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Neuropeptide Y (NPY) expression is limited to tissues of the central and peripheral nervous system. In the adrenal gland, NPY is found in a subset of cells of the adrenal medulla. Using in situ hybridization analysis, NPY mRNA expression was characterized during human fetal adrenal medullary development. We found a biphasic pattern of NPY mRNA expression during the development of the human adrenal medulla. NPY mRNA is detectable at the earliest evaluable time point (7.5 weeks of gestational age) through 18 weeks of gestational age, and is then not detectable until 8 months after birth. We also analyzed NPY mRNA expression in neuroblastoma tumors, which often arise in the adrenal medulla. Thirty-eight neuroblastoma tumors were analyzed for NPY mRNA expression using in situ hybridization. We found NPY mRNA expression in 30 of 38 tumors; 15 of 15 Stage IVS tumors from children under 1 year of age at diagnosis expressed NPY mRNA, whereas 0 of 4 Stage IV tumors from children less than 1 year of age at diagnosis expressed NPY mRNA. These data suggest that in children under 1 year of age at diagnosis, Stage IVS and Stage IV neuroblastoma may be marked by the presence or absence, respectively, of NPY mRNA expression. Moreover, since NPY is expressed for only a short period of time during embryogenesis, these tumors may arise from different neuroblast populations occurring during the course of adrenal medullary development.

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.

Class I major histocompatibility complex (MHC) antigen expression in neuroblastoma may play a role in the oncogenicity of this embryonal tumor of childhood. Since N-myc amplification in neuroblastoma tumors is associated with rapid tumor progression (33) and N-myc decreases Class I MHC antigen expression in rat neuroblastoma cells (21), we quantitated levels of N-myc mRNA and Class I MHC cell surface antigens in a panel of 24 human neuroblastoma cell lines. We found that N-myc expression is not invariably associated with low levels of beta 2-microglobulin (B2M) and Class I MHC antigen expression. As we considered that Class I MHC antigens may be regulated in association with the differentiation stage of the neuroblastoma tumor, we examined the expression of B2M during development of the human adrenal medulla, the tissue of origin of most neuroblastomas. We found that B2M is a marker of differentiated adrenal medullary cells, expressed late during the third trimester of development. Moreover, using morphological and immunological criteria, we found that B2M is expressed in differentiated tumor cells. These data suggest that the expression of B2M in neuroblastoma is associated with the stage of differentiation of the tumor cell and not N-myc expression. Furthermore, these findings suggest that neuroblastomas may correspond to the arrested differentiation of adrenal neuroblasts at different stages of development.

Levels of triiodothyronine, thyroxine, insulin, glucose and free fatty acids in the blood; contents of adrenaline, noradrenaline in adrenals and glycogen in the liver; activity of phenylethanolamine-N-methyltransferase in adrenals, hexokinase and glucose-6-phosphatase in the liver were studied in male Wistar rats and rats with inherited stress-induced arterial hypertension /ISIAH/. It was found that genetically caused rise of hypophyseal-thyroid systems activity in ISIAH-rats leads to a decrease of insulin blood level, activation of lipolysis and breach of glucose tolerance.

To assess the chronic effects of interleukin-1 (IL-1) and IL-2 on the hypothalamo-pituitary-adrenal axis in vivo, we administered recombinant human (rh) IL-1 alpha, rhIL-1 beta or rhIL-2 (2.0 micrograms/day) repetitively to adult male rats for 10 days. In rhIL-1 beta-treated rats, adrenocorticotropic hormone-like immunoreactivity (ACTH-LI) of the anterior pituitary appeared to increase first on day 3 followed by an increase of corticotropin-releasing hormone (CRH)-LI both in the hypothalamus and in the adrenal gland after day 7. At the end of the 10-day treatment, wet weights of the adrenal glands of rhIL-1 beta-treated rats increased significantly compared with those of control rats. Plasma ACTH levels in rhIL-1 beta-treated rats at the sampling time continued to be elevated throughout the experimental period. Under the same experimental design, rhIL-1 alpha increased plasma ACTH levels at the sampling time without changes in adrenal weight or in the peptide contents investigated. The same amount of rhIL-2 had no effect on these measured variables during the 10-day treatment. These data indicate that the repetitive administration of IL-1 beta resulted in chronic effects in the hypothalamo pituitary-adrenal axis to increase the activities in these organs during the treatment and, moreover, IL-1 possibly has a positive direct effect on the CRH-containing cells in the adrenal glands.

