scholarly journals Ectopic Agouti protein overexpression increases stimulated corticosterone production without effect on adenylate cyclase activity in mouse adrenal cells

2004 ◽  
pp. 613-618 ◽  
Author(s):  
AY Shevchenko ◽  
NM Bazhan ◽  
EN Makarova ◽  
TV Yakovleva ◽  
NR Karkaeva
Keyword(s):  
Adenylate Cyclase ◽  
Protein Overexpression ◽  
Dibutyryl Camp ◽  
Steroidogenic Enzymes ◽  
Steroidogenic Enzyme ◽  
Adrenal Cells ◽  
Agouti Protein ◽  
Corticosterone Release ◽  
Agouti Related Protein ◽  
Enzyme Reactivity

OBJECTIVE: The antagonism of Agouti protein (AP) and Agouti-related protein on melanocortin receptors suggests an inhibitory role in the regulation of steroidogenesis. However, we have previously demonstrated that ectopic AP overexpression increased restraint-induced corticosterone release and adrenal reactivity to ACTH in mice. A high steroidogenic response to ACTH may be a consequence of a stimulatory AP action on the adenylate cyclase (AC) and/or intracellular steroidogenic enzymes. The aim of the present study was to estimate the effect of ectopic AP overexpression on the activity of AC and steroidogenic intracellular enzymes. METHODS: ACTH and forskolin were used for AC stimulation, and dibutyryl cAMP and progesterone were used for stimulation of intracellular steroidogenic enzymes in isolated adrenal cells in male C57Bl/6J mice of two Agouti genotypes: A(y)/a (ectopic AP overexpression) and a/a (absence of AP in all tissues). RESULTS: ACTH and forskolin increased cAMP accumulation to the same extent in both A(y)/a and a/a mouse adrenal cells (P<0.001; ANOVA), but resulted in higher corticosterone production in A(y)/a mice (P<0.001 for ACTH and P<0.01 for forskolin; ANOVA). Dibutyryl cAMP- and progesterone-induced corticosterone production was higher in A(y)/a mice than in a/a mice (P<0.001 for dibutyryl cAMP and P<0.01 for progesterone; ANOVA). CONCLUSIONS: Ectopic AP overexpression increased stimulated corticosterone production and intracellular steroidogenic enzyme reactivity to cAMP without an effect on AC activity.

scholarly journals Analysis of mRNA expression for steroidogenic enzymes in the remaining adrenal cortices attached to adrenocortical adenomas.

2008 ◽  
Vol 158 (6) ◽  
pp. 867-878 ◽  
Author(s):  
Kazuto Shigematsu ◽  
Takehiro Nakagaki ◽  
Naohiro Yamaguchi ◽  
Kioko Kawai ◽  
Hideki Sakai ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Zona Fasciculata ◽  
Steroidogenic Enzymes ◽  
Steroidogenic Enzyme ◽  
Adrenocortical Adenomas ◽  
Aldosterone Production ◽  
Aldosterone Producing Adenoma ◽  
Cortical Nodules ◽  
Design And Methods

Design and methodsWe have recently demonstrated that the adrenal cortices attached to aldosterone-producing adenoma (APA) contained microscopic subcapsular micronodules suggestive of active aldosterone production. In this study, we used in situ hybridization to investigate the mRNA expression of steroidogenic enzymes in the adrenal cortices attached to cortisol-producing adenoma (CPA) and clinically silent adenoma (non-functioning adenoma; NFA), in addition to APA.ResultsMicroscopic subcapsular micronodules, which were several hundreds of micrometers in size and spheroid in shape, were observed in the cortices attached to CPA and NFA, as well as APA, at high frequency. Most of the cortical nodules in zona fasciculata to zona reticularis showed a suppressed steroidogenesis in the cortices attached to adenoma, but some expressed intensely all necessary steroidogenic enzyme mRNAs for cortisol synthesis.ConclusionsIt is thus necessary to keep in mind, on the occasion of subtotal adrenalectomy, that lesions with the potential to later develop into functional adrenocortical nodules may be present in other parts of the ipsilateral or contralateral adrenal cortices.

