3-ethyl and 3-methyl ethers of 19-iodocholesterol as potential adrenal scanning agents

19-Iodocholesterol-¹³¹I, though useful as an adrenal scanning agent, was found to be unstable. However, the substitution of methoxyl and ethoxyl groups for the hydroxyl group in the 3 position rendered these derivatives much more stable in solid form than the parent compound. They showed concentration in the adrenals of dogs similar to that of 19-iodocholesterol itself and therefore may be useful as adrenal scanning agents.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

Biochemical characterization of enkephalin-like immunoreactive peptides of adrenal glands

The total enkephalin-like immunoreactive peptide content of adrenal glands from dog, cattle, guinea pig and rat was investigated by radioimmunoassay using a (met⁵)-enkephalin antiserum. Dog adrenals contain the highest amount of peptides, cattle and guinea pig adrenals contain lesser amounts, and the rat adrenals had the least amount (0.05% that of the dog). Comparison of the (met⁵)-enkephalin content of the adrenal cortex and medulla with that of whole bovine adrenal gland indicates that the peptides are concentrated in the medulla. Analysis of the chromaffin granules from bovine adrenal medulla indicates this to be the primary storage site for (met⁵)-enkephalin-like peptides. Gel chromatography reveals a molecular heterogeneity of the immunoreactive peptides in all species tested; high molecular weight peptides account for a larger proportion of the immunoreactivity when compared with the low molecular weight peptides.     

Immunohistochemical localization of adrenal ferredoxin and distribution of adrenal ferredoxin and cytochrome P-450 in the rat adrenal

Adrenal ferredoxin, the iron-sulfur protein associated with cytochromes P-450 in adrenocortical mitochondria, has been localized immunohistochemically at the light microscopic level in rat adrenals by employing rabbit antiserum to bovine adrenal ferredoxin in both an unlabeled antibody peroxidase-antiperoxidase method and an indirect fluorescent antibody method. When sections of rat adrenals were exposed to the adrenal ferredoxin antiserum in both procedures, positive staining for adrenal ferredoxin was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. Marked differences in the intensity of staining, however, where observed among the three cortical zones: the most intense staining being found in the zona fasciculata, less in the zona reticularis, and least in the zona glomerulosa. Furthermore, differences in staining intensity were also observed among cells within both the zona fasciculata and the zona reticularis. In agreement with these immunohistochemical observations, determinations of adrenal ferredoxin contents by electron paramagnetic resonance (EPR) spectrometry in homogenates prepared from capsular and decapsulated rat adrenals revealed that the concentration of adrenal ferredoxin in the zona glomerulosa was lower than that in the zona fasciculata-reticularis. Similar results were obtained when the contents of cytochrome P-450 were determined in capsular adn decapsulated rat adrenal homogenates. These observations indicate that adrenal ferrodoxin and cytochrome P-450 are not distributed uniformly throughout the rat adrenal cortex.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Corticosteroidogenesis in the hypertrophic adrenal gland produced by betamethasone 17, 21-dipropionate in the rat fetus

Betamethasone 17, 21-dipropionate (BMDP), a potent glucocorticoid, produces adrenal hypertrophy in the rat fetus. The present study was performed to investigate the possible alterations of corticosteroidogenesis due to endogeneous substrates or exogenous pregnenolone in the incubation of homogenates of fetal hypertrophic adrenals caused by BMDP given to pregnant rats at day 19 of pregnancy.The corticosteroidal products and those levels per mg homogenate in an incubate of the hypertrophic adrenal homogenate did not differ from those of a normal adrenal. No accumulations of abnormal precursors or intermediates were found in the incubates of the hypertrophic adrenals. It is concluded from these findings that no qualitative alterations in the pathway of corticosteroidogenesis occurred in the hypertrophic adrenal glands caused by BMDP in the rat fetus. When the calculation was done per adrenal gland, the content of corticosterone in the incubate of the homogenate of the hypertrophic adrenal was remarkably higher than that found in a normal gland. This finding was compatible with the significant increase of the plasma corticosterone concentration in the fetuses with the adrenal hypertrophy caused by BMDP.     

New method for the synthesis of radioactive 19-idocholest-5-en-3beta-OL.

