Forskolin potentiates adrenocorticotropin-induced cyclic AMP production and steroidogenesis in isolated rat adrenal cells

Forskolin, a unique diterpene which directly activates the adenylate cyclase, stimulated production of both cyclic AMP and corticosterone in isolated rat adrenal cells, in vitro. This agent also potentiated the action of adrenocorticotropin and/or cholera toxin on cyclic AMP production and steroidogenesis at lower concentrations. It augmented both an early (cyclic AMP production) and a late (steroidogenesis) action of the hormone in the adrenal gland.     

Purification and characterization of a gamma-melanotropin precursor from frozen human pituitary glands.

A new melanocyte-stimulating peptide has been isolated from acid extracts of frozen human pituitary glands by salt/ethanol fractionation, Sephadex G-75 gel filtration and DEAE- and cM-cellulose ion-exchange chromatography. The peptide is glycosylated, has an N-terminal tryptophan residue and an apparent mol.wt. of 16000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its amino acid analysis closely resembles residues Trp-105 to Gln-29 predicted for the common precursor protein of bovine corticotropin and beta-lipotropin by Nakanishi, Inoue, Kita, Nakamura, Chang, Cohen & Numa [(1979) Nature (London) 278, 423-427]. This fragment is expected to have melanotropin activity due to the tetrapeptide -His-Phe-Arg-Trp- (residues -51 to -48) of the predicted sequence of the common precursor. It was found to have a molar potency of 1 X 10(-5) relative to alpha-melanotropin in the frog skin bioassay. These characteristics are consistent with the isolated melanotropin peptide being a non-corticotropin, non-lipotropin peptide of the human common precursor protein of corticotropin and lipotropin. The peptide neither potentiates the adrenal weight-maintenance activity of corticotropin-(1-24)-tetracosapeptide when administered to hypophysectomized rats, nor stimulates release of non-esterified fatty acids from isolated rat epididymal cells. A second N-terminal-tryptophan glycopeptide was also isolated, which had an amino-acid composition similar to that predicted for the bovine common precursor protein, residues Trp-105 to Gly-35.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.     

Prostacyclin: a potent stimulator of adrenal steroidogenesis.

The relative potencies of various prostaglandins were investigated in trypsin-dispersed cat adrenocortical cells. Prostacyclin proved to be the most potent steroidogenic prostaglandin, being 100-1000 times more potent than PGE2. This stimulant effect of prostacyclin was only partially dependent upon the presence of extracellular calcium and was associated with increased levels of cyclic AMP. These data suggest a possible role for prostacyclin in corticosteroidogenesis.     

Inhibition of steroidogenesis in rat adrenal cortex cells by a threonine analogue.

We have investigated the ability of amino acid analogues of serine and threonine to inhibit the increase in steroidogenesis elicited by addition of ACTH or cAMP in cells isolated from the rat adrenal cortex. We have found that the serine analogues, D, L-isoserine, alpha-methyl-D, L-serine and L-homoserine, are almost totally ineffective in inhibiting this process but that the threonine analogue, D, L-beta-hydroxynorvaline, at a concentration of 300 microM inhibits stimulated steroid hormone biosynthesis by ca 95%, while inhibiting overall protein synthesis by only ca 40%. This inhibition was found to occur in a dose-dependent manner and to be reversible by a stoichiometric concentration of threonine. These studies suggest that beta-hydroxynorvaline is functioning as a threonine analogue in our experimental system. Both the onset of inhibition by analogue and reversal of this inhibition by the natural amino acid occurred rapidly, without detectable lag. Since results obtained using cAMP as stimulant parallel those obtained using ACTH, the inhibitory effect of the analogue seems to occur subsequent to the synthesis of cAMP. Additionally, the analogue does not inhibit the conversion of pregnenolone to corticosterone, suggesting the site of action of analogue occurs prior to the synthesis of pregnenolone from cholesterol. Thus, the analogue may be exerting its effect on a protein that is synthesized subsequent to ACTH addition and is important in the acute phase of stimulated steroid hormone biosynthesis. Further, since ACTH action on adrenal cortex cells causes the activation of protein kinase A, which phosphorylates serine and threonine residues, it is possible that the effect of the analogue is to prevent the phosphorylation of a newly-synthesized protein.     