A novel pituitary peptide, designated 7B2, was shown to be present in the adrenal gland. 7B2-like immunoreactivity was mainly localized in the adrenal medulla, similarly to NPY and VIP. In order to elucidate the neural and humoral regulation of adrenal 7B2, NPY and VIP content, Wister rats were treated with reserpine (RES), ranitidine (RANT) or chlorpheniramine maleate (CPhM) for 7-10 days. The thyroid hormone excess and deficient states were experimentally produced with thyroxine (T4) treatment for 2 weeks, methylmercaptoimidazole (MMI) for 4 weeks, or a thyroidectomized state (Tx) for 4 weeks. Orchiectomy or neonatal monosodium glutamate (MSG) treatment was also done. 7B2, NPY and VIP contents were measured by specific radioimmunoassays. RES and RANT treatments caused significant 7B2 reduction (p less than 0.01) and adrenal NPY was significantly decreased by RES (p less than 0.05), while CPhM induced a VIP decreased (p less than 0.05). Orchiectomy did not affect the peptides concentrations, though MSG treatment did cause a reverse change in VIP and NPY. Although T4 administration did not cause any significant change, MMI treatment and Tx induced significant increase (p less than 0.05 or p less than 0.01) in these peptides. Gel or high performance liquid chromatographic analysis revealed the majority of each immunoreactivity coeluted with each standard. These results suggested that adrenal NPY seemed to be coregulated with catecholamine, while VIP was mainly affected by histaminergic control. Furthermore 7B2 might be modulated by both catecholaminergic and histaminergic nervous control. Thyroid hormone deficiency may also affect the amount of these peptides.

CRH, GH-releasing hormone (GHRH), somatostatin (SRIH), and peptide histidine methionine (PHM) were measured by RIA in extracts of normal adrenal glands and in extracts from adrenal and extraadrenal pheochromocytomas. In normal adrenal glands, immunoreactive (IR) CRH, IR-SRIH, and IR-PHM were detectable, while IR-GHRH was undetectable. In all 11 cases of adrenal pheochromocytomas, the tumors contained 2 or more of these four IR-peptides. In particular, IR-CRH was found in 73% (n = 8) of adrenal pheochromocytomas, IR-GHRH in 91% (n = 10), IR-SRIH in 91% (n = 10), and IR-PHM in 82% (n = 9) of adrenal pheochromocytomas. There was no significant correlation among the concentration of these peptides in each tumor, i.e. the concentrations of the IR-peptides were independent of each other. In contrast to the adrenal pheochromocytomas, none of these 4 IR-peptides was detectable in 5 extraadrenal pheochromocytomas. Gel filtration of pooled extracts from adrenal pheochromocytomas showed that the major component of the IR-CRH, IR-GHRH, IR-SRIH, and IR-PHM eluted in the position of their synthetic counterparts. Our results suggest that 1) the normal adrenal gland contains IR-CRH, IR-SRIH, and IR-PHM, but not IR-GHRH; 2) all of the adrenal pheochromocytomas we examined contained a number of hypothalamic releasing or inhibiting hormones; 3) their tissue concentrations were independent of each other; and 4) all of the extraadrenal pheochromocytomas we examined contained no such IR-peptides. The presence of hypothalamic hormones in adrenal pheochromocytomas and their absence in extraadrenal pheochromocytomas may be due to the differences in the chromaffin cells of their origin. Our data may be helpful in the differential diagnosis between adrenal and extraadrenal pheochromocytomas.

The innervation of the adrenal medulla has been investigated in normal Wistar rats from birth to old age and ultrastructural findings compared with biochemical markers of the cholinergic innervation of the adrenal gland and catecholamine storage. Morphological evidence of the immaturity of the innervation during the first postnatal week is provided and using quantitative morphometry the innervation of chromaffin cells is shown to reach a mean total of 5.4 synapses per chromaffin cell during the period 26 days to 12 weeks of age. The variation in contents of synaptic profiles is discussed in the light of recent work that demonstrates a major sensory as well as visceral efferent innervation of the gland. Adrenal medullary neurons usually occur in closely packed groups, intimately associated with Schwann cells. Axodendritic and axosomatic synapses on these neurons are described and the likely origin of axonal processes innervating the neurons discussed. In old age the density of innervation remains the same as in young adult animals even though the medulla shows evidence of hyperplasia and hypertrophy of individual chromaffin cells.

