RESUMO
Recently, we characterized leptin receptors in bovine adrenal medullary cells (Yanagihara et al. 2000). Here we report the stimulatory effect of leptin on catecholamine synthesis in the cells. Incubating cells with leptin (10 nM) for 20 min increased the synthesis of 14C-catecholamines from [14C]tyrosine, but not from L-3,4-dihydroxyphenyl [3-14C]alanine. The stimulation of catecholamine synthesis in the cells by leptin was associated with the phosphorylation and activation of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis. The incubation of cells with leptin resulted in a rapid activation of the mitogen-activated protein kinases (MAPKs). An inhibitor of MAPK kinase, U0126, nullified the stimulatory effect of leptin on the synthesis of 14C-catecholamines. Leptin potentiated the stimulatory effect of acetylcholine on 14C-catecholamine synthesis, whereas leptin failed to enhance the phosphorylation and activation of tyrosine hydroxylase induced by acetylcholine. These findings suggest that leptin stimulates catecholamine synthesis via the activation of tyrosine hydroxylase by two different mechanisms, i.e., one is dependent on tyrosine hydroxylase phosphorylation mediated through the MAPK pathway and the second is independent of enzyme phosphorylation.
Assuntos
Medula Suprarrenal/metabolismo , Catecolaminas/biossíntese , Leptina/farmacologia , Acetilcolina/farmacologia , Medula Suprarrenal/citologia , Medula Suprarrenal/efeitos dos fármacos , Animais , Butadienos/farmacologia , Bovinos , Células Cultivadas , Di-Hidroxifenilalanina/metabolismo , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Ionomicina/farmacologia , Ionóforos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
We report here the characterization and functional roles of the leptin receptor (ObR) in bovine adrenal medullary cells. The plasma membranes isolated from bovine adrenal medulla showed a single class of specific binding sites of (125)I-leptin with an apparent K(d) of 6.6 nM and B(max) of 62 fmol/mg protein. ObRa but not ObRb mRNA was detected in bovine adrenal medulla by reverse transcriptase-polymerase chain reaction. Incubation of cultured adrenal medullary cells with leptin (3-30 nM) for 20 min resulted in a significant increase in [(14)C]catecholamine synthesis from [(14)C]tyrosine without any change in catecholamine secretion. These findings suggest that leptin stimulates catecholamine synthesis through its receptors in bovine adrenal medullary cells.
Assuntos
Medula Suprarrenal/metabolismo , Proteínas de Transporte/metabolismo , Leptina/metabolismo , Receptores de Superfície Celular , Medula Suprarrenal/citologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Radioisótopos do Iodo , Dados de Sequência Molecular , Receptores para Leptina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
A method for simultaneous measurement of tyrosine hydroxylase (TH) activation and phosphorylation in permeabilised and intact bovine adrenal chromaffin cells (BACCs) was established. Permeabilised cells were stimulated with cyclic AMP (1--10 microM) in the presence of [32P]ATP and L-[carboxyl-(14)C]tyrosine. Intact BACCs were preincubated with 32P(i) for 3 h and stimulated with forskolin (1--5 microM) in the presence of L-[carboxyl-(14)C]tyrosine. On stimulation each well was covered with a sealed 'chimney' fitted with a small plastic cup containing 300 microl of 1.0 M NaOH that trapped the 14CO(2) released. TH activity was determined by measuring 14C radioactivity. TH phosphorylation was measured in the same cells by separating the solubilized proteins on SDS PAGE followed by autoradiography and/or HPLC analysis. It was found that H89, a protein kinase A inhibitor, significantly blocked both TH phosphorylation and activation in response to cyclic AMP in permeabilised cells. However, in intact cells, H89 was effective only in respect to forskolin-stimulated TH activity and did not block the forskolin-stimulated TH phosphorylation of Ser-40. The reason(s) for this lack of correlation between TH activation and phosphorylation is presently not understood.
Assuntos
Medula Suprarrenal/enzimologia , Bioensaio/métodos , Células Cromafins/enzimologia , Sulfonamidas , Tirosina 3-Mono-Oxigenase/metabolismo , Medula Suprarrenal/citologia , Animais , Bioensaio/instrumentação , Catecolaminas/biossíntese , Bovinos , Células Cultivadas/citologia , Células Cultivadas/enzimologia , Células Cromafins/citologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Digitonina/farmacologia , Inibidores Enzimáticos/farmacologia , Indicadores e Reagentes/farmacologia , Isoquinolinas/farmacologia , Permeabilidade/efeitos dos fármacos , Fosforilação , Solubilidade/efeitos dos fármacosRESUMO
The aim of this study was to determine the effect of angiotensin II (AII) on tyrosine hydroxylase (TOH) activity and phosphorylation in bovine adrenal chromaffin cells (BACCs). We report here that stimulation of BACCs with AII (100 nM) produced a significant increase in both TOH activity and phosphorylation over a period of 10 min. The increase in TOH activity was receptor-mediated. Tryptic phosphopeptide analysis by HPLC revealed that AII stimulated an increase in phosphorylation of three sites on TOH, Ser19, Ser31, and Ser40, with the largest increase being observed for Ser31 phosphorylation. Pretreatment of the cells with the protein kinase C inhibitor Ro 31-8220 (10 microM, 15 min) did not affect TOH activity or phosphorylation produced by AII. The inhibitor also did not affect the TOH activity or Ser40 phosphorylation produced by forskolin (10 microM, 10 min). In contrast, Ro 31-8220 fully inhibited the TOH activation as well as Ser31 and Ser40 phosphorylation of TOH produced by phorbol 12,13-dibutyrate (500 nM, 10 min). Removal of extracellular Ca2+ from the incubation medium inhibited the AII-induced TOH activity by 50% and significantly blocked Ser19 and Ser31 phosphorylation but did not affect Ser40 phosphorylation in response to AII. These results indicate that AII activates a complex and perhaps novel signaling pathway leading to the phosphorylation and activation of TOH. The TOH activation by AII appears to be partially independent of Ser40 phosphorylation, suggesting a potentially important role for Ser31 phosphorylation.
