[Barrett's esophagus: analyses from human and experimental animal studies]. / Barrett-Ösophagus: Erkenntnisse beim Menschen und aus experimentellen Tierstudien.

Whereas attention in the past has been focused on goblet cells as the primary marker for Barrett's esophagus (BE), the recent change in the definition now includes the non-goblet cell columnar cell-lined esophagus. In the present study the histological features of neoplasia of the lower esophagus and esophago-gastric junction in a German cohort were examined using immunohistochemical staining for MUC, CD10, intestinal and gastric type major tight junction proteins (claudins). Experimental studies using rat duodenogastric content reflux models have also been performed and data show that most neoplastic lesions of the esophageal glands in humans express gastric mucin phenotypes. Cardiac type mucosa was the main histological type in the surrounding mucosa of neoplastic lesions; however, most cardiac type mucosa has intestinal type tight junction proteins. BE with goblet cells has been reported to originate from stem cells located in the basal layer of esophageal squamous cell epithelium in previous models. However, the cardiac type mucosa seems to develop from the site of the stomach and not from the basal layer of esophageal squamous cell epithelium according to our model.

Is there any circadian variation consequence on acute case fatality of stroke? Takashima Stroke Registry, Japan (1990-2003).

BACKGROUND: Circadian periodicity in the onset of stroke has been reported. However, it is unclear whether this variation affects the acute stroke case fatality. Time of the day variation in stroke case fatality was examined using population-based stroke registration data. METHODS: Stroke event data were acquired from the Takashima Stroke Registry, which covers a stable population of approximately 55,000 in Takashima County in central Japan. During the period of 1990-2003, there were 1080 (549 men and 531 women) cases with classifiable stroke onset time. Stroke incidence was categorized as occurring at night (midnight-6 a.m.), morning (6 a.m.-noon), afternoon (noon-6 p.m.), and evening (6 p.m.-midnight). The 28-day case fatality rates and 95% confidence intervals (95% CI) were calculated by gender, age, and stroke subtype across the time blocks. After adjusting for gender, age at onset, and stroke severity at onset, the hazard ratios for fatal strokes in evening, night, and morning were calculated, with afternoon serving as the reference. RESULTS: For all strokes, the 28-day case fatality rate was 23.3% (95% CI:19.4-27.6) for morning onset, 16.9% (95% CI:13.1-21.6) for afternoon onset, 18.3% (95% CI:13.6-24.1) for evening onset, and 21.0% (95% CI:15.0-28.5) for the night onset stroke. The case fatality for strokes during the morning was higher than the case fatality for strokes during afternoon. This fatality risk excess for morning strokes persisted even after adjusting for age, gender, and stroke severity on onset in multivariate analysis. CONCLUSION: In the examination of circadian variation of stroke case fatality, 28-day case fatality rate tended to be higher for the morning strokes.

PGE2 signal via EP2 receptors evoked by a selective agonist enhances regeneration of injured articular cartilage.

OBJECTIVE: The effect of the prostaglandin E2 (PGE2) signal through prostaglandin E receptor 2 (EP2) receptors on the repair of injured articular cartilage was investigated using a selective agonist for EP2. METHODS: Chondral and osteochondral defects were prepared on the rabbit femoral concave in both knee joints, and gelatin containing polylactic-co-glycolic acid microspheres conjugated with or without the EP2 agonist was placed nearby. Animals were sacrificed at 4 or 12 weeks post-operation, and regenerated cartilage tissues and subchondral structure remodeling were evaluated by histological scoring. The quality of regenerated tissues was also evaluated by the immunohistochemical staining of EP2, type II collagen, and proliferating cell nuclear antigen (PCNA). As an evaluation of side effects, the inflammatory reaction of the synovial membrane was analyzed based on histology and the mRNA expression of matrix metalloproteinase3 (MMP3), tissue inhibitor of metalloproteinase 3 (TIMP3), and interleukin-1 beta (IL-1 beta). Also, the activity of MMP3 and the amount of tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein in joint fluid were measured. RESULTS: In both models, the EP2 agonist enhanced the regeneration of the type II collagen-positive tissues containing EP2- and PCNA-positive chondrocytes, and the histological scale of regenerated tissue and subchondral bone was better than that of on the control side, particularly at 12 weeks post-operation. No inflammatory reaction in the synovial membrane was observed, and no induction of pro-inflammatory cytokines was found in joint fluid. CONCLUSION: Selective stimulation of the PGE2 signal through EP2 receptors by a specific agonist promoted regeneration of cartilage tissues with a physiological osteochondral boundary, suggesting the potential usefulness of this small molecule for the treatment of injured articular cartilages.

