAP Quinase Quinase Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Ativação Enzimática/genética , Humanos , Células K562 , Mutação , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Compostos de Sódio/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/deficiência , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Metabolismo dos Carboidratos , AMP Cíclico/farmacologia , Deleção de Genes , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ácido 3-Hidroxibutírico/sangue , Adenilil Ciclases/metabolismo , Amônia/sangue , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Privação de Alimentos , Glucagon/farmacologia , Glucose/biossíntese , Glucose/metabolismo , Glucose-6-Fosfatase/genética , Hipoglicemia/genética , Fígado/enzimologia , Camundongos , Camundongos Knockout , Nitrogênio/sangue , Fenótipo , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ureia/sangueRESUMO
The chronic myelogenous leukaemia cell line K562 can be triggered in culture to differentiate along the erythrocytic pathway in response to a variety of stimulatory agents. In the presence of sodium butyrate, these cells differentiate to erythroblasts and acquire the capability to synthesize haemoglobin. We used this cell system to study alterations in the levels of several G-protein subunits during the cell differentiation programme and to assess the involvement of G(i)alpha2 in this process. Western immunoblot analysis revealed the presence of G(s)alpha1, G(s)alpha2, G(i)alpha2, G(q)alpha, Galpha(12), Gbeta1 and Gbeta2 in K562 cells. G(o)alpha, G(z)alpha, Galpha(13) and Galpha(16) were not detected. Although the levels of several G-protein subunits were altered after treatment with sodium butyrate, the most striking change was the robust increase in the levels of G(i)alpha2, which was accompanied by an increase in the mRNA for G(i)alpha2. Inactivation of G(i)alpha2 by adding Bordetella pertussis toxin to the cultures inhibited erythroblastic differentiation by as much as 62%, as measured by haemoglobin accumulation. Furthermore, the addition of an oligonucleotide anti-sense to G(i)alpha2 inhibited the sodium butyrate-induced robust increase in G(i)alpha2 levels, decreasing it to the basal levels seen in control cells; this treatment decreased the erythroblastic differentiation of the cells (as measured by haemoglobin expression) by 50%. Taken together, these findings imply that increased levels of G(i)alpha2 contribute to the sodium butyrate-induced erythroblastic differentiation of K562 cells.
Assuntos
Eritroblastos/metabolismo , Eritroblastos/patologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Butiratos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Humanos , Células K562 , Oligonucleotídeos Antissenso/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Among various G-protein subunits identified in atrial and ventricular membranes from newborn and adult rabbits, the most remarkable developmental and tissue-specific differences were observed in the amounts of the alpha-subunit of Go (Go alpha) and the beta-subunit (G beta). Go alpha was abundant in atrial membranes especially from newborn rabbits but was barely detectable in ventricles. In contrast, G beta was present in both atrial and ventricular membranes. In both tissues the level of G beta-2 was much higher than that of G beta-1. Starting just after birth, the high levels of Go alpha in atria at term decreased gradually to about half of the amount in term animals, at 30 days of age, while the amount of G beta remained relatively constant until 26 days after birth. The levels of G beta in ventricles slowly declined during neonatal development. Similar developmental patterns were also observed when these parameters were studied in membranes isolated from cultured myocytes. Northern blot analysis showed the presence of mRNA for Go alpha-1A isoform in RNA samples from atria but not those from ventricles. The developmental expression of Go alpha-1A mRNA was somewhat different from that of Go alpha protein, whereas the time course of G beta mRNA expression was similar to that of G beta protein. Among other G-protein subunits tested, the amount of the splice variant of Gs protein, Gs alpha-1, in atrial as well as ventricular membranes increased with age of animals, while the amounts of Gs alpha-2, Gi alpha 2 and G alpha q/11 did not change significantly during development. These results indicate differential localization and tissue-specific developmental expression of G-protein subunits in rabbit heart.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Átrios do Coração/citologia , Ventrículos do Coração/citologia , RNA Mensageiro/metabolismo , Coelhos , Fatores de TempoRESUMO
The mRNA levels for Gs alpha and G beta in liver from various age groups of rabbits were assessed by Northern and dot-blot hybridization assays using cDNA and oligonucleotide probes. The mRNA levels for both Gs alpha and G beta exhibited a transient 30-35% decrease at 3-6 h after birth, followed by a 3- to 3.5-fold increase which peaked at 2 days after birth, then gradually declined to adult levels at 4-6 weeks. The changes in mRNA levels paralleled the changes in protein levels [previously measured by immunoblotting (Kawai and Arinze, 1991)] in the first 2 days after birth, but not in the later periods of development. Beyond day 3, the increase in protein levels persists, reaching maximal levels at 4 weeks and equalling adult levels while their mRNA levels decrease to about 50% of the levels at day 2. In vivo administration of dexamethasone to neonatal rabbits increased hepatic mRNA levels for both Gs alpha and G beta by about 50%; the effect was moderate compared to the 2- to 3-fold increase in corresponding protein levels. The transcription rate for the Gs alpha gene increased by only 32% at day 2 after birth compared to term. A 68% increase in transcription rate for this gene was observed after the dexamethasone treatment. Taken together, these data indicate that the developmental and glucocorticoid-regulated expression of Gs alpha- and G beta-subunits in neonatal liver is modulated at the transcriptional level.
