scholarly journals Calmidazolium is a potent stimulator of steroidogenesis via mechanisms not involving cyclic AMP, calcium or protein synthesis

1992 ◽  
Vol 281 (1) ◽  
pp. 291-296 ◽  
Author(s):  
M S K Choi ◽  
B A Cooke
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Leydig Cells ◽  
Dose Range ◽  
Protein Translation ◽  
Side Chain ◽  
Dibutyryl Cyclic Amp ◽  
Adrenocortical Cells ◽  
Potent Stimulator ◽  
Chain Cleavage

This study reports an unexpected effect of calmidazolium on steroidogenesis. In contrast with previous work, which established that calmidazolium inhibits hormone-stimulated testosterone production in rat Leydig cells, the present study demonstrates that this compound is a potent stimulator of steroidogenesis when added by itself; this stimulation (approx. 10-fold in a 2 h incubation), was obtained over a narrow dose range (e.g.1-10 microM) in mouse and rat Leydig cells and in rat adrenocortical cells. The same concentrations of calmidazolium decreased basal cyclic AMP to undetectable levels in rat Leydig cells. Also, cyclic AMP stimulated with luteinizing hormone (LH), cholera toxin and forskolin was inhibited by calmidazolium (ED50 2 microM). In contrast with the actions of LH and cyclic AMP analogues on steroidogenesis, the effect of calmidazolium was not inhibited by removal of extracellular Ca2+, or by the addition of La3+ (a Ca(2+)-entry blocker), or the addition of cycloheximide (an inhibitor of protein translation). However, like dibutyryl cyclic AMP, calmidazolium-stimulated steroidogenesis was inhibited by aminoglutethimide, an inhibitor of cholesterol side-chain cleavage. Another calmodulin inhibitor, trifluoperazine, did not stimulate steroidogenesis. It is concluded that calmidazolium has a similar effect on steroidogenesis to LH, but by-passes the requirements for cyclic AMP, Ca2+, and protein synthesis. Calmidazolium is therefore a potentially important probe for elucidating the mechansims of control of steroidogenesis.

scholarly journals Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

1978 ◽  
Vol 170 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Felix H. A. Janszen ◽  
Brian A. Cooke ◽  
Maria J. A. Van Driel ◽  
Henk J. Van Der Molen
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Leydig Cells ◽  
Actinomycin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphodiesterase Inhibitor ◽  
Dibutyryl Cyclic Amp ◽  
Rat Testis ◽  
Stimulation Of

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [35S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of35S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.

scholarly journals The mechanism of action of lutropin on regulator protein(s) involved in Leydig-cell steroidogenesis

1979 ◽  
Vol 184 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Brian A. Cooke ◽  
L. Monica Lindh ◽  
Henk J. Van Der Molen
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Leydig Cell ◽  
Half Life ◽  
Leydig Cells ◽  
Protein Synthesis Inhibitor ◽  
Dibutyryl Cyclic Amp ◽  
Testosterone Production ◽  
Short Half Life ◽  
Regulator Protein

The dependence on lutropin of the synthesis of a proposed short-half-life protein regulator involved in Leydig-cell steroidogenesis was investigated. This was carried out by determining the effect of the protein-synthesis inhibitor cycloheximide, added before and during incubations with lutropin (and/or dibutyryl cyclic AMP), on the rate of testosterone production in suspensions of purified Leydig cells from adult rat testes. The Leydig cells were preincubated in Eagle's medium for 2.5h followed by 30min incubation with and without cycloheximide. The inhibitor was removed by washing the cells and then lutropin was added and testosterone concentrations were determined after incubation of the cells at 32°C. No significant effect of cycloheximide pretreatment on lutropin-stimulated steroidogenesis was found during 60min incubation. This was in contrast with the complete inhibiting effect of cycloheximide when it was added with the lutropin. The pretreatment experiments with cycloheximide were repeated in the presence of dibutyryl cyclic AMP and elipten phosphate (to inhibit cholesterol side-chain cleavage) followed by incubation with lutropin. After 5, 10, 20 and 60min of incubation, testosterone concentrations were 61±3, 46±3, 27±4 and 18±4% lower than in the cells pretreated without cycloheximide respectively (means±s.e.m., n=4–6). In the cells not pretreated with cycloheximide and in the absence of lutropin, testosterone production increased from 1.36±0.5 to 36.5±1.0ng/106 cells during 20min of incubation, after which no further increase occurred. Pretreatment of the cells with cycloheximide decreased these testosterone concentrations by 65, 46, 42 and 36% in the 5, 10, 20 and 60min incubations respectively (mean values, n=2–4). It is apparent from these results that inhibition of steroidogenesis only occurs if protein synthesis is inhibited in the presence of lutropin or cyclic AMP. A new hypothesis is put forward to explain these findings: it is proposed that lutropin affects the stability of a precursor of a regulator protein by converting it from a stable (inactive) to an unstable (active) form with a short half-life.