Morning surge in circadian periodicity of ischaemic stroke is independent of conventional risk factor status: findings from the Takashima Stroke Registry 1990-2003.

BACKGROUND: We examined the circadian periodicity of ischaemic stroke (IS) onset and its relationship with conventional risk factors using 14-year stroke registration data. METHODS: Ischaemic stroke event data were acquired from the Takashima Stroke Registry, which covers a stable population of approximately 55,000 in Takashima County in central Japan. During 1990-2003 there were 637 (353 men and 284 women) cases with classifiable onset time. IS incidence was categorized as occurring at night (midnight to 6 am), morning (6 am to noon), afternoon (noon to 6 pm), and evening (6 pm to midnight). The OR (with 95% CI) of having an IS in the morning, afternoon, and evening were calculated, with night serving as reference. RESULTS: There was significant diurnal variation in IS incidence (P < 0.001). The proportion of events was highest in the morning (40.7; 95% CI: 36.9-44.5), and lowest in the night (14.0; 95% CI: 11.5-16.9). In the morning an excess incidence of IS was observed in both genders, in subjects <65 years and > or =65 years, and in all IS subtypes. The morning excess of IS incidence was similar across seasons and days of the week. For all IS, morning excess was higher (odds ratio: 2.91; 95% CI: 2.29-3.70) compared to the night period. Similar trends persisted after adjusting for age, gender, and risk factors. CONCLUSION: In the examination of circadian variation of IS onset, a predominant morning peak independent of conventional risk factors was observed in a Japanese population with similar pattern across seasons of the year and days of the week.

An animal model of intrinsic dental erosion caused by gastro-oesophageal reflux disease.

OBJECTIVES: To explore the association between dental erosion and gastro-oesophageal reflux disease (GORD), we used an animal model of GORD. MATERIALS AND METHODS: We performed an operation to force gastro-duodenal contents reflux in male Wistar rats, and examined the teeth in the reflux rats at 15 or 30 weeks postoperatively. Dental erosion was evaluated based on a slightly modified index from a previous report. Estimation of pH was employed in the oesophageal and gastric contents. RESULTS: Macroscopically, dental erosion was only detected in the reflux rats. Histopathologically, dentin exposure was detected in three of the seven cases after 30 weeks. Alveolar bone destruction and osteomyelitis were also noted in severe cases. The pH of the oesophageal and stomach contents was 6.93 +/- 0.15 and 3.7 +/- 0.39, respectively. CONCLUSIONS: We confirmed the relationship between dental erosion and GORD. First step of dental erosion caused by GORD is the loss of surface enamel induced by regurgitation of an acidic liquid and acidic gas. Subsequently, further destruction of dental hard tissues and tooth supporting structure is accelerated by mixed juice with gastric and duodenal contents. The reflux animal model is a useful tool to examine the mechanism of dental erosion in GORD.

Intratumoural heterogeneity of intestinal expression reflects environmental induction and progression-related loss of induction in undifferentiated-type gastric carcinomas.