In 1970, ¹³¹I-19-iodocholest-5-en-3β-ol (VIII-¹³¹I) was synthesized and shown to concentrate in the adrenal cortex of dogs by Counsell $\text{et al}$. [1]. Beierwaltes and his colleagues at the university of Michigan have shown this radiopharmaceutical to be an effective adrenocortical scanning agent for the diagnosis of 5 types of adrenal disease in humans [2]. Radioactive 19-iodocholesterol is not available from commercial sources although it may be purchased from the University of Michigan Nuclear Pharmacy, Ann Arbor, as a radiochemical and converted to a radiopharmaceutical [3]. For this reason we undertook to synthesize it $\text{de novo}$ as described by Counsell $\text{et al}$. [1]. We found that 19-iodocholesterol prepared by this route contained an impurity, the amount of which varied from 20% to 60% as estimated by ¹³C nuclear magnetic resonance spectroscopy (CMR). We now describe a modification of this synthesis to yield VIII-¹³¹I of greater than 98% chemical purity.     

Effect of the stress of unilateral adrenalectomy on the depletion of individual cholesteryl esters in the rat adrenal

Normal rats were subjected to unilateral adrenalectomy and were killed 3 hr later. The concentration and composition of the cholesteryl esters in adrenals removed at operation and after death were compared. The esterified cholesterol concentration was lower in the adrenals obtained 3 hr after surgery. Cholesteryl arachidonate decreased in concentration significantly more than any other ester, followed in order of magnitude by linoleate and oleate. The cholesteryl ester concentration of adrenals removed from sham-operated rats 3 hr after surgery was also greatly reduced. On the basis of comparison with other work on the hydrolysis of cholesteryl esters by adrenal homogenates, it is concluded that the apparent selectivity in depletion of cholesteryl esters is due to differences in their rates of hydrolysis.     

Chloditan-induced changes in lipid peroxidation in the adrenals of dogs and guinea pigs

The effect of adrenocorticolytic drug chloditan (1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane;) on the content and the rate of lipid peroxidation in dog and guinea pig adrenals was studied. In adrenals of dogs and guinea the amount of malondialdehyde (MDA) was increased. In vitro spontaneous lipid peroxidation was delayed in adrenal homogenate of DDD-treated dogs. Spontaneous lipid peroxidation in adrenal homogenate was not decreased by feeding DDD in the guinea pig. DDD consumption did not affect on lipid peroxidation promoted by ascorbic acid and ferrous iron. The accumulation of MDA in dog and guinea pig adrenal homogenates was inhibited by NADPH.     

Adrenal glands of mouse and rat do not synthesize androgens.

Human adrenal glands produce considerable amounts of the C-19 steroids dehydroepiandrosterone (DHEA) and androstenedione. To investigate the capability of rodent adrenals to produce these steroids, cell suspensions of mouse and rat adrenal glands were incubated in the absence and presence of adrenocorticotropic hormone (ACTH). Corticosterone levels in the incubation medium increased dramatically in the presence of ACTH, but no significant amounts of 17-hydroxyprogesterone or androstenedione could be detected. This indicates that the adrenals of rat and mouse lack the enzyme 17 alpha-hydroxylase. Absence of plasma cortisol in the presence of high levels of corticosterone confirmed these data. Plasma levels of androstenedione were significantly decreased in castrated male rats as compared to levels observed in intact males, showing the contribution of the testes to the plasma content of androstenedione. Very low levels of androstenedione were observed in female, male and castrated male mice. Plasma concentrations of DHEA were not detectable in intact and castrated male mice and rats. It is concluded that rat and mouse lack the enzyme necessary to synthesize adrenal C-19 steroids and that the adrenals in these animals, therefore, do not contribute to plasma levels of androstenedione and DHEA.     

