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The effects of conditioned fear on the release of noradrenaline in the hypothalamic paraventricular nucleus (PVN) and the involvement of corticotropin-releasing factor (CRF) receptor type 1 (CRFR1) in conditioned fear-induced changes in noradrenaline release were examined by intracerebral microdialysis in rats. Conditioned fear was produced by placing animals into a box where they had previously been exposed to a 5-min period of electric footshock, 135 min prior to the start of experiment. Conditioned fear for 20 min produced a significant increase in the release of noradrenaline in the PVN. Intraperitoneal preadministration of a selective nonpeptidic CRFR1 antagonist, CRA1000, completely blocked the conditioned fear-induced release of noradrenaline. These results suggest that CRFR1 is involved in the release of noradrenaline in the hypothalamic PVN induced by conditioned fear.

There is a striking sex difference in the pattern of growth hormone (GH) secretion in rats. Our previous studies showed that short-term administration of pharmacological doses of testosterone or dihydrotestosterone (DHT) masculinized the GH secretory pattern in ovariectomized (OVX) rats. The locus where testosterone or DHT interacts with the somatotropic axis is believed to be the hypothalamus. To obtain insights into this phenomenon, we administered a single dose of DHT s.c. to adult OVX rats at 0.01, 0. 1 or 1 mg/rat. Blood GH concentrations were measured in unanaesthetized rats. Six to 12 h after the s.c. administration of all three doses of DHT, the GH secretory pattern revealed a male-like secretory pattern as shown by episodic bursts occurring at 2-3-h intervals with low or undetectable trough levels. When anterolateral deafferentation of the medial basal hypothalamus (ALC) was performed, the blood concentrations revealed irregularly occurring small fluctuations, instead of the usual high bursts, but the basal GH concentration was significantly higher than that of OVX-sham-operated rats. DHT treatment did not elicit pulsatile GH secretion or alter GH concentrations in OVX rats with ALC. When intact adult female rats received DHT at a dose of 1 mg/rat, the male-like GH secretory pattern was not induced. These results suggest that neural inputs from the anterolateral direction to the medial basal hypothalamus are necessary for the masculinizing effect of DHT on the GH secretory pattern in OVX rats, and that oestrogen in intact female rats prevents the masculinizing effect of DHT.

Glucocorticoids regulate growth hormone (GH) secretion by modulating both hypothalamic and pituitary function. At the level of the pituitary, glucocorticoids increase GH and GH-releasing hormone receptor (GHRH-R) gene expression. To test if glucocorticoids might also regulate the pituitary expression of the recently identified GH secretagogue (GHS) receptor, GHS-R; adult male rats were adrenalectomized or sham operated, and treated with the synthetic glucocorticoid (dexamethasone, 200 microg/day) or vehicle for 8 days. Pituitary GHS-R mRNA levels were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Adrenalectomy decreased pituitary GHS-R mRNA to 45% of vehicle-treated, sham-operated rats (P < 0.05). Administration of dexamethasone increased GHS-R mRNA levels in sham-operated as well as in adrenalectomized rats (199 +/- 24% (P < 0.05) and 369 +/- 48% (P < 0.01) of vehicle-treated controls). Addition of dexamethasone to primary rat pituitary cell cultures increased GHS-R mRNA levels in a dose- and time-dependent manner while the transcriptional inhibitor, actinomycin D, completely blocked the stimulatory action of dexamethasone. Taken together, these results suggest glucocorticoids directly increase pituitary GHS-R mRNA levels by stimulating GHS-R gene transcription.

Thyroid hormones regulate growth hormone (GH) secretion by actions both at the hypothalamus and at the pituitary gland. At the level of the pituitary, thyroid hormones increase GH and GH-releasing hormone receptor (GHRH-R) mRNA expression. To test if thyroid hormones might also regulate the pituitary expression of mRNA for the recently identified GH secretagogue (GHS) receptor, GHS-R, primary pituitary cell cultures from adult male rats were treated with triiodothyronine (T3) and GHS-R mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. T3 increased pituitary GHS-R mRNA levels in a dose- and time-dependent manner. The stimulatory action of T3 on GHS-R mRNA levels was also observed in the presence of the RNA synthesis inhibitor, actinomycin D, indicating that gene transcription is not required. Closer examination of the decay rates of GHS-R mRNA in the presence of actinomycin D revealed T3 extended the half-life of the GHS-R mRNA from 8 h (basal) to15 h, demonstrating that T3 increases GHS-R mRNA levels in vitro by increasing message stability.

GH synthesis and release from pituitary somatotropes is controlled by the opposing actions of the hypothalamic neuropeptides, GH-releasing hormone (GHRH), and somatostatin (SS). There is a striking sex difference in the pattern of GH secretion in rats. Early reports indicate that gonadal steroids have important imprinting effects during the neonatal period. Recently, our laboratory and others have reported that the GH secretory pattern is altered by short-term gonadal steroid treatment in adult rat, suggesting that gonadal steroids are also important determinants of the pattern of GH secretion during adult life. However, the site of action of gonadal steroids in the adult rat hypothalamus is still unknown. In this study, we used in situ hybridization in the adult male rat brain to determine whether GHRH neurons and/or SS neurons coexpress estrogen receptor α (ERα) and ERβ genes. In the medial basal hypothalamus of adult male rat, the ERα messenger RNA (mRNA) was located in medial preoptic area (MPA) and a...

Growth hormone (GH) secretagogues (GHSs), which stimulate GH secretion, are synthetic compounds that act through the GHS receptor (GHS-R) which has been recently cloned. We raised an antiserum in a rabbit against a synthetic peptide corresponding to amino acid residues 248-260 of the third intracellular loop of the rat GHS-R. A competitive immunoassay showed that the antiserum had a specific affinity for the target peptide. To confirm the specificity of the antiserum, the GHS-R cDNA was stably expressed in COS-7 cells. In Western blot analysis, the band was detected at 44 kDa in the extracts from COS-7 cells expressing GHS-R (COS-7/tf3-2) but not in those from wild-type COS-7 cells. Furthermore, while COS-7/tf3-2 cells were strongly immunostained for GHS-R, no GHS-R-like immunoreactivity was observed in wild-type COS-7 cells. Immunoreactive bands were also observed at approximately 46 kDa in the extracts from rat hypothalamus, pituitary and stomach by Western blot analysis. These studies are the first to show the existence of GHS-R protein in the stomach. The antiserum for the GHS-R is sensitive and specific, and it would be useful for clarifying the roles of GHS/ghrelin.

