The nature of the cytosolic activators of the adrenal steroid 21-hydroxylation system

The steroid 21-hydroxylase activity present in the microsomes of bovine adrenals is stimulated by components of the cytosol. The nature of these activators has been examined by two procedures. The first consisted of treating cytosol with increasing amounts of acetone. When the concentration of the organic solvent reached 50%, a precipitate, presumably proteinaceous, formed. The portion of the precipitate that was redissolvable in 0.05 m potassium phosphate buffer, pH 7.2, contained 7–15% of the stimulatory activity originally present in the cytosol. When the acetone concentration was raised to 90%, another active material precipitated. It was identified as oxidized glutathione (GSSG) and it accounted for about 5% of the activity in the cytosol. In an attempt to avoid the harmful effects of acetone, the second procedure employed only gel filtration and ion exchange resin chromatography. By these means the cytosol was separated into 11 protein fractions and a small molecular weight material. Forty six percent of the proteins and the same fraction of the stimulatory activity present in the original cytosol were recovered. Because all 11 protein fractions contained some stimulatory activity, the results suggested that the protein constituents of these fractions were relatively nonspecific. Yet, of the several known proteins which were tested for activity (bovine serum albumin, ovalbumin, human γ-globulin, bovine pancreatic ribonuclease, and pig insulin) only bovine serum albumin proved to be active. An additional 8% of the stimulatory activity of the cytosol was present in the fraction containing the low molecular weight components and this was all attributable to its GSSG content.     

Structural analysis of cloned cDNA for mRNA of microsomal cytochrome P-450(C21) which catalyzes steroid 21-hydroxylation in bovine adrenal cortex

We have isolated cDNA clones of the mRNA for cytochrome P-450 that catalyzes the steroid C-21 hydroxylation (P-450(C21)), which specifically catalyzes 21-hydroxylation of steroids in the microsomes of bovine adrenal cortex by using synthetic oligonucleotides as probes. Sequence determination of the cloned cDNA showed that it contains 2157 nucleotides and a poly(A) chain and that a single open reading frame of 1488 nucleotides codes for a polypeptide of 496 amino acids with a molecular weight of 56,113. The deduced amino acid composition is in agreement with that determined by direct amino acid analysis of purified P-450(C21) and the predicted primary structure contained amino acid sequences of N-terminal region and two internal tryptic fragments of the protein so far analyzed. Comparing the amino acid sequence with those of other forms of P-450 reveals that a conserved amino acid sequence containing a putative heme-binding cysteine is present in the equivalent position, proximate to the COOH terminus of the molecules and that P-450(C21) is phylogenically situated in an intermediate position between steroidogenic mitochondrial cytochrome P-450 which catalyzes the side-chain cleavage of cholesterol (P-450(SCC)) and drug-metabolizing microsomal P-450s. However, the amino acid sequence of P-450(C21) is much closer to that of drug-metabolizing P-450s than to that of P-450(SCC).     

Spironolactone and cytochrome P-450: impairment of steroid 21-hydroxylation in the adrenal cortex.

The effect of spironolactone administration on the activities of adrenal 21-hydroxylases was examined in male cortisol- and corticosterone-producing animals. Decreases in the activities of the 21-hydroxylases after spironolactone treatment occur only in those animals that predominantly produce cortisol rather than corticosterone and that have a high activity of adrenal steroid 17α-hydroxylase. The administration of spironolactone to cortisol-producing animals, namely, the guinea pig and the dog, caused a 50–75% loss in the activities of adrenal 21-hydroxylases with a concomitant decrease in the content of microsomal cytochrome P-450 and microsomal heme and in the activity of microsomal 17α-hydroxylase. Spironolactone treatment was also found to decrease the content of adrenal mitochondrial cytochrome P-450 in male guinea pigs but not male dogs. In contrast to its effect in cortisol-producing animals, the administration of spironolactone caused an increase in the activities of the microsomal 21-hydroxylases in the adrenals of corticosterone-producing animals such as the rat and the rabbit.     

