Apolipoprotein A-I is required for cholesteryl ester accumulation in steroidogenic cells and for normal adrenal steroid production.

In addition to its ability to remove cholesterol from cells, HDL also delivers cholesterol to cells through a poorly defined process in which cholesteryl esters are selectively transferred from HDL particles into the cell without the uptake and degradation of the lipoprotein particle. The HDL-cholesteryl ester selective uptake pathway is known to occur in human, rabbit, and rodent hepatocytes where it may contribute to the clearance of plasma cholesteryl ester. The selective uptake pathway has been studied most extensively in steroidogenic cells of rodents in which it accounts for 90% or more of the cholesterol destined for steroid production or cholesteryl ester accumulation. In this study we have used apo A-I-, apo A-II-, and apo E-deficient mice created by gene targeting in embryonic stem cells to test the importance of the three major HDL proteins in determining cholesteryl ester accumulation in steroidogenic cells of the adrenal gland, ovary, and testis. apo E and apo A-II deficiencies were found to have only modest effects on cholesteryl ester accumulation. In contrast, apo A-I deficiency caused an almost complete failure to accumulate cholesteryl ester in steroidogenic cells. These results suggest that apo A-I is essential for the selective uptake of HDL-cholesteryl esters. The lack of apo A-I has a major impact on adrenal gland physiology causing diminished basal corticosteroid production, a blunted steroidogenic response to stress, and increased expression of compensatory pathways to provide cholesterol substrate for steroid production.

NADPH production by the pentose phosphate pathway in the zona fasciculata of rat adrenal gland.

Biosynthesis of steroid hormones in the cortex of the adrenal gland takes place in smooth endoplasmic reticulum and mitochondria and requires NADPH. Four enzymes produce NADPH: glucose-6-phosphate dehydrogenase (G6PD), the key regulatory enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD), the third enzyme of that pathway, malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). However, the contribution of each enzyme to NADPH production in the cortex of adrenal gland has not been established. Therefore, activity of G6PD, PGD, MDH, and ICDH was localized and quantified in rat adrenocortical tissue using metabolic mapping, image analysis, and electron microscopy. The four enzymes have similar localization patterns in adrenal gland with highest activities in the zona fasciculata of the cortex. G6PD activity was strongest, PGD, MDH, and ICDH activity was approximately 60%, 15%, and 7% of G6PD activity, respectively. The K(m) value of G6PD for glucose-6-phosphate was two times higher than the K(m) value of PGD for phosphogluconate. As a consequence, virtual flux rates through G6PD and PGD are largely similar. It is concluded that G6PD and PGD provide the major part of NADPH in adrenocortical cells. Their activity is localized in the cytoplasm associated with free ribosomes and membranes of the smooth endoplasmic reticulum, indicating that NADPH-demanding processes related to biosynthesis of steroid hormones take place at these sites. Complete inhibition of G6PD by androsterones suggests that there is feedback regulation of steroid hormone biosynthesis via G6PD.

Voltage-gated transient currents in bovine adrenal fasciculata cells. I. T-type Ca2+ current.