scholarly journals Luteinizing Hormone/Human Chorionic Gonadotropin-Mediated Activation of mTORC1 Signaling Is Required for Androgen Synthesis by Theca-Interstitial Cells

2012 ◽  
Vol 26 (10) ◽  
pp. 1732-1742 ◽  
Author(s):  
Murugesan Palaniappan ◽  
K. M. J. Menon
Keyword(s):  
Chorionic Gonadotropin ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Steroidogenic Enzymes ◽  
Steroidogenic Enzyme ◽  
S6 Kinase ◽  
Ribosomal Protein S6 ◽  
Mtorc1 Signaling ◽  
Steroidogenic Acute Regulatory ◽  
I Cells

Abstract LH triggers the biosynthesis of androgens in the theca-interstitial (T-I) cells of ovary through the activation of a cAMP-dependent pathway. We have previously shown that LH/human chorionic gonadotropin (hCG) activates mammalian target of rapamycin complex 1 (mTORC1) signaling network, leading to cell proliferation. In the present study, we provide evidence that the LH/hCG-mediated activation of the mTORC1 signaling cascade is involved in the regulation of steroidogenic enzymes in androgen biosynthesis. Treatment with LH/hCG increased the expression of downstream targets of mTORC1, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E as well as steroidogenic enzymes. LH/hCG-mediated stimulation of the steroidogenic enzyme mRNA was blocked by the mTORC1 inhibitor, rapamycin. This inhibitory effect was selective because rapamycin failed to block hCG-mediated increase in the expression of Star mRNA levels. Furthermore, pharmacological targeting of mTORC1 with rapamycin also blocked LH/hCG- or forskolin-induced expression of cAMP response element-binding protein (CREB) and steroidogenic enzymes (P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase type 1, and 17α-hydroxylase/17,20 lyase) but produced no effect on steroidogenic acute regulatory protein levels. These results were further confirmed by demonstrating that the knockdown of mTOR using small interfering RNA selectively abrogated the LH/hCG-induced increase in steroidogenic enzyme expression, without affecting steroidogenic acute regulatory protein expression. LH/hCG-stimulated androgen production was also blocked by rapamycin. Furthermore, the pharmacological inhibition of mTORC1 or ribosomal protein S6 kinase 1 signaling prevented the LH/hCG-induced phosphorylation of CREB. Chromatin immunoprecipitation assays revealed the association of CREB with the proximal promoter of the Cyp17a1 gene in response to hCG, and this association was reduced by rapamycin treatment. Taken together, our findings show for the first time that LH/hCG-mediated activation of androgen biosynthesis is regulated by the mTORC1 signaling pathway in T-I cells.

Effects of C19 steroids on adrenal steroidogenic enzyme activities and their mRNA levels in guinea-pig fasciculata-glomerulosa cells in primary culture

1992 ◽  
Vol 132 (2) ◽  
pp. 269-276 ◽  
Author(s):  
P. H. Provencher ◽  
Y. Tremblay ◽  
A. Bélanger
Keyword(s):  
Guinea Pig ◽  
Primary Culture ◽  
Oxygen Tension ◽  
Enzyme Activities ◽  
Inhibitory Effect ◽  
Mrna Levels ◽  
Hydroxylase Activity ◽  
Steroidogenic Enzymes ◽  
Steroidogenic Enzyme ◽  
Steroid Synthesis