AIMS: Gene expression in tumours is regulated by environmental as well as genetic/epigenetic factors. This study assessed the environmental factors in intestinal expression of gastric cancers. METHODS AND RESULTS: We immunohistochemically examined intratumoural heterogeneity in the expression of Cdx2, MUC2, MUC5AC and MUC6 in 39 intramucosal and 49 extramucosally invasive undifferentiated-type gastric carcinomas (UGCs), consisting of signet ring cell carcinomas showing a layered structure (LS) in the mucosa and dedifferentiated tubular adenocarcinomas without LS and with minor tubular components (TC). The LS retains mucosal vertical polarity with superficial MUC5AC expression. Loss of this polarity was independent of intestinal expression and associated with extramucosal invasion. In LS(+) UGCs, intestinal expression was enhanced as the size of mucosal spread increased and was significantly reduced with deeper extramucosal invasion, whereas, in LS(-)/TC(+) UGCs, intestinal expression was frequent and predominant in the mucosa and was insignificantly reduced with deeper extramucosal invasion. CONCLUSIONS: In LS(+) UGCs, intestinal expression showed dynamic alteration probably by environmental induction and progression-related loss of induction, whereas it was relatively stable in LS(-)/TC(+) UGCs. Thus, intestinal expression in UGCs is not useful as a marker of tumour progression because it is also affected by environmental factors and genetic lineage.

A non-acromegalic case of multiple endocrine neoplasia type 1 accompanied by a growth hormone-releasing hormone-producing pancreatic tumor.

Cases of acromegaly due to GHRHproducing pancreatic endocrine tumors have been reported. Here we present a case of a 31-yr-old nonacromegalic man with hyperparathyroidism and elevated serum IGF-I with normal serum GH levels. Serum GH was not suppressed below 1 ng/ml by the glucose tolerance test and increased in response to TR H and GHRH administration. Magnetic resonance imaging (MRI) revealed pituitary hyperplasia and an abdominal computed tomography (CT ) scan showed a tumor in the pancreatic tail. Plasma concentration of GHRH was elevated. Based on these clinical data, multiple endocrine neoplasia (MEN) type 1 was suspected. Three enlarged parathyroid glands were removed and a distal pancreatectomy was performed. Pathological examination of the parathyroid glands and pancreatic tumor showed nodular hyperplasia and a well-differentiated endocrine tumor, respectively, both compatible with MEN features. Immunohistochemistry revealed positive immunoreactivity for GHRH, SS , insulin, glucagon, chromogranin A, and pancreatic polypeptide in the pancreatic tumor. After pancreatic surgery, elevated levels of GHRH and IGF-I were normalized and pituitary hyperplasia definitely decreased in size. In cases of pituitary hyperplasia with elevated IGF-I, ectopic GHRH syndrome must be considered even if physical features of acromegaly are absent. It is also important to measure plasma GHRH concentrations in order to give a diagnosis.

Biochemical and physiological studies on a kallikrein-like enzyme from the venom of Crotalus viridis viridis (prairie rattlesnake).

A kallikrein-like enzyme was isolated from Crotalus viridis viridis (Prairie rattlesnake) venom by Sephadex G-50, DEAE-Sephacel and heparin-Sepharose CL-6B column chromatography. The purified enzyme has a molecular mass of 32 kDa and an isoelectric point of 5.4. The enzyme catalyzed the hydrolysis of arginine esters, kallikrein substrates Pro-Phe-Arg-MCA and Z-Phe-Arg-MCA. The specificity of the enzyme's substrate requirement is demonstrated by the fact that no proteolytic activity was detected against either dimethyl casein or fibrinogen. The enzyme also cleaves kininogen analogs to release bradykinin. Although the enzyme induced contraction of the isolated rat uterus directly at high concentrations, more forceful contractions resulted when the reaction mixture of the enzyme and bovine plasma was applied to the uterus. The reaction mixture of 5.10(-11) M of the enzyme and plasma caused contractions equal to that of 10(-9) M of bradykinin. Additionally the enzyme demonstrated capillary permeability-increasing activity and hypotensive activity on the anesthetized rat, suggesting that the enzyme releases the dilator of the wall of capillaries from plasma. Uterine contraction, capillary permeability-increasing activity and arginine esterolytic activity were inhibited by diisopropyl fluorophosphate, indicating that the serine hydroxyl group is essential for enzymatic and biological activities. It was demonstrated that the NH2-terminal region of the enzyme has significant similarities in sequence with kallikrein-like enzymes from other snake venoms and porcine pancreatic kallikrein.

