Rat Adrenal Gland Research Articles

Rodent adrenocortical cells display high affinity binding sites and proteins for inhibin A, and express components required for autocrine signalling by activins and bone morphogenetic proteins

Inhibins are expressed in the adrenal cortex, but little is known of their binding or role in the adrenal. The aims of the present study were, first, to establish whether a mouse adrenocortical (AC) cell line expresses inhibins/activins and bone morphogenetic proteins (BMP), along with proteins required for inhibin to antagonise activin and BMP actions and, secondly, to characterise and compare inhibin binding sites and proteins in the rat adrenal gland and AC cells. AC cells were found to: (1) express mRNA for multiple BMPs (BMP-2, -3, -4, -6, -8a), growth/differentiation factors (GDF-1, -3, -5, -9), Lefty A and B, and the inhibin alpha, beta(A) and beta(B) subunits (2) secrete inhibin A and inhibin B and (3) express mRNA encoding the inhibin co-receptor, betaglycan, along with activin and BMP type I (ALK2-7) and type II (ActRII, ActRIIB, BMPRII) receptors, and binding proteins (follistatin, BAMBI, gremlin). When applied to sections of rat adrenal glands, [(125)I]inhibin A specifically bound to cells of the adrenal cortex, mainly in the zona reticularis. Scatchard analyses of in vitro [(125)I]inhibin A binding to dispersed rat adrenal cells and AC cells revealed sites of high affinity (K(d)(1) of 0.18 and 0.15 nM, respectively) and low affinity (K(d)(2) of 2.6 and 1.3 nM, respectively. Competition for [(125)I]inhibin A binding by activin A or B (30 nM) was negligible, whereas BMP-2, -6 and -7 competed for between 21 and 33% of specific inhibin A binding (IC(50) between 0.2 and 0.3 nM). Inhibin B crossreaction with inhibin A binding sites was < 8%. Multiple binding protein complexes (molecular weight ranging from 35 to > 220 kDa) were affinity labelled by [(125)I]inhibin A on both the primary rat adrenal and AC cells. The species of > 220 kDa were shown by immunoprecipitation to include betaglycan, the species of 105 kDa is consistent in size with type II receptors for activin/BMP, and that of 62 kDa co-migrates with the inhibin-follistatin complex. In summary, the results show that inhibin A binds selectively and with both high and low affinity to AC cells via multiple binding proteins, including a single betaglycan-like species. The results support the role of glycosylated betaglycan in the high affinity binding of inhibin A, but provide consistent evidence from two independent sources of adrenal cells that inhibin A interacts with several membrane proteins in addition to those currently understood to mediate the anti-activin/BMP actions of inhibin.

Identification of Morphine-6-glucuronide in Chromaffin Cell Secretory Granules

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.

Chronic intermittent hypoxia induces hypoxic sensitivity in adult rat adrenal medulla via oxidative stress

Catecholamine (CA) secretion from adult adrenal medulla is insensitive to direct effects of acute hypoxia. Given that adrenal medulla contributes to elevated plasma CA in rats exposed to chronic intermittent hypoxia (CIH), in the present study, we examined whether CIH promotes CA efflux by acute hypoxia from adrenals in adult rats and if so by what mechanisms. Experiments were performed on adult male rats exposed to 10d of CIH. In control animals, acute hypoxia had no effect, whereas nicotine (100 μM) evoked epinephrine (Epi) or norepinephrine (NE) efflux from ex vivo adrenal medulla. In sharp contrast, in CIH animals, acute hypoxia evoked robust efflux of both Epi and NE, whereas the effects of nicotine were attenuated or absent. Cytosolic and mitochondrial aconitase enzyme activities, indices of O2. −generation, were down-regulated in CIH adrenal medulla. Systemic administration of superoxide dismutase (SOD) mimetic, a scavenger of O2. − prevented acute hypoxia-evoked release of Epi and NE from CIH conditioned adrenal medulla. Furthermore, SOD mimetic prevented increases in arterial blood pressure and elevated plasma CA in CIH conditioned rats. These observations demonstrate that CIH up-regulates hypoxic sensing in adult adrenal medulla via mechanisms involving increased generation of O2. − and hypoxia-evoked CA secretion from adrenal medulla contributes, in part, to elevated blood pressure during CIH (Supported by NIH HL-25830).