ABSTRACT The present study examined the effects of steroids on steroidogenic enzyme activity in adrenal glands. Guinea-pig fasciculata-glomerulosa (FG) cells maintained in primary culture were exposed to steroids for 48 h. Although the treatment with androstenedione alone had no effect on 3β-hydroxysteroid dehydrogenase 4-ene-5-ene-isomerase (3β-HSD), 17-hydroxylase and 17,20-lyase activities, there was inhibition of 11-hydroxylase and 21-hydroxylase activities. When FG cells were exposed to 10 nmol ACTH/l for the last 24 h of incubation, ACTH alone had no effect on steroidogenic enzymes but, while combined with androstenedione, it further decreased 21-hydroxylase activity and stimulated 17-hydroxylase and 17,20-lyase activities. Cortisol, corticosterone, oestradiol and 11β-hydroxy androstenedione had no effect on steroidogenic enzyme activities while the inhibitory effect on 21-hydroxylase activity was only observed with androstenedione, testosterone and dihydrotestosterone. Addition of hydroxyflutamide, a pure antiandrogen, did not block the inhibitory effect of androstenedione on 21-hydroxylase and 11-hydroxylase activities. The reduction in oxygen tension from 19 to 2% which was aimed at examining the oxygen-mediated effects on steroidogenic enzymes, revealed that the reduction in 21-hydroxylase activity induced by androstenedione could not be prevented by low oxygen tension. An interaction of C19 steroids at the level of the enzymes is also suggested by our finding that androstenedione had no effect on basal and ACTH-stimulated steady-state 11-hydroxylase, 17-hydroxylase, 17,20-lyase and 21-hydroxylase mRNA levels. These results indicate that C19 steroids alter the adrenal steroidogenic enzyme activities in such a manner that C19 steroid synthesis is increased while glucocorticoid production is inhibited. The mechanism of action of C19 steroids does not involve gene expression for steroidogenic enzymes but probably a direct interaction with steroidogenic enzymes, namely 21-hydroxylase, 17-hydroxylase and 17,20-lyase. Our data suggest that C19 steroids may reduce the amount of 21-hydroxylase in the microsomal fraction which may have a major impact on the levels of microsomal P450 reductase available for 17-hydroxylase and 17,20-lyase activities. Journal of Endocrinology (1992) 132, 269–276

scholarly journals Agouti-Related Protein Antagonizes Glucocorticoid Production Induced through Melanocortin 4 Receptor Activation in Bovine Adrenal Cells: A Possible Autocrine Control

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 541-547 ◽  
Author(s):  
Mabrouka Doghman ◽  
Philippe Delagrange ◽  
Antonine Blondet ◽  
Marie-Claude Berthelon ◽  
Philippe Durand ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Melanocortin Receptor ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Related Protein ◽  
Peripheral Tissues ◽  
Adrenal Cells ◽  
Autocrine Control ◽  
Glucocorticoid Production ◽  
Agouti Related Protein

Abstract Agouti-related protein (Agrp), primarily expressed in the hypothalamus, is an endogenous antagonist of αMSH at the level of melanocortin 3 receptor (MC3-R) and MC4-R, but the adrenal gland represents the second major Agrp-expressing tissue. In adrenal fasciculata cells, the glucocorticoid secretion is under the control of ACTH, which binds specifically MC2-R, the only functional melanocortin receptor described in these cells to date. Nevertheless, using cultured bovine fasciculata adrenal cells, we report that Agrp has no antagonistic properties against ACTH at the level of MC2-R. In our studies, (Nle4, d-Phe7)-αMSH (NDP-αMSH) stimulated the production of cortisol in a dose-dependent manner, and these effects were abolished by Agrp or SHU9119, a synthetic antagonist of MC3-R and MC4-R. Using a more specific antagonist (JKC-363) and RT-PCR analysis, we can postulate that the effects of NDP-αMSH were mediated via MC4-R. These results are suggestive that adrenal glucocorticoid production could be regulated through MC4-R that may have some relevance in the physiology of adrenal cells. Moreover, Agrp might exert an autocrine control on adrenal cells because a protein with biological Agrp-like activity is secreted by these cells. This peptide could then modulate locally the functions of some peripheral tissues such as adrenals.

scholarly journals Pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors: distribution and involvement in the secretion of Podarcis sicula adrenal gland

2007 ◽  
Vol 196 (2) ◽  
pp. 291-303 ◽  
Author(s):  
Salvatore Valiante ◽  
Marina Prisco ◽  
Rosaria Sciarrillo ◽  
Maria De Falco ◽  
Anna Capaldo ◽  
...  
Keyword(s):  
Adrenal Gland ◽  
Adenylate Cyclase ◽  
Vasoactive Intestinal Polypeptide ◽  
Chromaffin Cells ◽  
Adrenal Glands ◽  
Podarcis Sicula ◽  
Adrenal Cell ◽  
Adrenal Cells ◽  
Pituitary Adenylate Cyclase

