Compartmentalization of corticotropin-dependent steroidogenic factors in adrenal cortex: evidence for a post-translational cascade in stimulation of the cholesterol side-chain split.

Neher, R.; Milani, Alessandro; Solano, Ángela R.; Podestá, Ernesto J.

doi:10.1073/pnas.79.6.1727

Cited by 29 publications

(31 citation statements)

References 28 publications

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“…However, this signal seems not to be specific for the cell which contains these reports. In fact, this concept was demonstrated in a cellfree system, in which a signal mediated by the activation of the adrenocorticotropin receptor in adrenal cells, is able to promote steroidogenesis in mitochondria from Leydig cells [30]. In the present studies we confirm these findings.…”

Section: Resultssupporting

confidence: 81%

Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion

Podestá¹,

Solano²,

Vedia

et al. 1984

European Journal of Biochemistry

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Electrofusion of rat adrenal and Leydig cells generated hybrids capable of synthesizing simultaneously both testosterone and corticosterone, under stimulation of lutropin or adrenocorticotropin. Evidence was obtained indicating that under such circumstances, heterologous lutropin receptor -adrenal adenylate cyclase complexes were formed.Cell fusion has a great importance in the generation of heterocarions and hybrid cell lines [I -31. The technique has also been used to study the mobility of cell membrane markers and receptors [4, 51. Fusion of cells, in homogenous or heterogenous populations, is achieved in vitro by treating cell suspensions with inactivated sendai virus, poly(ethy1ene glycol) or lysophosphatidylcholine [2, 6-81. A main limitation of these 'fusogenic agents' is however, the low yield and the scarce viability of the hybrids thus obtained.Electrical-field-induced cell fusion has been recently proposed as an alternative to the use of the above-mentioned fusogenic agents [9 -111. The technique involves the orientation of cells under an asymmetric, alternating current field, a phenomenon called dielectrophoresis [I 2 -171 and the induction of the fusion process by an electrical breakdown pulse [9 -111. Evidence indicates that, with such a fusion procedure, yields of viable hybrids are several orders of magnitude higher than with methods using chemical fusogenic agents [9, 101. Since the electrofusion procedure has been only recently introduced to studies on animal cells [lo], there is not enough evidence available on the possibility of producing, with these methods, cell hybrids which can maintain highly specialized biochemical processes. For Friend cells it has been shown that highly specialized biochemical processes such as stimulation of haemoglobin synthesis in the presence of dimethyl sulfoxide, can be induced in electrofused cells [IS, 191. This paper shows that electrofusion of rat adrenal and Leydig cells produced hybrids bearing the capacity to be stimulated by lutropin or adrenocorticotropin, thus synthesizing both testosterone and corticosterone. MATERIALS AND METHODSLeydig cells were prepared by collagenase digestion of adult rat testis as described [20-211, and rat adrenocortical cells from the zona fasciculata-reticularis, were obtained by trypsin digestion [22]. The cells were resuspended in 0.3 M mannitol and electrofused by a modification of published procedures [9, 101. Fusion was performed on a perspex cylindrical chamber, having two parallel platinum electrodes (40 pm separating space). The chamber was mounted on a glass slide to allow microscope observation of pearl-chain formation and cell fusion. Two electrode wires were connected in parallel to a function generator (Health, model IG-1271), and to a home-made pulse generator. The first one was used to produce a high-frequency alternating field, resulting in cell dielectrophoresis and pearl-chain formation as described elsewhere [9, 10,12-141. The second one allowed the application of short constant voltage pulses to induce...

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Section: Resultssupporting

confidence: 81%

Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion

Podestá¹,

Solano²,

Vedia

et al. 1984

European Journal of Biochemistry

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“…This protein was detected during the different purification steps by its biological activity e.g. stimulation of steroid synthesis in an in vitro recombination assay [34].…”

Section: Discussionmentioning

confidence: 99%

“…Progesterone from the latter incubation plus progesterone from mitochondria alone was subtracted from the progesterone of the complete incubation [34].…”

Section: In Vitro Recombination Assaymentioning

confidence: 99%

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Purification of a Novel 43‐kDa Protein (p43) Intermediary in the Activation of Steroidogenesis from Rat Adrenal Gland

Paz

Dada

Maciel

et al. 1994

European Journal of Biochemistry

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In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAEcellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/ PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in v i m recombination assay. This activity is inhibited by the use of phospholipase A, inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDSPAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein.We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin-induced stimulation of arachidonic acid release and steroid synthesis.

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“…Male Wistar rats (90-day-old) were used throughout. Adrenal gland or ZF cells were obtained from animals supplied with dexamethasone (10 µg/ml, ad libitum) in the drinking water for 16 h before they were killed, as previously described (Neher et al 1982). Animals were killed by decapitation and adrenal glands were excised and kept on ice.…”

Section: Animalsmentioning

confidence: 99%

An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones

Maciel¹,

Maloberti²,

Neuman³

et al. 2005

Journal of Molecular Endocrinology

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We have described that, in adrenal and Leydig cells, the hormonal regulation of free arachidonic acid (AA) concentration is mediated by the concerted action of two enzymes: an acyl-CoA thioesterase (MTE-I or ARTISt) and an acyl-CoA synthetase (ACS4). In this study we analyzed the potential regulation of these proteins by hormonal action in steroidogenic cells. We demonstrated that ACS4 is rapidly induced by adrenocorticotropin (ACTH) and cAMP in Y1 adrenocortical cells. The hormone and its second messenger increased ACS4 protein levels in a time and concentration dependent way. Maximal concentration of ACTH (10 mIU/ml) produced a significant effect after 15 min of treatment and exerted the highest increase (3-fold) after 30 min. Moreover, 35 S-methionine incorporation showed that the increase in ACS4 protein levels is due to an increase in the de novo synthesis of the protein. On the contrary MTE-I protein levels in Y1 and MA-10 cells did not change after steroidogenic stimuli. In contrast with the effect observed on protein levels, stimulation of both cell lines did not change ACS4 RNA levels during the first hour of treatment, indicating that the effect of both stimuli is exerted at the level of ACS4 protein synthesis.StAR protein induction has a key role on the activation of steroidogenesis since this protein increases the rate of the limiting step of the whole process. In agreement with the fact that the inhibition of ACS4 activity by triacsin C blocks cAMP-stimulated progesterone production by MA-10 Leydig cells, here we demonstrated that ACS4 inhibition also reduces StAR protein levels. Moreover, exogenous AA was able to overcome the effect of triacsin C on both events, StAR induction and steroidogenesis. These results were confirmed by experiments using ACS4-targeted siRNA which result in a reduction in both ACS4 and StAR protein levels. The concomitant decrease in steroid production was overcome by the addition of AA to the knocked-out cells. In summary, this study suggests that in adrenal and Leydig cells the hormonal action prompts the synthesis of a labile protein, ACS4, which activity is involved in the regulation of AA release and is essential for steroidogenesis and StAR protein induction.

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Compartmentalization of corticotropin-dependent steroidogenic factors in adrenal cortex: evidence for a post-translational cascade in stimulation of the cholesterol side-chain split.

Cited by 29 publications

References 28 publications

Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion

Production of steroid hormone and cyclic AMP in hybrids of adrenal and Leydig cells generated by electrofusion

Purification of a Novel 43‐kDa Protein (p43) Intermediary in the Activation of Steroidogenesis from Rat Adrenal Gland

An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones

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