In vivo administered reserpine increases piecemeal degranulation in rat adrenal chromaffin cells

The effects of the amine-depletory agent reserpine have been evaluated by transmission electron microscopy in chromaffin cells of the rat adrenal glands. The drug has been injected intraperitoneally in the animals at a dose of 0.5 mg/kg body weight in two administrations at 24-hr interval. The observed ultrastructural changes closely reminded of piecemeal degranulation (PMD), a slow and long-lasting secretory process previously described in normal and tumor pheochromocytes. Both adrenaline- and noradrenaline-storing cells presented the following microscopic features: high granule polymorphism, due to coexistence in the same cell of normal resting granules, granules with partially mobilized components, and large empty containers; absence of granule fusion; characteristic "haloed" pattern of residual secretory contents; great amount of 30-150 nm diameter, membrane-bound, electron-dense and -lucent vesicles, free in the cytoplasm or attached to granules; and multiple vesicles budding from the granule-limiting membranes. Morphometric analysis revealed that the frequency of all these microscopic parameters was found to be significantly increased in adrenal chromaffin cells from reserpinized rats in comparison to cells from control animals. These data suggest that reserpine, besides blocking the inward transport of catecholamines in chromaffin granules, might also stimulate a complex secretory reaction, which shares many common passages with bona fide PMD.

CCCP enhances catecholamine release from the perfused rat adrenal medulla

The present study was designed to investigate the effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, on secretion of catecholamines from the isolated perfused model of the rat adrenal gland and to establish the mechanism of its adrenomedullary secretion. The perfusion of CCCP (3 × 10 − 5 M) into an adrenal vein of for 90 min caused a great increase in catecholamine secretion. Tachyphylaxis to catecholamine-releasing effect of CCCP was not observed by repeated perfusion of it. The net catecholamine-releasing effects of CCCP were depressed by pretreament with pirenzepine (a selective muscarinic M 1-receptor antagonist), chlorisondamine (a selective neuronal nicotinic receptor antagonist), nicardipine (an L-type Ca 2+-channel antagonist), TMB-8 (an intracellular Ca 2+-antagonist), and the perfusion of EGTA plus Ca 2+-free medium, respectively. In the presence of CCCP (3 × 10 − 5 M), catecholamine secretory responses induced by ACh (5.32 × 10 − 3 M), high K + (5.6 × 10 − 2 M, a direct membrane depolarizer), DMPP (10 − 4 M, (a selective neuronal nicotinic receptor agonist), and McN-A-343 (10 − 4 M, (a selective muscarinic M 1-receptor agonist) were significantly enhanced. CCCP also significantly enhanced the catecholamine secretory responses evoked by Bay-K-8644 (10 − 5 M), L-type Ca 2+ channel activator, and cyclopiazonic acid (10 − 5 M), an inhibitor of Ca 2+-ATPase. Furthermore, the perfusion of FCCP (3 × 10 − 5 M), a similar mitochondrial uncoupler, into an adrenal vein of for 90 min also caused a great increase in catecholamine secretion in a similar pattern with CCCP. Taken together, the results demonstrate that CCCP causes the catecholamine secretion from the perfused rat adrenal medulla in a calcium-dependent fashion. It is thought that this catecholamine secretory enhancement of CCCP may be mediated by both cholinergic receptor stimulation and membrane depolarization, which are relevant to the cytoplasmic Ca 2+ increase by stimulation of the Ca 2+ influx as well as by the inhibition of Ca 2+ uptake into the cytoplasmic Ca 2+ stores (both endoplasmic reticulum and mitochondria in chromaffin cells). It also seems that protonophores, such as CCCP, suppress mitochondrial Ca 2+ uptake and increase the stimulated secretion of catecholamine by the secretagogues. These results indicate that mitochondria modulate catecholamine secretion by regulating the Ca 2+ mobilization for exocytosis.

