Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of translocation apparatus proteins.

Steroid-secreting cells possess abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. In this study we demonstrate that adrenal smooth microsomal subfractions enriched in these membranes also possess high levels of proteins belonging to the translocation apparatus, proteins previously assumed to be confined to morphologically identifiable rough endoplasmic reticulum (RER). We further demonstrate that these smooth microsomal subfractions are capable of effecting the functions of these protein complexes: co-translational translocation, signal peptide cleavage and N-glycosylation of newly synthesized polypeptides. We hypothesize that these elements participate in regulating the levels of ER-targeted membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally-regulated manner.

Induction of CYP1A1, but not CYP1A2, in adrenals of 3, 3'-methylcholanthrene-treated guinea pigs.

To test the inducibility of CYP1A homologs in guinea pig adrenal, the effects of 3,3'-methylcholanthrene, an archetypal inducer of CYP1A, were compared in guinea pig adrenal and liver. Western blot analysis showed that levels of both CYP1A1 (53 kDa) and CYP1A2 (56 kDa) increasedin liver microsomes of 3,3'-methylcholanthrene-treated guinea pigs. In adrenals, an immunoreactive protein comigrating with liver CYP1A1 was detected only after 3,3'-methylcholanthrene treatment. Protein comigrating with CYP1A2 was never detected in adrenal microsomes. A third inducible immunoreactive protein (57 kDa) was seen in liver, but not adrenal, after 3, 3'-methylcholanthrene treatment. Another immunoreactive protein (52 kDa), present constitutively in liver and adrenal microsomes, was not induced in either tissue by 3,3'-methylcholanthrene. The precise identities of the inducible 57-kDa and the noninducible 52-kDa proteins remain to be determined. However, the identity of the 53-kDa protein in the adrenal as CYP1A1 was confirmed by RT-PCR, Northern blot, and sequence analysis. Similar analyses demonstrated that, despite the fact that the 56-kDa protein was not detectable in adrenal microsomes, CYP1A2 mRNA was present in adrenals of control animals. Strikingly, CYP1A2 mRNA decreased in adrenal, but increased in liver, following 3,3'-methylcholanthrene treatment, underscoring differences in the regulation of CYP1A expression in the two tissues. Levels of ethoxyresorufin and methyoxyresorufin metabolism correlated with levels of CYP1A1 and CYP1A2 protein, respectively.

Molecular cloning of cDNA for guinea pig CYP1A2 comparison with guinea pig CYP1A1.

Guinea pig CYP1A2 cDNA was isolated by RT-PCR from liver tissue of 3,3'-methylcholanthrene-treated guinea pigs. It shares considerable sequence identity with guinea pig CYP1A1 (nt 77%, aa 65%), but differs in levels of constitutive expression, function, and inducibility. Western blot analysis of protein expressed by full-length cDNA in COS-1 cells identified CYP1A2 (56 kDa) and CYP1A1 (53 kDa) proteins in corun liver microsomes. CYP1A2 transfectants metabolized methoxyresorufin and ethoxyresorufin, while CYP1A1 transfectants metabolized only ethoxyresorufin. Constitutive expression of CYP1A2 mRNA (2.0 kb) and protein was much lower than that of CYP1A1 mRNA (2.6 kb) and protein, but the fold induction of CYP1A2 by 3,3'-methylcholanthrene was greater than that of CYP1A1. Changes in splicing of CYP1A2 pre-mRNA occur upon treatment with 3,3'-methylcholanthrene.

COSAS 2.0--a Macintosh-based stereological analysis system.

Quantitation of morphological data is a component of both basic and clinical research. Often it is necessary to extract three-dimensional information from two-dimensional images. Stereological analysis, the most reliable means of achieving this, is also increasingly used as a diagnostic tool in pathology. COSAS, a COmputerized Stereological Analysis System, is software which allows rapid recording of morphological data from two-dimensional images and performance of analyses necessary for both quantitation and three-dimensional interpretation. COSAS is reliable and easy to use. It was previously available in a VAX-based version (Cornacchia and Black, 1988). An improved version, COSAS 2.0, is now available for use on the Macintosh. COSAS 2.0 is considerably more "user friendly" and flexible than the VAX version of the program. COSAS 2.0 eliminates the necessity for the user to be linked with a large computer. COSAS 2.0 has the ability to interface with other commercially available software for scanned images (e.g., Adobe Photoshop, NIH-Image) and data manipulation (e.g., EXCEL, Delta Graphics). These features make it useful to both research and clinical laboratories. Although designed for the biologist, the system may be used to study cross sections of any solid. It could be of interest to material scientists, petrographers, and ceramicists, all of whom need to obtain quantitative information about three-dimensional spatial organization from two-dimensional sections or projections.

