Reorganization of the uncrossed visual pathways as revealed by Fos-like immunoreactivity in rats with neonatal monocular enucleation.

To elucidate the neuronal characteristics of the functional expansion in the uncrossed visual pathways (UXVPs), resulting from early monocular enucleation in rats, the feasibility of stimulus-dependent induction of the immediate early gene c-fos was examined immunohistochemically. In the UXVPs of rats with monocular enucleation at birth, patterned visual stimuli induced Fos-like immunoreactive (FLI) neurons much more densely in wide areas of the superficial layer throughout the superior colliculus (SC), and in the striate and extrastriate areas of the visual cortex (VC). In the UXVPs of rats monocularly enucleated after maturity, however, only a few stimulus-dependent FLI neurons were scattered in the restricted portions of the SC and the VC.

Expression of prostaglandin E receptor EP4 subtype in rat adrenal zona glomerulosa: involvement in aldosterone release.

We examined the role of prostaglandin (PG) E receptors in the secretion of aldosterone. PGE2 is known to exert its various biological functions by binding to PGE receptors. There are four subtypes of PGE receptors, EP1, EP2, EP3, and EP4. Among the PGE receptors EP2 and EP4 subtypes are coupled to Gs protein and stimulate adenylyl cyclase. In this study, PGE2 caused a dose-dependent increase in aldosterone production from the rat adrenal zona glomerulosa cells in vitro accompanied with an increase in intracellular cAMP concentration. A specific agonist for EP2, butaprost, did not increase the cAMP production or the aldosterone release, suggesting the possibility that EP4 mediates the secretion of aldosterone by PGE2. Northern blot hybridization analysis disclosed that EP4 gene was expressed in the rat adrenal gland but that EP2 gene was not. In situ hybridization revealed that EP4 mRNA is present abundantly in the zona glomerulosa of rat adrenal gland. These findings suggest that the PGE2-EP4 system is involved in the regulation of aldosterone secretion from the rat adrenal gland.

Mutation analysis of Gsalpha, adrenocorticotropin receptor and p53 genes in Japanese patients with adrenocortical neoplasms: including a case of Gsalpha mutation.

While the mechanisms of tumorigenesis for adrenocortical neoplasms remain unknown, several genes, such as Gsalpha, ACTH receptor (MC2-R), p53, and p16 tumor suppressor genes, are considered to be candidates for adrenocortical neoplasms. Mutation analysis studies have documented these genes in adrenocortical neoplasms, but these studies focused on the mutation of only one of these genes. In the present study we examined the mutations of three of these genes (Gsalpha, MC2-R, and p53) in adrenocortical neoplasms in Japanese patients. We amplified these genes using polymerase chain reaction and directly sequenced them in 30 functioning adrenocortical neoplasms. As for Gsalpha, we identified a heterogeneous substitution of glutamine to histidine at codon 227 and a gain of an Nru I restriction endonuclease site. The mutation was restricted to adenomatous tissue, and did not occur in the adjacent normal adrenal tissue or leukocytes of the patient. We did not find any mutations in MC2-R and p53. In conclusion, although the contribution of these three genes to adrenocortical tumorigenesis remains to be determined, it is suggested that the mutation of Gsalpha might play a role in functional adrenocortical neoplasms.

Prostaglandin E(2) (PGE(2)) induces the c-fos and c-jun expressions via the EP(1) subtype of PGE receptor in mouse osteoblastic MC3T3-E1 cells.

This study examined which subtype(s) of PGE receptors is involved in the induction of c-fos and c-jun by PGE(2) in MC3T3-E1 cells. We also investigated the possibility that the induction of these genes is involved in the growth and differentiation of this cell line. PGE(2) dose-dependently induced c-fos and c-jun mRNA expressions in MC3T3-E1 cells. Of the PGE analogs, 17-phenyl-omega-trinor PGE(2) (EP(1) agonist) and sulprostone (EP(1)/EP(3) agonist) were far more potent than butaprost (EP(2) agonist) and 11-deoxy PGE(1) (EP(2)/EP(4) agonist) in inducing c-fos and c-jun mRNA expressions. Since MC3T3-E1 cells do not express the EP(3) subtype, these results suggest that PGE(2) induces c-fos and c-jun mRNA expressions through the EP(1) subtype of its receptor. In order to study the functional relevance of these protooncogenes, we then studied the effect of inhibition of their synthesis by the use of antisense oligonucleotide. Alkaline phosphatase (ALP) suppression by 17-phenyl-omega-trinor PGE(2) was reversed by antisense oligonucleotide for either c-fos or c-jun. These results suggest that PGE(2), via the EP(1) subtype of the PGE receptor, negatively modulates the transition from proliferation to the matrix maturation stage through the induction of c-fos and c-jun. However, antisense oligonucleotide for c-fos or c-jun did not alter the prostaglandin G/H synthase-2 mRNA expression induced by EP(1). Thus, it is possible that c-fos and c-jun inductions do not account for all the EP(1)-mediated PGE(2) actions in MC3T3-E1 cells.

Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse.

The spleen has two main functions. The first is to provide a proper microenvironment to lymphoid and myeloid cells, whereas the second involves clearance of abnormal erythrocytes. Ad4BP/SF-1, a product of the mammalian FTZ-F1 gene (mFTZ-F1), was originally identified as a steroidogenic, tissue-specific transcription factor. Immunohistochemical examination of the mammalian spleens confirmed the expression of Ad4BP/SF-1 in endothelial cells of the splenic venous sinuses and pulp vein. In mFtz-F1 gene-disrupted (KO) mice, several structural abnormalities were detected in the spleen, including underdevelopment and nonuniform distribution of erythrocytes. Examination of the spleen of KO fetuses showed failure of development of certain tubular structures during embryogenesis. These structures are normally assembled by Ad4BP/SF-1 immunoreactive cells, and most likely form the vascular system during later stages of development. Other structural abnormalities in the spleen of the KO mice included defects in the tissue distribution of type-IV collagen, laminin, c-kit, and vimentin. These morphologic defects in the vascular system were associated with a decrease in the proportion of hematopoietic cells, although differentiation of these cells was not affected significantly. A high number of abnormal red blood cells containing Howell-Jolly bodies were noted in the KO mice, indicating impaired clearance by the splenic vascular system. We also detected the presence of an mRNA-encoding cholesterol side-chain cleavage P450 in the spleen, resembling the findings in steroidogenic tissues such as the gonads and adrenal cortex. The mRNA transcript was not involved in splenic structural defects as it was detected in the spleens of both normal and KO mice, indicating that the regulatory mechanism of the P450 gene in the spleen is different from that in steroidogenic tissues. Our results indicate that a lack of the mFtz-F1 gene in mice is associated with structural and functional abnormalities of the splenic vascular system.

[ACTH receptor, ACTH receptor anomaly, and familial glucocorticoid deficiency].

Familial glucocorticoid deficiency (FGD) is an autosomal recessive syndrome with hereditary adrenocortical unresponsiveness to ACTH. After the cloning of ACTH receptor or melanocortin-2 receptor (MC-2R) cDNA, several kinds of mutations in the receptor genes have been reported. However, the apparently normal ACTH receptor gene in some affected children suggests that the etiology of FGD is heterogeneous. In this short review, we describe the recent advances in the molecular biology of ACTH receptor genes, its post-receptor signal transduction in the adrenocortical cells, and the molecular genetics of the FGD and a related syndrome, Allgrove syndrome. We also discuss that this kind of work will help us to understand better about the molecular mechanism of the glucocorticoidogenesis in the human being.

Angiotropic large cell lymphoma which infiltrated to the adrenal glands presenting as reversible adrenal insufficiency.

A 72-year-old man who suffered from recurrent fever was found to have enlarged bilateral adrenal glands on computed tomographic scanning, combined with subclinical adrenal insufficiency. Based on the pathology of bone marrow aspiration, he was diagnosed to have an angiotropic large cell lymphoma (ALCL). Soon after the treatment with a combination chemotherapy, he achieved complete remission of ALCL and size and function of the adrenal glands were apparently normalized. ALCL should be included in the list of differential diagnoses of non-functioning bilateral adrenal swelling with non-specific symptoms such as fever.

Characterization of cationic amino acid transporter and its gene expression in rat hepatic stellate cells in relation to nitric oxide production.