Using a polyclonal antiserum raised against the first 34 amino acids of human parathyroid hormone-related peptide (PTHrP), we have localized PTHrP throughout the uro-genital tract of the human fetus aged between 8 and 40 weeks. Staining was present in the developing mesonephros, metanephros, gonads and in both the adrenal cortex and medulla. In particular, the developing mesonephric and metanephric renal tubules were intensely positive. Using Northern hybridization analysis we have detected a complex pattern of PTHrP mRNA transcripts ranging in size from 1.4 to 4.5 kb in early second trimester human fetal kidney. The presence of PTHrP in the mesonephros and metanephros provides evidence for a role for PTHrP in the regulation of fetal calcium metabolism. However, its presence in the gonad and adrenal gland invites the possibility of a wider role for PTHrP.

After removal of habitual partners, white female rats revealed a considerable increase in the corticosterone synthesis and secretion. An increase in the hormone secretion but not in its synthesis occurred in young male rats. In ageing male rats, emotional stress induced an increase in the corticosterone synthesis but the hormone did not enter their blood. High responsiveness of the adaptation systems was revealed in female rats through modification of their basal and stress-induced levels of corticosterone during a 72-hour exposure to light and darkness.

The basis for specific changes in differentiation that accompany ras-oncogene-mediated transformation are not understood. When short-term cultures of fibroblast-like cells from adult rat adrenal glands were transformed with Kirsten murine sarcoma virus (KiMSV), the original stromal characteristics of the target cells (metachromatic extracellular matrix, high collagen production, collagen incorporation into pericellular matrix and a fibroblastic morphology and growth pattern) diminished. In contrast, parenchymal differentiation markers (neutral lipid, delta 5, 3 beta-hydroxysteroid dehydrogenase, 21-hydroxylase and epithelial morphology) were enhanced. These changes in differentiation were initiated concurrently with the over-expression of the transforming protein v-p21, but were unrelated to the levels of v-p21 expression. They were independent of the immortalizing component of transformation mediated by v-K-ras, because they did not take place in spontaneously immortalized lines derived from the same target cells, unless the lines were also transformed with KiMSV. In normal embryonic development, stromal and parenchymal adrenocortical cells arise by divergent differentiation pathways from a common, multipotential mesenchymal precursor. The transformation-induced modulation from a predominantly stromal to a more-parenchymal phenotype is thus reminiscent of reversion to a more primitive, bipotential developmental stage.

The concentrations of two glucocorticoids (corticosterone and cortisol) were investigated in plasma and tissues of adult albino male rats and preweaning pups. An increase in corticosterone concentration in plasma and kidneys during maturation was shown. A steroid undistinguishable from cortisol in TLC, RIA and HPLC tests was detected in pups. A conclusion is made that cortisol takes part in hormonal regulation of immature rats.

Microtubule-associated protein 3 (MAP3, Mr 180,000), which in previous studies has been shown to be associated with glial processes and neurofilament-rich axons in rat brain, was examined in various non-neuronal rat tissues. Immunoblots of adult rat tissues (brain, liver, heart, spleen, adrenal medulla and kidney) showed that MAP3 is present in all organs tested. In addition we demonstrated that MAP3 is a heat-stable protein. Using immunohistochemistry, we established the localisation of MAP3 in various cell types. MAP3-containing cells appeared to have in common an asymmetric morphology with long processes that need structural support. In kidney MAP3 is limited to epithelial podocytes and in liver to Kupffer cells. In the adrenal gland, the cells of the cortex are devoid of MAP3 compared to the cells of the medulla. High concentrations of MAP3 are also found in cardiac muscle along the Z-disc and in the smooth muscle cells of the digestive tract. In spleen MAP3 is found in cells of the white pulp surrounding central blood vessels. A co-distribution of MAP3 with microtubules and intermediate filaments but not with microfilaments was found in each cell type examined. The widespread distribution pattern of MAP3 together with its molecular size and heat-stability indicate that MAP3 might be a member of the recently postulated family of homologous 200,000 Mr mammalian tissue MAPs. Potential functions for MAP3 in specific cell types are discussed.

