Boyd W. Harding

The acute action of adrenocorticotropic hormone on adrenal steroidogenesis

Abstract Using a soluble cholesterol-lecithin emulsion, some of the catalytic properties of the cholesterol side-chain cleaving system in rat adrenal mitochondria have been investigated to determine at which point this system is stimulated in vivo by adrenocorticotropic hormone (ACTH). A re-evaluation of cholesterol side-chain cleavage, 20α-hydroxycholesterol side-chain cleavage, and deoxycorticosterone 11β-hydroxylation in mitochondria isolated from normal rats, hypophysectomized rats, and hypophysectomized rats stimulated with ACTH shows that the V of cholesterol side-chain cleavage (1131 nmoles products/min per mg N) and 20α-hydroxycholesterol side-chain cleavage (938 nmoles products/min per mg N) are not affected by ACTH and are approx. 4 times greater than the V of deoxycorticosterone 11β-hydroxylation (250 nmoles corticosterone/min per mg N). Experiments testing the in vivo effects of ACTH on the levels of cytochrome P450-bound deoxycorticosterone and cholesterol in rat mitochondria indicate that the concentrations of both of these intermediates are increased in response to ACTH stimulation, and that cholesterol accumulates at its enzyme site only when hydroxylation is inhibited by anaerobiasis. We conclude from these data that ACTH may stimulate steroidogenesis by transporting cholesterol to the side-chain cleavage cytochrome P450 enzyme and that the transported cholesterol is rapidly metabolized.

Studies on adrenal cortical cytochrome P-450: I. Effects of substrates and electron donors on its catalytic, spectral, and electron paramagnetic resonance properties

Abstract The optical, electronic, and catalytic properties of cytochrome P-450 of bovine adrenal cortical mitochondria have been studied. 11-Deoxycortisol and other substrates for 11β-hydroxylation induce a characteristic difference spectrum in aerobic mitochondria showing absorption minima at 575, 530–535, and 420 mμ. Selective reduction of the hydroxylating enzymes in the absence of steroid substrate produces a typical hemoprotein reduced-minus-oxidized difference spectrum with α, β, and Soret absorption maxima at 575, 530–535, and 420 mμ, respectively. The kinetics of reduction and oxidation of this cytochrome have been correlated with oxygen uptake and steroid hydroxylation. Equilibration of the reduced hemoprotein with increasing carbon monoxide/oxygen ratios results in a progressive conversion of its 420mμ absorption band to the intense 450mμ absorption maximum characteristic of cytochrome P-450-CO. Electron paramagnetic resonance studies of aerobic mitochondria show signals ascribed to mitochondrial cytochrome P-450 at g values 2.42 and 2.26 which can be intensified by 11-deoxycortisol.

Role of the bovine adrenal cortical pyridine nucleotide transhydrogenase in steroid 11β-hydroxylation☆

Abstract Energy-controlled pyridine nucleotide transhydrogenase activity has been studied in mitochondrial and submitochondrial particles of bovine adrenal cortex. It has similar properties to those described in other tissues. The transhydrogenase activity of the submitochondrial particles can be utilized to support 11β-hydroxylation in a reconstructed hydroxylase system. The effect of hydroxylating conditions on the oxidation state of endogenous pyridine nucleotides in intact mitochondria, as determined by direct fluorometric measurements and enzyrnic assays in mitochondrial extracts, indicates that the energy-controlled pyridine nucleotide transhydrogenase is a participant, in the generation of intramitochondrial NADPH for the support of 11β-hydroxylation. It is concluded that this enzyme provides a link between the 11β-hydroxylase system and the respiratory chain and that its control by the energy state of the mitochondrion may serve as a mechanism for the regulation of corticoster-oidogenesis.

Substrate induced spin state changes in cytochrome P450

Abstract Deoxycorticosterone (DOC) produces 385 and 645 nm peaks and 420, 530 and 575 nm troughs, and 20αOH-cholesterol produces 420, 530 and 575 nm peaks and 385 and 645 nm troughs in the difference spectra of adrenal cortical submitochondrial particles. Electron spin resonance studies show that 20αOH-cholesterol produces an increase and 11-deoxycortisol or DOC produce a decrease in the low-spin signal of P450. These results suggest to us that the substrate induced difference spectra may be the result of a spin state shift in cytochrome P450.

Solubilization and properties of bovine adrenal cortical cytochrome P-450 which cleaves the cholesterol side chain

A cytochrome P-450 which cholesterol side-chain claving activity was solubilized from beef adrenal mitochondria with sodium deoxycholate and glycerol and purified by chromatography on DEAE-Sephadex. This preparation interacts spectrally with and performs side-chain cleavage of cholesterol and 20α-hydroxycholesterol but does not react with substrates for IIβ- or 18-hydroxylation. Absolute visible and ESR spectra indicate that this preparation is an equilibrium mixture of high- and low-spin statess of P-450 Fe3+. Cholesterol binding increase the relative proportion of the high-spin form, and 20α-hydroxycholesterol increases the relative proportion of the low-spin form of the cytochrome. Kinetic studies of the enzymes reducibility show that the rate of reduction of Fe3+ to Fe2+ is related to the proportion of high-spin P-450 present.

