RESUMO
Efficient post-Golgi trafficking depends on microtubules, but actin filaments and actin-associated proteins are also postulated. Here we examined, by inverse fluorescence recovery after photobleaching, the role of actin dynamics in the exit from the TGN of fluorescent-tagged apical or basolateral and raft or non-raft-associated cargoes. Either the actin-stabilizing jasplakinolide or the actin-depolymerising latrunculin B variably but significantly inhibited post-Golgi traffic of non-raft associated apical p75NTR and basolateral VSV-G cargoes. The TGN-exit of the apical-destined VSV-G mutant was impaired only by latrunculin B. Strikingly, the raft-associated GPI-anchor protein was not affected by either actin toxin. Results indicate that actin dynamics participates in the TGN egress of both apical- and basolateral-targeted proteins but is not needed for apical raft-associated cargo.
Assuntos
Actinas/metabolismo , Rede trans-Golgi/metabolismo , Animais , Transporte Biológico Ativo , Células COS , Chlorocebus aethiops , Cicloeximida/farmacologia , Recuperação de Fluorescência Após Fotodegradação , Técnica Indireta de Fluorescência para Anticorpo , Microscopia Confocal , Microscopia de Vídeo , Inibidores da Síntese de Proteínas/farmacologia , Fatores de Tempo , Transfecção , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/fisiologiaRESUMO
Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/fisiologia , Caveolinas/metabolismo , Extensões da Superfície Celular/metabolismo , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Actinas/metabolismo , Actinas/ultraestrutura , Córtex Suprarrenal/efeitos dos fármacos , Corticosteroides/biossíntese , Corticosteroides/metabolismo , Hormônio Adrenocorticotrópico/efeitos dos fármacos , Animais , Caveolina 1 , Caveolinas/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/ultraestrutura , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Células Tumorais CultivadasRESUMO
Although the role of arachidonic acid (AA) in the regulation of steroidogenesis is well documented, the mechanism for AA release is not clear. Therefore, the aim of this study was to characterize the role of an acyl-CoA thioesterase (ARTISt) and an acyl-CoA synthetase as members of an alternative pathway in the regulation of the intracellular levels of AA in steroidogenesis. Purified recombinant ARTISt releases AA from arachidonoyl-CoA (AA-CoA) with a Km of 2 micro m. Antibodies raised against recombinant acyl-CoA thioesterase recognize the endogenous protein in both adrenal tissue and Y1 adrenal tumor cells by immunohistochemistry and immunocytochemistry and Western blot. Stimulation of Y1 cells with ACTH significantly stimulated endogenous mitochondrial thioesterases activity (1.8-fold). Nordihydroguaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochondrial acyl-CoA thioesterases. ACTH-stimulated steroid synthesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA-CoA synthetase. The apparent IC50 for NDGA was reduced from 50 micro m to 25, 7.5 and 4.5 micro m in the presence of 0.1, 0.5 and 2 micro m triacsin C, respectively. Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells.
Assuntos
Glândulas Suprarrenais/citologia , Ácido Araquidônico/química , Coenzima A Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Hormônios/metabolismo , Tioléster Hidrolases/metabolismo , Aciltransferases/metabolismo , Animais , Western Blotting , Colesterol/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Imuno-Histoquímica , Cinética , Masoprocol/farmacologia , Camundongos , Mitocôndrias/metabolismo , Proteínas Recombinantes/metabolismo , Triazenos/farmacologiaRESUMO
Arachidonic acid is not freely stored in the cells. A number of different pathways for the mobilization of this compound have been proposed, including a novel mechanism that involves the release of arachidonic acid from arachidonoyl-CoA by a thioesterase with substrate specificity for very-long-chain fatty acids. In rat heart, the acyl-CoA thioesterase activity can be regulated by a mechanism that involves beta-adrenoceptors. In this paper we demonstrate that beta-adrenergic agonists also regulate the acyl-CoA thioesterase mRNA levels. Isoproterenol (10(-7)M)-a concentration known to exert physiological responses-increases in a time-dependent manner the acyl-CoA thioesterase mRNA levels, an effect blocked by a specific beta-adrenoceptor antagonist. In addition, our results show that cAMP is involved in this process. The acyl-CoA thioesterase mRNA levels are also increased by fasting, but not by di(2-ethylhexyl)phthalate, a peroxisome proliferator. These results may suggest the existence of a beta-adrenoceptor-activated regulatory pathway for arachidonic acid release in cardiac tissue.
Assuntos
Ácidos Graxos/metabolismo , Coração/fisiologia , Receptores Adrenérgicos beta/metabolismo , Tioléster Hidrolases/biossíntese , Agonistas Adrenérgicos beta/farmacologia , Animais , Ácido Araquidônico/metabolismo , Northern Blotting , Western Blotting , Dactinomicina/farmacologia , Imuno-Histoquímica , Isoproterenol/farmacologia , Perfusão , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
Nitric oxide (NO) synthase (NOS) expression was analyzed in rat adrenal zona fasciculata. Both neuronal NOS and endothelial NOS mRNAs were detected by RT-PCR, immunohistochemistry, and immunoblot analysis. The biochemical characterization of adrenal zona fasciculata NOS enzymatic activity confirmed the presence of a constitutive isoform. In a cell line derived from mouse adrenal cortex, only endothelial NOS expression was detected by both RT-PCR and immunoblot analysis. Nitrate plus nitrite levels in Y1 cell incubation medium were increased in the presence of L-arginine and the calcium ionophore A23187, but not D-arginine, indicating enzymatic activity. Moreover, a low, but significant, conversion of Larginine to L-citrulline, abolished by the NOS inhibitor, N(G)-nitro-L-arginine, was detected in Y1 cells. The effect of L-arginine on pregnenolone production was examined. L-Arginine decreased both basal and ACTH-stimulated pregnenolone production in Y1 cells. The inhibitory effect of L-arginine could be attributed to endogenously generated NO, because it was blocked by N(G)-nitro-L-arginine, and it was mimicked by the addition of a NO donor, diethylenetriamine-NO. An inhibitory effect of NO on pregnenolone production from 22Rhydroxycholesterol and on steroidogenic acute regulatory protein expression was also determined. Taken together, these results suggest that at least part of the adrenal NO could derive from steroidogenic cells and modulate their function.