We hypothesized that a change in composition of proteoglycans can regulate the bioactivity of fibroblast growth factor (FGF)-2 in the thyroid. In order to test this hypothesis, we established a simple and sensitive method for detecting FGF-2-binding heparan sulfates and characterized them in papillary thyroid carcinomas and normal thyroids. The thyroid extracts were applied to a Q-Sepharose anion exchange column. After the column was washed with 10 mM of phosphate buffer, 1 microgram of human recombinant FGF-2 was added onto the column. The column was eluted with a gradient of NaCl (0.3-1.5 M). Each fraction was blotted onto nitrocellulose membrane. Immunoreactivity of heparan sulfate and FGF-2 was revealed by the incubation of membranes with the specific antibodies, and quantitatively estimated by measuring the density of the color product. In normal thyroids, immunoreactivity of heparan sulfate was detected as two peaks at 0.7 and 0.9 M of NaCl. Heparan sulfate-containing fractions also showed FGF-2 immunoreactivity, indicating the complex formation of FGF-2 and heparan sulfate. In papillary thyroid carcinomas, immunoreactivity of heparan sulfate showed various elution profiles on Q-Sepharose chromatography, including single peak at 0.7 M of NaCl and the one similar to that of the normal thyroids. However, FGF-2 immunoreactivity was detected only in the fractions eluting at 0.7 M of NaCl. This loss of a subpopulation of FGF-2-binding heparan sulfate in human papillary thyroid carcinomas may lead to the increase of free FGF-2 bioavailable in extracellular matrix.

To obtain an insight into the influence of aging on leptin gene expression, the responses of leptin mRNA in retroperitoneal and epididymal adipose tissues and plasma leptin concentrations to 24-h food deprivation and refeeding were examined in 2-, 10- and 24-month-old normal rats. The basal level of leptin gene expression in retroperitoneal adipose tissue was significantly higher in 10- and 24-month-old rats than that in 2-month-old rats, while the level in epididymal adipose tissue was highest in 10-month-old rats for all three age groups. The basal concentrations of plasma leptin was significantly higher in 10- and 24-month-old rats than those in 2-month-old rats. The 24-h food deprivation was followed by a significant reduction in leptin mRNA expression in both retorperitoneal and epididymal adipose tissues for all three age groups. The leptin gene expression was restored to control levels 24 h following refeeding in the 2- and 10-month-old rats, but failed to be restored in the 24-month-old rats. In addition, the time course of recovery for leptin mRNA expression by refeeding to the control levels differed between the retroperitoneal and the epididymal adipose tissue in 2- and 10-month-old rats. The concentrations of plasma leptin 24 h following refeeding were compatible with the leptin mRNA levels in adipose tissues in three age groups. These results suggest that the expression of the leptin gene in response to food-deprivation and refeeding is influenced by an animal's age and that this expression is different for different regions of white adipose tissue.

Fibroblast growth factor (FGF)-2 is stored in the extracellular matrix (ECM). We hypothesized that FGF-2 is mobilized from the ECM and binds to receptors on the surface of FGF-2 responsive cells during thyroid enlargement. To test this hypothesis, we estimated levels of FGF-2 free from ECM in thyroids by comparing the efficiency of two methods for FGF-2 extraction (low salt and high salt). Because the high salt concentration (more than 1.5 M NaCl) is necessary to release FGF-2 from the normal ECM, FGF-2 extracted by low salt is indicative of ECM-free FGF-2. Human papillary thyroid carcinomas, normal part thyroid, and Graves' thyroid tissues were homogenized separately in an extraction buffer containing either 0 M NaCl (low salt) or 2.0 M NaCl (high salt), and the concentration of FGF-2 in the extracts was measured by enzyme-linked immunosorbent assay (ELISA). The yields of low and high salt extracts of immunoreactive (ir)FGF-2 from papillary carcinomas (low salt: 40.0 +/- 7.5, high salt: 233 +/- 53 ng/g tissue, mean +/- SE) were significantly higher than those of normal thyroid tissues extracted by the corresponding salt concentration (low salt: 14.6 +/- 1.8, high salt: 123 +/- 12 ng/g tissue). On the other hand, the extractable irFGF-2 from Graves' thyroid tissues (low salt: 25.2 +/- 2.5, high salt: 135 +/- 24 ng/g tissue) were not significantly different from that of normal thyroid tissues. However, the ratio of the extractable irFGF from carcinomas and Graves' thyroids by low salt to that by high salt (0 M/2 M ratio = 0.206 +/- 0.051, 0.209 +/- 0.025) were significantly higher than that of normal thyroids (0.120 +/- 0.014) (p < 0.05). These results suggest that intratissue ECM-free FGF-2 is increased in papillary thyroid carcinomas and Graves' thyroid tissues, and therefore a greater amount of FGF-2 may be available for stimulation of FGF-2 responsive cells.

Proliferation of vascular smooth muscle cells with the accumulation of proteoglycans in the extracellular matrix is one of the significant changes found in atherosclerotic lesions. In order to clarify the relationship between pericellular proteoglycan and cell growth, we established a simple method for quantitatively estimating the amount of pericellular proteoglycans and investigated the effects of various growth factors on the synthesis of pericellular proteoglycans by cultured A10 rat smooth muscle cells. Analysis of trypsin accessible [35SO4]-labeled material in the pericellular area of the A10 cell culture by Q-sepharose anion-exchange chromatography showed two peaks. One peak, eluted at 0.55 M NaCl, disappeared after treatment with 2 mU/ml of heparitinase, indicating that heparan sulfates (HS) were present. The other peak, which eluted at 0.65 M NaCl, disappeared with 20 mU/ml of chondroitinase ABC, indicating the presence of chondroitin sulfates and dermatan sulfates (CS/DS). We estimated the effects of several growth factors on the synthesis of the pericellular proteoglycans by measuring heparitinase- and chondroitinase-ABC-sensitive radioactivities. Although PDGF-AB significantly stimulated cell proliferation and the synthesis of pericellular CS/DS, its dose-dependent effect on the cell growth did not coincide with that on the proteoglycan synthesis. IGF-I (1 nM) increased pericellular CS/DS but not the cell number, while basic FGF (1 nM) and EGF (1 nM) increased the cell number but not pericellular CS/DS. All the growth factors we examined had no effect on the synthesis of pericellular HS. These results indicate that growth factors increase pericellular proteoglycans independently of their mitogenic effects.

GH secretion is regulated by hypothalamic somatostatin and GH-releasing factor. It has been postulated that GH feeds back on the hypothalamus and regulates its own secretion. We focused our attention on the action of GH in the hypothalamus in relation to GH secretion. Adult male rats were used throughout the studies, and the observation was made in conscious rats. Systemic administration of human GH induced c-fos gene expression, a marker of neuronal activity, in the hypothalamic arcuate nucleus (ARC) and the periventricular nucleus (PeV) in hypophysectomized male rats. The major cells in which c-fos gene expression was induced were neuropeptide Y (NPY) neurons in the ARC and somatostatin neurons in the PeV. GH receptor mRNA was demonstrated to be present in these neurons by in situ hybridization. The injection of a small dose of rat GH into the ARC or PeV inhibited GH secretion, whereas microinjection of IGF-I into these nuclei did not. Intracerebroventricular injection of NPY suppressed GH secretion, and this effect was abolished by anterolateral deafferentation of the medial basal hypothalamus (MBH), a procedure which disrupts the somatostatinergic input to the MBH. Taken together, these findings suggest that GH acts on NPY neurons in the ARC and somatostatin neurons in the PeV through GH receptor, and the activation of these neurons augments somatostatin release and inhibits GH secretion.