Changes in rat adrenal cyclic nucleotides during normal and neoplastic growth

In an attempt to correlate changes in cyclic nucleotide levels with in vivo growth of the rat adrenal gland we have measured adrenal cyclic AMP and cyclic GMP in normal, hyperplastic, and neoplastic rat adrenals. The first group of animals were subject to either unilateral adrenalectomy (ADX) or acute hypophysectomy 1 h prior to unilateral adrenalectomy (HADX). Cyclic nucleotides were measured in the contralateral adrenal post-operatively. In HADX rats cyclic GMP rose steadily throughout the 7 day study period, while ADX rats exhibited significant decreases in adrenal cyclic GMP. Cyclic AMP remained approximately 1.5 pm/mg tissue in HADX rats, while in ADX rats there was significant elevation of adrenal cyclic AMP at all time points. Cyclic GMP/cyclic AMP ratios remained constant in HADX animals; however, the growing adrenals of ADX animals exhibited depressed cyclic GMP/cyclic AMP ratios at all time periods.Adrenal hyperplasia was induced in a seond group of animals by a transplantable, corticotropin-secreting, pituitary tumor. Adrenals from age-matched animals served as controls. Adrenal cyclic AMP was significantly elevated in tumor-bearers at a time correspinding to the peak accumulation of adrenal weight, protein and DNA in these animals. In contrast, adrenal cyclic GMP in both tumor-beares and control animals fell steadily throughout the study period. Cyclic GMP/cyclic AMP ratios of control animals decreased from 2 to 3 weeks post-transplant remaining at the 3 week value during the period corresponding to rapid adrenal growth in tumor-bearers. The cyclic GMP/cyclic AMP ratio in the hyperplastic adrenals of tumor-bearers decreased steadily throughout their rapid growth period, suggesting a positive correlation between adrenal growth and depression of the cyclic GMP/cyclic AMP ratio.Cyclic nucleotide levels in neoplastic adrenals of rats bearing the transplantable adrenocortical carcinoma 494 were compared with cyclic nucleotides in normal rat adrenal glands. Cyclic AMP was not different in the two groups. However, the cyclic GMP content of neoplastic adrenals was significantly lower than that of normal adrenal tissue, causing a suppression of the cyclic GMP/cyclic AMP ratio in the neoplastic tissue. Thus, measurement of adrenal cyclic nucleotides in both hyperplastic and neoplastic rat adrenal glands suggests that adrenal growth in vivo may be characterized by a depression of the cyclic GMP/cyclic AMP ratio.     

Light and electron microscopic evidences of the presence of c-fos-like immunoreactivity in the rat adrenal cortex

Summary The presence and localization of c-fos-like immunoreactivity in the rat adrenal cortex has been demonstrated by immunocytochemical methods at both light and electron microscopic level. C-fos-like immunoreactivity was detected in the zona fasciculata and the zona reticulata, but not in the zona glomerulosa. Ultrastructurally, all products of c-fos-like immunoreaction were localized exclusively in the regions associated with the euchromatin in the nucleus of the immunoreactive cells. Moreover, a higher density of the immunoreactive cells in the adrenal cortex of pregnant rats was found with quantitative immunocytochemistry as compared to the non-pregnant. The characteristic zonation of c-fos-like immunoreactivity in the adrenal cortex suggest that the c-fos protein is involved in the normal function of the glucocorticoid-producing cells of mammalian adrenals. The numerical increase in the immunoreactive cells in pregnant rats implies that basal expression of the c-fos-like protein may vary with the functional state of the cortical cells.     

Studies on adrenal delta-aminolevulinic acid synthetase.

The presence of δ-aminolevulinic acid synthetase (EC 2.3.1.37) in rat and bovine adrenals has been demonstrated. When untreated animals are employed, the activity of δ-aminolevulinic acid synthetase in rat and bovine adrenal homogenates is comparable to the activity found in hepatic homogenates. Adrenal δ-aminolevulinic acid synthetase is localized in the mitochondrial fraction and appears to be refractory to induction by agents that induce the hepatic enzyme. Starvation of rats increased adrenal δ-aminolevulinic acid synthetase activity without altering the activity of the hepatic enzyme. Treatment of rats with adrenocorticotropin also dramatically increased adrenal δ-aminolevulinic acid synthetase activity. These results suggest that the adrenal enzyme may be controlled by factors that differ from those which regulate the activity of the hepatic enzyme.     

Light and electron microscopic evidences of the presence of c-fos-like immunoreactivity in the rat adrenal cortex

The presence and localization of c-fos-like immunoreactivity in the rat adrenal cortex has been demonstrated by immunocytochemical methods at both light and electron microscopic level. C-fos-like immunoreactivity was detected in the zona fasciculata and the zona reticulata, but not in the zona glomerulosa. Ultrastructurally, all products of c-fos-like immunoreaction were localized exclusively in the regions associated with the euchromatin in the nucleus of the immunoreactive cells. Moreover, a higher density of the immunoreactive cells in the adrenal cortex of pregnant rats was found with quantitative immunocytochemistry as compared to the non-pregnant. The characteristic zonation of c-fos-like immunoreactivity in the adrenal cortex suggest that the c-fos protein is involved in the normal function of the glucocorticoid-producing cells of mammalian adrenals. The numerical increase in the immunoreactive cells in pregnant rats implies that basal expression of the c-fos-like protein may vary with the functional state of the cortical cells.     