Assuntos
Dexametasona/farmacologia , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Fígado/metabolismo , RNA Mensageiro/biossíntese , Fatores Etários , Animais , Animais Recém-Nascidos , Sequência de Bases , DNA Complementar/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Dados de Sequência Molecular , RNA Mensageiro/genética , Coelhos , Transcrição Gênica/efeitos dos fármacosRESUMO
The effect of dexamethasone administration in vivo on the steady-state levels of G-protein subunits in liver of neonatal rabbits was investigated using specific antibodies to each subunit as well as bacterial toxin-mediated ADP-ribosylation assays. Parallel measurements were also made of the activity of adenylyl cyclase, as influenced by a variety of activators. Dexamethasone administration modulated the levels of G-protein subunits in liver in an age-dependent and subunit-specific manner but not in 24-h-old newborns. The inductive effect of dexamethasone was observed in animals older than 24 h, the greatest effect being on 2- to 3-day-old neonates. In 48-h-old animals the alpha-subunits Gs alpha-1, Gs alpha-2, Gi alpha and the beta-subunit G beta increased 2.0-, 2.1-, 4.3- and 2.8-fold, respectively, compared to the control. The increases were much less for older animals. Dexamethasone treatment also modulated effector-mediated stimulation of adenylyl cyclase activity in vitro and mimicked its effects on G-protein levels; the greatest increase (approximately 2-fold) in the activation of adenylyl cyclase occurred in membranes isolated from 2- to 3-day-old animals. In older animals there was either no effect of dexamethasone or a decrease in activity. The degree of change in enzyme activity paralleled the change in the amount of Gs alpha rather than of Gi alpha or G beta. These results suggest development-dependent regulation of hepatic G-proteins by glucocorticoids.
Assuntos
Dexametasona/farmacologia , Proteínas de Ligação ao GTP/biossíntese , Fígado/metabolismo , Fragmentos de Peptídeos/biossíntese , Adenilil Ciclases/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/efeitos dos fármacos , Regulação da Expressão Gênica , Immunoblotting , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/ultraestrutura , Fragmentos de Peptídeos/efeitos dos fármacos , CoelhosRESUMO
Ontogeny of trimeric GTP-binding regulatory proteins (G-proteins) and their subunits in rabbit liver during neonatal development was studied, by using bacterial-toxin-catalysed ADP-ribosylation of membrane proteins, immunoblot analysis to quantify the alpha-subunit (alpha s and alpha i) of stimulatory (Gs) and inhibitory (Gi) G-protein and the beta-subunit, and reconstitution assay with cyc- membranes (from Gs-deficient variant of S49 lymphoma cell) to measure Gs activity. Under optimal conditions of ADP-ribosylation, little cholera-toxin substrate (alpha s) was detected in membranes from liver of neonatal animals up to 24 h of age. Thereafter ribosylatable alpha s proteins, i.e. 45 kDa (alpha s-1) and 52 kDa (alpha s-2) proteins, were increasingly evident, reaching maximal levels in membranes from animals aged 4-6 weeks. The concentrations of alpha s-1 and alpha s-2, as determined by immunoblotting, were 6.1 +/- 0.8 and 2.7 +/- 0.4 pmol/mg of protein respectively at birth, and did not change during 0-24 h after birth. Thereafter they gradually increased to maximal levels of 22.1 +/- 1.3 and 10.5 +/- 0.7 pmol/mg of protein for alpha s-1 and alpha s-2 respectively, within 6 weeks. The beta-subunit also showed a similar 3-4-fold increase during the same age span. In contrast, the pertussis-toxin substrate (alpha i) was clearly evident even in membranes from term animals and in all age groups studied. Its developmental pattern, as assessed by ADP-ribosylation, was the same as that determined by immunoblot analysis. The functional activity of Gs in cholate extracts of membranes exhibited similar developmental pattern to that of cholera-toxin-mediated labelling. This activity also paralleled the concentrations of alpha s as measured by immunoblotting. These results suggest differential expression of G-protein subunits in liver during neonatal development.