Opiates and opioids have complex effects on the hypothalamic-pituitary-adrenal axis, and they stimulate the sympathetic nervous system. This study was designed to clarify the role of brain beta-endorphin in the mechanism by which stress increases plasma concentrations of adrenocorticotropin (ACTH), epinephrine (E), and norepinephrine (NE). Intracerebroventricular (i.c.v.) administration of beta-endorphin to rats significantly increased plasma ACTH levels at doses of 0.09, 0.3, and 1.5 nmol, and plasma E and NE levels at doses of 0.3 and 1.5 nmol. The rise of plasma ACTH, E, and NE levels by 0.3 nmol beta-endorphin was inhibited by intravenous (i.v.) administration of 2 mg/kg b.wt. naloxone. I.v. administration of anti-rat corticotropin-releasing hormone (CRH) rabbit serum completely blocked the beta-endorphin-induced ACTH secretion without affecting the secretion of E and NE. I.c.v. administration of anti-beta-endorphin rabbit gamma-globulin attenuated a 30-min restraint stress-induced rise of plasma ACTH levels without significant influence on the rise of E and NE levels, whereas i.v. administration of naloxone attenuated the restraint stress-induced rise of plasma ACTH, E and NE levels. These results suggest that i.c.v. administration of beta-endorphin stimulates the secretion of ACTH, E, and NE through opiate receptor, and that brain CRH mediates the beta-endorphin-induced secretion of ACTH. The results also suggest that brain beta-endorphin is, at least in part, involved in the restraint stress-induced stimulation of the hypothalamic-pituitary-adrenal axis, and that some opioids other than beta-endorphin are involved in the stimulatory mechanism of the autonomic nervous system and the adrenal medulla in the rat.

A 48-year-old woman with Cushing's syndrome due to bilateral adrenocortical adenomas is reported. The patient presented with a typical Cushingoid appearance. The serum cortisol level was elevated with loss of the diurnal rhythm and the plasma adrenocorticotropic hormone (ACTH) level was undetectable. Dynamic testing showed no suppression of urinary 17-OHCS by high-dose dexamethasone and no stimulation by metyrapone. An abdominal computed tomography (CT) scan showed bilateral adrenal tumors. Bilateral adrenalectomy was performed. The right adrenal gland contained a tumor that was encapsulated and consisted mainly of compact cells. The surrounding cortex was atrophic. The left adrenal gland contained an encapsulated tumor composed predominantly of clear cells. There were numerous small adrenocortical nodules in the surrounding cortex. Immunohistochemical analysis of steroidogenic enzymes (P450scc, 3β-HSD, P450c21, P450cl7 and P450cll) was performed. Immunoreactivity of all the enzymes was intense in the compact cells of the right adrenocortical adenoma, while the adjacent non-neoplastic cortex was negative for the enzymes. In the left adrenal tumor, the immunoreactivity of 3β-HSD was intense, while that of P450cl7 was weak. In the adrenocortical nodules, 3β-HSD activity was sporadically observed. G protein genes encoding Gs α and Gi2 were examined for activating mutations at codons 201 and 227 (Gs α) and codons 179 and 205 (Gi2 α) in the bilateral adrenal tumors, but no mutations were found. The bilateral adenomas of this patient showed marked differences in microscopic and immunohistochemical studies, suggesting that the capacity of steroidogenesis differs between the right and left tumors.(Internal Medicine 36: 804-809, 1997)

Recent evidence suggests that growth hormone-releasing peptides (GHRPs) mimic an unidentified native GH-releasing hormone (GHRH)-amplifying hormone. GHRH has been shown to stimulate food intake acting on the central nervous system. The present studies were conducted to test the hypothesis that GHRPs may also potentiate the central effect of GHRH on feeding in free-feeding rats. Intracerebroventricular (ICV) administration of picomole doses of a newly developed GHRP, KP-102, or human GHRH stimulated feeding, but the phenomenon was not reproduced by systemic injection. A prior ICV injection of a GHRH antagonist completely prevented the increase of food intake evoked by GHRH, but this pretreatment did not influence the increase in food intake induced by KP-102. When maximally effective doses of GHRH and KP-102 were co-administered ICV, the amount of food intake increased significantly compared with after ICV injection of a maximum dose of either peptide alone. These findings suggest that GHRPs stimulate food intake via a specific receptor for GHRPs in the central nervous system and amplify the central effect of GHRH on feeding.

Neuropeptide Y (NPY) may play a physiological role in the regulation of growth hormone (GH) secretion by acting via somatostatin (SS) in the periventricular nucleus (PeV), as well as via the GH-releasing factor in the arcuate nucleus (ARC) of the medial basal hypothalamus (MBH). The objectives of the present study were to determine the neuron structures and receptor subtypes necessary for mediating the inhibitory effect of NPY on GH secretion in unanesthetized male rats. To eliminate the influence of hypophyseotropic SS, anterolateral deafferentation (ALC) of the hypothalamus was performed. Intracerebroventricular (i.c.v.) administration of 1.17 nmol of NPY decreased the blood level of GH for 3–4 h in sham-operated rats, while the procedure was without effect in ALC rats. The i.c.v. administration of 1.17 nmol of a Y1-receptor agonist ([Leu31, Pro34]-NPY) or a Y2-receptor agonist (NPY 13–36 and NPY 3–36) similarly suppressed the blood GH level. The data support the hypothesis that neuron structures anterolateral to the MBH are required for NPY-induced inhibition of GH secretion that is mediated via Y1- and Y2- receptor subtypes. Combined with data of other investigators, SS is likely the neurohumoral mediator of the effect of NPY on GH secretion.

Growth hormone-releasing hexapeptide (GHRP) stimulates GH secretion by acting on both the pituitary and the hypothalamus through a poorly understood mechanism. To reveal the hypothalamic action of GHRP, rat brains were processed for in situ hybridization for c-fos mRNA as a marker of neuronal activity after systemic administration of a newly developed GHRP, KP-102. Hypophysectomized adult male Wistar rats were administered KP-102 through an indwelling right atrial cannula. KP-102 treatment was accompanied by transient expression of the c-fos gene selectively in the ventromedial and ventrolateral regions of the arcuate nucleus (ARC). The distribution of c-fos gene-expressing cells overlapped that of GRF mRNA-containing neurons in the ventrolateral region on adjacent sections, whereas few c-fos mRNA signals were detected in the dorsomedial region where somatostatin mRNA signals were localized. To confirm this observations, hypothalamic sections were subjected to double-label in situ hybridization. Twenty-three percent of c-fos mRNA-containing cells were GRF neurons, comprising 20% of the GRF neurons in the ARC. The remaining c-fos mRNA containing cells were unidentified. KP-102 thus appears to act on a subpopulation of GRF neurons and unidentified cells in the ARC to stimulate GH secretion.