Assuntos
Glândulas Suprarrenais/enzimologia , Angiotensina II/metabolismo , Células Cromafins/enzimologia , Serina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Células Cromafins/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/metabolismo , Indóis/farmacologia , Vias Neurais/fisiologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Receptores de Angiotensina/fisiologia , Fatores de TempoRESUMO
The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [gamma-32P]ATP, in the presence or absence of 10 microM Ca2+, 1 microM cyclic AMP, 1 microM phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca2+ increased the phosphorylation of Ser19 and Ser40. Cyclic AMP, and phorbol dibutyrate in the presence of Ca2+, increased the phosphorylation of only Ser40. Ser31 and Ser8 were not phosphorylated. The Ca2+-stimulated phosphorylation of Ser19 was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273-302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca2+-stimulated phosphorylation of Ser40 was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5-22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19-31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser19 and Ser40 were dephosphorylated by PP2A.
Assuntos
Glândulas Suprarrenais/enzimologia , Células Cromafins/enzimologia , Digitonina/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismo , Glândulas Suprarrenais/citologia , Animais , Cálcio/farmacologia , Bovinos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases , Proteínas/metabolismoRESUMO
The effects of the protein kinase C inhibitor CGP 41251 (31-benzoyl-staurosporine) on nicotinic responses of cultured bovine adrenal chromaffin cells have been investigated. CGP 41251 inhibited tyrosine hydroxylase activation by phorbol 12,13-dibutyrate, with an IC50 of < 0.3 microM and complete inhibition at 1 microM. In contrast, it had little effect on nicotine-stimulated tyrosine hydroxylase activity up to 1 microM, and did not fully inhibit it even at 10 microM. From 1 to 10 microM, CGP 41251 caused a similar concentration-dependent inhibition of tyrosine hydroxylase activity stimulated by nicotine, K+, forskolin and 8-Br-cyclic AMP. CGP 42700 (19,31-dibenzoyl-staurosporine), a structural analogue of CGP 41251 that lacks activity as a protein kinase C inhibitor, had no effect on tyrosine hydroxylase activity stimulated by any of the agonists. CGP 41251 had no effect on catecholamine secretion induced by nicotine. The results suggest phorbol ester-sensitive protein kinase C isozymes do not play a major role in nicotinic stimulation of tyrosine hydroxylase activity or catecholamine secretion in chromaffin cells.
Assuntos
Células Cromafins/enzimologia , Proteína Quinase C/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Animais , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The radioligand [3H]quinuclidinyl benzilate has been used to examine the muscarinic receptor population of the chick expansor secundariorum muscle during its post-hatch development. At hatch the muscle bound 0.165 fmol of the ligand, but during the following two weeks binding decreased rapidly. Further reduction in ligand binding to the adult stage was observed, but at a much slower rate. The developmental loss of binding sites within this tissue corresponds to previously demonstrated reduction in acetylcholine sensitivity.
Assuntos
Envelhecimento , Músculo Liso/metabolismo , Receptores Colinérgicos/metabolismo , Receptores Muscarínicos/metabolismo , Animais , Galinhas , Cinética , Quinuclidinil Benzilato/metabolismoAssuntos
Axônios , Gânglios Autônomos/metabolismo , Monoaminoxidase/metabolismo , Norepinefrina/metabolismo , Nervo Isquiático/lesões , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Gânglios Autônomos/enzimologia , Histocitoquímica , Ligadura , Microscopia de Fluorescência , Regeneração Nervosa , Proteínas do Tecido Nervoso/metabolismo , Ratos , Transmissão SinápticaRESUMO
1. The effects of a single dose of 6-hydroxydopamine (6-OHDA) compared with those of chronic reserpine treatment were studied in lamb sympathetic neurones and adrenal medulla by a combination of fluorescence histochemistry, electron microscopy and radiochemical assay.2. In sympathetic ganglia, 6-OHDA produced a rise in noradrenaline concentration within 24 h, and falls in tyrosine hydroxylase and monoamine oxidase activities, whereas reserpine caused a fall in noradrenaline, a rise in tyrosine hydroxylase activity and no change in monoamine oxidase activity. The fluorescence of intra- and postganglionic axons increased greatly within 24 h of 6-OHDA, and there was a corresponding accumulation of large dense-core vesicles within many axons whose neurotubules were disrupted. The changes were almost reversed after 3 weeks.3. In the vas deferens, the concentration of noradrenaline and tyrosine hydroxylase and monoamine oxidase activities had all fallen 24 h after 6-OHDA treatment and had started to recover 3 weeks later. In the adrenal medulla, 6-OHDA did not alter NA concentrations but increased tyrosine hydroxylase activity whereas reserpine depleted noradrenaline and increased tyrosine hydroxylase activity.4. The changes produced in sympathetic ganglia by 6-OHDA may be due both to a direct action on the axoplasmic transport of noradrenaline containing vesicles and indirectly to the reaction of the neurones to loss of the integrity of their axons.