BACKGROUND/AIMS: Nitric oxide is a potent mediator of hepatic sinusoidal hemodynamics and affects hepatic stellate cells (Ito cells, fat-storing cells). Although nitric oxide production may depend on the induction of inducible nitric oxide synthase and on transport of extracellular L-arginine, the precise mechanisms controlling nitric oxide production in stellate cells have not been well characterized. METHODS: Using stellate cells prepared from the male Wistar rat, kinetic analysis of L-arginine transport and reverse transcription-polymerase chain reaction for cationic amino acid transporter were carried out. The effect of tumor necrosis factor-alpha and interferon-gamma on L-arginine transport, mRNA expression of cationic amino acid transporter and inducible nitric oxide synthase, and nitric oxide production of stellate cells was assessed. RESULTS: The L-arginine transport system functioning in the transformed hepatic stellate cells was system y+, possibly mediated by cationic amino acid transporter-1 and cationic amino acid transporter-2B (Km approximately 50 microM). Tumor necrosis factor-alpha enhanced cationic amino acid transporter-2B mRNA expression and L-arginine transport, whereas cationic amino acid transporter-1 mRNA expression remained unchanged. Interferon-gamma induced the expression of inducible nitric oxide synthase mRNA without obvious changes in L-arginine transport. Interferon-gamma in combination with tumor necrosis factor-alpha induced nitric oxide production with an enhancement in cationic amino acid transporter-2B mRNA expression, inducible nitric oxide synthase mRNA expression, and L-arginine transport, while extracellular L-lysine competitively inhibited this nitric oxide production. CONCLUSIONS: In transformed hepatic stellate cells, tumor necrosis factor-alpha and interferon-gamma have a crucial role in nitric oxide production, and extracellular L-arginine transport and inducible nitric oxide synthase expression are regulated in a differential cytokine-specific manner. As the estimated Km of L-arginine transporter in transformed hepatic stellate cells is very similar to the physiological L-arginine concentration in portal vein, we assume that increased portal L-arginine concentration may easily affect sinusoidal blood flow through enhancement of autocrine nitric oxide production in transformed hepatic stellate cells of diseased liver.

Tumor necrosis factor receptors in the pituitary cells.

To clarify the site and mode of action of tumor necrosis factor (TNF) in the pituitary, we studied the effects, binding sites of TNF and its receptor mRNA in the two types of mouse pituitary-derived cell lines, AtT-20, ACTH-producing cells and TtT/GF, folliculo-stellate (FS)-like cells. First, we examined the expression of TNF receptor mRNA in these cells. Using Northern blot analyses with radiolabeled cDNA to murine TNF receptor p60 and p80 mRNAs as probes, we identified both types of mRNA in the poly(A)-containing RNA prepared from AtT-20 cells and p60 TNF receptor mRNA from TtT/GF. The identified mRNA were compatible in size with those detected in the immune-competent cells. Next, we studied the TNF-binding sites on these cells. Scatchard plot analysis of the significant binding of [125I]TNF revealed a single type of binding site with a Kd (dissociation constant) of 210 pM and 131 binding sites/cell on AtT-20. Similarly on TtT/GF, [125I]TNF showed 353 binding sites/cell with a Kd of 900 pM. [125I]TNF binding on both types of cells competed with TNF and lymphotoxin (TNF beta) in an equimolar fashion. Third, TNF stimulates ACTH synthesis in AtT-20 cells, while TNF increases immunoreactive interleukin (IL)-6 release from TtT/GF cells. These findings demonstrate that AtT-20 and TtT/GF cells are equipped with fully functional TNF receptor system, and suggest that ligand of the receptor, TNF alpha and/or TNF beta, can modulate ACTH synthesis and release as a direct hormonal effector on corticotrophs or indirect modulator through another paracrine mediator, such as IL-6 from FS cells.

CMO I deficiency caused by a point mutation in exon 8 of the human CYP11B2 gene encoding steroid 18-hydroxylase (P450C18).

Corticosterone methyloxidase I (CMO I) deficiency is an autosomal recessive disorder of aldosterone biosynthesis. To determine further the molecular genetic basis of CMO I deficiency, a patient of Turkish origin that suffered from CMO I deficiency was studied. Nucleotide sequencing of the PCR-amplified exons from the genomic DNA of this patient revealed a single point mutation CTG (leucine) CCG (proline) at codon 461 in exon 8 of CYP11B2, which is involved in the putative heme binding site of steroid 18-hydroxylase (P450(C18)). The expression study using a cDNA introducing the point mutation revealed that the amino acid substitution totally abolishes the P450(C18)p3 enzyme activities required for conversion of 11-deoxycorticosterone to aldosterone, even though the mutant product was detected in the mitochondrial fraction of the transfected cells. These results suggest that this point mutation causes CMO I deficiency.

Cooperative regulation of the human aromatase cytochrome P450 gene transcription by placenta-specific cis-acting elements.

