Involvement of an AP-1 complex in zone-specific expression of the CYP11B1 gene in the rat adrenal cortex.

The CYP11B1 gene, which encodes steroid 11 beta-monooxygenase, which is responsible for the synthesis of cortisol and corticosterone, the major glucocorticoids in mammals, is expressed specifically in the zona fasciculata of the adrenal cortex. We have analyzed the promoter region of the rat CYP11B1 gene by using a transient-expression system with adrenocortical Y1 cells and have identified a positive regulatory region. The region contained two adjacent sites for the binding of Y1-cell nuclear proteins: the binding site for an AP-1 transcription factor composed of JunD and a Fos-related protein, and the site for Ad4-binding protein (Ad4BP). The binding of the AP-1 factor to the regulatory region had a suppressive effect on that of Ad4BP in the nuclear extracts. Mutational analyses revealed that the transcriptional activation of the CYP11B1 gene promoter in Y1 cells was attributable to the AP-1 site but not to the Ad4 site. Subsequently, nuclear extracts of the zona fasciculata cells from the rat adrenal cortex were found to contain both AP-1 factor and Ad4BP, whose binding properties to the regulatory region were almost identical to those of the two factors in the Y1-cell nuclear extracts. Moreover, immunohistochemical analyses of rat adrenal cortices showed that the AP-1 factor was present in the nuclei of CYP11B1-expressing cells in the zona fasciculata but not in the nuclei of cells in the other zones. From these results, we propose that the AP-1 transcription factor found in this study plays an important role in the zone-specific expression of the CYP11B1 gene in rat adrenal cortex.

Magnetic and natural circular dichroism spectra of cytochgromes P-450(11) beta and P-450scc purified from bovine adrenal cortex.

Magnetic (MCD) and natural circular dichroism (CD) spectra various complexes of cytochrome P-450(11) beta (P-450(11) beta) and cytochrome P-450scc (P-450scc) from bovine adrenal cortex were measured from 250 nm to 700 nm. MCD and CD spectral contours of cytochromes P-450(11) beta and P-450scc in the Soret and visible regions were, as a whole, analogous to those of cytochromes P-450 from rabbit liver microsomes and also from Pseudomonas putida in their high-spin ferric, high-spin ferrous and ferrous-CO complexes. MCD spectrum of the low-spin ferric P-450scc free from the substrate, cholesterol, was very similar to that caused by addition of 20 alpha-hydroxycholesterol, a reaction intermediate. However, it was distinct from those of the low-spin ferric P-450(11) beta and P-450scc complexes caused by addition of external nitrogenous ligands. The electronic states of the heme in the low-spin ferric P-450 free from substrates seemed to be subtly different from those of low-spin complexes coordinated with external nitrogenous ligands. Soret CD spectra of ferric low-spin complexes were not so different from each other. Upon reduction of high-spin ferric P-450(11) beta or P-450scc, the Soret CD magnitudes increased significantly in contrast with those of other P-450s, the Soret CD magnitudes of which decrease upon reduction. This may reflect an increased proximity of the neighbouring aromatic groups upon reduction of high-spin P-450(11) beta or P-450scc. High substrate specificity of adrenal P-450s compared with liver P-450s can be explained in view of the above findings. THe CD spectra in the near ultraviolet region (250-350 nm) were found to be quite sensitive to the spin change for ferric P-450scc, while the MCD spectra in this region did not reflect substantially the spin state of the enzyme. MCD parameters of cytochrome P-450s were compared to those of other hemoproteins in diagrams describing selected MCD spectral values of hemoproteins so far available and were discussed in connection with the structures of the heme environment of P-450.

Resonance Raman spectra of bovine adrenal cytochrome P-450SCC.