Immunocytochemical localization of metabotropic (mGluR2/3 and mGluR4a) and ionotropic (GluR2/3) glutamate receptors in adrenal medullary ganglion cells.

The localization of metabotropic glutamate receptors of groups II (mGluR2/3) and III (mGluR4a) and the subunits 2 and 3 of alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ionotropic glutamate receptors (GluR2/3) was investigated with immunocytochemical methods in the rat adrenal gland. MGluR2/3, mGluR4a and GluR2/3 immunoreactivities were observed in large-sized, centrally located type I adrenal medullary ganglion neurons. Furthermore, the small-sized type II adrenal ganglion neurons identified by their immunoreactivity to brain nitric oxide synthase (bNOS), also expressed mGluR2/3, mGluR4a and GluR2/3. These cells were disposed in the peripheral portion of the adrenal medulla. None of the type I neurons were positively labeled for bNOS. These morphological observations suggest that activation of glutamate receptors in ganglion neurons may be instrumental in the control of adrenal endocrine systems as well as blood regulation.

Prehepatic Portal Hypertension Induces Alterations in Cytochrome Oxidase Activity in the Rat Adrenal Gland

One approach to assess neuroendocrine response to portal hypertension in short-term portal vein-stenosed rats consists in studying metabolic and functional activity patterns in adrenal glands using mitochondrial enzyme cytochrome c oxidase (COX) as a histochemical marker. Male Wistar rats were divided into two groups: a control group (Group I; n = 8), in which the animals did not undergo any operative intervention, and a triple calibrated portal vein stenosis group (TPVS) (Group II; n = 7). The sections of suprarenal glands were histochemically stained for COX and the optical densitometry was measured by a computer image analyzer attached to a microscope. In TPVS rats, COX activity in the adrenal gland cortex is lower than in control rats and affects the fascicular (52.30, 47.16–60.98, vs. 67.12, 60.31–73.89, p =. 002), glomerular (49.68, 46.19–53.56 vs. 70.47, 64.64–73.51, p <. 001), and reticular (47.35, 35.63–54.39, vs. 55.37, 49.76–58.97; p <. 05) layers. In contrast, COX activity in the adrenal gland medulla is similar in TPVS rats and in control rats (29.91, 29.54–31.18, vs. 29.67, 28.95–30.23). The changes in adrenocortical COX activity in short-term-TPVS rats could constitute a pathogenic factor for both splanchnic and systemic hyperdynamic circulations, described in this experimental model of prehepatic portal hypertension.