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fígado/crescimento & desenvolvimento , Adenosina Difosfato Ribose/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Western Blotting , Membrana Celular/metabolismo , Toxina da Cólera/metabolismo , Feminino , Proteínas de Ligação ao GTP/isolamento & purificação , Fígado/metabolismo , Substâncias Macromoleculares , Masculino , Peso Molecular , Toxina Pertussis , Coelhos , Fatores de Virulência de Bordetella/metabolismoRESUMO
The effect of age on catecholamine regulation of hepatic glycogenolysis and on hepatic adenylate cyclase was studied in male rats up to 24 mo of age. Epinephrine and norepinephrine stimulated glycogenolysis in isolated hepatocytes at all age groups studied. Isoproterenol, however, stimulated glycogenolysis only at 24 mo. In isolated liver membranes, usual activators of adenylate cyclase increased the activity of the enzyme considerably more in membranes from 24-mo-old rats than in membranes from either 3- or 21-mo-old rats. The Mn2+-dependent activity of the cyclase was increased by 2.9-fold in 3-mo-old animals and approximately 5.7-fold in 24-mo-old rats, indicating a substantial age-dependent increase in the intrinsic activity of the catalytic unit. The density of the beta-adrenergic receptor, as measured by the binding of [125I]-iodocyanopindolol to plasma membranes, was 5-8 fmol/mg protein in rats aged 3-12 mo but increased to 19 fmol/mg protein in 24-mo-old rats. Computer-aided analysis of isoproterenol competition of the binding indicated a small age-dependent increase (from 30% at 3 mo to 43% at 24 mo) in the proportion of beta-receptors in the high-affinity state. These observations suggest that beta-receptor-mediated hepatic glycogenolysis in the aged rat is predicated upon increases in the density of beta-receptors as well as increased intrinsic activity of the catalytic unit of adenylate cyclase.
Assuntos
Adenilil Ciclases/metabolismo , Envelhecimento/metabolismo , Fígado/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animais , Ligação Competitiva , Separação Celular , Ativação Enzimática , Glicogênio/metabolismo , Iodocianopindolol , Isoproterenol/metabolismo , Fígado/citologia , Fígado/enzimologia , Masculino , Pindolol/análogos & derivados , Pindolol/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
Scatchard plots of the binding of [3H]Gpp(NH)p to rabbit and guinea pig liver plasma membranes were curvilinear, and could be resolved by computer-assisted non-linear regression analysis into two sites which exhibit high and low affinities. The two sites appear to be of equal capacities. The Kd values for binding to rabbit liver membranes were 5.6 +/- 0.5 nM and 133 +/- 20 nM for the high and low affinity sites, respectively. The Kd values for binding to similar sites on guinea pig liver membranes were 7.6 +/- 0.9 nM and 289 +/- 75 nM. In addition to Ns and Ni which have been identified in liver membranes, one or more of the other GTP-binding proteins may contribute to the observed binding.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanilil Imidodifosfato/metabolismo , Fígado/metabolismo , Adenilil Ciclases/metabolismo , Adenilil Imidodifosfato/farmacologia , Animais , Sítios de Ligação , Ácido Edético/farmacologia , Ativação Enzimática/efeitos dos fármacos , Nucleotídeos de Guanina/metabolismo , Guanilil Imidodifosfato/farmacologia , Cobaias , Técnicas In Vitro , Cinética , Fígado/efeitos dos fármacos , Masculino , Coelhos , Ensaio RadioliganteRESUMO