scholarly journals The effect of lutropin on specific protein synthesis in tumour Leydig cells and in Leydig cells from immature rats

1978 ◽  
Vol 172 (1) ◽  
pp. 147-153 ◽  
Author(s):  
Felix H. A. Janszen ◽  
Brian A. Cooke ◽  
Maria J. A. Van Driel ◽  
Henk J. Van Der Molen
Keyword(s):  
Protein Synthesis ◽  
Leydig Cells ◽  
Sodium Dodecyl ◽  
Specific Protein ◽  
Side Chain ◽  
Chain Cleavage ◽  
Immature Rats ◽  
Cleavage Enzyme ◽  
Induced Proteins ◽  
Stimulation Of

The amount of35S incorporated into the various proteins after separation by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels was used as an estimate of their synthesis in the Leydig cells. Increased synthesis of proteins with apparent mol.wts. 27000 and 29000 was observed 3h after addition of lutropin to tumour Leydig cells. Incubation of Leydig cells from immature rats with lutropin (100ng/ml) for 2h or longer resulted in increased synthesis of proteins with apparent mol.wts. 11000, 21000, 27000 and 29000. At higher concentrations (≥100ng/ml) of lutropin there was a decrease in the synthesis of a protein with apparent mol.wt. 13000. The amount of lutropin required for the stimulation of protein synthesis in both types of Leydig cells was similar to that needed for stimulation of steroidogenesis. Lutropin-stimulated specific protein synthesis was not due to increased concentrations of testosterone, however, because (1) addition of testosterone to the cells had no effect on the synthesis of the proteins, and (2) inhibition of steroidogenesis with elipten phosphate (an inhibitor of the cholesterol side-chain-cleavage enzyme complex) did not abolish the effect of lutropin. The stimulation of specific protein synthesis was also not due to contaminating follitropin in the lutropin preparation. Addition of actinomycin D to the cells at the start of the incubation prevented the effect of lutropin on specific protein synthesis, indicating that mRNA synthesis may be needed for this effect of lutropin. Incubation of the cells with cycloheximide for 30min after labelling of the proteins did not result in a detectable decrease in the amounts of the lutropin-induced proteins, indicating that their half-life is longer than 30min.

scholarly journals A role for calmodulin in the regulation of steroidogenesis

1981 ◽  
Vol 90 (2) ◽  
pp. 402-407 ◽  
Author(s):  
PF Hall ◽  
S Osawa ◽  
CL Thomasson
Keyword(s):  
Cyclic Amp ◽  
Adrenal Tumor ◽  
Side Chain ◽  
Dibutyryl Cyclic Amp ◽  
Steroid Production ◽  
Steroid Synthesis ◽  
Side Chain Cleavage ◽  
Cleavage Activity ◽  
Chain Cleavage

Two approaches were used to study the possible role of calmodulin in the regulation of steroid synthesis by mouse adrenal tumor cells: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. Trifluoperazine inhibits three steroidogenic responses to both ACTH and dibutyryl cyclic AMP: (a) increase in steroid production, (b) increased transport of cholesterol to mitochondria, and (c) increased side-chain cleavage by mitochondria isolated from cells incubated with ACTH or dibutyryl cyclic AMP. When calmodulin is introduced into the cells via liposomes, steroid synthesis is slightly stimulated. When calmodulin extensively dialyzed against EGTA, this stimulation is abolished. Ca(2+) introduced via liposomes was also without effect. However, when both calmodulin and Ca(2+) are introduced via liposomes (either in separate liposomes or in the same liposomes), steroid synthesis is stimulated. This stimulation does not occur when either anticalmodulin antibodies or EGTA is also present in the liposomes or when trifluoperazine is present in the incubation medium. Calmodulin and Ca(2+) presented together in liposomes to the cells stimulate transport of cholesterol to mitochondria, and side-chain cleavage activity is greater in mitochondria isolated from cells previously fused with liposomes containing calmodulin and Ca(2+) than in mitochondria from cells fused with liposomes containing buffer only. These observations suggest that calmodulin may be involved in regulating the transport of cholesterol to mitochondria, a process which is stimulated by ACTH and dibutyryl cyclic AMP and which may account, at least in part, for the increase in steroid synthesis produced by these agents.