The role of the Krebs cycle in the generation of intramitochondrial reducing equivalents for the 11 beta-hydroxylation of deoxycorticosterone in isolated rat adrenal cells

Isolated rat adrenal cells were used to study the possible pathways of intramitochondrial NADPH generation for 11β-hydroxylation of 11-deoxycorticosterone. Pyruvate was efficiently utilized by the mitochondria as shown by evolution of ¹⁴CO₂ from [1-¹⁴C]- and [2-¹⁴C]pyruvate. Citrate, isocitrate, succinate, and malate were not utilized by intact cells due to their inability to permeate the plasma membrane. For every mole of corticosterone formed, 1.9 and 0.8 moles of ¹⁴CO₂ were formed from [1-¹⁴C]- and [2-¹⁴C]pyruvate, respectively, indicating that pyruvate dehydrogenase was quite active and supplied acetyl C?oA to the Krebs cycle. Fluorocitrate and 2,4-dinitrophenol inhibited 11β-hydroxylation of 11-deoxycorticosterone as well as the production of ¹⁴CO₂ from [2-¹⁴C]pyruvate. Comparison of data with the two inhibitors showed that for the same percentage of inhibition of ¹⁴CO₂ production, the inhibition of 11β-hydroxylation was greater with 2,4-dinitrophenol than with fluorocitrate. It is concluded that operation of the Krebs cycle may be essential for 11β-hydroxylation to occur primarily because NADH generated by the cycle provides ATP, via the respiratory chain, as well as the substrate for the energy-linked transhydrogenase that forms NADPH. The NADPH required for 11β-hydroxylation seems to be derived to a large extent via the energy-linked transhydrogenase.     

Cytochrome P450 and steroid 21-hydroxylation in microsomes from beef adrenal cortex

Cytochrome P450 in beef adrenal cortex microsomal preparations reacted with progesterone and with 17-hydroxyprogesterone at pH 7.4 to produce Type I spectral changes. The magnitude of the spectral shift produced by addition of progesterone or 17-hydroxyprogesterone was related to the concentration of cytochrome P450 (over P450 concentration range of 0.1 to 0.3 μM). Prior saturation of cytochrome P450 with 17-hydroxyprogesterone prevented further spectral shift with the addition of progesterone. On the other hand, saturation of cytochrome P450 with progesterone decreases the expected shift with 17-hydroxyprogesterone by more than 50% but did not prevent the shift. The difference spectra were diminished by more than 50% at pH 9.0.The addition of NADPH resulted in loss of the spectral shifts and production of 21-hydroxylated products, predominantly DOC and 11-deoxycortisol. These reactions were not inhibited by their specific products. The rate of 21-hydroxylation was linearly related to microsomal protein (and microsomal P450) concentration. The 21-hydroxylation of progesterone was competitively inhibited by 17-hydroxyprogesterone; inhibition of the 21-hydroxylation of 17-hydroxyprogesterone by progesterone was not demonstrated.     

beta-Endorphin stimulates corticosterone synthesis in isolated rat adrenal cells.

Studies are presented which demonstrate that β-endorphin induces corticosterone synthesis in isolated fasciculata cells. This activation of steroidogenesis has a lag period of 3 to 5 minutes and is cycloheximide-sensitive. The data suggest that β-endorphin exhibits steroidogenic activity by binding to the adrenocorticotropic hormone receptors of the cells.     

Column perifusion system for steroidogenesis in isolated rat adrenal cells

A perifusion system using a plastic column into which isolated rat adrenal cells had been installed was attempted. After ACTH or cAMP was administered to the column, the corticosterone concentration in the eluate was determined. ACTH in 10(-13) and 10(-12) M did not promote corticosterone production, whereas 10(-11) and 10(-10) M showed a dose dependent production of corticosterone. By iterative infusion of 10(-11) or 10(-9) M of ACTH, very clear responses to restimulation of ACTH were noted. Following the administrations of 10(-3) or 10(-2) M of dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP), the production of corticosterone increased dose-dependently. These results suggest that this perifusion system is effective for examining the effects of ACTH or cAMP on steroidogenesis of cells.     