Intraadrenal steroid metabolism in the guinea pig: guinea pig adrenal microsomes metabolize androstenedione in a manner distinct from liver microsomes.

Several immunochemical homologs of hepatic cytochromes P450 (CYPs) capable of steroid catabolism have been identified in the guinea pig adrenal cortex. Their predominance in males suggests a role in sex-differentiated metabolism of androgens. Therefore, we examined the ability of microsomes from male guinea pig adrenals and liver to metabolize androstenedione. Microsomes were incubated in the presence of radiolabeled steroids, the products of the reaction extracted, separated by TLC, and visualized by autoradiography. Metabolites were identified by comigration with commercially available standards in several solvent systems, in one and two dimensional TLC. Microsomes from both tissues metabolized androstenedione. However, the products formed differed markedly in the two tissues. Liver microsomes formed one major metabolite, testosterone. It represented 85% of the metabolized androstenedione. 6 beta-Hydroxylated androstenedione and testosterone each comprised 3-4% of the liver metabolites. In addition, at least 10 other products were formed, but taken together they constituted less than 8% of the metabolized androdostenedione. Adrenal microsomes, on the other hand, produced several major metabolites: 16 alpha-, 16 beta-, and 6 beta-hydroxy-androstenedione, plus one unidentified product constituted 93% of the metabolized androstenedione. 16 alpha-Hydroxylation of androstenedione was 60 fold, 16 beta-hydroxylation 12 fold, and 6 beta-hydroxylation 2.5 fold greater in adrenal than in liver microsomes. The unidentified product, which was the least polar, was formed exclusively by adrenal microsomes. The hydroxylation reactions performed by adrenal tissue are consistent with the presence in adrenal microsomes of immunochemical homologues of members of the CYP1A, 2B, 2C and 3A families which have known steroid hydroxylation functions in liver. The Kms of the formation of 16 alpha-, 16 beta- and 6 beta-hydroxyandrostenedione by adrenal microsomes are in the range reported for steroid hydroxylation reactions in rat liver tissue. Their distinct values suggest that these hydroxylation steps are performed by different CYPs. However, assignment of site-specific steroid hydroxylation reactions to individual hepatic CYP homologs in the adrenal requires further investigation and is being pursued using combined techniques of biochemistry and molecular biology.

Immunocytochemical characterization of intrarenal adrenal tissue.

Heterotopic adrenal tissue has been reported in multiple sites, but its functionality has seldom been assessed. In this case, immunocytochemistry was used to characterize adrenocortical tissue present in the subcapsular region of a nephrectomy specimen and to determine its potential for steroidogenesis. Immunodetectable cytochrome P450scc was detectable in the adrenal gland, but not in the renal tissue, clarifying the demarcation between the two tissue types. The high level of this key enzyme in steroid synthesis in the adrenocortical cells suggested that they were capable of producing steroids.

Estrogen, not testosterone, creates male predominance of a P4501-related cytochrome in adult guinea pig adrenals.