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are regulatory neuropeptides of the hypothalamus–hypophyseal–adrenal axis, acting via the common receptors VPAC1 and VPAC2 and the selective PACAP receptor PAC1. In the adrenal glands of the Italian wall lizard, Podarcis sicula, the presence of VIP in chromaffin cells, and the VIP-stimulated release of catecholamine and aldosterone in vivo, was previously shown. To examine the localization of both peptides and receptors and their mRNAs in the adrenal gland of P. sicula, immunohistochemistry and in situ hybridization were performed: PACAP and its mRNA were detected in chromaffin cells, VPAC1 was found associated with steroidogenic tissue, VPAC2 and PAC1 with chromaffin tissue. Using ‘far western blot’ technique, we showed the presence of specific binding sites for VIP/PACAP in the adrenal glands of the lizard. The effects of both VIP and PACAP on the adrenal cells of the lizard were examined in vitro in adrenal cell co-cultures: both VIP and PACAP enhanced catecholamine, corticosterone and aldosterone release from adrenal cell co-culture in a time- and dose-dependent manner. The catecholamine release was inhibited by PAC1 antagonist and in VPAC2 immunoneutralized adrenal cells. The effects of VIP and PACAP on aldosterone secretion were counteracted by VPAC1 antagonist administration in vitro. Corticosterone secretion elicited by VIP was not blocked by VPAC1 antagonist, while the PACAP-induced release of corticosterone was blocked by the antagonist. Overall, our investigations indicate that these neuropeptides of the secretin superfamily can act not only as neurotransmitters but also as autocrine and paracrine regulators on chromaffin and cortical cells, being important mediators of the non-cholinergic system in the lizard adrenal gland.

THE DEVELOPMENT OF ADRENOCORTICOTROPHIN-SENSITIVE ADENYLATE CYCLASE ACTIVITY IN THE FOETAL RABBIT ADRENAL: A CORRELATED BIOCHEMICAL AND MORPHOLOGICAL STUDY

1976 ◽  
Vol 71 (3) ◽  
pp. 333-341 ◽  
Author(s):  
JANET D. M. ALBANO ◽  
PATRICIA M. JACK ◽  
TERESA JOSEPH ◽  
R. P. GOULD ◽  
P. W. NATHANIELSZ ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Morphological Changes ◽  
Smooth Endoplasmic Reticulum ◽  
Major Change ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Steroidogenic Enzymes ◽  
Ultrastructural Studies ◽  
Steroidogenic Tissue ◽  
Lamellar Type

SUMMARY Proliferation of the smooth endoplasmic reticulum, the site of some hydroxylating steroidogenic enzymes in foetal adrenocortical cells, is the first major change in the process of their differentiation into steroidogenic tissue. This was observed in our ultrastructural studies on foetal rabbit adrenals to begin at about day 19 of development. Morphological changes in the mitochondria, the site of production of other steroidogenic enzymes, occurred at about day 24. The elongated or rod-shaped forms of the earlier stages became flattened and rounded by this time, while the cristae were transformed from a flattened lamellar type of the earlier stages to the tubulo-vesicular form of the adult. Other changes observed included an increase in microvilli and in cell size, with a concomitant increase in thickness of the gland. Adenylate cyclase activity in foetal adrenal homogenates was assessed in response to sodium fluoride (NaF) and ACTH. All preparations responded to NaF. While good responses to ACTH were observed at days 24, 27, 28 and in the neonate, there was a lack of any significant response in the day 19 gland. Foetal ACTH was depressed by administration of cortisol, and the effects of this treatment on both the morphological changes and adenylate cyclase activity was reassessed. The response of foetal adrenals to ACTH was depressed by this treatment and differentiation of the mitochondria was arrested. These results suggest a circumscribed period for the development of ACTH-sensitive adenylate cyclase coinciding with the time at which final differentiation of the mitochondria is completed. Furthermore, both the differentiation of the mitochondria and the development of ACTH-sensitive adenylate cyclase in the foetal adrenal may be dependent on foetal ACTH secretion.