ACTH activation of glycolysis in the rat adrenal gland

Abstract A number of substrates of glycolysis, the pentose phosphate pathway, the Krebs cycle and the pyridine and adenine nucleotides (including cyclic AMP) have been measured before and after ACTH stimulation in hypophysectomized rats. Crossovers in substrate concentrations at the level of phosphofructokinase and glyceraldehyde phosphate dehydrogenase were found, indicating an activation of these enzymes in the stimulated adrenals. Elevations in cyclic AMP and a decrease in the ATP ADP · AMP ratio were also found, either of which could explain phosphofructokinase activation.

Biosynthesis of adrenal corticosteroids: energy metabolism and the hydroxylases

Abstract Evidence for the existence of at least two different cytochrome P450s concerned with corticosteroid hydroxylation has been presented. Metyrapone, a competitive inhibitor of steroid 11β-hydroxylation, affects the ligand fields of the iron of these mixed function oxidases. This agent interferes with the binding of 11β-hydroxylation substrates at the same or interacting sites at low concentrations and with other substrates at high concentrations. Because of the parallel changes in optical properties of the oxygenase, oxygen uptake and oxidation of reduced pyridine nucleotides upon addition of steroid substrates, in addition to their competitive interaction with metyrapone, it seems likely that they also react with the ligand field of cytochrome P450 to alter its redox properties. This presumably increases its rate of oxidation of electron donors and its rate of reaction with oxygen and hydroxylation of substrate. Such a substrate initiated event could be the site of primary control in steroidogenesis and might activate energy metabolism in general by reducing the level of citrate and ATP, and thereby, producing the activation observed in phosphofructokinase. However, the failure to observe an affect of cholesterol on the above parameters even though its oxygenase has very similar properties as other steroid oxygenases, as judged by its action spectrum, argues against such a substrate mediated control mechanism as a general phenomenon for control of corticosteroid hydroxylations. Since subcellular adrenal preparations generally show a high rate of synthesis of corticosteroids from endogenous precursor (cholesterol), when fortified with suitable electron donors, it is likely that the failure to demonstrate cholesterol effects on the cytochrome P450 of these particles is because the oxygenase is already saturated with endogenous cholesterol. Under such circumstances, the rate limiting reations would, most logically, be concerned with the generation of reducing equivalents and not with the provision of corticoid intermediates. The possibility exists that in the isolation of the subcellular particles the cholesterol hydroxylating enzyme is altered in some manner or a compartment is destroyed that permits it to interact with its substrate, a process normally mediated by ACTH. In this case, the primary action of ACTH would be either on the activation or synthesis of a “permease” or a specific synthetase. However, ability of NADPH-generator systems to bypass the stimulatory effect of ACTH on corticoid secretion in certain in vitro studies (8.10) argues against this possibility and further supports the hypothesis that places the control of corticosteroidogenesis on a regulation of energy metabolism. Basically similar conclusions have been drawn by many other investigators (51), differing only in the site at which the control is placed. The studies reported here suggest that phosphofructokinase, but not phosphorylase, could be a regulatory site. Whether or not it is primary, secondary, or concurrent to regulation at other sites is not known.

The effect of ether stress and cycloheximide treatment on cholesterol binding and enzyme turnover of adrenal cortical cytochrome P450scc

Relative rate constants for the formation of pregnenolone from cytochrome P450scc bound cholesterol in adrenal cortical mitochondria of stressed, stressed plus cycloheximide treated and dexamethasone treated rats were calculated from the ratios of initial rates of pregnenolone formation and the pregnenolone induced difference spectrum. In mitochondria from adrenals removed under aerobic conditions in vivo, the rate constant for the enzyme in stressed rats is twice a high as the rate constant for the enzyme from the stressed plus cycloheximide group, and four times as high as that for the enzyme from dexamethasone treated rats. Anoxia for 5 min in the intact gland increases the rate constant in all groups. Pregnenolone difference spectra are higher in mitochondria from stressed plus cycloheximide treated rats than in mitochondria from stressed rats, when adrenals are removed aerobically. It is concluded that ACTH increases cholesterol binding to cytochrome P450scc, by increasing either the enzymes affinity for its substrate or the availability of cholesterol and in addition promotes turnover of the enzyme. Both of these effects of ACTH are inhibited by cycloheximide.

The effect of theophylline on adenosine 3',5'-monophosphate-dependent protein kinase and ACTH1-24 stimulated steroidogenesis in bovine adrenal cortical cells.

Abstract Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH 1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH 1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH 1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH 1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced 3 H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.

Increased levels of a specific cyclic-AMP binding protein in acth stimulated bovine adrenal cortical cells

Abstract Ion exchange chromatography of bovine adrenal cortex cytosol on DEAE-cellulose reveals two major cAMP binding protein fractions. Isolated adrenal cortical cell cytosol was found to contain these same two binding proteins although in different proportions. These two binding proteins can be distinguished by the inhibition of [ 3 H]-cAMP binding by a spectrum of cAMP analogues. The isolated adrenal cortical cells show an increase in total cAMP binding protein activity when stimulated with ACTH 1−24 . This increase in total binding protein activity is due almost exclusively to one of the two binding proteins. Cycloheximide (10 μM) inhibits this ACTH stimulated cAMP binding protein.