Familial Cushing's syndrome due to ACTH independent bilateral macronodular adrenocortical hyperplasia occurring in siblings is reported. The proband was a 69-year-old woman who presented with a typical Cushingoid appearance. The serum cortisol level was elevated, with a loss of diurnal rhythm, and the plasma ACTH level was undetectable. Dynamic testing showed no suppression of urinary 17-OHCS by high dose dexamethasone and no stimulation by metyrapone. An abdominal CT scan showed bilateral adrenal enlargement. The patient died of a subarachnoid haemorrhage, and autopsy revealed a massively thickened adrenal cortex composed of nodules up to 3.5 cm in diameter. A pituitary adenoma was not found. We learned that the patient's elder brother was also diagnosed at 59 years of age with Cushing's syndrome due to bilateral macronodular adrenocortical hyperplasia. His plasma cortisol levels were not suppressed by high dose dexamethasone and the plasma ACTH level was undetectable. Screening of the available family members by administering 1 mg dexamethasone at midnight and performing abdominal CT scan revealed impaired suppressibility of serum cortisol associated with enlarged bilateral adrenal glands in a 64-year-old sister and a 54-year-old brother. The 64-year-old sister was considered as a possible 'affected' case in the early stages of development, because the basal level of ACTH was not suppressed and hyperplasia of the bilateral adrenal glands as revealed by CT scan was less evident.

In rats with anterolateral deafferentation of the medial basal hypothalamus, the growth hormone (GH) level in the blood showed irregular and small fluctuations instead of the usual high bursts and low trough level, and the baseline GH level was higher than that in sham-operated rats. Continuous infusion of a glucose solution to operated rats increased the baseline level, GH pulse and pulse amplitude. I.v. bolus injection of the glucose solution resulted in a significant but transient increase in GH level. Insulin-induced hypoglycemia decreased the blood GH level in operated rats more effectively than in sham-operated ones and that was prevented by simultaneous infusion of glucose. Since SS influence on GH secretion had been largely eliminated in rats with anterolateral deafferentation of the medial basal hypothalamus, it is highly unlikely that the effects of hyperglycemia or hypoglycemia on GH secretion were the consequence of altered SS secretion.

Central glucoprivation evoked by the intracerebroventricular administration of 2-deoxy-D-glucose (2DG) induces eating and suppresses growth hormone (GH) secretion in rats. To elucidate the hypothalamic mechanism of these phenomena, the induction of c-fos gene expression was examined by in situ hybridization using rats with centrally administered 2DG. Autoradiography on X-ray film showed that c-fos gene expression was transiently induced in discrete hypothalamic regions; namely the paraventricular nucleus, arcuate nucleus (ARC), the surrounding regions of the third ventricle dorsal to the ARC, and the periventricular nucleus (PeV). The time course of the expression was different in these nuclei. Double-label in situ hybridization for c-fos mRNA and neuropeptide Y (NPY) or somatostatin mRNAs revealed that 20% of the NPY neurons in the ARC expressed the c-fos gene, while a small population of somatostatin neurons (6.1% in the ARC and 2.6% in the PeV) expressed the c-fos gene following 2DG administration. Since NPY is an orexigenic neuropeptide and has an inhibitory effect on GH secretion, the data suggest that the activation of a subpopulation of NPY neurons in the ARC contributes, in part, to the increased food intake and suppression of GH secretion after central glucoprivation evoked by 2DG.

The ontogeny of growth hormone receptor (GHR) gene expression was studied in the rat hypothalamus. Total RNA from the hypothalamus of rats at different developmental stages (embryonic day 15–56 days of age) was characterized using a 32 P-labeled RNA probe derived from the extracellular domain of the rat GHR cDNA. Two RNA species, 4.5 kilobases (kb) encoding for GHR and 1.2 kb encoding for GH-binding protein, were detected in hypothalamic tissue from embryonic day 15 to 56 days of age. Their levels were low at embryonic day 15 and increased toward 3 days of age. The level of 4.5-kb transcript preferentially increased from 7 days after birth, and it was maintained until 35 days of age. Thereafter, the level of 4.5-kb transcript declined. The ratio between the 4.5- and 1.2-kb transcripts was less than 2.0 from embryonic day 15 to 3 days after birth, while it was larger than 4 after 7 days of age. There was no sex difference in the levels or the ratios of the transcripts of the GHR gene from 7 to 56 days of age. The findings indicate that the 4.5-kb transcript preferentially processed postnatally in the rat hypothalamus.

Intracerebroventricular (i.c.v.) injection of somatostatin (SS) or arginine-vasopressin (AVP) elicits barrel rotation (BR) in rats. To identify the potential neuron structures involved in this characteristic behavior, the regional expression of the c-fos gene in rat brain after i.c.v. injection of SS (10 micrograms) or AVP (1 micrograms) was examined by hybridization histochemistry. The c-fos expression could serve as a marker of neuronal activity and/or neural transmission. Following SS-induced BR, c-fos gene expression was observed in the lingula, uvula, nodulus, simplex, centralis, and culmen of the cerebellum, while following AVP-induced BR, c-fos gene expression was observed in the first four of the above-mentioned regions of the cerebellum, but not in the centralis or culmen. In these regions, the c-fos mRNA signals were observed on the granular layer. Expression of the c-fos gene was immediately and transiently induced and was not observed in rats in which BR was not evoked after SS or AVP injection. In both control rats and SS- or AVP-injected rats, the c-fos gene expression was induced in the piriform cortex and the flocculus of the cerebellum. The findings suggest that BR is a manifestation of behavior induced by massive transsynaptic activation of the granular cells in the cerebellum.

To examine whether somatostatin (SS) exerts influences on the steady state levels of GH-releasing factor (GRF), the effect of SS on GH gene transcription was examined in rats. This approach was used because it has been shown that GRF stimulates GH gene transcription independent of GH release, and SS does not inhibit basal or GRF-stimulated GH gene transcription. Therefore, it is assumed that an effect of SS on GH gene transcription would be mediated by a change in GRF levels. Adult female Sprague-Dawley rats were provided with right atrial cannulae. Studies were performed using unanesthetized rats. Pituitary GH gene transcription was measured by transcription assay. An iv administration of antiserum to rat GRF 150 min previously significantly decreased GH gene transcription compared with that in control rats given normal goat serum. A continuous infusion of SS (300 micrograms/kg.h) via the cannula for 150 min significantly decreased GH gene transcription compared with that in control rats receiving 0.9% NaCl. When GRF (3 micrograms/kg.h) was given simultaneously with SS (300 micrograms/kg.h), GH gene transcription increased significantly compared with that in rats receiving SS infusion alone. After the withdrawal of SS infusion, GH gene transcription rapidly and significantly increased. The data suggest that SS reduces the steady state levels of GRF.

There is increasing evidence to suggest that supraphysiological doses of glucocorticoids suppress growth hormone secretion in vivo by augmenting somatostatin release from the hypothalamus; previously, we reported an increase in hypothalamic somatostatin content in dexamethasone-treated rats. To further examine whether the production of somatostatin really is augmented, hypothalamic somatostatin mRNA levels were determined by the Northern blot technique in female rats receiving 330 μg of dexamethasone daily for three days. In two series of experiments, hypothalamic somatostatin mRNA levels in dexamethasone-treated rats were significantly (psd)% and 153±38% of the controls. In the dexamethasone-treated rats, plasma growth hormone levels were markedly suppressed compared with those of the controls. These results further support the hypothesis that pharmacological doses of glucocorticoids increase the production and release of somatostatin from the hypothalamus and thus inhibit growth hormone secretion, overriding the direct stimulatory effect of glucocorticoids on growth hormone production at the pituitary level.