The whole cell version of the patch clamp technique was used to identify and characterize voltage-gated Ca2+ channels in enzymatically dissociated bovine adrenal zona fasciculata (AZF) cells. The great majority of cells (84 of 86) expressed only low voltage-activated, rapidly inactivating Ca2+ current with properties of T-type Ca2+ current described in other cells. Voltage-dependent activation of this current was fit by a Boltzmann function raised to an integer power of 4 with a midpoint at -17 mV. Independent estimates of the single channel gating charge obtained from the activation curve and using the "limiting logarithmic potential sensitivity" were 8.1 and 6.8 elementary charges, respectively. Inactivation was a steep function of voltage with a v1/2 of -49.9 mV and a slope factor K of 3.73 mV. The expression of a single Ca2+ channel subtype by AZF cells allowed the voltage-dependent gating and kinetic properties of T current to be studied over a wide range of potentials. Analysis of the gating kinetics of this Ca2+ current indicate that T channel activation, inactivation, deactivation (closing), and reactivation (recovery from inactivation) each include voltage-independent transitions that become rate limiting at extreme voltages. Ca2+ current activated with voltage-dependent sigmoidal kinetics that were described by an m4 model. The activation time constant varied exponentially at test potentials between -30 and +10 mV, approaching a voltage-independent minimum of 1.6 ms. The inactivation time constant (tau i) also decreased exponentially to a minimum of 18.3 ms at potentials positive to 0 mV. T channel closing (deactivation) was faster at more negative voltages; the deactivation time constant (tau d) decreased from 8.14 +/- 0.7 to 0.48 +/- 0.1 ms at potentials between -40 and -150 mV. T channels inactivated by depolarization returned to the closed state along pathways that included two voltage-dependent time constants. tau rec-s ranged from 8.11 to 4.80 s when the recovery potential was varied from -50 to -90 mV, while tau rec-f decreased from 1.01 to 0.372 s. At potentials negative to -70 mV, both time constants approached minimum values. The low voltage-activated Ca2+ current in AZF cells was blocked by the T channel selective antagonist Ni2+ with an IC50 of 20 microM. At similar concentrations, Ni2+ also blocked cortisol secretion stimulated by adrenocorticotropic hormone. Our results indicate that bovine AZF cells are distinctive among secretory cells in expressing primarily or exclusively T-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage-gated transient currents in bovine adrenal fasciculata cells. II. A-type K+ current.

In whole cell patch clamp recordings on enzymatically dissociated adrenal zona fasciculata (AZF) cells, a rapidly inactivating A-type K+ current was observed in each of more than 150 cells. Activation of IA was steeply voltage dependent and could be described by a Boltzmann function raised to an integer power of 4, with a midpoint of -28.3 mV. Using the "limiting logarithmic potential sensitivity," the single channel gating charge was estimated to be 7.2 e. Voltage-dependent inactivation could also be described by a Boltzmann function with a midpoint of -58.7 mV and a slope factor of 5.92 mV. Gating kinetics of IA included both voltage-dependent and -independent transitions in pathways between closed, open, and inactivated states. IA activated with voltage-dependent sigmoidal kinetics that could be fit with an n4h formalism. The activation time constant, tau a, reached a voltage-independent minimum at potentials positive to 0 mV. IA currents inactivated with two time constants that were voltage independent at potentials ranging from -30 to +45 mV. At +20 mV, tau i(fast) and tau i(slow) were 13.16 +/- 0.64 and 62.26 +/- 5.35 ms (n = 34), respectively. In some cells, IA inactivation kinetics slowed dramatically after many minutes of whole cell recording. Once activated by depolarization, IA channels returned to the closed state along pathways with two voltage-dependent time constants which were 0.208 s, tau rec-f and 10.02 s, tau rec-s at -80 mV. Approximately 90% of IA current recovered with slow kinetics at potentials between -60 and -100 mV. IA was blocked by 4-aminopyridine (IC50 = 629 microM) through a mechanism that was strongly promoted by channel activation. Divalent and trivalent cations including Ni2+ and La3+ also blocked IA with IC50's of 467 and 26.4 microM, respectively. With respect to biophysical properties and pharmacology, IA in AZF cells resembles to some extent transient K+ currents in neurons and muscle, where they function to regulate action potential frequency and duration. The function of this prominent current in steroid hormone secretion by endocrine cells that may not generate action potentials is not yet clear.

Hormone-sensitive lipase deficiency in mice causes lipid storage in the adrenal cortex and impaired corticosterone response to corticotropin stimulation.

Hormone-sensitive lipase (HSL, E.C.3.1.1.3, gene designation Lipe) is reportedly the major cholesteryl esterase of adrenal cortex. Because of the potential importance of cholesteryl ester hydrolysis in steroidogenesis, gene-targeted HSL-deficient mice were assessed for adrenal cortical morphology and function. Compared with control animals, HSL deficiency results in a marked accumulation of lipid droplets both in zona glomerulosa and zona fasciculata. In the zona fasciculata, lipid accumulation was observed progressively from the outer to the inner regions, culminating near the corticomedullary junction with the formation of syncytial-lipoid structures having the appearance of degenerative cells. These morphological changes did not significantly alter the basal levels of circulating corticosterone, but following ACTH stimulation, corticosterone levels were decreased (P < 0.001). The observation of normal basal corticosterone and aldosterone levels demonstrates that some free cholesterol for steroid synthesis can be produced independently of HSL. Taken together, these results indicate that HSL-deficient mice accumulate lipid droplets in such a way as to impair acute ACTH stimulation of corticosterone secretion. Such observations are also found in some forms of congenital adrenal hyperplasia. By extension, HSL deficiency may be a cause of hereditary adrenocortical hypofunction in humans.