Kinin-releasing enzyme from the venom of Bitis arietans (puff adder).

A kinin-releasing enzyme was isolated from Bitis arietans (puff adder) venom by Sephadex G-100 and DEAE-cellulose column chromatographies. The kinin-releasing enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion. Its molecular mass is approximately 45 kDa with an isoelectric point of 6.5. Kinin-releasing enzyme possesses proteolytic activity which hydrolyzes the Leu6-Cys7, His10-Leu11 and Ala14-Leu15 bonds of the B chain of oxidized insulin and the A alpha and B beta chain of fibrinogen. Kinin-releasing and benzoyl-L-arginine ethyl ester hydrolytic activities of this enzyme were inhibited by diisopropyl fluorophosphate, suggesting that the serine hydroxyl group is involved in enzymatic activities.

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus).

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.

Chronic central infusion of ghrelin increases hypothalamic neuropeptide Y and Agouti-related protein mRNA levels and body weight in rats.

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 microg/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.

Diastolic paradoxic jet flow in patients with hypertrophic cardiomyopathy: evidence of concealed apical asynergy with cavity obliteration.

In 20 of 198 patients with hypertrophic cardiomyopathy, Doppler color flow imaging revealed diastolic paradoxic jet flow across the obliterated left ventricular apex toward the base that suggested the presence of a discrete apical chamber. This prospective study characterized echocardiographic, ventriculographic and scintigraphic findings in these patients, as well as their clinical features. Although echocardiography did not directly show the apical chamber in 13 of the 20 patients, left ventriculography always revealed a small apical outpouching separated from the major basal cavity. Systolic bulging of the apex was always followed by early diastolic shrinkage together with persistent cavity narrowing between the two chambers. After the systolic jet flow, the paradoxic jet flow lasted for 366 +/- 160 ms after aortic valve closure and always extended into the diastolic filling period. The maximal velocity of the paradoxic jet flow occurred during isovolumetric relaxation and the mean velocity was 2 +/- 0.8 m/s, indicating a higher diastolic pressure in the apical chamber than in the main ventricle. Compared with patients who manifested cavity obliteration alone, patients with a paradoxic jet flow more often developed systemic embolism (p less than 0.01), ventricular tachycardia (p less than 0.05) and thallium perfusion abnormalities localized to the apical region (p less than 0.01). Thus, paradoxic jet flow could be an important marker of concealed apical asynergy and the risk of adverse clinical events. The higher diastolic apical pressure suggested by the flow may contribute to the development of an apical aneurysm, even in the absence of fixed coronary artery disease.

New antibody immobilization method via functional liposome layer for specific protein assays.

A specific protein assay system based on functional liposome-modified gold electrodes has been demonstrated. To fabricate such assay system, a liposome layer was initially grown on top of a gold layer. The liposome layer contained two kinds of functional molecules: biotin molecules for the binding sites of streptavidin and N-(10,12-pentacosadiynoic)-acetylferrocene molecules for the facile redox probe in electrochemical detections. Then, streptavidin was attached on the functional liposme-modified layer using the interaction of streptavidin-sbiotin complex. On the streptavidin-attached surface, antibody molecules, anti-human serum albumin antibodies could be immobilized without any secondary antibodies. AFM imaging of the streptavidin-attached liposome surface revealed a uniform distribution of closely packed streptavidin molecules. In situ quartz-crystal microbalance and electrochemical measurements demonstrated that the wanted antibody-antigen reactions should occur with high specificity and selectivity. Our specific antibody assay system, based on a functional liposome modified electrode, can be developed further to yield sophisticated structures for numerous protein chips and immunoassay sensors.

Effect of prostaglandin E1 on fractional distribution of cardiac output and organ blood flow in man: a simultaneous and non-invasive determination using double dose thallium-201 scintigraphy.

