3β-Hydroxysteroid dehydrogenase activity in bovine adrenal cortex mitochondria: conversion of oxygenated 3β-hydroxy-sterols

Adrenal cortex 3β-hydroxysteroid dehydrogenase (3β-HSD) is able to convert many C₁₉ and C₂₁ 3β-OH-5-ene steroids into products with a 3-keto-4-ene structure. In the present investigation we describe the conversion of a number of C₂₇ and C₂₄ 3β-5-ene sterols by adrenal milochondrial 3β-HSD. Among these substrates were (20S)-5-cholestcne-3β,20-diol. (22R)-5-cholestene-3β,22-diol and (20R,22R)-5-cholestene-3β,20.22-triol, compounds occurring as intermediates in the cholesterol side-chain cleavage reaction. Cholesterol itself was not converted to a measurable extent.     

Effect of cholesterol and protein content on membrane fluidity and 3β-hydroxysteroid dehydrogenase activity in mitochondrial inner membranes of bovine adrenal cortex

The steroid biosynthetic enzymes in the adrenal cortex are localised in endoplasmic reticulum and mitochondrial membranes. For some of the enzymes in endoplasmic reticulum the activity appears to be modulated by lipid fluidity, (21-hydroxysteroid hydroxylase and 3β-hydroxysteroid dehydrogenase). A mechanism for the regulation of corticosteroid biosynthesis mediated by the membrane fluidity has been suggested. Therefore a study of the mitochondrial inner membrane of the bovine adrenal cortex has been undertaken in comparison with a previous study of the endoplasmic reticulum. The kinetic parameters of the 3β-hydroxysteroid dehydrogenase were studied as a function of pH and temperature. No thermal transition can be observed in the Arrhenius plot for this enzyme in contrast with the results obtained for the microsomal enzyme. Membrane fluidity using, as fluorescent probes, diphenylhexatriene and a set of n-(9-anthroyloxy) fatty acids has been also studied as a function of temperature with or without addition of cholesterol. No thermal transition in the lipid phase can be observed. The addition of cholesterol to total mitochondrial membrane as to a lipid extract of the membrane decreases fluidity to the same extent as it does with microsomes. The presence of a large amount of protein in mitochondria has an effect which is additive to that of the cholesterol.     

Membrane dopamine β-hydroxylase: a precursor for the soluble enzyme in the bovine adrenal medulla

• 1.1. Searching for endogenous proteolytic activities converting the membrane form of dopamine β-hydroxylase (dopamine β-monooxygenase, DBH) into the soluble and releasable form, DBH was monitored enzymatically and immunologically in aqueous and detergent-solubilized extracts of the adrenomedullary fractions.
• 2.2. Degradation of the soluble DBH and acidic chromogranins by activation of endogenous proteases occurred during lysis in H₂O.
• 3.3. Shifts in the hydrophobicity of the membrane DBH were also apparent. Loss in enzyme protein or activity was, on the other hand, not observed for bufier-dialysed CG (pH 5–6).
• 4.4. Limited proteolysis within the membrane phase was, however, indicated by the shift towards dominance of the intermediate hydrophobic DBH in the buffer-dialysed CG.
• 5.5. By two-dimensional, crossed immunoelectrophoresis with cationic detergent the microsomal DBH was immunologically identical to the granule-bound enzyme but differed from the latter in molecular heterogeneity and in susceptibility to proteolytic solubilization by endogenous protease activities.
• 6.6. DBH in the membranes of the chromaffin granules was proteolytically solubilized at pH 6–8 and the soluble DBH further degraded at pH 5.
• 7.7. The results indicate that a post-translational conversion of the amphiphilic DBH into the soluble form, initiated at the level of the microsomes, may continue within the light and the heavy granule fractions which contain several DBH-converting and degrading proteolytic activities with acid optima.