Resonance Raman spectra were observed for a mitochondria-type cytochrome p-450 (P-450SCC) for the first time. Reduced P-450SCC at pH 7.4 exhibited the V4 line at 1342 cm-1, which is an unusually low frequency compared with an ordinary protohemoprotein but is common to the family of cytochrome P-450, suggesting the coordination of a strong pi-donor such as thiolate anion at the fifth coordination position of the heme iron. The anomaly was preserved for the CO-complex of the reduced form. The V10 line of oxidized P-450SCC with a substrate was observed at 1617 cm-1. This frequency and those of other structure-sensitive bands implied that the heme iron of oxidized P-450SCC adopts the hexa-coordinate high-spin structure, in contrast with the high-spin type cytochrome P-450 purified from phenobarbital- or 3-methylcholanthrene-treated rabbit liver microsomes which presumably have a penta-coordinate structure. In the presence of 20alpha-hydroxycholesterol, oxidized P-450SCC gave the V10 line at 1637 cm-1, i.e., at a frequency similar to that of low-spin type cytochrome P-450. The alkaline-denatured P-420SCC preparation in the presence of both dithiothreitol and EDTA, but not the P-450SCC gave the V10 line at 1637 cm-1, i.e., at a frequency similar to that of low-spin type cytochrome P-450. The alkaline-denatured P-420SCC preparation in the presence of both dithiothreitol and EDTA, but not the P-450SCC.

Zone-specific expression of aldosterone synthase cytochrome P-450 and cytochrome P-45011 beta in rat adrenal cortex: histochemical basis for the functional zonation.

Zonal distribution of aldosterone synthase cytochrome P-450 and cytochrome P-45011 beta in rat adrenocortex was investigated immunochemically using specific antibodies to these enzymes. Localization of aldosterone synthase cytochrome P-450 (cytochrome P-450aldo), a recently identified enzyme that converts deoxycorticosterone to aldosterone in rat adrenocortex was strictly confined to two or three outermost cell layers in the zona glomerulosa. In contrast, cytochrome P-45011 beta, which forms corticosterone, but not aldosterone, from deoxycorticosterone, was localized in the zona fasciculata-reticularis and not in the zona glomerulosa. Neither enzyme was detected in the medulla or the capsule. The functional zonation of adrenocortex with respect to aldosterone and corticosterone syntheses is, thus, ascribable to the localization of cytochromes P-450aldo and P-45011 beta in the respective zones. When rats were maintained under Na-depleted conditions for 10 days, the zona glomerulosa cells containing cytochrome P-450aldo proliferated to 10-15 layers, the thickness of which was 5-7-fold that in the nonstimulated rats. Proliferation of the cytochrome P-450aldo-positive cells into the zona fasciculata-reticularis was also observed along with arterial walls. Under these conditions, no significant change in the distribution of cytochrome P-45011 beta was noted. These results indicate that the angiotensin-II stimuli, which had been elicited by the low Na treatment, promoted proliferation of the glomerulosa cells, resulting in increased expression of cytochrome P-450aldo in rat adrenocortex.

Development of functional zonation in the rat adrenal cortex.

In an attempt to elucidate the mechanism(s) through which the functional adrenal cortex is established, we analyzed immunohistochemically the expression of various markers for the adrenocortical zones, i.e. the zona glomerulosa (zG), the zona fasciculata (zF), and the zona reticularis (zR), as well as markers for the medulla, and further examined the distribution and behavior of DNA-synthesizing cells in rat adrenal glands during development. The results showed that 1) separation of the cortex and medulla, and the development of functional zonation in the cortex began at around the time of birth, 2) at fetal stages when cortical zonation was not established, DNA-synthesizing cells were found scattered throughout the gland, where they proliferated without significant migration, and 3) after birth in the adrenal cortex with established cortical zonation, DNA-synthesizing cells were localized near the undifferentiated zone between zG and zF, and then they migrated centripetally. Cell death appeared to occur in the innermost portion of the cortex, where many resident macrophages are present. These findings illustrate basic processes underlying adrenal development and suggest that the undifferentiated region is apparently the stem cell zone of the adrenal cortex that maintains the cortical zonation.

Regulation of aldosterone synthase cytochrome P-450 in rat adrenals by angiotensin II and potassium.