A tight linkage between generalized resistance to thyroid hormone (GRTH) and the thyroid hormone receptor-beta (TR beta) gene is indicated. We evaluated a family with GRTH for the TR beta gene. We found that a new point mutation, consisting of a cytosine to adenine replacement at nucleotide position 1642, resulted in substitution in codon 448 in the T3-binding domain of TR beta. This base substitution was found in only one allele of affected members, but not in unaffected members of the family. The in vitro translation products of this mutant TR beta gene demonstrated significantly reduced T3-binding affinity. Previously, others have reported a kindred with GRTH, in that the same codon was subjected to proline to histidine replacement due to a mutation consisting of a cytosine to adenine replacement at nucleotide position 1643. There appeared to be a significant phenotypic difference between our kindred and that described by others.

The neuronal expression of the protooncogene c-fos could serve as a marker of neural activity. To identify the brain sites responding to GH, rat brains after systemic administration of recombinant human GH (rhGH) were processed for hybridization histochemistry for c-fos mRNA. Adult male Wistar rats were hypophysectomized 10 days before rhGH administration. After hypophysectomy, rats received sc cortisone acetate (0.5 mg/kg BW) and L-T4 (20 microgram/kg BW) daily. Four international units (1.33 mg) of rhGH were given iv through an indwelling right atrial cannula. Vehicle was administered to control animals. The rhGH treatment was accompanied by expression of the c-fos gene in the arcuate nucleus (ARC) of the hypothalamus. The accumulation of the c-fos mRNA was transient, reaching maximum values at 60 min and decreasing thereafter to reach control levels within 120 min after rhGH injection. Among control animals, c-fos gene expression was not detected in the ARC. The c-fos mRNA was also detected in the paraventricular nucleus after rhGH administration; however, it was comparable to that in control animals. When rhGH was administered twice at 40-min intervals, c-fos gene expression was induced in the periventricular nucleus (PeV) as well as the ARC 40 min after the second rhGH injection. Throughout the studies, c-fos mRNA was not detected other than in the ARC, paraventricular nucleus, and PeV in the hypothalamus. In the ARC, distribution of the cells expressing the c-fos gene appears to overlap at least in part with somatostatin (SS) mRNA-containing cells. In the PeV, it appeared to correlate generally with the distribution of SS mRNA-containing cells. The data suggest that GH feeds back on neurons of hypothalamic PeV and ARC expressing SS mRNA, and that c-fos expression is involved in the feedback mechanism.

Idiopathic pituitary GH deficiency appears to result from neonatal disruption of hypophyseal portal vessels in the majority of patients. To examine the mechanism of GH deficiency associated with the disease, the effect of pituitary stalk section on GH secretion was studied in rats. Adult male rats were subjected to stalk section without inserting an impermeable membrane between the cut ends. They were studied 3 to 4 weeks after surgery. In stalk-sectioned rats, pituitary weight, body weight and hypothalamic SRIH content were significantly reduced as compared with sham-operated rats. Hypothalamic GHRH content, plasma T3, T4, corticosterone and testosterone levels, and weights of testes remove and adrenal glands were comparable in the two groups. Plasma GH profiles of sham-operated rats showed characteristic periodic pulses occurring at 2.5-3 h intervals with intervening trough period. In stalk-sectioned rats, plasma GH levels were low with small fluctuations, but GH levels were significantly higher than trough levels of sham-operated rats. The amount of GH secreted during a 6-h period as measured by planimetry was significantly reduced. To ascertain the regeneration of hypophyseal portal vessels, post SRIH rebound in GH secretion, which requires the presence of endogenous GHRH, was examined. Withdrawal of exogenous SRIH infusion triggered a large rebound GH secretion whose magnitude did not differ between groups. In stalk-sectioned rats, GH response to met-enkephalin analogue, FK 33-824, was not observed, whereas prolactin response to the secretagogue was observed in the majority of rats. It appears that in stalksectioned rats, hypophyseal portal circulation is re-established, but GHRH release from the hypothalamus is impaired in the face of sufficient supply of other hypophysiotropic hormones.

The effect of electrical stimulation of the hypothalamic periventricular nucleus (PVN) on plasma GH profile was studied in unanesthetized female Wistar rats. A bipolar concentric electrode was implanted into the PVN, hypothalamic ventromedial nucleus (VMH), or intervening area between the PVN and VMH. Serial blood specimens were collected from an indwelling right atrial cannula. Plasma GH levels were reduced significantly during electrical stimulation of PVN, and a large rise of plasma GH levels followed after cessation of stimulation. An identical plasma GH profile was observed in response to the repeated stimulation. This rebound secretion of GH was completely inhibited by the administration of rat GRF antiserum. The effect of electrical stimulation of VMH on plasma GH levels was similar to that of PVN stimulation. However, the stimulation of hypothalamic area intervening between PVN and VMH was not followed by a surge of GH secretion. Since a continuous exposure of somatotrophs to GRF even in a concurrent presence of somatostatin (SS) is known to induce attenuation of the GH response to GRF through receptor effect, the results suggest that the release of endogenous GRF is augmented following the cessation of electrical stimulation of neurons providing hypophysiotropic SS.

The effect of intermittent infusions of somatostatin (SS) on growth hormone (GH) secretion was studied in unrestrained adult male rats deprived largely of SS influence on the medial basal hypothalamus by anterolateral deafferentation (AL-cut). In addition, the influence of hypothalamic surgery on the plasma GH response to beta-endorphin (beta-END) was observed. In sham-operated rats, high-amplitude GH pulses separated by low baseline levels occurred at 185 min intervals. In rats with AL-cut, GH pulses were difficult to identify upon visual appraisal and baseline plasma GH levels became significantly higher than those of sham-operated rats. When AL-cut was performed unilaterally (half-AL-cut), low amplitude GH pulses separated by elevated baseline GH levels occurred at frequent intervals. The amount of GH secreted during 6 h was significantly reduced in rats with AL-cut or half-AL-cut as compared to that of sham-operated rats. The plasma GH response to intracerebroventricular injection of beta-END (4 micrograms) was abolished in AL-cut rats, and the response was significantly reduced in half-AL-cut rats as compared to that of sham-operated rats. When AL-cut rats were subjected to repeated infusions of SS (30 micrograms/kg b. wt./h, 150 min) separated by 30 min control periods, a large rebound of GH secretion was observed after removal and the amount of GH secreted during 6 h became comparable to that of sham-operated rats. The results suggest that SS plays important roles in the dynamic secretion of GH.