     

Mode of action of sulfoxides in preventing loss of activity of 11β-hydroxylase in cultured bovine adrenocortical cells

Previously, loss of 11β-hydroxylase activity when adrenocortical cells are incubated with the pseudosubstrate cortisol was found to be reduced when the concentration of oxygen was lowered, or when butylated hydroxyanisole (BHA) or dimethyl sulfoxide (Me₂SO) were included in the medium. In the present experiments, we tested the hypothesis that Me₂SO protects 11β-hydroxylase by scavenging OH^? radicals. Substances known to react with OH^? at high rates and non-toxic enough to be used at concentrations of 10–100 mM, including several alcohols, benzoate and radioprotectant thiols, did not prevent loss of activity of 11β-hydroxylase in the presence of 50 μM cortisol. Two of the alcohols, ethanol and glycerol, as well as Me₂SO, were radioprotective in cultured bovine adrenocortical cells. Therefore free OH^? radicals do not appear to be involved in loss of 11β-hydroxylase activity. When sulfoxides other than dimethyl sulfoxide were tested for their ability to protect 11β-hydroxylase in the presence of cortisol, several aryl sulfoxides, particularly dibenzyl sulfoxide, as well as dipropyl sulfoxide, were active at concentrations to $\text{1}\text{200}$ of that required for Me₂SO. Previously, we have demonstrated that 11β-hydroxylase inhibitors, particularly metyrapone, effectively protect against loss of 11β-hydroxylase activity in the presence of pseudosubstrates and therefore we examined whether sulfoxides may act by directly inhibiting 11β-hydroxylase. Me₂SO showed an ED₅₀ for inhibition of 11β-hydroxylase activity of > 1 M, in contrast to its ED₅₀ for protection of 34 mM. For metyrapone, however, the ED₅₀ for inhibition of the enzyme (250 nM) was close to that for protection of activity (270 nM). The other sulfoxides showed ED₅₀-values for inhibition of 11β-hydroxylase that were substantially higher than the ED₅₀-values for protection. Sulfoxides may have a mixed mode of action in protection of 11β-hydroxylase activity, as previously shown for phenols; they may protect by radical scavenging, but may also need to bind close to the active site of the enzyme where destructive radicals may be formed.     

Dopamine β-hydroxylase of bovine adrenal medullae. A rapid purification procedure

1. A rapid purification procedure for dopamine β-hydroxylase from bovine adrenal-medulla chromaffin granules is presented. The homogeneity of the purified enzyme was demonstrated by means of three independent criteria. The specific activity of the enzyme compares favourably with that obtained by more involved procedures. 2. The stability of the enzyme was investigated and storage in polypropylene tubes was found preferable to storage in glass. 3. The soluble and particulate forms of dopamine β-hydroxylase appear to be identical, since membrane-bound and membrane-enclosed forms of the enzyme exhibit similar properties as regards size, charge and amino acid composition. 4. Ca²⁺ was found to stimulate the release of dopamine β-hydroxylase from bovine chromaffin granules in vitro. 5. An endogenous inhibitor of the enzyme was found in the chromaffin granules. This inhibitor was not inactivated either by heating at 100°C or by pretreatment with p-chloromercuribenzoate or Cu²⁺ ions.     

Discrepancy between the histochemically and biochemically determined 3β-hydroxysteroiddehydrogenase activity in the calf adrenal cortex

Summary In sections of the calf adrenal cortex the histochemically determined3-hydroxysteroiddehydrogenaae activity is lower in the zona glomerulosa than in the zona fasciculata. Biochemically the activity of this enzyme was found in mitochondrial as well as in microsomal fractions. The mitochondrial, respectively the microsomal fractions of the two zones showed identical enzyme activities. So there is a discrepancy between the histochemical results on tissue sections and biochemical results obtained with isolated subcellular fractions.However, if the histochemical determination of 3-hydroxysteroiddehydrogenase activity is carried out on sections of mitochondrial and microsomal pellets of the two zones the results are in agreement with the biochemical findings. Therefore the observed discrepancy rather seems to be related with the state of tissue—intact or cell fractions—than with the used histochemical method.     

Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.     

Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.     