Changes in the levels of aldosterone synthase cytochrome P-450, a recently identified enzyme in rat adrenals, were studied in response to the renin-angiotensin system and K stimuli. As examined by an immunoblot technique, the zona glomerulosa mitochondria from rats fed on a low Na-normal K diet (8.6 mmol Na+ and 207 mmol K+/kg of diet) or a low Na-high K (0.2 M KCl in drinking water) diet for 4-10 days contained significantly higher amounts of aldosterone synthase cytochrome P-450 than those from rats fed on a normal diet (86 mmol Na+ and 207 mmol K+/kg of diet). Activities of the enzyme were also found to increase by about 10-fold on day 10. In concert with these changes, both plasma renin activity and plasma aldosterone concentration increased, indicating that the renin-angiotensin system was activated in these rats. Feeding with a normal Na-high K diet also induced significantly higher levels of both amount and activity of aldosterone synthase cytochrome P-450 together with an elevated serum K concentration on day 4, though they all decreased to near the control level on the following days. On the other hand, when enalapril malate, an angiotensin I-converting enzyme inhibitor, was administered to the low Na-normal K rats, the increases in the amount and activity of the enzyme as well as in plasma aldosterone concentration were suppressed altogether. However, the enalapril administration to the low Na-high K rats suppressed the increases only partially. These results indicate that the aldosterone synthase cytochrome P-450 is an ultimate target of the regulation of aldosterone biosynthesis by angiotensin II and K.

A novel cell layer without corticosteroid-synthesizing enzymes in rat adrenal cortex: histochemical detection and possible physiological role.

A stratum of cells that did not contain both aldosterone synthase cytochrome P450 (cytochrome P450aldo) and cytochrome P45011 beta was found immunohistochemically between the zona glomerulosa and the zona fasciculata of the rat adrenal cortex. As cytochromes P450aldo and P45011 beta are the enzymes responsible for the biosynthesis of aldosterone and corticosterone, respectively, the cells there are considered to be incapable of synthesizing both aldosterone and corticosterone. Furthermore, the cells are regarded as inert in producing adrenal androgens, because rat adrenal cortex is known to lack steroid 17 alpha-hydroxylase. Thus, the stratum is composed of cells that do not synthesize any of the major corticosteroids in significant quantities. It was 5-10 cells thick under normal feeding conditions, but diminished to 4-5 cells thick when animals were maintained under Na restriction, which is known to stimulate the secretion of angiotensin-II. When the distribution of 5-bromo-2'-deoxyuridine-labeled nuclei in the adrenocortex from BrdU-administered rats was examined, the stained nuclei were concentrated in and around the cell stratum. The pulse-chase experiments showed that the labeled cells migrated out of this layer and into the zonae fasciculata-reticularis. On the basis of these findings, we suggest that the newly discovered cell layer is the progenitor cell zone of the rat adrenal cortex.

Complexes of cytochrome P450 with metyrapone. A convenient method for the quantitative analysis of phenobarbital-inducible cytochrome P450 in rat liver microsomes.

Phenobarbital-inducible cytochrome P450 variants have a higher affinity (app. Ks = 0.6 microM). The difference in affinity has enabled us to quantitate, by metyrapone-induced difference spectral analysis, phenobarbital-inducible cytochromes P450 in liver microsomal membranes. The result obtained by this simple, rapid technique compare well with those found by more sophisticated techniques such as radioimmunoassay and immunoprecipitation.

Cytochrome P-45011 beta and P-450scc in adrenal cortex: zonal distribution and intramitochondrial localization by the horseradish peroxidase-labeled antibody method.

In an attempt to elucidate the regulation mechanism(s) of adrenocortical steroidogenesis, cytochrome P-450scc and cytochrome P-45011 beta were localized in bovine adrenal glands by the direct peroxidase-labeled antibody method. At the light microscopic level, parenchymal cells of the zona fasciculata and the zona reticularis stained heavily for both cytochromes, while the parenchymal cells of zona glomerulosa stained lightly for both. At the electron microscopic level, these two cytochromes were associated with the matrix side of the inner mitochondrial membranes of parenchymal cells from all three zones of the adrenal cortex. The association of cytochrome P-450 with the inner mitochondrial membrane, in a manner similar to that previously reported for adrenodoxin and adrenodoxin reductase (F Mitani, Y Ishimura, S Izumi, K Watanabe, Acta Endocrinol 90:317, 1979), establishes that the steroid monooxygenase systems exist at this site. The degree of immunocytochemical staining within a single cell varied from one mitochondrion to another: some stained intensely along the entire inner membrane, including the cristae, some stained only along segments of the inner membrane, and some did not stain at all. This heterogeneity in staining was observed in mitochondria stained in situ as well as in isolated mitochondria. These findings suggest that there is a heterogeneity in steroidogenesis among mitochondria contained within a single cell of the adrenal cortex.