Aromatase cytochrome P450 catalyses the reaction to convert androgens to estrogens by coupling with NADPH-cytochrome P450 reductase in the endoplasmic reticulum. The human aromatase cytochrome P450 gene (CYP19) is expressed in a variety of tissues under regulation of tissue-specific promoters. Previously, we localized a cell-type specific transcriptional enhancer element between -242 and -166 relative to the major cap site of the gene, by transient expression analysis in human BeWo choriocarcinoma cells. In the present study, we demonstrate that the enhancer element consists of two subelements, element I (located between -238 and -200), and element II (located between -196 and -176) as analysed by DNase I footprinting using the nuclear extracts of BeWo cells. The gel mobility shift assay shows that each of these subelements binds specific nuclear factor(s). The transient expression of the bacterial chloramphenicol acetyltransferase gene constructs involving the subelements in BeWo cells reveals that the elements activate reporter gene expression synergistically when present together, nevertheless each of the elements by itself also has an enhancer activity. The transient expression analysis further shows that element I is responsible for the transcriptional synergism with the binding site of a nuclear factor-interleukin-6 (NF-IL-6) (also known as CCAAT enhancer/binding protein beta), which is located between -2141 and -2115 relative to the major cap site of the gene. These results suggest that the enhancer element plays important roles in sustaining the high levels of CYP19 expression in placental cells in cooperation with other cis-acting transcritional regulatory elements.

The role of the phrenic nerves in stress response in upper abdominal surgery.

Previous studies have failed to demonstrate a block of the endocrine response to upper abdominal surgery by thoracic epidural analgesia. To clarify the bases for this failure, we compared the effects of epidural analgesia of different dermatome levels up to C8-T2 or C3-4. The patients who received general anesthesia alone showed significant increases of adrenocorticotropic hormone (ACTH) and arginine vasopressin (AVP) immediately after skin incision. The patients with C8-T2 blocked developed significant increases in these hormones, not after the skin incision, but after the intraabdominal procedure. Of the eight patients with C3-4 block, six developed no such responses throughout the study period. The responses of oxytocin (OXT) and prolactin (PRL) were more susceptible to epidural analgesia and were blocked at the C8-T2 level. Growth hormone (GH) showed no correlation with surgical procedures and epidural block. These findings indicate that the nociceptive neural information during upper abdominal surgery is conveyed by the sensory fibers included in both the thoracic and lumbar spinal nerves that innervate the abdominal wall and the intraabdominal viscera, and by the phrenic nerves that innervate the diaphragm. The rationale for postulating the involvement of the phrenic nerves can be referred to the embryonal descent of the diaphragm from the C3-5 myotomes that serves as the upper wall of the abdominal cavity.

Effect of IL-1 receptor antagonist and antiserum to TNF-alpha on LPS-induced plasma ACTH and corticosterone rise in rats.

Using an antiserum against tumor necrosis factor (TNF)-alpha and an interleukin (IL-1) receptor antagonist, we studied putative roles of these cytokines in mediating the endotoxin-induced elevation of plasma adrenocorticotropic hormone (ACTH) and corticosterone levels in freely moving rats. Intravenous administration of Escherichia coli lipopolysaccharide (LPS) increased plasma ACTH and corticosterone levels in a dose-dependent manner. The plasma corticosterone reached to its highest level among a series of experiments after the administration of even the smallest dose (0.03 microgram/kg) tested. Plasma ACTH and corticosterone levels in these rats were completely inhibited by the intravenous administration of anti-murine TNF-alpha-rabbit antiserum (anti-TNFAS) after the administration of LPS but not by the intravenous administration of IL-1 receptor antagonist (IL-1RA). On the other hand, both recombinant human IL-1RA and anti-TNFAS significantly inhibited plasma ACTH increase stimulated with 10 micrograms/kg LPS. These findings indicate that 1) when the plasma corticosterone increase induced by intravenous LPS remains below its maximum, the effect is exclusively mediated by TNF-alpha, and 2) when a larger amount of LPS is administered, both IL-1 beta and TNF-alpha participate at least in part in the hypothalamic-pituitary-adrenal axis activation.

Endocrine-paracrine interaction in communication between the immune and endocrine systems. Activation of the hypothalamic-pituitary-adrenal axis in inflammation.