Guinea pig adrenal microsomes possess a distinctive cytochrome P450 that is immunochemically related to P4501 and correlates with microsomal capacity for xenobiotic metabolism. This 52K protein and the capacity for metabolizing compounds such as ethylmorphine are located in the zona reticularis, are suppressed by ACTH, and are predominant in adult males. The protein is undetectable and the enzyme activity is low in young prepubertal animals. In males, both increase with age. However, in females, the protein remains undetectable, and ethylmorphine demethylase activity remains low into adulthood. Despite this clear sex difference through puberty and into sexual maturity, we recently observed that in female retired breeders, both the 52K cytochrome P450 and the capacity for metabolism of ethylmorphine appear at levels equal to those in males of comparable age. As estrogen levels are low in female retired breeders, we decided to investigate whether estrogen plays a role in maintaining the low levels of this protein and of xenobiotic metabolism seen in younger females. In a series of gonadectomy and hormone replacement experiments, we demonstrated that estrogen suppressed the levels of both protein and enzyme activity in adult guinea pigs. Ovariectomy resulted in the appearance of the 52K cytochrome P450 and of ethylmorphine demethylase in female adrenal microsomes at levels comparable to those seen in adult males. Estrogen replacement suppressed the increase in both protein concentration and enzyme activity. In hemiovariectomized cycling females, compensatory hypertrophy of the remaining ovary occurred, and the characteristic low levels of the 52K P450 and enzyme activity were maintained. Furthermore, estrogen treatment of male guinea pigs suppressed levels of both the 52K P450 and ethylmorphine demethylase activity in male adrenals. These experiments demonstrate that estrogen plays a significant role in the regulation of this protein. Testosterone, on the other hand, was not required to maintain the higher levels of 52K P450 and correlated enzyme activity in adult males. The levels of both were the same in normal, castrated, and sham-operated males, treated with testosterone or vehicle alone or left untreated. In fact, castration of prepubertal males resulted in a rapid rise to adult male levels of both immunodetectable protein and enzyme activity, implying that some suppressive agent of testicular origin effects the gradual increase that normally occurs with age in males.(ABSTRACT TRUNCATED AT 400 WORDS)

A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment.

Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers.

Cytochrome P450s in the guinea pig adrenal that are immunologically similar to liver forms: estrogen suppression explains male-female differences.

Several cytochrome P450s have been identified in guinea pig adrenal microsomes which are distinct from the known steroidogenic P450s, c17 and c21, and are immunochemically related to cytochrome P450s found in liver. One, a 52 K protein related to P450 I (CYP1), occurs almost exclusively in males, is localized to the inner zone, and is suppressed by ACTH. Its levels correlate with microsomal capacity for xenobiotic metabolism. The others, related to P450s II and III (CYP2 and 3), are more predominant in males, but not exclusive to them, are found in both the inner and outer zones, and are not suppressed by ACTH. Their functions remain to be elucidated. The male predominance of the CYP1-related protein has recently been shown to be due to suppression of the protein in females by estrogen. To determine if estrogen is also involved in the regulation of the CYP2-related proteins, ovariectomized and sham-operated animals were treated with a long-acting estrogen, estradiol valerate, or with the vehicle alone. These P450s reached male levels in ovariectomized females treated only with the vehicle. Their enhanced levels were suppressed by treatment with estrogen. Estrogen treatment also suppressed the levels of the P450s seen in sham-operated females. Endogenous estrogen produced similar effects. In hemi-ovariectomized females the contralateral ovary hypertrophied, a state in which estrogen levels would be maintained or increased. In these females no increase occurred in the immunodetectable P450s. In normal females, estrogen levels are low in prepubertal animals, rise at the time of puberty and drop again after ovarian cycling is completed. The CYP2-related proteins were present in adrenal microsomes of prepubertal females, but were suppressed after puberty. On the other hand, post-estrous females, in whom estrogen levels would be low, acquired male levels of these proteins in their adrenal microsomes. P450c17 and P450c21, as well as 3ß-hydroxysteroid dehydrogenase, were not affected by surgery or estrogen. Taken together, these experiments indicate that suppression by estrogen in females can account, in large part, for the predominance of several immunochemical homologs of liver P450s in adult male guinea pig adrenals.

Differential ACTH response of immunodetectable HMG CoA reductase and cytochromes P450(17 alpha) and P450(21) in guinea pig adrenal outer zone cell types, zona glomerulosa and zona fasciculata.