Isolation of two distinct cytochromes P-45011 beta with aldosterone synthase activity from bovine adrenocortical mitochondria.

Two distinct forms of cytochrome P-45011 beta, with apparent molecular weights of 48,500 (48.5K) and 49,500 (49.5K), have been isolated from bovine adrenocortical mitochondria. Their amino acid sequences up to the 19th position from the N-terminus were only different at the 6th position (Val and Ala for the 48.5K and 49.5K enzymes, respectively). Each sequence was assignable to a distinct cDNA clone for cytochrome P-450(11) beta (Kirita, S., et al. [1988] J. Biochem. 104, 683-686), indicating that the two proteins originate from different genes in bovine adrenocortical cells. Both forms of cytochrome P-450(11) beta were capable of catalyzing aldosterone synthesis as well as the 11 beta- and 18-hydroxylation of 11-deoxycorticosterone. Thus, at least two distinct cytochrome P-450(11) beta species exist in the adrenal cortex and participate in steroidogenesis.

Rotation and protein-protein interactions of cytochrome P-450 in the inner membrane of adrenocortical mitochondria.

Rotational diffusion of the total cytochrome P-450 (P-450scc plus P-45011 beta) in bovine adrenocortical mitochondria was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the hemo.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions of cytochrome P-450 with other membrane proteins. The absorption anisotropy decayed within 1 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the presence and absence of deoxycorticosterone (DOC), a substrate for cytochrome P-45011 beta. The observed value for the normalized time-independent anisotropy r(infinity)/r(0) and the average rotational relaxation time phi are r(infinity)/r(0) = 0.88 and phi = 233 microseconds when DOC is absent, and r(infinity)/r(0) = 0.65 and phi = 350 microseconds when DOC is present. Judging from the phi value, rotating P-450 is not a monomeric molecule, but would be a small microaggregate with an average diameter of about 120 A. A significantly high value of r(infinity)/r(0) implies co-existence immobile populations of cytochrome P-450. Based on the assumption that the heme angle tilts 55 degrees from the membrane plane (Gut et al. (1983) J. Biol. Chem. 258, 8588-8594), 65% (when DOC is present) or 88% (when DOC is absent) of cytochrome P-450 in mitochondria is immobilized within the experimental time range of 2 ms due to the presence of immobile protein microaggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on cytogenesis in adult rat adrenal cortex: circadian and zonal variations and their modulation by adrenocorticotropic hormone.

Circadian rhythms and zonal variations in the cell proliferation of adult rat adrenal cortex were studied by following the cells in the DNA-synthesizing stage (S-phase) as assessed by 5-bromo-2'-deoxyuridine incorporation into the cell-nuclei and/or by visualizing proliferating cell nuclear antigen. The S-phase cells were observed throughout the day in two regions of the adrenal cortex: (i) a region from the inner half of the zona glomerulosa to near the outer margin of the zona fasciculata, and (ii) the outer one-fourth portion of the zona fasciculata. Very little change in number was observed in the former region between day and night, while a burst of cell proliferation occurred in early morning at 3-4 a.m. in the latter region. A prominent rise in the plasma adrenocorticotropic hormone (ACTH) concentration preceded the burst of cell proliferation by about 4 h. Upon raising the plasma ACTH concentration by administration of ACTH or metyrapone, prominent cell proliferation also occurred in the same portion of the zona fasciculata 4-6 h after the provoked ACTH surge. Thus at least two sites in rat adrenal cortex are responsible for cytogenesis in this endocrine organ, and respond differentially to day/night cycles and circulating ACTH levels.