Phenylethanolamine-N-methyltransferase and dopamine-β-hydroxylase immunoreactivity and the occurrence of noradrenaline and adrenaline in the nervous system of the snail Helix aspersa

Summary The presence of dopamine--hydroxylase (DBH) and phenylethanol-amine-N-methyltransferase (PNMT) immunoreactivity in specific neurones of the snail Helix aspersa has been demonstrated. In addition, high performance liquid chromatography and electrochemical detection have revealed the presence of noradrenaline and adrenaline in the snail central nervous system, although the major catecholamine is dopamine. These results suggest that adrenaline, and perhaps noradrenaline, have transmitter or modulatory functions in the snail nervous system.     

β2-Adrenoceptor agonists in the regulation of mitochondrial biogenesis

The stimulation of mitochondrial biogenesis (MB) via cell surface G-protein coupled receptors is a promising strategy for cell repair and regeneration. Here we report the specificity and chemical rationale of a panel of β₂-adrenoceptor agonists with regards to MB. Using primary cultures of renal cells, a diverse panel of β₂-adrenoceptor agonists elicited three distinct phenotypes: full MB, partial MB, and non-MB. Full MB compounds had efficacy in the low nanomolar range and represent two chemical scaffolds containing three distinct chemical clusters. Interestingly, the MB phenotype did not correlate with reported receptor affinity or chemical similarity. Chemical clusters were then subjected to pharmacophore modeling creating two models with unique and distinct features, consisting of five conserved amongst full MB compounds were identified. The two discrete pharmacophore models were coalesced into a consensus pharmacophore with four unique features elucidating the spatial and chemical characteristics required to stimulate MB.     

The physiological significance of 11β-hydroxysteroid dehydrogenase in the rat lung

We have examined the interconversion of cortisone (E) and cortisol (F) in rat lung homogenate and microsomal fraction and in the isolated rat lung perfused with Krebs bicarbonate solution containing 4.5% albumin. In the perfused lung the apparent K_m was 5.1 μM E and the V_max was 9nmol·g⁻¹ · min⁻¹. The ability of the lung to reduce E to F was enhanced both by 7 days prior exposure of the rat to an ambient temperature of 2°C and by starvation of the rat for 3 days. The activity was inhibited by adrenalectomy and castration of 7 days duration. Whereas little steroid oxidation occurred in the perfused lung, preparations of lung homogenatcs and microsomal fraction readily reduced or oxidised the 11-position of the corticoid molecule depending on the preponderance of either NADPH or NADP, respectively. We conclude, that the predominance of the reductive reaction in the whole rat lung under physiological conditions reflects the very active pentose-phosphate shunt in the lung, which produces NADPH. We suggest that this ability of the lung to activate E to F may exert a fine control over the arterial concentrtion of unbound, physiologically active, 11-hydroxylated steroid.     

Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

Summary The localization of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.Supported by grants from the Ministry of Education Science and Culture, Japan     

Immunocytochemical localization of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method

Summary The subcellular locilazations of tryrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

The thioredoxin system in aging muscle: key role of mitochondrial thioredoxin reductase in the protective effects of caloric restriction?

Cellular redox balance is maintained by various antioxidative systems. Among those is the thioredoxin system, consisting of thioredoxin, thioredoxin reductase, and NADPH. In the present study, we examined the effects of caloric restriction (2 mo) on the expression of the cytosolic and mitochondrial thioredoxin system in skeletal muscle and heart of senescent and young rats. Mitochondrial thioredoxin reductase (TrxR2) is significantly reduced in aging skeletal and cardiac muscle and renormalized after caloric restriction, while the cytosolic isoform remains unchanged. Thioredoxins (mitochondrial Trx2, cytosolic Trx1) are not influenced by caloric restriction. In skeletal and cardiac muscle of young rats, caloric restriction has no effect on the expression of thioredoxins or thioredoxin reductases. Enforced reduction of TrxR2 (small interfering RNA) in myoblasts under exposure to ceramide or TNF-alpha causes a dramatic enhancement of nucleosomal DNA cleavage, caspase 9 activation, and mitochondrial reactive oxygen species release, together with reduced cell viability, while this TrxR2 reduction is without effect in unstimulated myoblasts under basal conditions. Oxidative stress in vitro (H2O2 in C2C12 myoblasts and myotubes) results in different changes: TrxR2, Trx2, and Trx1 are induced without alterations in the cytosolic thioredoxin reductase isoforms. Thus aging is associated with a TrxR2 reduction in skeletal muscle and heart, which enhances susceptibility to apoptotic stimuli but is renormalized after short-term caloric restriction. Exogenous oxidative stress does not result in these age-related changes of TrxR2.     

