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.

Neuropeptide Y expression in the developing adrenal gland and in childhood neuroblastoma tumors.

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.

Efecto del ejercicio físico agudo sobre la distribución y el metabolismo de la vitamina A / Effect of acute physical exercise on the distribution and metabolism of vitamin A

Se evaluó el efecto del ejercio físico agudo sobre la distribución y el metabolismo de la vitamina A en un modelo experimental animal. Se estudiaron las concentraciones plasmáticas, hepaticas, renales, testiculares y de glándulas suprarrenales de la vitamina A y de sus principales formas moleculares: retinol y ésteres de retinol, en animales sometidos a ejercicio físico durante 2 h sin previo entrenamiento. Se observó una disminución en la concentración hepática y testicular de la vitamina A y un incremento de ésta en los riñones de los animales ejercitados. Se demostró, además, el incremento del catabolismo hepático del retinol bajo las condiones experimentales

Chronic effects of interleukin-1 on hypothalamus, pituitary and adrenal glands in rat.

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.

Beta 2-microglobulin expression in human embryonal neuroblastoma reflects its developmental regulation.

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.

[Endocrine-metabolic relations in rats with genetic arterial hypertension]. / Endokrinno-metabolicheskie otnosheniia u kryss geneticheski obuslovlennoi arterial'noi gipertenziei.

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.

Changes in adrenal neuropeptides content [peptide 7B2, neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP)] induced by pharmacological and hormonal manipulations.

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.

Immunoreactive corticotropin-releasing hormone, growth hormone-releasing hormone, somatostatin, and peptide histidine methionine are present in adrenal pheochromocytomas, but not in extra-adrenal pheochromocytoma.

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 gland. IV. Innervation of the rat adrenal medulla from birth to old age. A descriptive and quantitative morphometric and biochemical study of the innervation of chromaffin cells and adrenal medullary neurons in Wistar rats.

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.

Parathyroid hormone-related peptide in the human fetal uro-genital tract.

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.

V-K-ras transformation induces reversion to an earlier developmental form in adult rat adrenal cells.

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.

[Changes in the content of glucocorticoids in the plasma and tissues of rats in postnatal ontogeny]. / Izmenenie soderzhaniia gliukokortikoidov v plazme i tkaniakh krys v postnatal'nom ontogeneze.

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.

[Sex differences in the age-related dynamics of the reactivity to physiological stressors in white rats]. / Polovye razlichiia vozrastnoi dinamiki reaktivnosti k fiziologicheskim stressoram u belykh krys.

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.

Microtubule-associated protein 3 (MAP3) expression in non-neuronal tissues.

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.

Autometallographic demonstration of gold in the adrenal gland of rats exposed to sodium aurothiomalate. A light and electron microscopic study.

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.

Genetic evidence for alpha 2-adrenergic receptor subtypes in rat brain, heart and adrenal gland.

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)

[Analysis of the mechanism of the stress-protective action of delta sleep-inducing peptide]. / Analiz mekhanizma stressprotektivnogo deistviia peptida, indutsiruiushchego delta-fazu sna.

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.

Plasma cortisol and adrenal ascorbic acid levels after ACTH treatment with a high intake of ascorbic acid in the guinea pig.

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.

High molecular weight forms of basic fibroblast growth factor recognized by a new anti-bFGF antibody.

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.