Conjoint action of phosphatidylinositol and adenylate cyclase systems in serotonin-induced facilitation at the crayfish neuromuscular junction

1989 ◽  
Vol 62 (6) ◽  
pp. 1251-1259 ◽  
Author(s):  
D. Dixon ◽  
H. L. Atwood
Keyword(s):  
Protein Kinase ◽  
Neuromuscular Junction ◽  
Adenylate Cyclase ◽  
Early Phase ◽  
Single Application ◽  
Dibutyryl Camp ◽  
Fluorescent Marker ◽  
Epsp Amplitude ◽  
Two Phases ◽  
Crayfish Neuromuscular Junction

1. Pulsatile application of serotonin (5-HT) leads to facilitation of excitatory postsynaptic potentials (EPSPs) in crayfish "opener" neuromuscular preparations. The facilitation resulting from a single application of serotonin shows two phases: an early, rapidly decaying phase, and a less intense, long-lasting phase of 1- to 2-h duration. A previous study implicated the phosphatidylinositol system as an essential component in serotonin-induced facilitation, especially the early phase. The present study was conducted to determine the roles of the adenylate cyclase and phosphatidylinositol systems in both phases of serotonin-induced facilitation. 2. Relatively brief applications of agents known to affect the intracellular concentration of cAMP (forskolin, 1 microM; and IBMX, 100 microM) cause an increase in EPSP amplitude, which persists for 1-2 h. 3. The duration of the less intense, long-lasting phase of serotonin-induced facilitation is prolonged in the presence of 1 microM IBMX. This concentration of IBMX does not affect EPSP amplitude by itself. A membrane-permeant analog of cAMP (applied in concentrations less than or equal to 1 mM) is also not effective in altering EPSP amplitude. However, when dibutyryl cAMP is applied in the presence of 1 microM IBMX, EPSP amplitude is increased (60-80%). 4. Localized presynaptic injection of the "Walsh Inhibitor" (PKI), which inhibits cAMP-activated protein kinase, blocks the less intense, long-lasting phase of serotonin-induced facilitation at synapses near the site of injection. Normal facilitation develops at synapses within the same preparation remote from the site of injection. Distribution of the injected inhibitor within the axon can be visualized by tagging PKI with a fluorescent marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1-induced corticosterone release occurs by an adrenergic mechanism from rat adrenal gland

1992 ◽  
Vol 263 (3) ◽  
pp. E461-E466 ◽  
Author(s):  
A. R. Gwosdow ◽  
N. A. O'Connell ◽  
J. A. Spencer ◽  
M. S. Kumar ◽  
R. K. Agarwal ◽  
...  
Keyword(s):  
Adrenal Gland ◽  
Incubation Period ◽  
Interleukin 1 ◽  
Primary Cultures ◽  
Adrenal Cells ◽  
Adrenergic Antagonist ◽  
Corticosterone Release ◽  
Local Release ◽  
Dose Dependent ◽  
Pituitary Adrenal Axis

Interleukin-1 (IL-1) has been shown to stimulate corticosterone release from the adrenal gland directly, and indirectly through activation of the hypothalamic-pituitary-adrenal axis. The aim of this paper was to determine whether IL-1-stimulated corticosterone release occurs indirectly through the local release of catecholamines from the rat adrenal gland. To accomplish this, experiments were conducted on both quartered rat adrenal glands and primary cultures of dispersed adrenal cells. Incubation of quartered adrenals with adrenocorticotropic hormone (ACTH, 10(-12) to 10(-8) M) or IL-1 beta (10(-12) to 10(-8) M) resulted in dose-dependent increases (P less than 0.05) in corticosterone release. Corticosterone release stimulated by 10(-8) M doses of ACTH and IL-1 beta began to rise 30 min after incubation and peaked at 2 h. In primary cultures of adrenal cells, IL-1 alpha and IL-1 beta elevated corticosterone release after a 24-h incubation period. ACTH elevated corticosterone levels at 4 and 24 h. The stimulatory effect of IL-1 on corticosterone release was mimicked by epinephrine (10(-6) M), and was selectively blocked by the alpha-adrenergic antagonist phentolamine (10(-5) M). The beta-adrenergic antagonist propranolol (10(-5) M) did not change IL-1-induced corticosterone release. Neither phentolamine nor propranolol had an effect on ACTH-stimulated corticosterone release. Both IL-1 alpha and IL-1 beta significantly increased (P less than 0.05) epinephrine levels after a 24-h incubation period compared with media-treated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

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