Sources of ammonia for urea synthesis in isolated rat liver cells

l-Leucine inhibits urea synthesis in rat hepatocytes from a number of nitrogen sources, including ammonia. The inhibition by l-leucine is largely overcome by addition of 1 mM l-ornithine, suggesting that the main site of l-leucine action is at ornithine transcarbamylase, rather than at glutamate dyhydrogenase. l-Norvaline is a more potent inhibitor of urea synthesiss than is l-leucine, but again the inhibition is largely counteracted by l-ornithine. Addition of aminooxyacetate and l-norvaline strongly suppresses the formation of glucose and lactate from l-asparagine, suggesting that an alternate pathway of aspartate metabolism, the purine nucleotide cycle, in not a major pathway. Hadacidin, an inhibitor of adenylosuccinate synthetase, an enzyme of the purine nucleotide cycle, has no effect on urea synthesis in rat liver cells.     

Pentose cycle and reducing equivalents in rat mammary-gland slices

1. Slices of mammary gland of lactating rats were incubated with glucose labelled uniformly with (14)C and in positions 1, 2, 3 and 6, and with (3)H in all six positions. Glucose carbon atoms are incorporated into CO(2), fatty acids, lipid glycerol, the glucose and galactose moieties of lactose, lactate, soluble amino acids and proteins. C-3 of glucose appears in fatty acids. The incorporation of (3)H into fatty acids is greatest from [3-(3)H]glucose. (3)H from [5-(3)H]glucose appears, apart from in lactose, nearly all in water. 2. The specific radioactivity of the galactose moiety of lactose from [1-(14)C]- and [6-(14)C]-glucose was less, and that from [2-(14)C]- and [3-(14)C]-glucose more, than that of the glucose moiety. There was no randomization of carbon atoms in the glucose moiety, but it was extensive in galactose. 3. The pentose cycle was calculated from (14)C yields in CO(2) and fatty acids, and from the degradation of galactose from [2-(14)C]glucose. A method for the quantitative determination of the contribution of the pentose cycle, from incorporation into fatty acids from [3-(14)C]glucose, is derived. The rate of the reaction catalysed by hexose 6-phosphate isomerase was calculated from the randomization pattern in galactose. 4. Of the utilized glucose, 10-20% is converted into lactose, 20-30% is metabolized via the pentose cycle and the rest is metabolized via the Embden-Meyerhof pathway. About 10-15% of the triose phosphates and pyruvate is derived via the pentose cycle. 5. The pentose cycle is sufficient to provide 80-100% of the NADPH requirement for fatty acid synthesis. 6. The formation of reducing equivalents in the cytoplasm exceeds that required for reductive biosynthesis. About half of the cytoplasmic reducing equivalents are probably transferred into mitochondria. 7. In the Appendix a concise derivation of the randomization of C-1, C-2 and C-3 as a function of the pentose cycle is described.     

Uptake and 25-hydroxylation of vitamin D3 by isolated rat liver cells

The physiological roles played by hepatocytes and nonparenchymal cells of rat liver in the metabolism of vitamin D3 have been investigated. Tritium-labeled vitamin D3 dissolved in ethanol was administered intravenously to two rats. Isolation of the liver cells 30 and 70 min after the injection showed that vitamin D3 had been taken up both by the hepatocytes and by the nonparenchymal liver cells. The relative proportion of vitamin D3 that accumulated in the nonparenchymal cells increased with time. Perfusion of the isolated rat liver with [3H] vitamin D3 added to the perfusate confirmed the ability of both cell types to efficiently take up vitamin D3 from the circulation. By a method based on high pressure liquid chromatography and isotope dilution-mass fragmentography it was found that isolated liver cells in suspension had a considerable capacity to take up vitamin D3 from the medium. About 2.5 fmol of vitamin D3 were found to be associated with each hepatocyte or nonparenchymal cell after 1 h of incubation. 25-Hydroxylation in vitro was found to be carried out only by the hepatocytes. The rate of hydroxylation was about the same whether the cells were isolated from normal or rachitic rats (3.5 and 4 pmol of 25-hydroxyvitamin D3 formed per h per 10(6) cells, respectively). The possibility that the nonparenchymal cells might serve as a storage site for vitamin D3 in the liver is discussed.     