In the guinea pig adrenal the cells of the outer zone secrete high levels of steroid and respond to ACTH with increased synthesis of cholesterol and steroid. The outer zone consists of two cell types: zona fasciculata (ZF) and zona glomerulosa (ZG). To determine the relative contribution of ZF and ZG to the outer zone's ACTH response, purified populations of each cell type were prepared from ACTH-treated and control animals. Levels of proteins potentially involved in the ACTH response were measured by ELISA. HMG CoA reductase, the rate limiting enzyme of cholesterol synthesis, and two cytochrome P450s, P450(17 alpha) and P450(21), were studied. P450(17 alpha) is required for production of cortisol, but not for corticosterone or aldosterone. P450(21) is required for production of all of these corticosteroids. ZF cells had 4-5 fold greater concentration of all three proteins, but the proteins in ZG cells showed a greater response to ACTH (approximately 3 fold). The response of ZG cells to ACTH and their possession of P450(17 alpha) is consistent with observations made in vitro that ZG cell populations synthesize cortisol and respond to ACTH with increased output of cortisol as well as of corticosterone and aldosterone. In ZG cells to a greater extent than ZF cells, the response to ACTH involved increases in levels of enzymes of both cholesterol and steroid synthesis, suggesting that new protein synthesis is an important component of their ACTH response. On the other hand, the fact that ZF cells can increase steroid output in response to ACTH with a lesser increase in these proteins suggests a different mechanism of regulation. Mobilization of stored cholesterol may be more important in the ACTH-responsiveness of the lipid-filled ZF cells than in the lipid-poor ZG cells.

Immunodetectable cytochromes P450 I, II, and III in guinea pig adrenal--hormone responsiveness and relationship to capacity for xenobiotic metabolism.

Cytochrome P450s, proteins involved in metabolism of sterols, steroids, and a variety of foreign compounds, have been grouped into families based on amino acid sequence. We have identified a microsomal cytochrome P450 in guinea pig adrenal immunochemically related to P450c,d (P450I), induced in rat liver by methylcholanthrene. The inner zone localization, male predominance, and suppression by ACTH of this protein correspond to the ability of the adrenal microsomes to metabolize ethylmorphine. Its immunoreactivity, localization, and regulation distinguish it from P450(17) alpha (P450XVII) and P450(21) (P450XXI), known microsomal steroid hydroxylases. To examine whether other cytochrome P450s homologous to those in liver might be present in the guinea pig adrenal, microsomes were reacted with antibodies to hepatic P450s from families II and III. Each probe detected proteins in microsomes, but not in mitochondria, which were in the lower mol wt range of cytochrome P450s (47-50K). The immunoreactivity of all was diminished in animals treated with spironolactone, a compound which destroys cytochrome P450s in the adrenal, but not in liver. All were present in both outer and inner zones and in both males and females. None was suppressed by ACTH in the inner zone. Thus, only the previously described 52K protein reactive with anti-P450I corresponds in both distribution and ACTH response to the capacity for xenobiotic metabolism in guinea pig adrenal microsomes. On the other hand, unlike the 52K protein, the newly discovered proteins related to P450s II and III were all suppressed in the outer zone following dexamethasone treatment, suggesting that they might be related to dexamethasone-suppressible functions, such as metabolism of sterols and steroids.

Effects of age, adrenocorticotropin, and dexamethasone on a male-specific cytochrome P450 localized in the inner zone of the guinea pig adrenal.

Guinea pig adrenal microsomes have a capacity for xenobiotic metabolism which is localized to the inner zone, greater in adult males then females, greater in older than in younger males, and suppressed by ACTH. In this paper we show that a cytochrome P450 distinct from P450(21) and P450(17) alpha, which is localized to the inner zone, is male specific, increases with age, and is suppressed by ACTH. This is the first report of a sex-dependent cytochrome P450 in the adrenal. It is immunochemically related to the two members of the P450I family, P450 c and d, neither of which has been reported to be hormonally regulated. The correlation of this cytochrome P450 with the ability of the microsomes to metabolize ethylmorphine suggests that it accounts for this capacity. Whether metabolism of foreign compounds and/or male-specific steroid hydroxylation are its functions in vivo remains to be determined.

A cytochrome P450 immunochemically related to P450c,d (P450I) localized to the smooth microsomes and inner zone of the guinea pig adrenal.