beta-Adrenergic receptors in guinea-pig liver plasma membranes were characterized by radioligand binding, using l-[3H]dihydroalprenolol ([3H]DHA), l-3-[125I]iodocyanopindolol ([125I]CYP) and dl-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2- one hydrochloride [( 3H]CGP-12177). The binding of both [125I]CYP and [3H]CGP-12177 to membranes exhibited high affinity (Kd = 3.5 +/- 0.2 pM for [125I]CYP and 0.75 +/- 0.10 nM for [3H]CGP-12177) and stereospecificity; the maximal binding sites were 130 +/- 15 and 137 +/- 8 fmoles/mg protein respectively. Catecholaminergic agonists competed for these binding sites in the order l-isoproterenol greater than l-epinephrine greater than l-norepinephrine, which is typical for beta 2-adrenergic receptors. The binding data are supported by parallel experiments on adenylate cyclase activation by catecholamines, and on antagonism of this activation by beta 1- and beta 2-selective blockers. The binding of [3H]DHA was excessive (Bmax = 21.4 pmoles/mg protein), exhibited low affinity (Kd = 34.6 nM), and lacked stereospecificity. When liver membranes were incubated at 50 degrees for 40 min in the presence of an agonist, l-isoproterenol, the binding of [3H]DHA to the heat-treated membranes exhibited high affinity (Kd = 1.07 +/- 0.17 nM) and the Bmax was reduced to 139 +/- 22 fmoles/mg protein. In such membranes, as opposed to native membranes, stereospecificity was evident and catecholaminergic agonists competed for the binding sites in the order typical for beta 2-adrenergic receptors. However, agonist competition of the binding to the heat-treated membranes could not be modulated by guanine nucleotides, indicating a loss of communication between the receptor and the guanine nucleotide regulatory protein.
Assuntos
Alprenolol/análogos & derivados , Di-Hidroalprenolol/metabolismo , Temperatura Alta , Fígado/metabolismo , Receptores Adrenérgicos beta/metabolismo , Adenosina Difosfato Ribose/metabolismo , Toxina Adenilato Ciclase , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Toxina da Cólera/farmacologia , Guanilil Imidodifosfato/farmacologia , Cobaias , Iodocianopindolol , Isoproterenol/farmacologia , Masculino , Pindolol/análogos & derivados , Pindolol/metabolismo , Propanolaminas/metabolismo , Receptores Adrenérgicos beta/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Adrenergic receptors in human liver plasma membranes were characterized by radioligand binding assays. The binding of [3H]dihydroalprenolol [( 3H]DHA) to partially purified membranes was rapid, of high affinity, saturable, and stereospecific. The binding of [125I]iodocyanopindolol to the same membranes was also saturable and stereospecific, but extremely slow, and at 37 C required about 6 h for equilibration. The maximum number of binding sites from six livers determined with these two beta-receptor ligands was 36-83 fmol/mg protein. Catecholaminergic agonists competed for these binding sites in the order typical for beta 2-adrenergic receptors. IPS 339 [(tertiarybutylamino-3-ol-2-propyl)oximino-9-fluorene hydrochloride], a beta 2-selective antagonist, was at least 3 orders of magnitude more potent in inhibiting the binding of [3H]DHA than the beta 1-antagonist, atenolol. Computer-aided analysis of the competition curves as well as Hofstee transformations of the binding data indicated the predominance of the beta 2-subtype. The GTP analog guanyl-5'-yl-imidodiphosphate, decreased the binding affinity of the agonist, l-isoproterenol, indicating the modulation of agonist-promoted coupling of the receptors to guanine nucleotide regulatory proteins. The maximum number of binding sites for the binding of [3H]prazosin and [3H]dihydroergocryptine were the same (60-70 fmol/mg protein), indicating that the majority of the alpha-receptors are of the alpha 1-subtype. Competition experiments with prazosin and yohimbine confirmed the predominance of the alpha 1-receptor subtype, although the presence of alpha 2-receptors cannot be completely ruled out. These results indicate that adrenergic receptors in human liver plasma membranes are predominantly of the beta 2- and alpha 1-subtypes.