Arecoline inhibits catecholamine release from perfused rat adrenal gland1

To study the effect of arecoline, an alkaloid isolated from Areca catechu, on the secretion of catecholamines (CA) evoked by cholinergic agonists and the membrane depolarizer from isolated perfused rat adrenal gland. Adrenal glands were isolated from male Sprague-Dawley rats. The adrenal glands were perfused with Krebs bicarbonate solution by means of a peristaltic pump. The CA content of the perfusate was measured directly using the fluorometric method. Arecoline (0.1-1.0 mmol/L) perfused into an adrenal vein for 60 min produced dose- and time-dependent inhibition in CA secretory responses evoked by acetylcholine (ACh) (5.32 mmol/L), 1.1-dimethyl-4-phenyl piperazinium iodide (DMPP) (100 micromol/L for 2 min) and 3-(m-choloro-phenyl-carbamoyl-oxy)-2-butynyl trimethyl ammonium chloride (McN-A-343) (100 micromol/L for 2 min). However, lower doses of arecoline did not affect CA secretion of high K(+) (56 mmol/L); higher doses greatly reduced CA secretion of high K(+). Arecoline also failed to affect basal catecholamine output. Furthermore, in adrenal glands loaded with arecoline (0.3 mmol/L), CA secretory response evoked by Bay-K-8644 (10 micromol/L), an activator of L-type Ca(2+) channels, was markedly inhibited, whereas CA secretion by cyclopiazonic acid (10 micromol/L), an inhibitor of cytoplasmic Ca(2+)-ATPase, was not affected. Nicotine (30 micromol/L), which was perfused into the adrenal gland for 60 min, however, initially enhanced ACh-evoked CA secretory responses. As time elapsed, these responses became more inhibited, whereas the initially enhanced high K(+)-evoked CA release diminished. CA secretion evoked by DMPP and McN-A-343 was significantly depressed in the presence of nicotine. Arecoline dose-dependently inhibits CA secretion from isolated perfused rat adrenal gland evoked by activation of cholinergic receptors. At lower doses arecoline does not inhibit CA secretion through membrane depolarization, but at larger doses it does. This inhibitory effect of arecoline may be mediated by blocking the calcium influx into the rat adrenal medullary chromaffin cells without the inhibition of Ca(2+) release from the cytoplasmic calcium store. There seems to be a difference in the mode of action of nicotine and arecoline in rat adrenomedullary CA secretion.

Nestin expression in normal adrenal gland and adrenocortical tumors.

Human adrenocortical cells have been shown to express cytokeratins and vimentin. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous system and that has been recently reported in rat adrenal gland as well. Using immunohistochemical and biochemical approaches, the present study demonstrates that nestin is constantly expressed in situ in the cortex of normal human adrenal glands. Nestin expressing cells were prevalently located in the zona reticularis but some positive cells could be spotted in the zona fasciculata as well. Moreover, patches of nestin-positive cells have been constantly detected on sections of cortical adenomas. In contrast, adrenal carcinomas displayed a variable number of nestin-immunoreactive cells that in some cases were virtually absent. Samples of renal clear cell carcinoma metastasis in the adrenals were also examined which did not show nestin-immunoreactivity. We propose that a positive nestin-immunoreaction could be useful in differential diagnosis of clear cell tumors in adrenal glands.

Lithium‐induced gene expression of inducible cyclic adenosine monophosphate early repressor in the rat adrenal gland

Lithium has acute and chronic effects on the hypothalamic-pituitary-adrenal gland (HPA) axis that are important for both therapeutic (e.g., treatment of mood disorders) and experimental (e.g., as the toxin in conditioned taste aversion studies) applications. We visualized lithium-induced activation of the HPA axis in rats by the adrenal expression of inducible cAMP early repressor (ICER), which is activated by elevated intracellular cAMP. We have shown that 1) intraperitoneal lithium chloride (LiCl) induces transient expression of ICER and c-fos mRNAs in the rat adrenal cortex and increases plasma level of corticosterone; 2) the cortical expression of ICER mRNA by LiCl occurs in a dose-dependent manner; 3) adrenal induction of ICER expression is delayed compared with c-fos expression; 4) dexamethasone pretreatment (4 mg/kg) blocks corticosterone release and adrenocortical ICER induction either by systemic LiCl (76 mg/kg) or by restraint stress; and 5) intracerebroventricular LiCl (127 microg/5 microl) is sufficient for adrenocortical, but not medullary, ICER induction. These results suggest that adrenocortical ICER expression could serve as a reliable marker for lithium-induced activation of the HPA axis. Understanding the activation of immediate-early genes such as c-fos or ICER in response to a single LiCl injection is an important first step in understanding the long-term changes in gene expression elicited by lithium that are involved in its therapeutic and toxic effect. The pattern and mechanism by which lithium stimulates ICER transcription in the adrenal gland would serve as a useful model system in future studies of lithium.