On the requirement for protein synthesis during corticotropin-induced stimulation of cholesterol side chain cleavage in rat adrenal mitochondrial and solubilized desmolase preparations.

Following simple homogenization, significant amounts of mitochondrial-derived, cholesterol side chain cleaving enzyme (desmolase) activity are recovered in rat adrenal 105 000 X g-supernatant fraction. Corticotropin administration enhances soluble desmolase activity, and cycloheximide potentiates this effect. The lipid droplet fraction which has no desmolase activity markedly enhances pregnenolone synthesis in the soluble desmolase preparations, presumably by supplying free cholesterol substrate. Corticotropin particularly with cycloheximide pretreatment, enhances lipid fraction activity. Thus increased cholesterol availability may largely explain the corticotropin effect on the soluble desmolase system. Since protein synthesis is required for corticotropin activity in intact mitochondria, but not in calcium-swollen mitochondria or the soluble enzyme system, the labile protein apparently required during corticotropin action may function to overcome a "barrier" which exists only in the intact mitochondria and restrains cholesterol side chain cleavage.     

Human transcortin synthesis by a cell-free translation of hepatic mRNA

To evaluate the site of synthesis and to characterize the translated transcortin, poly (A)-containing RNA (mRNA) from human liver was translated in a cell-free system derived from rabbit reticulocyte lysate. The in vitro synthesized product was identified as transcortin by immuno-precipitation with its specific antiserum. This translated transcortin could be displaced from the antibody by unlabeled purified transcortin obtained from plasma. Furthermore, when the translation mixture was applied to a cortisol-Sepharose column, the translated transcortin was bound to the matrix in a specific manner, indicating that this product binds to cortisol. The molecular weight of the translated transcortin was estimated to be 45,700 by its mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis, while that of plasma transcortin was 53,800. The difference in molecular weight between the translated transcortin and plasma transcortin was probably due to the presence of pre-sequence (signal peptide) in addition to the absence of carbohydrate moiety in the former. In conclusion, human liver mRNA directed the synthesis of transcortin, and the translated transcortin binds to cortisol in spite of the absence of carbohydrate moiety.     

Tributyltin disturbs bovine adrenal steroidogenesis by two modes of action

Tributyltin, an environmental pollutant, affected adrenal steroid hormone biosynthesis by two modes of action. Treatment of bovine adrenal cultured cells with 10-100 nM tributyltin for 48 h suppressed cortisol and androstenedione secretion, but induced the accumulation of 17alpha-hydroxyprogesterone and deoxycortisol, indicating that the P450(C21) and P450(11beta) activities were specifically suppressed. Direct inhibition of the enzymatic activities due to tributyltin was not observed in isolated organelles of untreated cells at concentrations less than 10 microM. Western blotting experiments using specific antibodies against steroidogenic enzymes showed that treatment with 1-100 nM tributyltin caused a decrease in cellular P450(C21) and P450(11beta) protein levels, and real-time PCR experiments showed that the decrease in protein content was attributable to decreases in mRNA of the enzymes. Tributyltin at concentrations higher than 100 nM suppressed all steroid biosynthesis in the adrenal cells. This suppression was closely correlated to the decrease in steroidogenic acute regulatory protein. Since nanomolar concentrations of tributyltin disturbed steroidogenesis in mammalian cells, there is the possibility that steroid hormone synthesis in polluted wild animals is affected by this compound.     

Fetal rat adrenal steroidogenesis and steroid transfer to adrenalectomized mother.