Zonal differences in adrenocortical lipid peroxidation: role of alpha-tocopherol.

Studies were done to evaluate the relationship between alpha-tocopherol (alpha-T) concentrations and lipid peroxidation (LP) in vitro in microsomal preparations from the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Microsomes were incubated with ferrous ion (Fe2+) to promote free radical production, and alpha-T levels and LP were monitored after various incubation times. alpha-T concentrations were far lower in inner than outer zone preparations and were rapidly depleted from inner zone microsomes by incubation with Fe2+. Coinciding with alpha-T depletion was a large and rapid increase in LP. With outer zone microsomes, alpha-T depletion required more than 30 min, and very little LP was demonstrable during this period. However, once alpha-T depletion occurred, LP was rapidly initiated and reached levels similar to those obtained with inner zone preparations. Inhibition of LP by MnCl2 prevented the Fe(2+)-induced declines in alpha-T in both zones. The results demonstrate the importance of alpha-T as a modulator of adrenal LP and indicate that the zonal differences in LP are largely attributable to the differences in alpha-T concentrations.

Characterization of the steroidogenic responsiveness and ultrastructure of purified zona fasciculata/reticularis cells from bovine adrenal cortex before and after primary culture.

Analysis by electron microscopy indicated that after 3 days of primary culture, purified bovine adrenal zonal fasciculata/reticularis (ZF/ZR) cells showed improved integrity of their ultrastructure, with an increased density of lipid droplets and smooth endoplasmic reticulum. The basal cortisol output was significantly (P less than 0.05) greater on day 3 of culture than for the freshly isolated cells in six out of seven experiments. Similarly, in six experiments with ACTH (1 nmol/l) and five experiments with angiotensin II (10 nmol/l), the stimulated cortisol secretion was significantly (P less than 0.01 for all 11 experiments) higher on day 3 of culture than in freshly isolated cells. No significant increase in cortisol secretion above basal was observed with noradrenaline at any concentration in the freshly isolated cells, whereas a dose-dependent increase in cortisol secretion was observed on day 3 of culture in all of four experiments. These findings were supported by cyclic (c) AMP output measured in one such experiment. Thus the basal cAMP output and that stimulated by ACTH (1 nmol/l) were significantly higher after culture (P less than 0.001, n = five wells for basal comparison; P less than 0.05, n = three wells for ACTH at 1 nmol/l). In agreement with the cortisol results, cAMP production was unaffected by any concentration of noradrenaline in the freshly isolated cells, whereas a dose-dependent rise was found after culture. Angiotensin II at all concentrations had no effect on cAMP production in freshly isolated or cultured cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterisation of calcineurin from adrenal cell cytoskeleton: identification of substrates for Ca2+-calmodulin-dependent phosphatase activity.

Ca2+-calmodulin-dependent protein phosphatase activity is found in cytoskeletons of Y-1 mouse adrenal and bovine fasciculata cells. The activity is inhibited by three inhibitors of calmodulin (trifluoperazine, W-7 and pimozide) with EC50 in the low micromolar range. Protein phosphatase activity is inhibited by vanadate, fluoride, Zn2+ and pyrophosphate, stimulated by Mn2+ and found to be tightly bound to the cytoskeleton. Substrates for endogenous phosphatase activity were defined by one- and two-dimensional polyacrylamide gels. Phosphatase activity was seen with proteins that are substrates for both cyclic AMP-dependent and cyclic AMP-independent kinase enzymes. One specific Ca2+-calmodulin-dependent phosphatase, namely calcineurin, was purified to near homogeneity from cytoskeletons of Y-1 cells. The enzyme was found to be a heterodimer (MW 61,000 and 16,000) and the smaller subunit was shown to cross-react with antibodies raised against calcineurin from bovine brain. The purified enzyme catalyzes dephosphorylation of proteins (phosphorylase kinase and casein), phosphoamino acids (tyr greater than thre greater than ser) and a synthetic substrate (p-nitrophenyl phosphate). In addition, a new application of membrane transfer was devised by which the purified enzyme was incubated with a Western blot of cytoskeleton following incubation with [32P]ATP. This method defined four specific substrates of the enzyme (MW 150,000, 55,000, 35,000 and 30,000). Anti-calcineurin revealed that only a single Ca2+-calmodulin-dependent phosphatase is found in adrenal cell cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