Changes in P2Y2 receptor localization on adrenaline- and noradrenaline-containing chromaffin cells in the rat adrenal gland during development and aging

Using immunohistochemistry, the occurrence and age-related changes of the P2Y2 receptor was investigated in the adrenal gland of rat at different ages, ranging from embryonic day E16 to 22 months. Immunoreactivity for the P2Y2 receptor was present in chromaffin cells and nerve fibres at all ages examined. Double labeling with the antibody against phenyl ethanolamine-N-methyltransferase, which marks adrenaline-producing chromaffin cells, revealed that only a few of the P2Y2-immunoreactive cells were adrenaline producing at embryonic day E16, the vast majority being noradrenaline-containing cells. However, immunoreactivity for adrenaline-containing cells in the P2Y2 receptor-labeled chromaffin cells increased with increasing age and at 1 week post-natal almost all chromaffin cells were positive for both P2Y2 and phenyl ethanolamine-N-methyltransferase, while noradrenaline-containing cells were minimal. At 2 weeks, there was a dramatic drop in P2Y2-immunoreactive chromaffin cells and this was maintained in adult rats, noradrenaline-containing cells dominating. In the aging rat adrenals, P2Y2 receptor-immunoreactivity was localized in subpopulations of both adrenaline and noradrenaline-producing cells. Intrinsic neurones were also visible that were positively labeled with the P2Y2 receptor antibody in the adrenals of both adult and aging rats. P2Y2-immunoreactive nerve fibres formed a plexus around the adrenal cortical cells of zona glomerulosa in the post-natal, but not in adult or aging rats.In conclusion, this study suggests that ATP, acting through P2Y2 receptors, may influence the phenotypic expression of chromaffin cells during the development and aging of the rat adrenal gland. However, during early development, when the chromaffin cells are actively dividing and during aging, when the adrenal medullary cells are known to show hyperplastic lesions, ATP acting through P2Y2 receptors may be involved in other physiological activities, such as proliferation and/or differentiation of the chromaffin cells associated with their adrenaline or noradrenaline phenotype.

Differential expression and function of beacon in the rat adrenal cortex and medulla

Beacon gene is overexpressed in obese rats, and beacon was found to stimulate food intake. Evidence has been recently provided that beacon is also expressed in the endocrine glands of normal rats, including adrenal cortex, of which it appears to regulate secretory activity. To further characterize the role of beacon in the rat adrenals, we investigated the level of beacon expression in the adrenal zona glomerulosa (ZG), zona fasciculata-reticularis (ZF/R) and medulla (AM), and the in vitro secretory responses to beacon[47-73] (hereinafter, beacon) of adrenocortical and adrenomedullary tissues. Real-time polymerase chain reaction revealed similar high levels of beacon mRNA in the ZG and ZF/R, and significantly lower (-80%) levels in AM. Immunocytochemistry showed that the distribution of beacon protein followed that of beacon mRNA. Quantitative high pressure liquid chromatography demonstrated that beacon (5x10(-7) M) reduced by about 56% the in vitro total steroid-hormone production from ZG and ZF/R tissues, without affecting catecholamine secretion from AM specimens. The beacon-induced lowering in the secretory activity of adrenal cortex depended on similar reductions (from 50-64%) in the production of the main adrenocortical hormones (pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxy-corticosterone and aldosterone), thereby suggesting an inhibitory action of beacon in the early step of steroidogenesis (i.e. the conversion of cholesterol to pregnenolone). The hypothesis is advanced that beacon is to be considered an autocrine-paracrine negative regulator of mineralo- and glucocorticoid synthesis in the rat adrenal gland.