Sex differences in adrenocortical structure and function. XIV. Heparin-releasable liver-lipase like activity in rat adrenals as affected by gonadectomy and testosterone or estradiol replacement

Heparin-releasable liver-lipase like activity was studied in adrenals of intact, gonadectomized and gonadectomized-testosterone or estradiol replaced rats. Besides acid lipase, rat adrenals contain a neutral lipase activity. From both male and female adrenals about 70% of this activity is extractable by heparin containing medium. Activity of this lipase in 8000 g supernatant was higher in female than male adrenals. Orchiectomy of 12 weeks duration increased neutral lipase activity in rat adrenal, an effect reversed to normal level by testosterone replacement. On the other hand ovariectomy had no effect while estradiol replacement resulted in an increased activity of the neutral lipase of rat adrenal gland. The results showed a distinct sex-difference in heparin-releasable liver-lipase like activity of rat adrenals which depends on the inhibitory action of testosterone on the enzyme activity.     

Adrenal renin: a possible local regulator of aldosterone production

Extrarenal renin has been identified in a number of tissues, including the brain, the submaxillary gland, uterus, ovary, vascular endothelium, testes, pituitary gland, and the adrenal cortex. In some tissues, including the adrenal cortex, all of the components of the renin-angiotensin system have been identified; however, no specific physiologic role has been clearly demonstrated for these extrarenal renin-angiotensin systems. We have studied the role of the renin-angiotensin system in the adrenal cortex of the rat and have found that renin is localized and synthesized in the zona glomerulosa cells. Its production can be influenced by alterations in electrolyte balance, as well as the genetic background of the rat. In adrenal capsular explant cultures, a converting enzyme inhibitor can lower angiotensin II production and reduce the stimulation of aldosterone by potassium, suggesting that this system is involved in the aldosterone response to potassium. In addition to rat adrenals, renin has been identified in human adrenal tissue and human adrenal tumors, including aldosteronomas, and a patient with hypertension has been reported to have an adrenal tumor that appeared to be secreting renin into the circulation.     

Increase in free cholesterol content of the adrenal cortex after stress: radioautographic and biochemical study

The purpose of this investigation was to quantify free cholesterol biochemically and in radioautographs of 3H-digitonin cholesterol complex in fasciculata cells of control and stressed rat adrenal cortex. Stress was induced by ether, laparotomy, and adrenal and intestinal handling. Control rats were anesthetized with nembutal. All animals were killed ten minutes from the beginning of anesthesia. The adrenals were excised and either fixed in glutaraldehyde containing 3H-digitonin or homogenized for biochemical determination of free cholesterol. The plasma corticosterone level of each animal was measured. The fixed adrenals were processed, using different methods of dehydration and embedment, for light and electron microscopic radioautography. The mean number of silver grains (mean) per unit area of zona fasciculata was counted from light microscopic radioautographs. Crystals of cholesterol-digitonide complex were more numerous in stressed fasciculata cells, particularly over SER. Silver grains were localized over or close to the crystals. The mean for stressed rats was significantly higher than control values, indicating more free cholesterol in fasiculata cells of stressed rats. The results were not affected by either the method of dehydration or the type of embedding medium used. The morphologic results were substantiated by biochemical findings of increase in free cholesterol in adrenals of stressed rats. Plasma corticosterone was significantly high in stressed rats. The increase in free cholesterol in stimulated fasciculata cells is consistent with a previously reported increase in cholesterol esterase activity after ACTH stimulation.     

Adrenal and liver metabolites of 18-hydroxy-11-deoxycorticosterone in the rat. Identification of reduced compounds (18-OH-TH-DOC)

The presence of reduced metabolites of 18-hydroxy-11-deoxycorticosterone has been investigated in the adrenals of 23 day-old and adult rats and in the liver of adult rats. By thin-layer chromatography a fraction of the adrenal steroid extract migrating like tetrahydro-corticosterone has been isolated. By gas chromatography-mass spectrometry several isomers of 3,18,21-trihydroxy-pregnan-20-one (18-OH-TH-DOC) have been separated in this fraction and identified by comparison with authentic samples which have been chemically and enzymatically synthesized. The major tetrahydrogenated metabolite in the adult and prepuberal rat adrenals is 3β,18,21-trihydroxy-5α-pregnan-20-one (18-OH-TH-DOC II). The 3α,18,21-trihydroxy-5β-pregnan-20-one has been found only in the prepuberal rat adrenal. A third tetrahydrogenated isomer has been tentatively identified as 3α,18,21-trihydroxy-5α-pregnan-20-one. Quantitative measurements by mass fragmentography show that adrenal reductase activity on 18-hydroxy-11-deoxycorticosterone is higher than on corticosterone. The 18-OH-TH-DOC II has been identified in the liver of adult male rat.     

Immunocytochemical localization of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method.

The subcellular localizations of tryrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

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.