ACTH-induced ultrastructural changes in the zona fasciculata of the hamster adrenal cortex. Are intraadrenal thrombi regulators of corticosteroid secretion?

Correlated stereological and functional studies were performed on the effect of massive ACTH doses on adrenal cortex of the female hamster. ACTH resulted in a marked increase in adrenal gland weight at day 6 of treatment followed by a drop at day 9. Stereology showed significant enlargement of the zona fasciculata (ZF) cells with the highest value at day 6 and subsequent drop at day 9 of treatment. This hypertrophy was due to a notable increase in the volume of mitochondrial, SER, Golgi apparatus and lipid droplet compartments. Cortisol secretion by adrenal slices and homogenates was also highest at day 6 of ACTH administration and notably lower at day 9. At day 6 of ACTH treatment in outer ZF thrombi were seen. In their vicinity the subendothelial space was dilated and endothelial cells dissociated from the basal lamina. Numerous erythrocytes were also visible among dissociated ZF cells. At day 9 of experiment in outer part of ZF numerous spaces devoid of parenchymal cells appeared. The earlier authors considered the "empty spaces" or "holes" in hyperstimulated adrenal cortex as a sign of holocrine secretion of steroid hormones. The present findings enable us to introduce a new hypothesis on the development of these spaces. In our opinion in hyperstimulated adrenal cortex numerous thrombi may be formed leading thus to the degeneration of adrenocortical cells. Thus, the appearance of the "empty spaces" or "holes" in the gland is not connected with the holocrine secretion but with the regulation of the number of secretory cells in adrenal cortex by the thrombi-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent changes in the function and morphology of mitochondria of rat adrenal zona fasciculata.

The function and morphology of adrenal zona-fasciculata (ZF) mitochondria were studied in 4-, 10- and 16-month-old rats, since in this species ageing causes a marked decline in glucocorticoid secretion coupled with high levels of circulating ACTH. Dispersed intact ZF cells displayed a significant age-dependent impairment of their basal pregnenolone (PREG) secretion, but isolated ZF mitochondria showed an increased capacity to convert cholesterol to PREG (the first rate-limiting step of steroid synthesis). These data are in keeping with the contention that the age-related deficit of rat ZF secretion is located prior to the activity of intramitochondrial cholesterol side-chain cleaving enzymes (cytochrome-P450scc). Stereology showed a notable age-dependent increase in the number of mitochondria per unit cell-volume, coupled with a marked decrease in their average volume. The width of the mitochondrial intermembrane space remained unchanged, but its average volume strikingly decreased. This last finding fits well with the enhanced capacity of mitochondria to produce PREG, since intermembrane space is an aqueous barrier to the translocation of free cholesterol from the outer membrane to the cristae, where cytochrome-P450scc is located. In conclusion, the hypothesis is advanced that all these age-related functional and morphological mitochondrial changes are an ACTH-dependent compensatory response enabling ZF cells to partially counteract their decreased glucocorticoid secretory capacity, which in turn is due to the impaired utilization of intracytoplasmic stores of cholesterol esters.

Comparative stereological studies on zonation and cellular composition of adrenal glands of normal and anencephalic human fetuses. II. Cellular composition of the gland.