Assuntos
Fígado/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/metabolismo , Idoso , Ligação Competitiva , Membrana Celular/metabolismo , Di-Hidroalprenolol/metabolismo , Di-Hidroergotoxina/metabolismo , Feminino , Guanilil Imidodifosfato/farmacologia , Humanos , Iodocianopindolol , Cinética , Masculino , Pessoa de Meia-Idade , Pindolol/análogos & derivados , Pindolol/metabolismo , Prazosina/metabolismo , Ensaio Radioligante , Simpatolíticos/metabolismo , Simpatomiméticos/metabolismo , Ioimbina/metabolismoRESUMO
[32P]ADP-ribosylation of membrane proteins catalyzed by either cholera toxin or pertussis toxin was markedly enhanced by NADP+. The effect was concentration dependent; with 20 microM [32P]NAD+ as a substrate maximal enhancement was obtained at a concentration of 0.5-1.0 mM NADP+ for rabbit and guinea-pig liver membranes and 0.1 mM NADP+ for human erythrocyte membranes. NADP+ appears to act by inhibiting the degradation of NAD+ by NAD+-glycohydrolase (NADase) present in membrane preparations, probably as an alternate substrate for the enzyme. Among inhibitors tested (NADP+, isonicotinic acid hydrazide, imidazole, nicotinamide, L-arginine methyl ester and HgCl2) to suppress the enzyme activity, NADP+ was the most effective and, at 10 mM, inhibited hepatic NADase activity by about 90%. The effect of NADP+ was much greater than that of other known effectors of ADP-ribosylation such as Mg2+ and phosphate, or the NADase inhibitors, isonicotinic acid hydrazide and isonicotinamide. In membranes which contain substantial activities of NADase the inclusion of NADP+ in the assay system is necessary to achieve maximal ADP-ribosylation of membrane proteins.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Toxina da Cólera/farmacologia , Proteínas de Membrana/metabolismo , NAD+ Nucleosidase/metabolismo , NADP/farmacologia , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Membrana Eritrocítica/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Cobaias , Humanos , Masculino , Cloreto de Mercúrio/farmacologia , NAD+ Nucleosidase/antagonistas & inibidores , CoelhosRESUMO
Guanine nucleotide-dependent modulation of agonist binding to the beta-receptor reflects coupling of the receptor to the nucleotide regulatory protein. Similarly, guanine nucleotide-dependent stimulation of adenylate cyclase can be used as an index of coupling between the regulatory protein and the catalytic unit of the cyclase. Using both approaches we have studied coupling in the beta-adrenergic receptor-adenylate cyclase system in rabbit liver during neonatal development. With [3H]dihydroalprenolol as ligand, the Bmax was relatively unchanged (200-300 fmol/mg of protein) between birth and end of day 1 and was similar to adult values. Guanyl-5'-yl imidodiphosphate-dependent shift in agonist (l-isoproterenol) competition curves was biphasic, decreasing from 10-fold in membranes isolated from animals at term to about 6-fold in membranes from 6-h-old neonates, and increasing progressively in older animals to a maximal measurable value of 42-fold in the adult. The ability of guanyl-5'-yl imidodiphosphate, GTP, GTP plus isoproterenol, NaF, or forskolin to activate adenylate cyclase was also biphasic and age-dependent. With Mn2+ the measured activity was not at any time greater than the activity at term. Pretreatment of membranes with cholera toxin resulted in differential levels of enhancement of adenylate cyclase activity wherein much lower enhancement was observed in membranes from neonatal animals. With [32P]NAD as substrate, cholera toxin-catalyzed ADP-ribosylation of membranes indicated development-dependent accumulation of Ns peptides. From these results we suggest that there is a decreased efficiency in the coupling of the beta-adrenergic receptor to hepatic adenylate cyclase in early neonatal life. The molecular basis for the biphasic nature of the coupling is presently unclear.