Conversion of cholesterol to pregnenolone mobilizes cytochrome P-450 in the inner membrane of adrenocortical mitochondria: protein rotation study.

Rotation of cytochrome P-450 was examined in bovine adrenocortical mitochondria before and after an enzymatic transformation of cholesterol into pregnenolone by cytochrome P-450scc in the presence of malate. Rotational diffusion was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate substrate-dependent intermolecular interactions of cytochrome P-450 with other redox components. Rotational mobility of cytochrome P-450 was significantly dependent on the decrease in cholesterol content by side chain cleavage reaction catalyzed by cytochrome P-450scc. In a typical experiment, the observed value for the normalized time-independent anisotropy r(infinity)/r(0) was decreased from 0.78 in control mitochondria to 0.60 after conversion of 21% of cholesterol to pregnenolone, while no significant change was observed for the average rotational relaxation time phi of about 700 microseconds. Significantly high values of r(infinity)/r(0) = 0.78 and 0.60 imply co-existence of mobile and immobile populations of cytochrome P-450. Since we observed that the heme angle tilted 55 degrees from membrane plane, 22% (control mitochondria) and 40% (after conversion of cholesterol to pregnenolone) of cytochrome P-450 in mitochondria are calculated to be mobile in the preparation. The significant mobilization of cytochrome P-450scc molecules caused by the conversion of cholesterol to pregnenolone is likely due to changes in protein-protein interactions with its redox partners, since the lipid fluidity was kept unchanged by the cholesterol depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cytochrome P450 activities in adrenocortical mitochondria from normal rats and human neoplastic tissues.

From our immunocytochemical studies, cytochrome P450scc and P45011 beta systems were localized on the matrix side of inner membrane of the mitochondria in the parenchymal cells of adrenal cortex. However, the degree of immunocytochemical staining varied from one mitochondrion to another within a single cell; some stained intensely along the entire inner membrane, some stained only along segments of the inner membrane, and some did not stain at all. The ratio of stained to unstained mitochondria was approximately unity in untreated rats, while stained mitochondria greatly increased upon ACTH administration suggesting that the population of cytochrome P450 system-containing mitochondria increased upon long-term ACTH action. By the combined use of flash photolysis and substrate difference spectroscopy, quantitative determination of substrate-bound and free forms of P450scc and P45011 beta in mitochondria became possible. Increases in total amounts of P450scc and cholesterol resulted from the long-term ACTH administration to rats. The ratio of cholesterol-bound P450scc to its free form was essentially unchanged under these conditions. On the other hand, amounts of P45011 beta were not increased significantly by long-term ACTH administration. The ratio of DOC-bound P45011 beta to the free form increased significantly, however, by long-term ACTH administration and also in the mitochondria from patients suffering from Cushing's syndrome.

Expression of cytochromes P450aldo and P45011 beta in rat adrenal gland during late gestational and neonatal stages.

The development of the rat adrenal gland during late gestational and neonatal stages was studied by following the expression of aldosterone synthase cytochrome P450 (P450aldo) and glucocorticoid-synthesizing cytochrome P450 (P45011 beta). Cells expressing P450aldo, a functional marker for the mineralocorticoid-synthesizing zona glomerulosa, were not detected until day 20 of fetal age, i.e., 2 days before birth, although the zona glomerulosa cells were histologically recognizable at the 18th day of gestation. The intensity of P450aldo staining thereafter became stronger with age in the outer portion of the cortex. Cells expressing P45011 beta, a marker for the glucocorticoid-producing zona fasciculata, were present in the fetal adrenals on the 18th day. P45011 beta-positive cells were distributed over the whole adrenal gland and intermingled with the cells containing tyrosine hydroxylase, a marker enzyme for medullary cells. The P45011 beta-positive and tyrosine hydroxylase-positive cells began to separate on the 20th day, and were completely resolved from each other around the third day after birth. Expression of P450aldo and P45011 beta, together with that of tyrosine hydroxylase, thus serves as a suitable marker for studying the development of the adrenal gland.