In our previous paper (Bocian-Sobkowska et al., 1997) we demonstrated a striking difference in development of zonation in adrenals of normal and anencephalic human fetuses. The purpose of the present study was to characterize, by means of stereology, the cellular composition of developing adrenals in the same case. Studies were performed on 11 pairs of adrenal glands from normal fetuses and 10 from anencephalic fetuses. In the studied period of development (24 to 39 weeks of intra-uterine life) the average volume of cells in normal glands increased as follows: zona glomerulosa (ZG) from 355 to 870 microns3; zona fasciculata (ZF) from 779 to 1200 microns3; fetal zone (FZ) from 2004 to 2380 microns3: and medulla (M) from 600 to 970 microns3. In anencephalic fetuses, the appropriate values were: ZG-380-680 microns3; ZF-460-680 microns3; FZ-1820-1680 microns3; and M-870-1400 microns3. At the end of the studied period the number of ZG cells in normal fetuses was two fold higher than in anencephalics, ZF cells-6-fold and in FZ-5-fold higher, while in the M the number of cells was nearly equal in both groups. During the whole investigated period of intra-uterine development the total number of adrenocortical cells in normal glands increased ca 2.5-fold, while in anencephalic glands only ca 0.5-fold, reaching at the end ca 40% of normal value. In both normal and anencephalic adrenals the number of ZG and M cells was highly correlated with ZG/M cell ratio, being slightly higher in normal glands. No such relation was demonstrated for cells of the remaining adrenocortical zones.

Adrenomedullin and calcitonin gene-related peptide inhibit aldosterone secretion in rats, acting via a common receptor.

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) did not affect either basal or ACTH-stimulated secretion of a1dosterone and corticosterone by dispersed rat capsular and inner adrenocortical cells, respectively. However, both peptides strongly depressed angiotensin-II (ANG- II)-stimulated a1dosterone production by capsular cells, the minimal effective concentration was 10(-7) M. The inhibitory effect of both ADM and CGRP was reversed by CGRP8-37, a specific CGRP1 receptor antagonist; a complete reversal was obtained with a CGRP8-37 concentration of 10(-6) M. Our findings indicate that ADM and CGRP specifically interfere with the intracellular mechanisms transducing the secretagogue signal of ANG-II, and suggest that the ADM effect is mediated by CGRP receptors

The effects of monensin on Golgi complex of adrenal cortex and steroidogenesis.

The ultrastructural and biochemical changes produced by monensin on zona fasciculata cells of the rat adrenal cortex are described. In this study we used adrenal cells in culture, adrenal slices and the intact animal. Monensin (1 microM) was added to the culture medium containing the cells, and to the incubation medium containing the adrenal slices, and was injected intravenously to the intact animal (0.65 mg/kg body weight). The ultrastructural alterations were similar in the three experimental conditions, and consisted of Golgi complex disorganization with dilated cisternae or large smooth vesicles. Quantitative analysis showed a significant increase of the relative volume of the Golgi area. The biochemical study demonstrated a significant decrease of corticosterone concentrations in culture medium after monensin addition, and in adrenal glands from treated rats. These results showed that monensin alters the fine structure of adrenal cortex Golgi complex and inhibits corticosteroidogenesis, which supports the probable role of the Golgi complex in the regulation of steroidogenesis.

A morphometric study of the reversal of ACTH-induced hypertrophy of rat adrenocortical cells after cessation of treatment.

The effect of a prolonged (7-day) ACTH administration on rat zona fasciculata cells and its reversal after cessation of treatment was investigated by morphometry. ACTH treatment caused a notable cell hypertrophy, which was mainly due to the increase in the volume of the mitochondrial compartment and to smooth endoplasmic reticulum (SER) proliferation, and a conspicuous rise in the basal level of corticosterone. After cessation of ACTH administration, rat zona fasciculata cells underwent a time-dependent atrophy, so that after 5 days they resembled those of control animals, and the blood concentration of corticosterone reverted to the base-line value. The cell atrophy was provoked by the decrease in the volumes of the mitochondrial compartment and SER, and was associated with a striking time-dependent accumulation of dense bodies. Stereology demonstrated that during the first two days after ACTH withdrawal the decrease of SER prevailed over that of the mitochondrial compartment, while the reverse occurred during the remaining three days. The increase in the volume of dense-body compartment, though largely due to the accumulation of residual bodies, was mainly coupled with a rise in the volume of the microautophagic-vacuole compartment during the first two days after ACTH cessation and with an increase in that of the macroautophagic-vacuole compartment during the following three days. The hypothesis is advanced that both micro- and macroautophagy play a role in the reversal of ACTH-induced hypertrophy of rat zona fasciculata cells after cessation of treatment, the first process being mainly involved in the elimination of SER, and the second one in the degradation of mitochondria.