A 57-year-old female was admitted to our hospital because of Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). On admission, disturbance of consciousness and hyponatremia were recognized. The patient's endocrinological data showed low levels of adrenocorticotropic hormone (ACTH) (less than 5 pg/ml) and cortisol (5.9 μg/dl). Other anterior pituitary hormones were normal. Plasma ACTH and cortisol did not respond to the corticotropin releasing factor (CRF) test. A diagnosis of isolated ACTH deficiency was made. This is a rare case of isolated ACTH deficiency complicated with hematological malignancies.(Internal Medicine 36: 819-821, 1997)

A 39-year-old man with lipoprotein lipase (LPL) deficiency (height 177.7 cm, body weight 67 kg, and body mass index 21.2 kg/m2) showed severe hypertriglyceridemia (2,032 mg/dl). LPL activity and concentration were markedly low in postheparin plasma. LPL gene analysis revealed a homozygous mutation, Asp204 --> Glu in exon 5. Fasting plasma glucose (81 mg/dl) and insulin (2.7 microU/ml) levels were normal. Plasma glucose pattern during oral glucose (75 g) tolerance test was normal, however 30 minutes after glucose-loading the insulin secretion unexpectedly increased to 89.4 microU/ml. These data suggested that chylomicronemia might be related to a hyper-response of insulin secretion to glucose without obesity.

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Like the synthetic growth hormone secretagogues (GHSs), ghrelin specifically releases growth hormone (GH) after intravenous administration. Also consistent with the central actions of GHSs, ghrelin-immunoreactive cells were shown to be located in the hypothalamic arcuate nucleus as well as the stomach. Recently, we showed that a single central administration of ghrelin increased food intake and hypothalamic agouti-related protein (AGRP) gene expression in rodents, and the orexigenic effect of this peptide seems to be independent of its GH-releasing activity. However, the effect of chronic infusion of ghrelin on food consumption and body weight and their possible mechanisms have not been elucidated. In this study, we determined the effects of chronic intracerebroventricular treatment with ghrelin on metabolic factors and on neuropeptide genes that are expressed in hypothalamic neurons that have been previously shown to express the GHS-R and to regulate food consumption. Chronic central administration of rat ghrelin (1 μg/rat every 12 h for 72 h) significantly increased food intake and body weight. However, it did not affect plasma insulin, glucose, leptin, or GH concentrations. We also found that chronic central administration of ghrelin increased both neuropeptide Y (NPY) mRNA levels (151.0 ± 10.1% of saline-treated controls; P < 0.05) and AGRP mRNA levels (160.0 ± 22.5% of saline-treated controls; P < 0.05) in the arcuate nucleus. Thus, the primary hypothalamic targets of ghrelin are NPY/AGRP-containing neurons, and ghrelin is a newly discovered orexigenic peptide in the brain and stomach.

Studies in mammals have shown that synthetic Met-enkephalin derivatives, called growth hormone-releasing peptides (GHRPs), stimulate growth hormone (GH) release. The present study was conducted to determine whether the GHRP, KP-102, specifically stimulates GH release in a teleost. Tilapia (Oreochromis mossambicus) were given a single intraperitoneal injection of KP-102 (D-Ala-D-beta;-Nal-Ala-Trp-D-Phe-Lys-NH(2)) or bovine GHRH(1-29)-amide or vehicle and blood was sampled at 1, 6 and 12 h after injection. KP-102 was administered at two doses of 1 ng/g and 10 ng/g body weight, whereas GHRH (positive control) was administered at a single dose of 10 ng/g body weight. Plasma levels of tilapia GH and prolactins (tPRL(177) and tPRL(188)) were determined by radioimmunoassay. As expected, GHRH injection significantly (P

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Like the synthetic GHSs, ghrelin specifically releases GH following intravenous administration. Also consistent with the central actions of GHSs, ghrelin-immunoreactive cells were shown to be located in the hypothalamic arcuate nucleus as well as the stomach. However, the central actions of ghrelin have not been elucidated. Here, we used radioactive in situ hybridization histochemistry to examine the effects of central administration of rat ghrelin on neuropeptide genes that are expressed in hypothalamic neurons that were previously shown to express GHS-R. We found that central administration of ghrelin increased both agouti-related protein (AGRP) mRNA levels (245.8 +/- 28.3% of the saline-treated controls; p < 0.01) in the hypothalamus and food intake (5.7 +/- 0.9 g ghrelin vs. 1.9 +/- 0.5 g saline; p < 0.05). On the other hand, 1 microg of rat ghrelin central administration did not alter the episodic GH release of freely moving adult male rats. Thus, ghrelin has an alternative role in stimulating food intake via an increase of AGRP rather than the release of GH from the pituitary.

Survivin is a newly discovered inhibitor of the apoptosis protein, IAP, expressed during development and in human cancers. The effector cell protease receptor-1 (EPR-1) gene is oriented in the opposite direction on the same DNA double strand. Thus, the Survivin and EPR-1 (Survivin/EPR-1) genes exist in a head-to-head configuration. It is not clear whether mutual expression of the Survivin/EPR-1 genes occurs in both normal cells and cancer cells. Here, we investigated the mutual expression of the Survivin/EPR-1 genes in 12 normal peripheral blood (PB) specimens, seven normal bone marrow (BM) specimens, five lymph node (LN) specimens, and seven leukemic cell lines, and 27 patients with malignant lymphoma (ML), four with acute lymphocytic leukemia (ALL), three with acute myelocytic leukemia (AML), and four with chronic myelocytic leukemia in blastic crisis (CML-BC). Using Northern blot analysis, small amounts of EPR-1 mRNA were detected in normal PB, normal BM and LN specimens, but no Survivin mRNA was detected. However, Survivin mRNA was detected in two of the 12 normal PB, six of the seven normal BM and one of the five LN specimens using reverse transcription and polymerase chain reaction (RT-PCR). Expression of both the Survivin and EPR-1 genes was detected in six of the seven cell line samples by Northern blot, and in all of them by RT-PCR. Mutual expression of the Survivin and EPR-1 genes was detected in three of the four CML-BC samples, 15 of the 27 ML samples, two of the four ALL samples, and all three AML samples using the RT-PCR method. No EPR-1 expression with or without Survivin expression was clearly detected in eight of the nine diffuse large B-cell lymphoma (DLB) specimens, two of the six follicular center lymphoma specimens, one of the four specimens of nodular sclerosis of Hodgkin's lymphoma, two of the four ALL specimens or one of the four CML-BC specimens. The data presented here show that disrupted expression of the Survivin/EPR-1 genes occurred in many kinds of hematologically malignant cells. This may be of biological importance.

The effects of a 6-day intracerebroventricular (i.c.v.) infusion of KP-102, a growth hormone-releasing peptide (GHRP), on food intake and body weight gain were observed in free-feeding rats that were or were not subjected to intermittent electric footshock stress during the 6 days. Food intake and body weight were significantly lower in rats exposed to a 60-min period of footshock twice a day for 6 days compared to non-stressed rats. A 6-day, i.c.v. infusion of KP-102 significantly and steadily increased food intake and body weight in free-feeding non-stressed rats compared to control rats receiving saline i.c.v. In rats exposed to intermittent footshock stress during the 6-day infusion, KP-102 treatment stimulated feeding behavior and resulted in significantly higher body weight compared to stressed rats that received i.c.v. infusion of saline. These results indicate that during a 6-day continuous i.c.v. infusion of GHRP, KP-102, food intake and body weight steadily increased without attenuation of the GHRP effect in both non-stressed rats and those subjected to intermittent stress.