Taking advantage of the fact that the distribution of Thallium-201 in an organ almost parallels the blood flow to the organ, we devised a non-invasive method of simultaneously determining blood flow and its distribution in human organs. With this method, we investigated the effect of exogenous prostaglandin E1 (PGE1) on the central and peripheral circulation. Scintigraphy was performed after two injections of 201Tl chloride, one at rest and one after the administration of PGE1 or saline. In eight subjects given PGE1 (0.2 microgram X kg-1 X min-1, iv), blood pressure fell, whereas both heart rate and cardiac output rose. The fractional distribution was increased in the heart (+13.3 +/- 2.98%, mean +/- SD), but decreased in the liver (-10.2 +/- 1.95%) and gastro-intestinal tract (-7.09 +/- 2.65%). No significant change was observed in the kidneys or leg. The blood flow in all these organs was increased. In the six control subjects given saline, there was no change in these central and peripheral haemodynamics. Thus, the intravenous infusion of PGE1 in man causes uneven blood flow distribution in heart, liver, kidney, gastro-intestinal organs and leg muscles, despite increased blood flow to all these organs. The method used in this study is relatively non-invasive and useful in evaluating organ blood flow in man, and seems to be widely applicable to physiological and pharmacologic research.

Subcutaneous adipocytes promote the differentiation of squamous cell carcinoma cell line (DJM-1) in collagen gel matrix culture.

Cancer cell-stromal cell interaction plays a crucial role in the malignant growth of cancer cells. In the skin, the main stromal cell types consist of dermal fibroblasts and subcutaneous adipocytes. Fibroblasts are shown to promote the invasive growth of various cancer cell types. The interaction between cancer cells and stromal adipocytes, however, has not been sufficiently studied even in cutaneous carcinoma. To address the effects of adipocytes on the biologic behavior of cancer cells, we examined the growth and differentiation of a squamous cell carcinoma cell line of the skin (DJM-1), using a three-dimensional collagen gel matrix culture with a cutaneous environmental factor, air exposure. The growth was estimated by the uptake of bromodeoxy-uridine (BrdU) for 24 h. The BrdU indices of DJM-1 cells in stromal-cell-free, fibroblast-containing, and adipocyte- containing conditions were 19.7 +/- 1.9%, 19.8 +/- 2.8%, and 4.7 +/- 1.4%, respectively, whereas the BrdU index on the gel containing both fibroblasts and adipocytes was 10.4 +/- 3.3%. In terms of differentiation, DJM-1 cells cocultured with adipocytes constructed the best-organized stratified layer with a cornified-like structure in all conditions above. The differentiation markers involucrin and cytokeratin 10 were immunohistochemically detected in this structure of DJM-1 cells. Adipocyte-induced phenomena were not affected distinctively by air exposure. These results indicate that adipocytes, but not fibroblasts, promote the differentiation of squamous cell carcinoma cells (DJM-1) and inhibit their growth. These adipocyte-induced phenomena were not completely inhibited by fibroblasts. In conclusion, we suggest that stromal adipocytes may be involved in the differentiating mechanisms of cutaneous carcinoma cells.

Reconstruction of thyroid follicles from isolated porcine follicle cells in three-dimensional collagen gel culture.

Thyroid follicles, an essential functional unit of the thyroid, are ball-like structures and exist in the extracellular matrix in vivo. Thus far, the follicles have not been reconstructed in any culture system. The presumed reason for that was that the in vitro environment for the follicle cells in monolayer culture markedly differed from their environment in vivo. We, therefore, considered that isolated follicle cells had to be localized in a three-dimensional environment of extracellular matrix, specifically collagen, to reconstruct thyroid follicles in vitro. At first, follicle cells were completely isolated. These cells were cultured in the three-dimensional collagen gel. An intracytoplasmic cavity first developed in individual cells. A single cell with the cavity then underwent cell division, and the follicle consisting of two cells was reconstructed. This gradually grew to be a large ball-like structure through proliferation of the component cells, and they exhibited morphological polarity specific for thyroid follicle cells. In addition, these cells clearly produced thyroid hormones. To the best of our knowledge, this report is the first instance of reconstruction of thyroid follicles in an in vitro culture system. This culture system is more useful than the monolayer culture system in that this system provides a more physiological environment for investigations of differentiation of follicle cells. Further experiments using this method will probably provide a new clue to the mechanism of thyroid folliculogenesis.