Studies of 11β-hydroxylation by beef adrenal mitochondria

The 11β-hydroxylations of androstenedione (Δ⁴A), 11-deoxycortisol (S) and deoxycorticosterone (DOC) were studied using mitochondria from calf or heifer adrenal tissue. Standard assay conditions were: non-radioactive androstenedione (30.0μM) 11-deoxycortisol (24.5 μM) or deoxycorticosterone (26.0 μM) plus 6.0 × 10⁴ d.p.m. ¹⁴C-steroid, 0.2 mM NADPH, 1.0 mM Mg²⁺ 1.0 mM Ca²⁺ and the mitochondrial fraction equivalent to 20 mg of adrenal tissue in a final vol. of 3ml of 0.1 M HEPES buffer. pH 7.4. Incubations were performed at 37°C for 4min. Product formation under these conditions was identical to product formation measured when the NADPH and Ca²⁺ were replaced with 10mM malate. The 11β-hydroxylation of Δ⁴A showed a requirement for NADPH and oxygen, indicating that the enzyme involved is a mixed-function oxidase. The K_m values for calf adrenal mitochondria were 3.8, 8.5 and 8.0μM for Δ⁴A, S and DOC, respectively. For heifer adrenal mitochondria, the K_m values were 12 and 15μM respectively, for Δ⁴A and S. Competition studies in which equal amounts of two substrates were incubated simultaneously, revealed that Δ⁴A, S and DOC did not compete for the same enzymatic site, but were hydroxylated to the same degree in the presence or absence of each of the other two precursors. The 11β-hydroxylations of S and DOC were stimulated by Mg²⁺ at a concentration of 1.0 mM, while the 11β-hydroxylation of Δ⁴A was inhibited by this concentration of Mg²⁺. In experiments in which the mitochondria were preheated at 50°C for 6 min, the 11β-hydroxylation of Δ⁴A, under standard assay conditions, was 96% of the unheated value, while the 11β-hydroxylation of S and DOC was 77 and 59%, respectively, of the unheated values. These studies indicate that there are three substrate specific 11β-hydroxylases in beef adrenal mitochondria.     

The discovery of potent inhibitors of aldosterone synthase that exhibit selectivity over 11-β-hydroxylase

Aldosterone, the final component of the renin–angiotensin–aldosterone system, plays an important role in the pathophysiology of hypertension and congestive heart failure. Aldosterone synthase (CYP11B2) catalyzes the last three steps of aldosterone biosynthesis, and as such appears to be a target for the treatment of these disorders. A sulfonamide–imidazole scaffold has proven to be a potent inhibitor of CYP11B2. Furthermore, this scaffold can achieve high levels of selectivity for CYP11B2 over CYP11B1, a key enzyme in the biosynthesis of cortisol.     

The presence of antibodies to human dopamine-β-hydroxylase in commercially available antisera

Three criteria have been used to demonstrate the presence of antibodies to human dopamine-β-hydroxylase in commercially available antisera directed against various human serum fractions. These criteria are the inhibition of enzyme activity, complement fixation and binding of ¹²⁵I-labelled dopamine-β-hydroxylase to the immobilized antisera. The level of antibody present in some of these antisera was sufficient to allow their use in the radioimmunoassay of the enzyme. The possibility of other useful antibodies occurring in these and similar antisera is suggested.