The central administration of corticotropin-releasing hormone (CRH) to experimental animals sets into motion a coordinated series of physiological and behavioral events that promote survival during threatening situation. A large body of evidence suggest that CRH in the central nucleus of the amygdala (CEA) induces fear-related behaviors and is essential to fear conditioning; however, evidence of CRH-mediated activation of the amygdala under physiological situation is still limited. We report here a study of the impact of a psychological stressor on hypothalamic and amygdala CRH systems in the rat. Non-footshocked rats placed in a floored compartment surrounded by footshocked rats were defined as the psychological stress group. Rats were exposed to psychological stress for 15 min, and then sacrificed 1.5 and 3 h after cessation of stress. We found that our psychological stressor induced an increase in both CRH mRNA levels, as assessed by in situ hybridization histochemistry, and CRH content, as assessed by micropunch RIA, in the CEA. Exposure to the psychological stressor also caused a significant increase in CRH mRNA levels with a trend for an increase in CRH content in the dorsolateral subdivision of the bed nucleus of the stria terminalis (BNST) which is anatomically associated with the CEA. In contrast, psychological stress induced a small, but significant increase in type-1 CRH receptor (CRHR-1) mRNA in the hypothalamic paraventricular nucleus (PVN), while it failed to elevate either PVN CRH mRNA levels or content, CRH content in the median eminence (ME), or levels of plasma ACTH or corticosterone (CORT). Thus, in the context of a psychological stressor, the activation of the amygdala CRH system can occur without robust activation of the hypothalamic CRH system. In the light of previous data that the psychological stress-induced loss of sleep was reversed by the central administration of a CRH antagonist, these data suggest that CRH in the CEA may contribute to the psychological stress-evoked fear-related behavior such as hyperarousal. These data also indicate that in response to a psychological stressor, the amygdala CRH system is much more sensitive than is the CRH system emanating from the PVN.

We examined the effect of insulin-like growth factor-I (IGF-I) on GH-releasing factor (GRF) receptor expression in the primary rat anterior pituitary cell culture. The levels of GRF receptor mRNA were dose-dependently reduced by IGF-I treatment for 24 h. To clarify whether altered levels of GRF receptor mRNA contribute to GRF receptor concentrations, we examined the GH response to GRF in vitro. There was no difference in basal GH secretion between control and IGF-I pretreated cells, while GRF-stimulated GH secretion in cells pretreated with IGF-I for 24 h was significantly lower than that in control cells. Moreover, specific [125I] Tyr10-human GRF binding to pituitary cells was reduced significantly by IGF-I treatment. These results suggest that IGF-I acts directly on the pituitary and participates in the regulation of GRF receptor expression.

OBJECTIVE AND IMPORTANCE: Adrenocorticotropin (ACTH)-secreting pituitary adenomas causing Cushing's disease are often difficult to identify because of their variable locations and their small size. This report presents histological evidence of an ectopic ACTH-secreting adenoma located entirely within the cavernous sinus. CLINICAL PRESENTATION: A 62-year-old woman presented with central obesity, hypertension, and osteoporosis. Endocrinological evaluation suggested the presence of an ACTH-secreting pituitary adenoma; however, imaging studies, including dynamic magnetic resonance imaging, did not reveal any visible lesions in the pituitary gland. Bilateral cavernous sinus sampling demonstrated a large central/peripheral ACTH gradient, with a right/left ACTH gradient. The patient was treated as having pituitary-dependent Cushing's disease, until she died suddenly as a result of acute respiratory failure. INTERVENTION: In a postmortem histological examination, an ACTH-secreting adenoma was found in the right cavernous sinus, which was completely surrounded by dura mater and had no direct connection with the pituitary gland. CONCLUSION: Although they are rare, such adenomas located in the cavernous sinus should be recognized as one of the reasons for inaccurate cavernous sinus sampling and the failure of transsphenoidal surgery for patients with ACTH-dependent Cushing's syndrome.

A severely malnourished 87-yr-old man presented with hypoglycemia. Serum GH levels were elevated, and serum levels of insulin-like growth factor I (IGF-I), IGF-binding protein-3, and GH-binding protein were extremely reduced. The patient’s GH was biologically active. Administration of GH for 4 consecutive days resulted in a slight increment in serum IGF-I levels, but no elevation of serum IGF-binding protein-3. The expression of GH receptor messenger ribonucleic acid in the liver was greatly reduced. An autopsy revealed a Rathke’s cleft cyst confined to the sella turcica. Immunohistochemical studies for GH showed that there was nothing to suggest a tumor overproducing GH. In addition, TSH levels were elevated in the presence of normal thyroid hormone levels, and there was a cluster of cells showing strong immunohistochemical staining for the TSH β-subunit in the pituitary. In this patient, the decreased expression of GH receptor messenger ribonucleic acid in the liver may have been responsible for the GH resistance, which was probably caused by malnutrition.

Experimental evidence suggests that differential pituitary sensitivity to hypothalamic signals exerts a role in mediating both age and sex dependent patterns of growth hormone (GH) release and synthesis. One mechanism by which pituitary sensitivity to hypothalamic GH regulators could be modified is by the differential synthesis of their pituitary receptors. In the present report we therefore studied the age and sex dependency of the expression of receptors for two known stimulators of GH release, growth hormone-releasing hormone (GHRH) and the synthetic peptidyl and non-peptidyl GH secretagogues (GHSs). Pituitary GHRH receptor (GHRH-R) and GHS receptor (GHS-R) mRNA levels were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) in male and female rats at postnatal day 1, 10, 30 and 75. We also examined the age- and sex-dependent expression of the GHS-R in whole hypothalamic extracts, since the GHS-R is also expressed in a variety of nuclei within the hypothalamus and has been linked to central regulation of the GH-axis. Pituitary GHRH-R mRNA concentrations were age-dependent; the highest levels were observed in d1 pituitaries and then declined with age, reaching a nadir by d30. These results are in concordance with the age-related decline in pituitary GHRH sensitivity. In contrast, the ontogenic pattern of GHS-R expression was bimodal; GHS-R mRNA concentrations in d1 and d30 pituitaries were approximately twice those at d10 and d75. These results mirror the transient increase in GHS sensitivity observed around the onset of puberty, suggesting that gonadal steroids mediate GHS-R expression. GHRH-R mRNA levels were comparable in males and females within each age while GHS-R mRNA levels were gender dependent. At d30, male GHS-R mRNA levels were ≈30% greater than in their female counterparts. This was reversed at d75, when females had 89% more GHS-R mRNA per pituitary and 65% more per somatotrope than did age-matched males. These sexual differences further support a role for gonadal steroids in the modulation of pituitary GHS-R synthesis. The ontogenic and gender-specific pattern of hypothalamic GHS-R expression differed from that observed for the pituitary. Hypothalamic GHS-R mRNA levels increased with age but exhibited no significant sex difference at each age tested. Taken together, these data demonstrate that changes in the levels of pituitary GHS-R mRNA, but not GHRH-R mRNA, are associated with changes in the gonadal steroid environment, thereby implicating the GHS/GHS-R signalling system as a control point in the establishment and maintenance of sexually dimorphic patterns of GH secretion.