In addition to their capacity for steroid synthesis, guinea pig adrenal microsomes have a well documented ability to metabolize foreign compounds. The capacity for metabolism of foreign compounds is localized to the smooth endoplasmic reticulum-filled cells of the inner zone. However, it has not been clear whether they possess cytochrome P450(s) specific for this function, distinct from the two known steroid hydroxylases, P450(21) and P450(17)alpha. Multiple prominent protein bands in the mol wt range of known cytochrome P450s are seen on sodium dodecyl sulfate gels of guinea pig adrenal microsomes. Most are more intense in smooth microsomes, where the concentration of cytochrome P450 is highest. However, one band (52K) appears unique to the smooth microsomes. This band is also characteristic of microsomes obtained from the inner zone. This protein and two others (54K and 50K) are concentrated in the membrane pellet after carbonate treatment of the microsomes, indicating that they are integral membrane proteins. All three decrease in intensity after treatment of the animals with spironolactone, a compound known to cause depletion of adrenal cytochrome P450s. On Western blots of microsomal proteins the 54K and 50K proteins react with antibodies specific for P450(17) alpha and P450(21), respectively. The 52K protein, characteristic of the smooth microsomes and inner zone, does not react with anti-P450(21) or anti-P450(17) alpha, but does react with polyclonal antibody raised against microsomal cytochrome P450s induced by methylcholanthrene in rat liver (P450c,d). These results suggest that there is at least one additional cytochrome P450 in adrenal microsomes which is immunochemically distinct from P450(21) and P450(17) alpha. Its localization to the smooth microsomes and inner zone microsomes correlates with the high activity for ethylmorphine metabolism detected in these fractions. This suggests that this cytochrome P450, which is immunochemically related to members of the P450I subfamily, may be associated with the ability of guinea pig adrenal microsomes to metabolize foreign compounds.

Acyl-coenzyme A: cholesterol acyltransferase and cholesterol ester hydrolase in the outer and inner cortices of the guinea pig adrenal: effects of adrenocorticotropin and dexamethasone.

The guinea pig adrenal cortex can be separated into two zones with contrasting lipid contents: a lipid-rich outer cortex and a lipid-poor inner cortex. Cytoplasmic lipid droplets contain cholesterol esters and are thought to act as a reservoir for steroid precursor. Such lipid droplets are numerous in the outer cortex, but accumulate in the inner region only after treatment with ACTH. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) esterifies cholesterol to fatty acids for storage in this pool, while cholesterol ester hydrolase (CEH) cleaves these esters, increasing free cholesterol. However, the relationship between intracellular cholesterol content and the activity of these enzymes is not clear. In this study we examine the capacity to esterify cholesterol (ACAT) and to hydrolyze cholesterol esters (CEH) in the lipid-rich and lipid-poor regions of the guinea pig adrenal in control animals and animals treated with ACTH or dexamethasone (DEX). For all conditions, the lipid-filled outer cortex possessed more ACAT and CEH activity than did the inner. Although ACAT and CEH in the outer cortex were relatively insensitive to ACTH compared to the enzymes in the inner zone, they were significantly suppressed after DEX treatment. This implies that in the basal state, ACAT and CEH may be almost maximally stimulated in the outer cortex, and that in these cells, cholesterol esterification and hydrolysis may occur at a high rate even in the basal state. The inner cortex responded to ACTH with increased ACAT activity, but was little affected by DEX. In the inner zone, CEH activity was not affected by ACTH or DEX. The lower level of these enzymes in the inner cortex correlates with the paucity of lipid droplets in these cells in the basal state, while the increase in ACAT, but not CEH, in this zone after ACTH treatment correlates with the accumulation of small lipid droplets in inner cortical cells of ACTH-treated animals.

3-Hydroxy-3-methylglutaryl coenzyme A reductase in outer versus inner cortices of the guinea pig adrenal: effects of adrenocorticotropin and dexamethasone.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, a rate-limiting enzyme in cholesterol biosynthesis, was examined in the lipid-filled outer cortical cells and smooth endoplasmic reticulum-filled inner cortical cells of guinea pig adrenals. The specific activity of HMG CoA reductase was higher in microsomes obtained from the outer cortex, but was stimulated by ACTH and suppressed by dexamethasone (DEX) in both regions. Immunoblots of the microsomal proteins revealed a higher concentration of immunoreactive HMG CoA reductase (mol wt 94K) in microsomes from outer cortical cells. Changes in the intensity of this band corresponded to changes in activity after treatment with ACTH and DEX. However, quantitative immunoassay revealed that changes in activity in the inner zone after ACTH and in both zones after DEX were greater than could be accounted for by changes in immunodetectable protein, implying that other regulatory factors play a role in these cases. Incubation of outer and inner cortical tissues in vitro showed that outer cortical tissue had a greater capacity to incorporate [14C]acetate into cellular cholesterol (free and esterified) and into secreted steroid than did inner cortical tissue. ACTH enhanced the incorporation of [14C]acetate by outer cortical tissues into secreted steroid, but had little effect on incorporation by inner cortical tissues. Assay of the medium indicated that outer cortical tissues secreted much more steroid and increased secretion in response to ACTH, whereas inner cortical tissues secreted little steroid and were little affected by ACTH. Taken together, these results show that outer cortical cells have a greater capacity for cholesterol synthesis and that enhancement of this capacity after ACTH treatment is correlated with an increase in HMG CoA reductase protein. On the other hand, while the level of HMG CoA reductase immunodetectable protein and activity in inner cortical cell microsomes is considerable and appears to increase after ACTH treatment, it is not translated into an ability to synthesize cholesterol. This suggests that the activity of the immunodetectable HMG CoA reductase in the inner zone is suppressed in vivo and that the prominent smooth endoplasmic reticulum found in inner cortical cells has additional functions. The inability of the inner zone to synthesize cholesterol accounts in part for its low steroid production.