Plural cells organize thyroid follicles through aggregation and linkage in collagen gel culture of porcine follicle cells.

Some follicle cells organize thyroid follicles through proliferation of single cells in collagen gel culture. The aim of this study was to clarify whether two or more (plural) cells can form follicles through aggregation and linkage in this culture system. To address this question, we performed collagen gel culture of porcine follicle cells, using cell labeling with PKH 2 dye. A mixture of dye-labeled and nonlabeled cells was cultured in collagen gel. Organized follicles consisted of both dyed and nondyed cells. This suggested that plural cells reconstructed follicles through aggregation and linkage. To further confirm this finding, cells embedded in collagen gel were cultured in inhibition of cell proliferation with 2 micrograms/ml aphidicolin. Forty to 60 percent of the cells formed follicles, which did not grow larger. Electron microscopy showed that intracytoplasmic cavities appeared in the cells. In contrast, 20-30% of the embedded cells developed into cavity-embracing single cells, which remained signet rings. These results indicate that follicle cells organize follicles in collagen gel culture by two means: through cell division of cavity-embracing single cells and through aggregation and linkage of plural cells.

Growth hormone induces expression of the c-fos gene on hypothalamic neuropeptide-Y and somatostatin neurons in hypophysectomized rats.

The neuronal expression of the protooncogene c-fos may serve as a marker of neural activity. We previously examined brain sites upon which GH exerts an immediate early influence in rats and determined that the c-fos gene was transiently expressed in the hypothalamic periventricular nucleus (PeV) and arcuate nucleus (ARC) after recombinant human GH (rhGH) administration. As the distribution of c-fos messenger RNA (mRNA)-containing cells appeared to overlap with that of somatostatin (SS) neurons in both the PeV and ARC, we hypothesized that GH exerts a feedback effect on hypothalamic SS neurons. To extend this hypothesis, we characterized the neurons expressing the c-fos gene in response to rhGH administration in hypophysectomized rats. Adult male Wistar rats were hypophysectomized 10 days before use. After hypophysectomy, rats received daily sc injections of cortisone acetate (0.5 mg/kg BW) and L-T4 (20 micrograms/kg BW). Four international units (1.33 mg) of rhGH were given iv through an indwelling right atrial cannula. The vehicle was given to the control animals. Coronal sections of the hypothalamus were processed for in situ hybridization after rhGH or vehicle administration. To estimate the localization of neurons expressing the c-fos gene, the adjacent hypothalamic sections, 30 microns in thickness, were processed for hybridization histochemistry for SS, neuropeptide-Y (NPY), or GRF mRNA. In the ARC, the distribution of c-fos mRNA-containing cells appeared to overlap with that of NPY and partially with that of SS mRNA-containing cells, but it clearly differed from the distribution of GRF mRNA-containing cells. In the PeV, distribution of the cells expressing the c-fos gene was comparable to that of SS mRNA-containing cells. To further ascertain the distribution, hypothalamic sections, 6 microns in thickness, were processed by double label in situ hybridization using a 35S-labeled c-fos cRNA probe and a digoxigenin-labeled NPY or SS cRNA probe. In the ARC, 65% of the c-fos gene-expressing cells were NPY neurons. In the PeV, 60% of the c-fos gene-expressing cells were SS neurons. NPY is known to act within the hypothalamus and inhibit GH secretion via SS in rats, and the NPY neurons in the ARC have been shown to project to SS neurons in the PeV. Our findings suggest that the feedback effect of GH on the hypothalamus is mediated not only by SS neurons in the PeV, but also by NPY neurons in the ARC.

Systemic administration of recombinant human growth hormone induces expression of the c-fos gene in the hypothalamic arcuate and periventricular nuclei in hypophysectomized rats.

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.

Somatostatin reduces transcription of the growth hormone gene in rats.

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.