A complementary DNA (cDNA) clone - cA2-47 - corresponding to a new alpha 2-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the alpha 2-receptor poly(A+) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5' half of the coding region of this cDNA at 37 degrees C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet alpha 2-receptor genomic probe failed to hybridize to any rat brain mRNAs. Under lower stringency conditions, hybridization of the full-length cDNA, cA2-47, to selected rat tissue poly(A+) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined.(ABSTRACT TRUNCATED AT 250 WORDS)

An antibody against basic fibroblasts growth factor (bFGF) was raised using purified bovine pituitary bFGF. Western blot analysis revealed immunoreactive bands at 18, 24, 30-33 and 46 kDa in immunoaffinity purified extracts of pituitary and adrenal gland using this antibody. A similar staining pattern was obtained with ovary extracts with the exception of the missing 18 kDa band. A second anti-bFGF antibody raised against a synthetic peptide comprising the 24 N-terminal amino acids of bFGF reacted with the 18 kDa and the 46 kDa band of immunoaffinity purified ovary and adrenal gland extracts.

The influence of ascorbic acid (AA) intake on plasma cortisol (PC) and tissue AA levels after ACTH treatment was examined using guinea pigs. ACTH produced an 8- to 10-fold rise in PC levels (p less than 0.0001) over the 4-hour experimental period compared to gel-injected control animals. The magnitude of rise in PC was similar at both normal (0.50 g/kg diet) and high (10 g/kg diet) AA intakes. A 30-100% higher (p less than 0.0001) level of AA in tissues (adrenals, liver, and kidneys) and plasma was observed with the high AA diet. ACTH resulted in a 26-30% lower level of AA in the adrenals (p less than 0.0001), but not in other tissues or plasma. PC and adrenal AA responses were unrelated (r = -0.326). These results suggest that ACTH alters AA only in the adrenals, the PC response to and percent decrease in adrenal AA levels with ACTH are not related to AA status, and the absolute level of AA in the adrenals is not critical for steroidogenesis.

The presence of gold was investigated in sections of the adrenal glands from rats which had been exposed to intraperitoneal sodium aurothiomalate (32 to 120 mg). Gold was histochemically detected in cortical endocrine cells, chromaffin cells and in fibroblasts and macrophages of both the cortex and medulla. Invisible traces of gold were silver enhanced by autometallography making them readily visible at both the light and electron microscopic levels. The intracellular staining intensity was dose-dependent. In general, the number as well as the staining intensity of individual cells, were highest in the zona glomerulosa and zona reticularis. In gold-containing cells the silver-amplified deposits were present in lysosomes.

By RIA there were studied the contents of corticosterone, ACTH, beta-endorphin and insulin in the blood plasma, met- and leu-enkephalin in different regions of the rat brain and in the adrenal glands after a 6-hour immobilization. The stress increased the content of corticosterone, ACTH, beta-endorphin, but not insulin in the blood plasma, and the levels of met-enkephalin in the adrenal glands, but decreased the met-enkephalin contents in the striatum. The injection of DSIP (0.1 mg/kg, i/p) blocked partly the elevation of corticosterone only. The authors propose, that stress-protective action of DSIP is realized with the involvements of the hypothalamo-pituitary-adrenal gland system.

The hydroxylation of testosterone and 5alpha-dihydrotestosterone at C-2 was measured quantitatively in vivo and in vitro in rats by using "metabolically labile" tritium labelled substrates. The enzyme activity was dependent on the sex and age of the animals. There was a good correlation between in vivo and in vitro experiments. Whereas testosterone was hydroxylated only in liver, 5alpha-dihydrotestosterone was hydroxylated in adrenals, kidney, lung and prostate as well. Hydroxylation was not sex-dependent in adrenals and lungs.

A comparison of human adult and fetal adrenals with respect to their levels of glyceryl ether lipids and other lipid components is reported. Fetal glands contained significantly lower levels of alk-1-enyl phosphoglycerides and of cholesterol. Neutral glyceryl ether diesters, and ethanolamine and choline phosphoglycerides were isolated from adult adrenal tissue. The composition of the O-alkyl glycerol groups in these lipid fractions was obtained by means of gas chromatography of the trimethylsilyl ethers and diacetyl derivatives; O-alk-1-enyl glycerols were analyzed as their diacetates. About one-half of the alkyl and alk-1-enyl glycerol moieties present in glyceryl ether diesters contained hydrocarbon side chains with 20, 22, or 24 carbon atoms. Long hydrocarbon chains (C19-24) were also found in the O-alkyl glycerol moieties present in the total lipids of fetal adrenals.