Lipoprotein requirements for secretion of ultraviolet-absorbing corticosteroids by guinea pig adrenocortical cells in vitro: inner versus outer cortices; zona glomerulosa versus zona fasciculata.

Most studies of lipoprotein requirements for steroid secretion by the adrenal have examined the mixed cell population of the whole gland; none have examined lipoprotein requirements of guinea pig adrenocortical cells. In this study the effect of exogenous lipoprotein on the ability of cells from each of the different regions of the guinea pig adrenal cortex to synthesize and secrete steroids has been analyzed in vitro, under baseline and ACTH-stimulated conditions. Most studies have assessed the effects of lipoprotein on one or a few selected steroids. In this study the effects of lipoprotein and ACTH were examined both by an assay for fluorogenic steroids and by HPLC analysis of the spectrum of UV-absorbing steroids. Guinea pig outer adrenocortices, containing zona glomerulosa and zona fasciculata, maintained in vitro as fragments or as isolated cells, secreted at least 5 times more steroid than the inner cortex, predominantly zona reticularis, and were dependent upon lipoproteins for their secretion. Corticosterone (B) and cortisol (F) were the predominant products of both zones. Aldosterone (Aldo), 18-hydroxycorticosterone (18-OH B), deoxycorticosterone (DOC), 11 beta-hydroxyandrostenedione (11 beta-OH And), androstenedione (And), and deoxycortisol were less abundant products of the outer cortex, while the inner cortex secreted only very small amounts of these steroids. Each of the outer cortical cell types secreted a distinct spectrum of steroids. Aldo, 18-OH B, and DOC were characteristic of glomerulosa cells, but B was most prominent. Fasciculata cells secreted primarily F, with 11 beta-OH And as their next most prominent product. Low density lipoprotein (LDL) enhanced steroid secretion by glomerulosa cells to a greater extent than that by fasciculata cells, but the stimulation of LDL utilization by ACTH was greatest for fasciculata cells. LDL and ACTH also influenced the pattern of steroids secreted by each cell type. Addition of LDL to glomerulosa cells enhanced secretion of DOC and B, but not that of Aldo or 18-OH B. In fasciculata cell cultures, LDL enhanced secretion of both F and 11 beta-OH And. ACTH, particularly in the presence of LDL, stimulated secretion by glomerulosa cells of Aldo and 18-OH B, as well as that of F, And, and 11 beta-OH And. The combined presence of ACTH and LDL in fasciculata cell cultures preferentially stimulated secretion of F and B.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone and relaxin secretion in relation to the ultrastructure of human luteal cells in culture: effects of human chorionic gonadotropin.