On the 22nd day of gestation in rats, fetuses of acutely adrenalectomized mothers were injected subcutaneously with 0.43 muCi 4-14C-progesterone in 0.05 ml saline. Ten and 20 min after injection to fetuses, samples were taken to determine the 14C-progesterone metabolites in the plasma and adrenal glands. After extraction of the samples taken, the metabolites were separated by two-dimensional thin-layer chromatography and identified by autoradiography. 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 11beta-hydroxyprogesterone were identified in the plasma of injected fetuses, and, in far smaller amounts, in the plasma of their mothers. The plasma of noninjected fetuses also contained very small amounts of these corticoids. The fetal adrenal glands contained far smaller amounts of radioactive steroids than the fetal plasma did. The results obtained show that steroids of fetal origin can cross the placenta in and out, constituting evidence that the fetal adrenal glands are the only source of the plasma corticoids of their adrenalectomized mothers.     

Prostaglandin E2 potentiates granulocyte-macrophage colony stimulating factor-induced histamine synthesis in bone marrow cells: role of cAMP.

Histamine synthesis in response to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) by murine hematopoietic cells is strikingly potentiated by prostaglandin E2 (PGE2). This synergy is mediated by an increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP), since: (a) exogeneous and endogeneous cAMP generated either by forskolin or IBMX potentiate GM-CSF-induced histamine synthesis, (b) the maximal potentiating effects of PGE2 and cAMP are not cumulative, and (c) GM-CSF together with PGE2 enhances intracellular cAMP content in a bone marrow population enriched for GM-CSF target cells. cAMP and PGE2 enhance histidine decarboxylase activity induced by GM-CSF showing that both factors act on histamine synthesis rather than on its release. Conversely, histamine synthesis promoted by Interleukin 3 (IL-3), the unique cytokine sharing this property with GM-CSF, is not modulated by PGE2 or cAMP, suggesting two distinct mechanisms for the induction of this biological activity in hematopoietic cells.     

Activation of steroidogenesis and adenylate cyclase by adenosine in adrenal and Leydig tumor cells.

Steroidogenesis by Y-1 adrenal tumor cells in culture is stimulated by ATP, adenyl-5'-yl imidodiphosphate (App(NH)), adenosine 5'(beta, alpha-methylene)triphosphate (App(CH2)p), ADP, AMP, NAD, FAD, and adenosine but not by adenine or other nucleoside triphosphates. ATP, App(NH)p, App(CH2)p, and adenosine are active in the micromolar range. Like adrenocorticotropic hormone (ACTH), the onset of stimulation is immediate and occurs to the same extent. Also active are 2'- and 5'-deoxyadenosine and 2-chloroadenosine whereas adenine xyloside, L-riboside, or arabinoside have very low activity. Stimulation is accompanied by rounding of the cells. Dipyridamole, an inhibitor of adenosine transport, increased the response to low concentrations of adenosine, suggesting that adenosine acts externally. Stimulation of steroidogenesis by adenosine or phosphorylated adenosine compounds fails to occur in the presence of crystalline adenosine deaminase, and the effect of the enzyme on adenosine, ATP, or NAD stimulation is reversed by the competitive inhibitor erythro-9-[3-(nonane-2-ol)]adenine. This suggests that the enzyme acts specifically on adenosine and a requirement for the conversion of the above compounds to adenosine seems probable. The inhibition of cAMP effects by adenosine deaminase suggests that some of its effects are also mediated by conversion to adenosine. Similar stimulation is seen in I-10 Leydig tumor cells, but an ACTH-resistant mutant of Y-1 cells, called OS-3, is relatively resistant to adenosine. Adenosine and 2-chloroadenosine stimulate adenylate cyclase in membranes from Y-1 and I-10 cells at concentrations slightly greater than are effective for steroidogenesis. Other nucleosides are ineffective. Like the NH2-terminal 24 residues of adrenocorticotropic hormone (1-24 ACTH), the adenosine effect in Y-1 membranes is rapid and is on the Vmax intercept (versus ATP) and not on the Km. In contrast to steroidogenesis, adenosine is only a partial agonist for adenylate cyclase. It effect occurs in the presence of ITP, GTP, or guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Theophylline inhibits adenosine-stimulated steroidogenesis. Inhibition of adenylate cyclase occurs in the same concentration range but is of the mixed type.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.