The regional distribution of kassinin-like immunoreactivity (KLI) was investigated by radioimmunoassay in central and peripheral tissues of the cat. In the cat brain KLI was found to have a widespread distribution with the highest concentrations present in the substantia nigra followed by the hypothalamus and caudate nucleus. Moderate levels were detected in the spinal cord, brainstem and thalamus but only low levels were found in the frontal cortex and cerebellum. In the periphery KLI was present in moderate amounts in the pituitary but in only low amounts in the dorsal root ganglion. KLI was present throughout the gastrointestinal tract with the highest concentration in the duodenum. The distribution of KLI was found to resemble closely that of substance P-like immunoreactivity (SPLI) although the ratio of SPLI to KLI varied widely throughout the tissues tested. In the frontal cortex and cerebellum the concentration of SPLI was at least 5-fold higher than that of KLI whereas in the pituitary gland, caudate nucleus and terminal ileum the concentrations of SPLI were only 50% higher. High pressure liquid chromatographic analysis of individual tissue extracts was used in order to characterize the KLI. In the caudate nucleus, substantia nigra and hypothalamus the major immunoreactive peak co-eluted with synthetic substance K. In contrast, in the spinal cord and terminal ileum the major peak of KLI co-eluted with synthetic neuromedin K. These results demonstrate that KLI is widely distributed throughout cat tissues and that this distribution closely resembles that of SPLI. However, it also appears that there are important qualitative differences in the KLI between the areas tested.

The levels of enkephalin-containing polypeptides (ECPs) in the adrenal glands of normal tensive rats (WKY) and spontaneously hypertensive rats (SHR) were compared. Innervated and denervated adrenals from both types of rats showed very similar levels of ECPs. The only difference observed was a small increase in the 18 kdal ECP and a concomitant decrease in the 12 kdal and 5.3 kdal ECPs in the innervated SHR rat adrenal gland. From these data it appears that the adrenal ECPs are not a major contributor to hypertension in the SHR rat nor does hypertension, at this age, affect the ECP levels.

The changes in the level of sulfhydryl fractions in total adrenals were measured with the aid of Ellman's reagent [Sedlák and Lindsay 1968] in rats exposed either to stress by forced immobilization or by the injury in Noble-Collip drum. It was found that nonprotein sulfhydryl fraction (NP-SH) in rat adrenals is represented exclusively by SH-glutathione (GSH). Moreover, the increase of the concentration of GSH in adrenals was related to the time and number of exposures to stressor. The maximum enhancement of GSH in adrenals occurred when the animals were subjected to stress daily for 7 days. It is suggested that GSH may be involved in the steroidogenesis in the inhibition of nonenzymatic decarboxylation of DOPA and in the proteosynthesis in adrenals.

Our study compares the properties of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from a human metastatic virilizing carcinoma and that from normal adrenal glands obtained from kidney donors. Optimal conditions for enzyme assay were obtained when a 50-mM imidazole buffer (pH 7.2) containing 5 mM EDTA, 250 mM NaCl, 1 mM phenyl-methylsulfonylfluoride, 0.1 mM leupeptin, and 5.5 mM dithiothreitol (DTT) was used. A 30-min preincubation period preceding addition of substrates enhanced reductase activity by 1.75-fold. In crude microsomal preparations, Km values were similar for both tumor and normal tissues and varied between 4 and 5 microM (S)HMG-CoA. The presence of NaF in homogenization and incubation media decreased the maximum velocity, but not the Km. A partially purified rat liver phosphorylase phosphatase preparation or a similar preparation from the carcinoma restored to maximal levels the reductase activity of microsomes prepared in the presence of NaF. A Km of 96 microM NADP was found for the carcinoma microsomal preparation. Preincubation of microsomes in the presence of monothioglycerol or DTT resulted in an increased reductase activity, suggesting a possible inactive enzyme precursor(s) consisting of disulfide-linked units. Reactions of the DTT-activated enzyme incubated in the presence of increasing amounts of NADPH showed sigmoidal kinetics. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a 92.5K mol wt protein band that reacted with a rat antireductase antibody. Reductase activity in different regions of the carcinoma varied from 679-1763 pmol/mg protein, with an average of 1146 pmol. In three normal adrenal glands we found values of 23.4, 48.1, and 36 pmol. We concluded that the expression of HMG-CoA reductase activity was elevated in human adrenal carcinoma.