Recent reports indicate that a gain-of-function mutation in fibroblast growth factor receptor 3 (FGFR-3) inhibits cell growth in the cartilaginous growth plates. These results suggest that FGFR-3 may be the receptor transducing growth inhibitory signals. Using reverse transcription-PCR we examined seven papillary thyroid carcinomas to determine FGFR-3 expression. Six out of the seven papillary carcinomas expressed FGFR-3. To clarify the role of FGFR-3 in thyroid carcinoma, FGFR-3 was overexpressed in an established human papillary thyroid carcinoma cell line. High levels of FGFR-3 protein were identified in cells stably transfected with the vector containing FGFR-3 cDNA. The specific binding of 125I-FGF-2 of these cells was threefold higher than that of control cells. Growth rates of cells overexpressing FGFR-3 were similar to those of control cells. However, cells overexpressing FGFR-3 continued to grow beyond the density at which control cells stopped proliferating. These results suggest that FGFR-3 in thyroid carcinoma is not involved strongly in the cell proliferation mechanism but may contribute to the malignant extension of some of the carcinomas by modifying cell contact signaling.

Growth hormone secretagogues (GHSs) are synthetic peptidyl and nonpeptidyl compounds that are believed to stimulate the release of GH by a direct effect on the pituitary somatotrope and by stimulation of growth hormone-releasing hormone (GHRH) release and the suppression of somatostatin (SRIH) tone. Recently, the receptor for these pharmacologic agents was cloned and its expression localized to the pituitary and hypothalamus. The elucidation of an unique GHS receptor (GHS-R) suggests there is a yet to be identified endogenous ligand which could exert an important role in regulation of GH secretion. It is clearly established that GH acts to regulate its own production by feeding back at the level of the hypothalamus to downregulate GHRH and upregulate SRIH synthesis and by induction of IGF-I, which acts at the pituitary to block somatotrope responsiveness to GHRH. If the endogenous GHS/GHS-R signaling system is important in regulating GH release, it might be reasoned that changes in circulating GH concentrations would also directly or indirectly (via generation of IGF-I) modify GHS-R production. To test this hypothesis we used RT-PCR to examined pituitary and hypothalamic GHS-R mRNA levels in the spontaneous dwarf rat (SDR), an animal model characterized by the absence of GH due to a point mutation in the GH gene. In the absence of GH feedback regulation, SDR pituitary GHS-R mRNA levels were 385 ± 61% greater (p < 0.01) than those observed in normal controls while SDR hypothalamic GHS-R mRNA levels were not significantly different from those in normal rats. Three-day subcutaneous infusion of rat GH by osmotic pump reduced SDR pituitary GHS-R mRNA levels to 55 ± 9% of vehicle-treated controls (p < 0.05) but did not significantly alter hypothalamic GHS-R mRNA levels. To test if the changes in GHS-R mRNA levels observed following GH treatment were due to elevation of circulating IGF-I concentrations, SDRs were infused with recombinant human IGF-I. Replacement of IGF-I did not significantly alter either pituitary or hypothalamic GHS-R mRNA levels, indicating that GH acts independent of circulating IGF-I to regulate pituitary GHS-R expression in the SDR model.

The possibility that vasopressin (VP) is involved in stress-induced suppression of immune function was examined in rats. Intermittent electrical footshock for 60 min suppressed the proliferative response of splenic T cells to the mitogen concanavalin A as well as natural killer (NK) cytotoxicity, and the former change was partially, and the latter was completely, blocked by intracerebroventricular (i.c.v.) preadministration of a V1 receptor antagonist. The footshock-induced suppression of the T cell proliferative response was completely abolished by coadministration of a corticotropin-releasing hormone (CRH) receptor antagonist and the V1 receptor antagonist. The i.c.v. administration of VP suppressed the proliferative response of splenic T cells and NK cytotoxicity in an adrenal-independent manner. These effects were completely reversed by i.c.v. preadministration of the V1 receptor antagonist. These results suggest that brain VP, in conjunction with CRH, suppresses immune function through the V1 receptor in rats under stress.

We evaluated the effect of intracerebroventricular (i.c.v.) administration of interleukin (IL)-1beta on the rectal temperature and the release of noradrenaline (NA) in the hypothalamic paraventricular nucleus (PVN) of the rat. IL-1beta increased rectal temperature at doses ranging from 300 pg to 300 ng, whereas it, at doses ranging from 3 ng to 300 ng, significantly stimulated the release of NA in the PVN measured by intracerebral microdialysis. The stimulatory effect of IL-1beta on the release of NA was blocked by the subcutaneous injection of indomethacin. These findings suggest that IL-1beta stimulates the release of NA in the PVN via prostaglandin, and that the release of NA in the PVN is not necessarily related to the increase in body temperature.

T-cell malignant lymphoma of the gastrointestinal tract is rare. The genotype of gastric T-cell lymphoma remains unclear. The aim of this study was to elucidate the pathogenesis of a case of primary gastric T-cell lymphoma by using cytogenetics and molecular biology. Gastric biopsy specimens and lymphoma cells in the ascites were examined by immunocytology, cytogenetic analysis, and Southern blot analysis. The histological diagnosis of the gastric lymphoma was diffuse large cell type. T-cell markers were positive in immunocytochemistry of the gastric lymphoma cells and in FACS analysis of lymphoma cells in the ascites. All lymphoma cells in the ascites had complex abnormal karyotypes containing t(8;14)(q24;q32). Southern blot analysis revealed rearrangement of the IgH and C-MYC genes of the lymphoma cells in both the stomach and the ascites, but no comigration of the C-MYC with the JH locus could be detected. The TCR-β and -γ genes were in their germ-line configurations. In this patient, although the phenotype was T-cell lymphoma, the karyotype t(8;14)(q24;q32) and genotype had the characteristics of B-cell lymphoma. The unique B-cell genotype configuration and the C-MYC activation suggested that the cellular origin of this rare case of malignant lymphoma with a T-cell phenotype was quite immature lymphocytes.

Human umbilical cord blood (UCB) is currently considered as a third source of hematopoietic stem cells for transplantation, following the bone marrow and growth-factor-mobilized peripheral blood (MPB). To evaluate the potential benefits of UCB, we performed a comparative study of the properties of the stem cells in UCB and MPB samples. CD 34+ cell determination and CFU-GM colony assay showed a lower frequency of committed progenitors in UCB than in MPB. In contrast, a higher of the CD 34+ CD 38- subset in UCB suggested that more primitive, multipotent progenitors are enriched in UCB than in MPB. Phenotypic analysis of UCB lymphocytes revealed a reduced level of T cell subsets, especially cytotoxic CD 8+ lymphocytes, which might minimize graft versus host disease in clinical practice. In conclusion, UCB is an attractive alternative source for stem cell transplantation, but ex vivo expansion of stem/progenitor cells could be effective for attaining rapid and safer hemopoietic reconstruction.