Assuntos
Adenilil Ciclases/metabolismo , Fígado/metabolismo , Receptores Adrenérgicos beta/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Membrana Celular/metabolismo , Toxina da Cólera/farmacologia , Di-Hidroalprenolol/metabolismo , Feto , Guanosina Trifosfato/farmacologia , Isoproterenol/farmacologia , Cinética , Fígado/crescimento & desenvolvimento , Masculino , Coelhos , Receptores Adrenérgicos beta/efeitos dos fármacosRESUMO
Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein.
Assuntos
Animais Recém-Nascidos/metabolismo , Carboxiliases/metabolismo , Mitocôndrias Hepáticas/enzimologia , Fosfoenolpiruvato Carboxilase/metabolismo , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Cobaias , Imunodifusão , Fosfoenolpiruvato Carboxilase/imunologia , Fosfoenolpiruvato Carboxilase/isolamento & purificação , Inanição/enzimologiaRESUMO
Glycogenolysis in hepatocytes isolated from fed guinea pigs was much more enhanced by the beta-agonist, isoproterenol, than by equimolar concentrations of the alpha-agonists, phenylephrine and norepinephrine. The stimulatory effects of catecholamines occurred with the following order of potency: isoproterenol greater than epinephrine greater than norepinephrine. This order of potency is characteristic of beta 2-adrenergic receptors. That beta 2-receptors are responsible for mediating catecholamine stimulation of glycogenolysis in guinea-pig hepatocytes was further deduced from the inhibition of agonist-stimulated glycogenolysis by beta-receptor sub-type-selective antagonists. Thus, IPS 339, a beta-antagonist which has higher affinity at beta 2-sites than at beta 1-sites, was three orders of magnitude more potent in inhibiting isoproterenol-stimulated glycogenolysis than either atenolol or practolol, both of which are beta 1-selective antagonists. The beta 2-agonists zinterol and procaterol also stimulated glycogenolysis in hepatocytes and their effects were inhibited by propranolol and IPS 339, but not by practolol. Furthermore, activation of phosphorylase in these hepatocytes by isoproterenol, epinephrine, and norepinephrine also occurred with the potency order expected for beta 2-receptors. These results are in sharp contrast to those obtained with rat hepatocytes and emphasize that species differences occur in the regulation of hepatic glycogenolysis by catecholamines.
Assuntos
Catecolaminas/farmacologia , Glicogênio Hepático/metabolismo , Receptores Adrenérgicos beta/fisiologia , Receptores Adrenérgicos/fisiologia , Animais , Epinefrina/farmacologia , Cobaias , Técnicas In Vitro , Isoproterenol/farmacologia , Masculino , Norepinefrina/farmacologia , Fenilefrina/farmacologia , Ratos , Ratos Endogâmicos , Receptores Adrenérgicos beta/efeitos dos fármacos , Especificidade da EspécieRESUMO
[3H]Dihydroalprenolol was used to study beta-adrenergic binding sites in plasma membranes isolated from rabbit liver. Specific binding was measured at 25 degrees C as the difference between total binding and binding in the presence of 2 microM dl-propranolol or 10 microM l-isoproterenol. Binding was saturable and stereoselective. The maximum number of binding sites (Bmax) was 434 +/- 41 fmol/mg of protein. The Kd for this binding as determined by Scatchard analysis was 1.39 +/- 0.09 nM. This value agreed well with the Kd value (1.27 +/- 0.12 nM) determined by kinetic analysis. The potency order for the displacement of bound [3H]dihydroalprenolol was isoproterenol greater than epinephrine greater than norepinephrine, indicative of beta 2-receptors. Use of beta 1- and beta 2-subtype-selective inhibitors also supported the interpretation that the binding characteristics are those of beta 2-receptors. Computer-aided analysis of this inhibition indicated that the beta-receptors in this membrane are predominantly, if not exclusively, of the beta 2-subtype. That these receptors are responsible for mediating catecholamine stimulation of hepatic glycogenolysis was deduced from the inhibition of agonist-stimulated glycogenolysis, in isolated hepatocytes, by beta-receptor subtype-selective antagonists. Thus, the hydrochloride of (t-butylamino-3-ol-2-propyl)oximino-9 fluorene, a beta-antagonist which has higher affinity at beta 2-sites than at beta 1-sites, was 3 orders of magnitude more potent in inhibiting isoproterenol-stimulated glycogenolysis than either atenolol or practolol, both of which are beta 1-selective antagonists. These results resemble the inhibition of [3H]dihydroalprenolol binding in plasma membranes. The glycogenolytic effects of catecholamines occurred with the potency order isoproterenol greater than epinephrine greater than norepinephrine. Thus, both by radioligand binding studies and by metabolic studies, the functional adrenergic receptor in the rabbit liver is shown to be of the beta 2-subtype.