The adrenal components of C. mrigala are embedded in the pronephric cephalic kidney around the post cardinal vein. The cortical cells responded positively to the lipids, ascorbic acid, delta 5-3 beta-HSD, G-6-PD, MAO, acid and alkaline phosphatase tests. The presence of intense MAO activity may suggest the possible involvement of monoamines in the adrenocortical function. Localization of lipids and delta 5-3 beta-HSD show the sites of corticosteroid synthesis. In the chromaffin cells, MAO, acid and alkaline phosphatase activity was moderate whereas they gave a strong reaction to ascorbic acid test in comparison to the cortical cells. Noradrenaline (NA) and adrenaline (A) storing cells were differentiated adopting glutaraldehyde silver, dichromate and iodate techniques. NA and A storing cells are almost totally depleted of their contents after reserpine treatment. The histochemical response of the adrenal gland of this species is largely comparable to that of higher vertebrates.

Phasic changes of biogenic amines were revealed in brain structures after isolated action of vibration; 15-min vibration increased the content of catecholamines in the hypothalamus and decreased the content of serotonin. 30-min vibration decreased the amount of biogenic amines in the hypothalamus and the cortex. Vibration of a longer duration (1 hr) increased the amount of catecholamines in the brain. After 2-hr vibration the content of catecholamines returned to the control level but the concentration of serotonin still remained above it. Combined action of vibration and noise (2 hrs) decreased the content of catecholamines and increased the amount of serotonin in cerebral structures. Dopamine and serotonin tended to decrease in the blood and adrenals whereas in the urine their amount increased. Multiple combined action of vibration and noise induced the most obvious changes.

The hypothesis that increased generation of lipid peroxides (LP) causes articular cartilage damage in older patients and in those with osteoarthritis was tested by directly measuring LP tissue levels in various layers of human articular cartilage. The LP content was significantly greater in the superficial than in the deeper portion of the cartilage, but lower in cartilage than in liver, kidney, adrenal glands and synovium. When LP were related to the total lipid content of these tissues, a high peroxide per lipid ratio was obtained for articular cartilage. The relevance of these findings to the mechanism of cartilage fibrillation is discussed.

The molecular forms of opioid peptides in human adrenal have not been well characterised. These peptides are predominantly derived from the proenkephalin A precursor, which has the sequence of Met-enkephalin(Arg6,Phe7) as its carboxyl terminus. We have looked in the present study at the subcellular distribution and the molecular form of immunoreactivity to this sequence in post-mortem human adrenal medulla and in phaeochromocytoma. In the human adrenal homogenates, the immunoreactivity distributes on a sucrose gradient in a manner consistent with localisation in chromaffin granules. On chromatography, the immunoreactivity from adrenal medulla is predominantly in the heptapeptide form; the intermediate (3000-4000) molecular weight material is only a minor component of immunoreactivity, in contrast to bovine tissue extracts where this is the major form of immunoreactivity. In the phaeochromocytoma extracts, the heptapeptide sequence again predominates over a minor amount of intermediate sized material. The results are discussed in terms of post-mortem changes, precursor processing and the function of the adrenal medulla.

The s.c. administration of chlorpromazine (CPZ) caused an elevation of plasma glucose and cyclic AMP levels in fed intact mice, but not in fed adrenalectomized mice. The pretreatment with reserpine partially blocked the elevation of plasma glucose and cyclic AMP. The pretreatment with 6-hydroxydopamine or alpha-methyl-p-tyrosine failed to inhibit the increase of plasma glucose and cyclic AMP induced by CPZ. These findings suggest that CPZ acts on the central nervous system and increases plasma glucose and cyclic AMP through epinephrine release from the adrenal medulla. The elevations of plasma glucose and cyclic AMP induced by epinephrine were enhanced by CPZ. CPZ increased plasma glucose and cyclic AMP significantly at high room temperatures (28 and 33 degrees C) at which CPZ did not decrease body temperature. The mechanism of CPZ action is discussed in relation to the increase in the plasma glucose and cyclic AMP levels.