There are bidirectional communications between the immune and endocrine systems. Cytokines produced in inflammatory foci cause changes in the endocrine system, including activation of the hypothalamic-pituitary-adrenal (HPA) axis. Hormones produced in the endocrine system, especially glucocorticoids, affect the immune system to modulate its function. This is an important endocrine system for the defence mechanism. In addition, bacterial lipopolysaccharide produces cytokines in the brain and endocrine organs which are considered to act through the paracrine mechanism to regulate the HPA axis. Endocrine-paracrine interaction is important for the defence mechanism of the organism.

Bacterial lipopolysaccharide-induced expression of interleukin-6 messenger ribonucleic acid in the rat hypothalamus, pituitary, adrenal gland, and spleen.

Whereas the stimulatory effect of interleukin-6 (IL-6) on the hypothalamic-pituitary-adrenal (HPA) axis is well established, its mode of action in this axis has yet to be fully elucidated. To further study the role of IL-6 in the HPA axis, we compared the expression of IL-6 messenger RNA (mRNA) in the rat hypothalamus, pituitary, and adrenal gland with that in the spleen after ip or intracerebroventricular (icv) administration of bacterial lipopolysaccharide (LPS). After either ip or icv administration, LPS induced the expression of IL-6 mRNA, which consists of 1.2 kilobases (kb) and 2.4 kb subclasses, in all these tissues of the HPA axis as well as in the spleen. Although we used 100 times less amount of LPS for the icv administration than that used for ip LPS, plasma ACTH levels in both the conditions rapidly reached comparable levels. This icv dose induced IL-6 mRNA expression in the hypothalamus faster than ip dose but also stimulated IL-6 mRNA expression in the hypothalamus, pituitary, and adrenal gland more effectively and smoothly than the ip LPS dose did. Northern blot analysis revealed that in the hypothalamus, pituitary, and adrenals, the predominant subclass of IL-6 mRNA was not 1.2 kb but 2.4 kb. In contrast, this subclass was the minor component in the spleen induced under the same circumstances. These findings indicate that IL-6-synthesizing cells in the HPA axis differ in character from those in the spleen, and that LPS applied in vivo may modulate IL-6 expression in these cells directly and/or indirectly through secondarily activated functions in the neuronal or endocrine systems.

Human corticotropin-releasing hormone (hCRH) test: sex and age differences in plasma ACTH and cortisol responses and their reproducibility in healthy adults.

The effectiveness and safety of MCI-028, a synthetic human corticotropin-releasing hormone (hCRH), as a diagnostic drug were examined in 65 healthy male and 24 healthy female adult volunteers. Mean maximum concentrations of plasma ACTH and cortisol after intravenous administration of 100 micrograms of MCI-028 were 3.0 and 2.0 times their basal concentrations, respectively, and there were no significant age or sex differences in the responses. Good reproducibility was observed in the responses in 59 male subjects who received a second administration after 1 to 2 weeks. Although slight adverse reactions such as mild and transient hot flushing were observed, these were not serious.

Evaluation of hypothalamo-pituitary-adrenocortical function in children by human corticotropin-releasing hormone (MCI-028) test.

A dose of 1.5 micrograms/kg of MCI-028, human corticotropin-releasing hormone (hCRH), was administered intravenously to 38 children with non-endocrine short stature with normal function in the hypothalamo-pituitary-adrenocortical axis and to 71 children with a disorder in the same axis. Blood levels of adrenocorticotropic hormone (ACTH) and cortisol were determined to evaluate the axis. The 95% confidence limits of peak responses of ACTH and cortisol in non-endocrine short stature were between 17.2 and 135.3 pg/ml, and between 13.1 and 35.6 micrograms/dl, respectively, and were used as standards for children. When compared with these standards, the hormonal responses in children with various disorders in the hypothalamo-pituitary-adrenocortical axis were as follows: in two children with Cushing's syndrome caused by adrenal tumor, ACTH values were decreased and were not responsive to hCRH, while cortisol values, though within the normal limit, were not responsive; in children with primary adrenal insufficiency or congenital adrenal hyperplasia, cortisol values were decreased and not responsive, whereas ACTH values tended to be increased and ACTH response high except for 21 alpha-hydroxylase deficiency of congenital adrenal hyperplasia. In two cases of pituitary dwarfism complicated with ACTH deficiency, both ACTH and cortisol values were decreased and poorly responsive; and in children who were receiving glucocorticoid, both ACTH and cortisol values tended to be decreased and to respond poorly to hCRH. As for side effects, hot flushing was observed among 8.0% of the subjects after administration of hCRH. But this symptom was not severe and no other side effects of clinical importance were observed.(ABSTRACT TRUNCATED AT 250 WORDS)