Time-dependent changes in adrenal cortex ultrastructure and corticosterone levels after noise exposure in male rats.

In response to a stressful stimulus, there is a marked activation of the hypothalamic-pituitary-adrenal axis leading to a release of adrenocorticotropic hormone. This, in turn, acts on the zona fasciculata of the adrenal cortex to increase corticosterone plasma levels. Given the frequency of chronic intermittent noise exposure in man, we selected loud noise to evaluate concomitant changes in the ultrastructure of the adrenal cortex and corticosterone release. Following chronic (21 days, 6 h per day) loud white noise exposure (100 dBA, 0-26 KHz), we found the zona fasciculata to be most sensitive to time-dependent ultrastructural changes. These consisted of modifications in cell compartments involved in hormone synthesis and release. On the other hand, we found a progressive increase in corticosterone plasma levels which reached a plateau 9 days after noise exposure. The significance of these changes, in relation to phenomena like sensitization to repetitive stress, are discussed. Furthermore, the present data suggest that chronic loud noise exposure might potentially lead to endocrine dysfunctions.

[Structure of the adrenal gland in rat strain with hereditary stress-induced arterial hypertension nursed by Wistar dams]. / Strukturnye osobennosti nadpochechnika krys linii s nasledstvennoi indutsirovannoi stressom arterial'noi gipertenziei pri vskarmlivanii samkami linii Wistar.

The comparative investigation of the adrenal structure in two groups of rats of Inherited Stress-Induced Arterial Hypertension (ISIAH) strain was conducted. The animals of the first group were nursed by normotensive Wistar rats, while those of the second (control) group were reared by their own mothers. The volume of the adrenal medulla in rats of the first group was found to exceed that in the second group. In the adrenal zona glomerulosa and zona fasciculata of 3-week-old rats of the first group parenchymal-stromal ratio and the average volume of adrenocorticocytes were lower than those in the animals of the second group. This was accompanied by a decrease in mitochondrial volume density and accumulation of lipid droplets in the cytoplasm, indicating a reduction of hormone-producing activity of endocrinocytes in the animals the first group as compared to controls. By 6 month of age, arterial pressure and quantitative parameters of adrenal medulla and cortex in the animals the first group were..... as compared to those in the second group. It is suggested that the nursing of ISIAH rat pups by normotensive Wistar dams had modulating effect on the adrenal structure and therefore on arterial hypertension development.

Development of monoclonal antibodies against human CYP11B1 and CYP11B2.

1. The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate. 2. Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41-52 for the CYP11B2 and amino acids 80-90 for the CYP11B1 enzyme. 3. The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.

Heterogeneous levels of oxidative phosphorylation enzymes in rat adrenal glands.

Mitochondria are organelles that produce ATP and reactive oxygen species, which are thought to be responsible for a decline in physiological function with aging. In this study, we morphologically and biochemically examined mitochondria in the rat adrenal gland. Immunohistochemistry showed that the rank order for intensity of immunolabelling for complex IV was zona reticularis > zona fasciculata >> adrenal medulla, whereas for complex V α and ß subunits, it was zona fasciculata > zona reticularis and adrenal medulla. The immunolabelling for complex I was homogeneous in the adrenal gland. The difference in immunolabelling between complexes I and IV indicates that the ratio of levels of complex I to that of complex IV in the zona reticularis was smaller than that in the zona fasciculata and the adrenal medulla. Electron microscopy revealed that aging rats had zona reticularis cells with many lysosomes and irregular nuclei. The result suggests that the level of proteins involved in oxidative phosphorylation is coordinated within the complex, but differs between the complexes. This might be responsible for degeneration of zona reticularis cells with aging.