Two separate ferredoxins that differ in molecular mass by about 1.5 kDa were isolated from both pig kidney and bovine adrenal mitochondria. The proteins had different biochemical and immunological properties and appeared to be distinct gene products. The smaller ferredoxin from pig kidney (renodoxin, Mr approximately equal to 13,500) was very similar to bovine adrenodoxin (Mr = 14,048). Both proteins had nearly identical N-terminal amino acid sequences and electron-transfer activities. However, renodoxin and adrenodoxin expressed distinct antigenic determinants, although they were immunologically cross-reactive. The larger kidney (approximately equal to 15-kDa) and adrenal (approximately equal to 15.3-kDa) ferredoxins were biochemically similar to each other but they had lower specific activities and their N-terminal sequences were different when compared to renodoxin and adrenodoxin. Each of the four ferredoxins had a visible absorption spectrum characteristic of a [2Fe-2S] chromophore. But in addition, the larger ferredoxins displayed a prominent A276 peak due to their higher tyrosine content and the presence of tryptophan, which is absent in adrenodoxin and renodoxin. Consequently, the larger ferredoxins were termed Trp-ferredoxin. Using antibody to pig kidney Trp-ferredoxin, the larger adrenal and kidney ferredoxins were found to be very similar immunologically; however, the Trp-containing and adrenodoxin-type ferredoxins did not cross-react in immunoblot analysis. Nevertheless, it was shown from competition ELISA and activity-inhibition analysis that the two ferredoxin types had limited common antigenic determinants. Trp-ferredoxin was the major iron-sulfur protein in kidney whereas adrenodoxin was the dominant molecular form in adrenal gland.

Mach-Zehnder interferometric measurements were used to assess the extent of section thickness variability (inter- and intrasection) associated with cryostat microtomy of adrenal sections over a typical working range of 10-20 micron. Sections were obtained using a Bright's Cambridge rocking type and a Damon rotary type cryostat microtome to allow comparative analyses. The effective thickness of tissue sections after being mounted onto slides by flash drying was reduced by 90% relative to microtome section thickness setting. A linear relationship between measured thickness and microtome setting was obtained with both instruments. Thickness variability between replicate sections over the range of microtome settings approximated 11% for the rocking microtome and 5% with the rotary microtome. Average intrasection variability was found to be 7% for rocking microtome sections and 4% for sections obtained with the rotary microtome. However, this variability is a negligible source of error in cytophotometric analyses, providing replicate sections are used and an adequate number of measurements are made on mask-delimited individual cells or tissue specimen areas.

The effect of hypophysectomy and hormonal replacement on the density of peripheral benzodiazepine binding sites (PBS) in rat adrenal gland and kidney was studied. In the adrenal gland, hypophysectomy caused a significant decrease of 3-fold in PBS density. In the kidney, in contrast, hypophysectomy did not affect PBS density. In the adrenal gland, adrenocorticotropic hormone (ACTH) administered to hypophysectomized rats caused a significant increase of more than 5-fold in PBS density compared to untreated hypophysectomized rats, and of more than 1.6-fold compared to intact rats. In contrast, the hormones pregnant mare serum gonadotropin (PMSG), diethylstilbestrol (DES), and hydrocortisone (HC), administered to hypophysectomized rats, failed to restore PBS density in the adrenal gland. In the kidney, HC administered to hypophysectomized rats caused an increase of 1.4-fold in PBS density compared to untreated hypophysectomized and intact rats. In contrast, the hormones ACTH, PMSG, and DES, administered to hypophysectomized rats, did not affect PBS density in the kidney. None of the hormones tested altered the equilibrium dissociation constant of PBS in either the adrenal gland or the kidney. These findings indicate that PBS density in rat adrenal gland and kidney is hormonally modulated.

A method for the separation and determination of ascorbic acid (AsA) and erythorbic acid (ErA) in animal tissues is described. It employs high-performance liquid chromatography on a LiChrosorb-NH2 column in conjunction with a mixture of acetonitrile, acetic acid and water as an eluant. Application of the method, which is sensitive, rapid and simple, to the analyses of AsA and ErA in animal tissues such as liver, adrenals, spleen, kidneys and heart gave satisfactory results. Dehydro-AsA and dehydro-ErA in rat liver could be determined after reduction with H2S. The method was shown to be useful for the routine analyses.

Cytochrome b561 from bovine adrenal medulla chromaffin granules was purified in octylglucoside by hydrophobic chromatography. The apparent molecular weight was determined to be 30,000 by gel electrophoresis in sodium dodecyl sulfate. This molecular weight differs from that previously reported for cytochrome b561 purified by other procedures. The granule cytochrome was compared to mitochondrial cytochrome b and, in spite of similar molecular weights and optical spectra, these two proteins were found to be different by the following criteria: 1) amino acid composition, 2) NH2-terminal analysis, 3) interaction with antimycin, and 4) reduction with durohydroquinone.