Age‐associated changes in mRNA levels of Phox2, norepinephrine transporter and dopamine β‐hydroxylase in the locus coeruleus and adrenal glands of rats

Age-related changes in the gene expression of the transcription factors, Phox2a and 2b, and two marker proteins, norepinephrine transporter (NET) and dopamine beta-hydroxylase (DBH), of noradrenergic neurons were characterized in the locus coeruleus (LC) and adrenal glands using in situ hybridization. Analysis of changes was performed in rats that were 1-23 months of age. Compared to 1-month-old rats, there was a 62% increase of Phox2a messenger RNA (mRNA) in the LC of 3-month-old rats, and a decline of 37% in 23-month-old rats. In contrast, levels of Phox2b mRNA in the LC remained unchanged in 3-month-old rats, but declined to a 30% reduction in 23-month-old rats. Interestingly, mRNA levels of NET in the LC decreased with increasing age to a reduction of 29%, 30% and 43% in 3-, 8- and 23-month-old rats, respectively. Similarly, DBH mRNA in the LC declined with increasing age to a 56% reduction in 23-month-old rats. mRNA levels of Phox2a, Phox2b, NET and DBH in the adrenal medulla of 23-month-old rats were significantly lower than those of 1-month-old rats. Semi-quantitative reverse transcription assays of the same genes yielded data similar to in situ hybridization experiments, with beta-actin mRNA levels being unchanged across the ages. Taken together, these data reveal that reduced Phox2 mRNAs in the LC and adrenal medulla of aging rats are accompanied by a coincidental decline in mRNA levels of NET and DBH and suggest a possible relationship between Phox2 genes and the marker genes in noradrenergic neurons after birth.

Influence of naloxone on catecholamine release evoked by nicotinic receptor stimulation in the isolated rat adrenal gland

The present study was designed to investigate the effect of naloxone, a well known opioid antagonist, on the secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane-depolarization in the isolated perfused rat adrenal glands, and to establish its mechanism of action. Naloxone (10(-6) approximately 10(-5) M), perfused into an adrenal vein for 60 min, produced dose- and time-dependent inhibition of CA secretory responses evoked by ACh (5.32 x 10(-3) M), high K+ (5.6 x 10(-2) M), DMPP (10(-4) M) and McN-A-343 (10(-4) M). Naloxone itself also failed to affect the basal CA output. In adrenal glands loaded with naloxone (3 x 10(-6) M), the CA secretory responses evoked by Bay-K-8644, an activator of L-type Ca2+ channels, and cyclopiazonic acid, an inhibitor of cytoplasmic Ca(2+)-ATPase, were also inhibited. In the presence of met-enkephalin (5 x 10(-6) M), a well known opioid agonist, the CA secretory responses evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly inhibited. Taken together, these results suggest that naloxone greatly inhibits the CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as that by membrane depolarization. It seems that these inhibitory effects of naloxone does not involve opioid receptors, but might be mediated by blocking both the calcium influx into the rat adrenal medullary chromaffin cells and the uptake of Ca2+ into the cytoplasmic calcium store, which are at least partly relevant to the direct interaction with the nicotinic receptor itself.

Characterization of the Sulfhydryl-Sensitive Site in the Enzyme Responsible for Hydrolysis of 2- Arachidonoyl-Glycerol in Rat Cerebellar Membranes

We have previously reported that the endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is hydrolyzed in rat cerebellar membranes by monoglyceride lipase (MGL)-like enzymatic activity. The present study shows that, like MGL, 2-AG-degrading enzymatic activity is sensitive to inhibition by sulfhydryl-specific reagents. Inhibition studies of this enzymatic activity by N-ethylmaleimide analogs revealed that analogs with bulky hydrophobic N-substitution were more potent inhibitors than hydrophilic or less bulky agents. Interestingly, the substrate analog N-arachidonylmaleimide was found to be the most potent inhibitor. A comparison model of MGL was constructed to get a view on the cysteine residues located near the binding site. These findings support our previous conclusion that the 2-AG-degrading enzymatic activity in rat cerebellar membranes corresponds to MGL or MGL-like enzyme and should facilitate further efforts to develop potent and more selective MGL inhibitors.