Assuntos
Glicogênio Hepático/metabolismo , Fígado/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos/metabolismo , Animais , Ligação Competitiva , Fracionamento Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Di-Hidroalprenolol/metabolismo , Cinética , Masculino , CoelhosRESUMO
The capacity of foetal and neonatal liver to oxidize short-, medium- and long-chain fatty acids was studied in the guinea pig. Liver mitochondria from foetal and newborn animals were unable to synthesize ketone bodies from octanoate, but octanoylcarnitine and palmitoylcarnitine were readily ketogenic. The ketogenic capacity at 24 h after birth was as high as in adult animals. Hepatocytes isolated from term animals were unable to oxidize fatty acids, but at 6 h after birth production of 14CO2, acid-soluble products and acetoacetate from 1-14C-labelled fatty acids was 40-50% of the rates at 24 h. At 12 h of age these rates had already reached the 24 h values and did not change during suckling in the first week of life. The activities of hepatic fatty acyl-CoA synthetases, which were minimal in the foetus or at term, increased to maximal values in 12-24 h. The data show that the capacity for beta-oxidation and ketogenesis develops maximally in this species during the first 6-12 h after birth, and appears to be partly dependent on the development of fatty acid-activating enzyme.
Assuntos
Animais Recém-Nascidos/metabolismo , Ácidos Graxos/metabolismo , Fígado/metabolismo , Animais , Animais Lactentes , Coenzima A Ligases/metabolismo , Cobaias , Corpos Cetônicos/biossíntese , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Mitocôndrias Hepáticas/metabolismo , Oxirredução , Fosfoenolpiruvato/biossínteseRESUMO
Liver mitochondria isolated from fetal and newborn guinea pigs synthesized phosphoenolpyruvate at 4-6 nmol/min per mg protein with 2 mM malate, succinate, and alpha-ketoglutarate as substrates. These rates were 90-110% of that by adult liver mitochondria and were not substantially altered in the second half of gestation or within 24 h after birth. Both palmitoyl- and octanoylcarnitine were inhibitory to phosphoenolpyruvate synthesis in adult and fetal preparations, but free octanoate was inhibitory only in adult liver mitochondria.
Assuntos
Animais Recém-Nascidos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Fosfoenolpiruvato/biossíntese , Animais , Caprilatos/farmacologia , Carnitina/análogos & derivados , Carnitina/farmacologia , Cobaias , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Palmitoilcarnitina/farmacologia , Succinatos/metabolismoRESUMO
Gluconeogenesis from (U-14C)-lactate occurred in hepatocytes prepared from term fetuses which lack cytosolic phosphoenolpyruvate carboxykinase and was almost completely inhibited by 3-mercaptopicolinate but was relatively insensitive to amino-oxyacetate. 12 h after birth when up to 32% of the total hepatic phosphoenolpyruvate carboxykinase activity was detectable in the cytosol, glucose synthesis was increased 4.4-fold in hepatocytes from fasted neonates and was partially (37%) sensitive to amino-oxyacetate. In livers of fasted 24-hour-old neonates total phosphoenolpyruvate carboxykinase activity was distributed between the mitochondria and the cytosol in the ratio of 60:40. In hepatocytes prepared from such animals, amino-oxyacetate inhibited glucose synthesis by about 56%, suggesting that up to half of the carbon flow from lactate to glucose was via the formation of phosphoenolpyruvate in the mitochondria. These studies indicate an important role for mitochondrial phosphoenolpyruvate carboxykinase in neonatal gluconeogenesis.