Serotonin stimulates calcium influx in isolated rat adrenal zona glomerulosa cells.

To investigate the role of calcium as a second messenger in serotonin-stimulated aldosterone secretion, radiolabelled calcium influx studies were carried out in purified rat adrenal zona glomerulosa cells using 45CaCl2. The results show that serotonin caused calcium influx within 45 seconds of addition and this continued for up to 105 seconds. Angiotensin II also caused calcium influx; however, the effect was significantly smaller than that of serotonin. Serotonin-stimulated calcium influx could be inhibited by the calcium antagonist verapamil and by methysergide, a selective serotonin receptor type-1/2 antagonist. The data indicate that serotonin directly stimulates calcium uptake in zona glomerulosa cells via calcium channels which are coupled to specific serotonin receptors.     

Solubilization of membrane-bound adenylate cyclase of isolated rat adrenal cortex cells.

The membrane-bound adenylase cyclase (ATP pyrosphosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Milipore filters with pores 0.22 micron in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5'-guanylyl-imidodiphosphate at a concentration of 1 . 10(-6)M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5'-guanylyl-imidodiphosphate.     

Further studies on corticosteroidogenesis VI. Pyruvate and malate supported steroid 11β-hydroxylation in rat adrenal gland mitochondria

Pyruvate and pyruvate plus ATP have been shown to support 11β-hydroxylation of 11-deoxycorticosterone into corticosterone in incubated rat adrenal gland mitochondria. Corticosterone production with pyruvate plus ATP was not as great as with malate plus P_i, malate plus ATP or malate plus pyruvate. Respiratory chain inhibitors, trans-aconitate, oxaloacetate, arsenite and the uncoupler 2,4-dinitrophenol, inhibited corticosterone formation. On the other hand, cysteine sulfinate and pyruvate, which led to the removal of excess metabolic oxaloacetate formed from malate oxidation, increased rat adrenal mitochondrial O₂ consumption as well as corticosterone production from 11-deoxycorticosterone. P_i and ATP also appeared to act in the same way in that these agents brought about a greater conversion rate of oxaloacetate into pyruvate. Pyruvate, resulting from the oxidation of malate, accumulated in the incubation system only when arsenite was added. Arsenite additions to malate and isocitrate inhibited the conversion of 11-deoxycorticosterone into corticosterone except when the 11β-hydroxylation of 11-deoxycorticosterone was supported with exogenous NADPH in Ca²⁺-swollen mitochondria. These results as well as the observations that NAD-linked malate dehydrogenase ( -malate: NAD⁺ oxidoreductase (decarboxylating), EC 1.1.1.39) is at least 10 times as active as the NADP-linked enzyme ( -malate: NADP⁺ oxidoreductase (decarboxylating), EC 1.1.1.39) in sonicated rat adrenal gland mitochondria, led to the conclusion that under our incubation conditions malate was mainly oxidized via the NAD-linked malate dehydrogenase. The fact that in malate incubations pyruvate did not accumulate because of its further metabolism in rat adrenal gland mitochondria, does not support the possibility that these mitochondria are the source of pyruvate for a “malate shuttle” originally thought to occur in rat adrenal gland⁷. This shuttle would have depended on the formation of pyruvate from malate in rat adrenal gland mitochondria followed by extrusion of the pyruvate formed intramitochondrially into the cytoplasm of the cell.