An accurate and precise procedure for the rapid analysis of ketoconazole in the lung, liver, plasma and adrenal gland of the rat has been developed. Separation of the drug from endogenous substances was achieved by solid-phase extraction followed by reversed-phase chromatography on a Novapak C18 column using a mobile phase of methanol--acetonitrile--0.02 M phosphate buffer (pH 6.8) (35:30:35). Depending on the tissue, the recoveries of ketoconazole and clotrimazole, which was used as an internal standard, ranged between 85.0 to 93.6% and 79.3 to 84.1%, respectively. The reproducibility of the assay was between 3 and 4%, depending on the tissue involved. The sensitivity of the procedure permitted the monitoring of ketoconazole levels for up to 24 h in the adrenal and up to 48 h for plasma, lung and liver, following oral administration of 150 mg/kg of the drug to male rats.

Recently, we described the presence of six immunoreactive (ir) morphinans in bovine adrenal and hypothalamus and identified one as morphine [Goldstein, A., Barrett, R. W., James, I. F., Lowney, L. I., Weitz, C. J., Knipmeyer, L. L. & Rapaport, H. (1985) Proc. Natl. Acad. Sci. USA 82, 5203-5207]. We now report that ir morphinans corresponding to the previously reported peak 1 (morphine), peak 4, and peak 5 are consistently present in extracts of bovine hypothalamus and variably present in extracts of bovine adrenal and rat brain. We no longer detect the previously reported peaks 2, 3, or 6, and we have established that they were contamination artifacts. Peak 1 is coeluted with morphine in two distinct reversed-phase HPLC systems, as is peak 4 with codeine. We have purified peak 1 and peak 4 compounds from bovine hypothalamus and determined their identities by gas chromatography/mass spectrometry (GC/MS): peak 1 is confirmed to be morphine and peak 4 is codeine.

An antibody to the low density lipoprotein (LDL) receptor was prepared by immunization of rabbits with a partially purified receptor preparation from bovine adrenal cortex. Immunoglobulin G (IgG) isolated from the serum of immunized rabbits blocked the binding of 125I-LDL to intact bovine adrenal membranes as well as to the partially purified bovine adrenal LDL receptor. The anti-receptor IgG also blocked the binding, uptake, and degradation of 125I-LDL by monolayers of human fibroblasts and thus prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase. A monovalent Fab fragment of th anti-receptor IgG retained the ability to inhibit LDL receptors of partially purified bovine adrenal cortex and monolayers of human fibroblasts. By immunofluorescence staining, the anti-receptor IgG was shown to bind to normal fibroblasts in discrete foci that were linearly arranged on the cell surface. No such foci were seen on fibroblasts from a patient with the receptor-negative form of homozygous familial hypercholesterolemia. The rabbit anti-receptor IgG also blocked the binding of 125I-LDL to membranes from dog and rat liver, but it did not prevent LDL binding to rabbit adrenal membranes or rabbit fibroblasts. The immunologic cross-reactivity of LDL receptors from bovine adrenal cortex, human fibroblasts, canine liver and adrenal gland, and rat liver indicates that the structure of this receptor has been widely conserved among animal species and tissues.

The substrate for ADP-ribosyltransferase from Clostridium botulinum was purified from the cytosol of bovine adrenal gland. Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of DEAE-Sepharose and phenyl-Sepharose, gel filtration on a TSK-gel G3000SW column, and Mono Q fast protein liquid chromatography. On DEAE-Sepharose chromatography, the substrate activity was eluted in two separate peaks, and electrophoretic analyses revealed that the substrates in the two peaks are of similar molecular weight but different isoelectric points. The major peak of the substrate was further purified. It was purified about 1,800-fold with a recovery of 2.2% by the above procedures. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation showed a single protein band at Mr 22,000. The purified protein served as a substrate for botulinum ADP-ribosyltransferase and was maximally ADP-ribosylated to the extent of about 0.7 mol of ADP-ribose/mol of protein. A guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was co-purified with the ADP-ribosylation substrate, and the purified protein maximally bound about 0.5 mol of GTP gamma S/mol. GTP gamma S binding was effectively competed by GTP and GDP but not by GMP, ATP, and ADP. Thus, the ADP-ribosylation substrate is a GTP-binding protein. This protein, designated Gb (b for botulinum), is widely distributed in various tissues. It was rich in brain, pituitary, and adrenal glands, and poor in heart, smooth, and skeletal muscles.