Blocking BRE expression in Leydig cells inhibits steroidogenesis by down-regulating 3β-hydroxysteroid dehydrogenase

Conversion of cholesterol to biologically active steroids is a multi-step enzymatic process. Along with some important enzymes, like cholesterol side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD), several proteins play key role in steroidogenesis. The role of steroidogenic acute regulatory (StAR) protein is well established. A novel protein, BRE, found mainly in brain, adrenals and gonads, was highly expressed in hyperplastic rat adrenals with impaired steroidogenesis, suggesting its regulation by pituitary hormones. To further elucidate its role in steroidogenic tissues, mouse Leydig tumor cells (mLTC-1) were transfected with BRE antisense probes. Morphologically the BRE antisense cells exhibited large cytoplasmic lipid droplets and failed to shrink in response to human chorionic gonadotropin. Although cAMP production, along with StAR and P450scc mRNA expression, was unaffected in BRE antisense clones, progesterone and testosterone yields were significantly decreased, while pregnenolone was increased in response to human chorionic gonadotropin stimulation or in the presence of 22(R)OH-cholesterol. Furthermore, whereas exogenous progesterone was readily converted to testosterone, pregnenolone was not, suggesting impairment of pregnenolone-to-progesterone conversion, a step metabolized by 3beta-HSD. That steroidogenesis was compromised at the 3beta-HSD step was further confirmed by the reduced expression of 3beta-HSD type I (3ss-HSDI) mRNA in BRE antisense cells compared with controls. Our results suggest that BRE influences steroidogenesis through its effects on 3beta-HSD action, probably affecting its transcription.

Biochemical Characterisation and Immunohistochemical Localisation of the Secretogranin II‐Derived Peptide EM66 in the Hypothalamus of the Jerboa (Jaculus orientalis): Modulation by Food Deprivation

The neuroendocrine protein secretogranin II is the precursor of several neuropeptides, including secretoneurin and a novel 66-amino acid peptide, EM66, the sequence of which has been highly conserved across the vertebrae phylum. The presence of EM66 has been detected in the adult and fetal human adrenal gland, as well as the rat pituitary and adrenal glands. The present study aimed to explore a possible neuroendocrine role of EM66 by analysing its occurrence and distribution within the jerboa hypothalamus, and its potential implication in the control of feeding behaviour. High-performance liquid chromatography analysis of jerboa hypothalamic extracts combined with a radioimmunoassay of EM66 revealed a single peak of immunoreactive material exhibiting the same retention time as recombinant EM66. Immunocytochemical labelling showed that EM66-producing neurones are widely distributed in several hypothalamic regions, including the preoptic area, the suprachiasmatic, supraoptic, parvocellular paraventricular and arcuate nuclei, and the lateral hypothalamus. Food deprivation for 5 days induced a significant increase in the number of EM66-containing neurones within the arcuate nucleus (105% increase) and the parvocellular aspect of the paraventricular nucleus (115% increase), suggesting that EM66 could be involved in the control of feeding behaviour and/or the response to stress associated with fasting. Altogether, these data reveal the physiological plasticity of the EM66 system in the hypothalamus and implicate this novel peptide in the regulation of neuroendocrine functions.

The rapid release of corticosterone from the adrenal induced by ACTH is mediated by nitric oxide acting by prostaglandin E2.