This report describes the first study to correlate the ultrastructure of long-term monolayer cultures of human luteal cells with their secretion of relaxin and progesterone under basal and human chorionic gonadotropin-stimulated conditions. In culture from 14 to 28 days, cells from both corpora lutea of the menstrual cycle and corpora lutea of pregnancy took on characteristics of granulosa luteal cells, particularly after exposure to human chorionic gonadotropin. Relaxin was detectable in the luteal cell cultures only at early time points. The effect of human chorionic gonadotropin on media relaxin levels differed in cells of the cycle and cells of pregnancy. In the cells of the cycle, relaxin was detectable only on day 2 and was decreased by human chorionic gonadotropin (p less than 0.01). In cells of pregnancy, relaxin was detectable for the first 4 days of culture and was not affected by human chorionic gonadotropin. Progesterone was detectable in all the luteal cell cultures and was enhanced by human chorionic gonadotropin (10 and 50 IU/ml) after 24 days of exposure. At earlier time points in both the luteal cells of the cycle and the luteal cells of pregnancy, the human chorionic gonadotropin-induced increases in progesterone levels were not as consistent. However, in all cases of progesterone enhancement, smooth-surfaced endoplasmic reticulum was increased in the human chorionic gonadotropin-treated cells compared with corresponding controls, consistent with more active steroid production. In addition, gap junctions, considered to be responsive to trophic hormones, were increased in the treated cells. In conclusion, this long-term monolayer culture of human luteal cells, as monitored by ultrastructural and hormonal changes, retained the differentiated function of progesterone secretion and exhibited responsiveness to human chorionic gonadotropin. Therefore, morphologic and functional aspects of progesterone secretion may be investigated more closely with use of this long-term luteal cell culture system.

Stereological analysis of peroxisomes and mitochondria in intestinal epithelium of patients with peroxisomal deficiency disorders: Zellweger's syndrome and neonatal-onset adrenoleukodystrophy.

Peroxisomes, participants in lipid metabolism, have been shown to be altered in liver in two metabolic diseases in which long-chain fatty acids accumulate in tissues: Zellweger's syndrome and neonatal adrenoleukodystrophy (ALD). The intestine also plays a role in lipid metabolism, and we have had the opportunity to compare peroxisomes in normal intestinal epithelium with those from patients with Zellweger's syndrome and neonatal ALD at the electron microscopic level by using the combined techniques of cytochemistry and stereological analysis. Peroxisomes were numerous in intestinal epithelium of the normal individuals. They were ellipsoidal in shape with average diameters of 0.37 by 0.56 micron and filled with coarsely granular, DAB+ content. Peroxisomes in the intestinal epithelium of the ALD patient were similar in appearance and number but smaller in size (0.28 by 0.44 micron). Peroxisomes of normal appearance were absent from the intestinal epithelium of patients with Zellweger's syndrome; DAB+ content, however, was observed in rare, membrane-bound structures of much smaller size (0.12 by 0.19 micron). In liver of patients with Zellweger's syndrome, peroxisomes are lacking; in neonatal ALD they are abnormal in appearance and greatly reduced in number. The presence of rare minute peroxisomes in the intestinal epithelium in Zellweger's syndrome and of small peroxisomes in this epithelium in neonatal ALD indicate that peroxisomes in the intestinal epithelium are affected in these diseases, but to a lesser extent than in the liver. In the ALD intestinal epithelium, DAB+ material was also seen in long, sinuous, tubular or cisternal elements intermingled and occasionally in continuity with peroxisomes. It is suggested that these represent the early stages of peroxisome formation, the peroxisomal reticulum as originally envisioned by Lazarow, while the rare structures seen in Zellweger's represent rudiments of such a reticulum. Lamellar inclusions and clear spaces occurred in the cytoplasm adjacent to these structures indicating either that material accumulated there had been extracted during fixation or that these regions are more susceptible to autolysis. Mitochondria are also involved in lipid metabolism and have been reported to be abnormal in Zellweger's tissue. No qualitative differences were observed in the mitochondria of the intestinal epithelia examined in this study. Although quantitation revealed a greater mean volume, number, and surface density of mitochondria in the intestinal epithelia of neonatal ALD, it was not a statistically significant difference in all cases.

Peroxisomal defects in neonatal-onset and X-linked adrenoleukodystrophies.

Accumulation of very long chain fatty acids in X-linked and neonatal forms of adrenoleukodystrophy (ALD) appears to be a consequence of deficient peroxisomal oxidation of very long chain fatty acids. Peroxisomes were readily identified in liver biopsies taken from a patient having the X-linked disorder. However, in liver biopsies from a patient having neonatal-onset ALD, hepatocellular peroxisomes were greatly reduced in size and number, and sedimentable catalase was markedly diminished. The presence of increased concentrations of serum pipecolic acid and the bile acid intermediate, trihydroxycoprostanic acid, in the neonatal ALD patient are associated with a generalized diminution of peroxisomal activities that was not observed in the patient with X-linked ALD.