Suppressed expression of the cytochrome P45017α protein in the testicular feminized (Tfm) mouse testes

1993 ◽  
Vol 139 (1) ◽  
pp. 127-NP ◽  
Author(s):  
C. Le Goascogne ◽  
N. Sananès ◽  
M. Gouézou ◽  
E. E. Baulieu ◽  
P. Robel
Keyword(s):  
Cytochrome P450 ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Side Chain ◽  
Dibutyryl Cyclic Amp ◽  
Chain Cleavage ◽  
Left And Right ◽  
The Right ◽  
Old Mice

ABSTRACT The testes of testicular feminized (Tfm) mice synthesize and secrete abnormally low amounts of testosterone, as a consequence of selectively decreased cytochrome P45017α activity. To investigate the mechanism of this deficiency, three steroidogenic enzymes were immunolabelled in the testes of normal and Tfm adult (2·5–6 month old) mice. Cholesterol side-chain cleavage cytochrome P450 (P450scc) and Δ5-3β-hydroxysteroid dehydrogenase (3β-HSD) were detected in the Leydig cells of both normal and Tfm mice whereas, in contrast to normal mice, only a small proportion of Leydig cells were immunostained for cytochrome P450-17α-hydroxylase,C17→20 lyase (P45017α) in the testes of Tfm mice. The numbers of cells differed from male to male and interestingly were markedly higher in the right testis. Explants of testes from Tfm mice were kept in organ culture at 32 °C for 45 h, with or without dibutyryl cyclic AMP (100 or 500 μmol/l). All Leydig cells remained positive for P450scc and 3β-HSD, and P45017α became detectable in the majority of Leydig cells in both left and right testes, showing that the lack of expression of P45017α protein in Tfm mouse testes in vivo is not structural but is a regulatory phenomenon. Journal of Endocrinology (1993) 139, 127–130

scholarly journals Resveratrol Stimulates Cortisol Biosynthesis by Activating SIRT-Dependent Deacetylation of P450scc

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3258-3268 ◽  
Author(s):  
Donghui Li ◽  
Eric B. Dammer ◽  
Marion B. Sewer
Keyword(s):  
Protein Expression ◽  
Fold Increase ◽  
Pyridine Nucleotide ◽  
Nucleotide Metabolism ◽  
Side Chain ◽  
Mitochondrial Enzyme ◽  
Adrenocortical Cells ◽  
Protein Levels ◽  
Chain Cleavage ◽  
Cleavage Enzyme

In the human adrenal cortex, cortisol is synthesized from cholesterol by members of the cytochrome P450 superfamily and hydroxysteroid dehydrogenases. Both the first and last steps of cortisol biosynthesis occur in mitochondria. Based on our previous findings that activation of ACTH signaling changes the ratio of nicotinamide adenine dinucleotide (NAD) phosphate to reduced NAD phosphate in adrenocortical cells, we hypothesized that pyridine nucleotide metabolism may regulate the activity of the mitochondrial NAD+-dependent sirtuin (SIRT) deacetylases. We show that resveratrol increases the protein expression and half-life of P450 side chain cleavage enzyme (P450scc). The effects of resveratrol on P450scc protein levels and acetylation status are dependent on SIRT3 and SIRT5 expression. Stable overexpression of SIRT3 abrogates the cellular content of acetylated P450scc, concomitant with an increase in P450scc protein expression and cortisol secretion. Mutation of K148 and K149 to alanine stabilizes the expression of P450scc and results in a 1.5-fold increase in pregnenolone biosynthesis. Finally, resveratrol also increases the protein expression of P450 11β, another mitochondrial enzyme required for cortisol biosynthesis. Collectively, this study identifies a role for NAD+-dependent SIRT deacetylase activity in regulating the expression of mitochondrial steroidogenic P450.

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