The adrenal cortex is a major stress organ in mammals that reacts rapidly to a multitude of external and internal stressors. Adrenocorticotropin (ACTH) is the main stimulator of the adrenal cortex, activating corticosteroid synthesis and secretion. We evaluated the mechanism of action of ACTH on adrenals of male rats, preserving the architecture of the gland in vitro. We demonstrated that both sodium nitroprusside (NP), a nitric oxide (NO) donor, and ACTH stimulate corticosterone release. NO mediated the acute response to ACTH because Nomega-nitro-l-arginine methyl ester, a NO synthase inhibitor, and hemoglobin, a NO scavenger, blocked the stimulation of corticosterone release induced by ACTH. NP stimulated prostaglandin E release, which in turn stimulated corticosterone release from the adrenal. Additionally, indomethacin, which inhibits cyclooxygenase, and thereby, prostaglandin release, prevented corticosterone release from the adrenal induced by both NP and ACTH, demonstrating that prostaglandins mediate acute corticosterone release. Corticosterone content in adrenals after incubation with ACTH or NP was lower than in control glands, indicating that any de novo synthesis of corticosterone during this period was not sufficient to keep up with the release of the stored hormone. The release induced by ACTH or NP depleted the corticosterone content in the adrenal by approximately 40% compared with the content of glands incubated in buffer. The mechanism of rapid release is as follows: NO produced by NO synthase activation by ACTH activates cyclooxygenase, which generates PGE(2), which in turn releases corticosterone stored in microvesicles and other organelles.

Cerebellin in the rat adrenal gland: gene expression and effects of CER and [des-Ser1]CER on the secretion and growth of cultured adrenocortical cells

Cerebellin (CER) is a regulatory peptide, originally isolated from rat cerebellum, which derives from the cleavage of precerebellin (Cbln), three types of which (Cbln1-3) have been identified in humans and rats. CER is also expressed in several extra-cerebellar tissues, including adrenal gland, and evidence has been provided that CER exerts a modulatory action on human and rat adrenal gland. Hence, we have investigated the expression of Cbln1-3 mRNAs and CER protein-immunoreactivity (IR) in the various zones of rat adrenal glands, and the effects of CER and its metabolite [des-Ser(1)]CER (des-CER) on the secretion and growth of cultured rat adrenocortical cells. Reverse transcription-polymerase chain reaction showed high and low expression of Cbln2 mRNA in zona glomerulosa (ZG) and zona fasciculata-reticularis, respectively. Cbln1 was not expressed, and Cbln3 mRNA was detected only in ZG. No Cbln expression was found in adrenal medulla. Immunocytochemistry demonstrated the presence of CER-IR exclusively in the adrenal cortex, the reaction being more intense in ZG. As expected, ACTH (10(-8) M) markedly enhanced corticosterone secretion and lowered proliferation rate of cultured adrenocortical cells. CER was ineffective, while des-CER exerted an ACTH-like effect, but only at the lowest concentration (10(-10) M). Taken together, these findings allow us to conclude that CER is expressed in rat adrenal cortex, and to suggest that CER conversion to des-CER by endopeptidases is needed for CER to exert its autocrine-paracrine regulatory functions.

Effects of acupuncturing Tsusanli (ST36) on expression of nitric oxide synthase in hypothalamus and adrenal gland in rats with cold stress ulcer

To study the protective effect of acupuncturing Tsusanli (S(T)36) on cold stress ulcer, and the expression of nitric oxide synthase (NOS) in hypothalamus and adrenal gland. Ulcer index in rats and RT-PCR were used to study the protective effect of acupuncture on cold stress ulcer, and the expression of NOS in hypothalamus and adrenal gland. Images were analyzed with semi-quantitative method. The ulcer index significantly decreased in rats with stress ulcer. Plasma cortisol concentration was up regulated during cold stress, which could be depressed by pre-acupuncture. The expression of NOS1 in hypothalamus increased after acupuncture. The increased expression of NOS2 was related with stress ulcer, which could be decreased by acupuncture. The expression of NOS3 in hypothalamus was similar to NOS2, but the effect of acupuncture was limited. The expression of NOS2 and NOS3 in adrenal gland increased after cold stress, only the expression of NOS1 could be repressed with acupuncture. There was no NOS2 expression in adrenal gland in rats with stress ulcer. The protective effect of acupuncturing Tsusanli (S(T)36) on the expression of NOS in hypothalamus and adrenal gland can be achieved.