Inhibitory effect of oxytocin on accelerated colonic motility induced by water-avoidance stress in rats.

Recent studies have indicated that brain and gut activities are interrelated and exposure to several stressors, such as water-avoidance stress, stimulates the motor function of the gut through corticotropin-releasing factor (CRF)-signalling pathways in the brain. Central oxytocin is known to attenuate stress responses, including CRF expression in the brain. Here, we examined whether central oxytocin attenuated the acceleration of colonic motility induced by water-avoidance stress. A force transducer was attached to the distal colon of male rat, and the colonic motility and faecal pellet output were recorded while the rats were exposed to water-avoidance stress. Intracerebroventricular (i.c.v.) injections of oxytocin (5, 50 and 500 pmol) and the oxytocin receptor antagonist tocinoic acid (25 microg) were administered before exposure to water-avoidance stress, and the effect of oxytocin on colonic motor function was determined. Centrally administered oxytocin inhibited the accelerated colonic motility induced by water-avoidance stress. The effective dose ranged between 5 and 50 pmol on i.c.v. injection. Oxytocin also decreased the number of CRF-positive cells in the paraventricular nucleus and corticosterone release. The inhibitory effect of oxytocin on accelerated colonic motility was blocked by pretreatment with oxytocin receptor antagonist. Furthermore, centrally administered tocinoic acid enhanced the acceleration of colonic motility. These results suggested that endogenous central oxytocin may contribute to the regulation of colonic function and inhibit the brain CRF-signalling pathways targeting the gut, resulting in the inhibition of stress-induced colonic contractions.

Distribution of aquaporin-9 in the rat: an immunohistochemical study.

The aim of this study was to identify the distribution of aquaporin-9 by immunohistochemistry in rat tissues using specific antipeptide antiserum which we recently produced. Anti-aquaporin-9 antibody was raised in New Zealand white rabbits immunized with a conjugate of synthetic aquaporin-9 peptide with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. Aquaporin-immunoreactivity was visualized in the anterior pituitary, central nervous system, retina, thyroid gland, gastrointestinal tract, liver lung, pancreas and testis. When using antiserum preincubated with synthetic peptides or rat hypothalamus homogenate, which contains aquaporin-9 peptide, no significant stain of the hypothalamus was detected. These findings suggest that aquaporin-9 is widely distributed and that the method used is valuable in studying the distribution of aquaporin-9 in rats.

Comparison of new different assay systems for thyrotropin receptor antibodies with reference to thyroid-stimulating antibodies and thyroid stimulation-blocking antibodies in Graves' disease.

The aim of our study was to evaluate the diagnostic sensitivity of the new thyrotropin receptor antibody (TRAb) assays (Cosmic TRAb CT, ELISA and Yamasa DYNOtest TRAb). TRAb was positive in 43 of 44 (97.7%) untreated patients with Graves' disease by both TRAb CT and/or ELISA and NYNOtest TRAb. Thus the new TRAb assays were clearly more sensitive than the conventional assay (positivity: 85%). There was a strong positive correlation between the data obtained in TRAb CT and/or ELISA and those obtained in DYNOtest TRAb (r = 0.942, p < 0.0001). There was a significant correlation between the new TRAb and TSAb (r = 0.696, p < 0.0001). Although there was a significant correlation between the new TRAb and thyroid stimulation-blocking antibody (TSBAb), the correlation coefficient was low (r = 0.605, p < 0.0001). The increased sensitivity of the new TRAb assays for Graves' disease provides an advantage over conventional assay.

Radioimmunoassay for somatostatin receptor type 2.

OBJECTIVE: To develop radioimmunoassay for somatostatin receptor type 2 (SSTR2) and search for its presence in certain rat tissues. METHODS: Anti-SSTR2 antiserum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic SSTR2 with bovine serum albumin. Radioiodination of SSTR2 was performed by chloramin T method followed by purification of radioiodinated material on Sephadex G-25 column. RESULTS: The obtained antibody did not crossreact with SSTR1, SSTR3, SSTR4, SSTR5, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. The assay was performed with a double antibody system. SSTR2 was extracted from the tissues with acid acetone. The dilution curve of acid acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue SSTR2 was about 89 %, and the intra-assay and inter-assay variations were 4.9 % and 7.8 %, respectively. SSTR2 was found in the hypothalamus, cerebrum, cerebellum, pituitary, stomach and testis. CONCLUSIONS: These data suggest that this assay system is suitable for the estimation of SSTR2 in the tissues.

[Implication on thyroid function tests].

We tried to investigate the present problems, concerning the reference individual and interval in thyroid function tests and find the solutions for them. We are now using healthy adults for the reference individual and interval. Recently, we found the sex-difference and age-related changes for reference individual and interval in free T3 measurement. We raised the questions on whether there are any sex-differences and/or age-related changes or not. Surprisingly, there were almost no detailed data about them especially in Japan. Therefore, we examined the Europe data, and found out some kind of sex-differences and age-related changes. We propose the following examinations using many Japanese population in order to provide a precise and proper reference individual and interval: 1. Whether there are any sex-difference in thyroid function tests? 2. Whether there are age-related change in thyroid function tests, for instance, simply dividing population into the immature, adult and the aged?

Radioimmunoassay for hypocretin-2.

OBJECTIVE: To develop radioimmunoassay for hypocretin-2 (Hcrt-2). And search for its presence in certain rat tissues. METHODS: Anti-Hcrt-2 serum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic Hcrt-2 with bovine serum albumin. Radioiodination of Hcrt-2 was performed by chloramine T method, followed by purification of radoiodinated material on Sephadex G-25 column. RESULTS: The obtained antibody did not cross react with hypocretin-2, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. The assay was performed with a double antibody system. Hcrt-2 was extracted from the tissues with acid acetone. The dilution curve of acid acetone extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue Hcrt-2 was about 85 % and the intra-assay and inter-assay variation were 5.6 % and 8.0 %, respectively. Hcrt-2 was found in the hypothalamus, cerebrum, brain stem and testes. CONCLUSIONS: The obtained data suggest that the assay system developed is suitable to measure Hcrt-2 in tissues and that Hcrt-2 is mainly found in the hypothalamus.

Radioimmunoassay for orexin A.

A radioimmunoassay for orexin A has been developed. Anti-orexin A antiserum was raised in New Zealand white rabbits immunized with a conjugate of synthetic orexin A with bovine serum albumin. This antibody did not crossreact with orexin B, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. Radioiodination of orexin A was performed with the chloramin T method, followed by purification of radioiodinated material on Sephadex G-25 column. Orexin A was extracted from tissues using acid-acetone. The assay was performed with a double antibody system. The dilution curve of acid-acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue orexin A was about 80%,and the intra-assay and inter-assay variations were 5.2% and 7.8%, respectively. Orexin A was found in the hypothalamus, cerebrum and testis. These data suggest that this assay system is suitable for the measurement of tissue orexin A and that orexin A is found in the central nervous system and testis.

Effects of orexin A on thyrotropin-releasing hormone and thyrotropin secretion in rats.

Effects of orexin A on secretion of thyrotropin-releasing hormone (TRH) and thyrotropin (TSH) in rats were studied. Orexin A (50 microg/kg) was injected iv, and the rats were serially decapitated. The effects of orexin A on TRH release from the rat hypothalamus in vitro and on TSH release from the anterior pituitary in vitro were also investigated. TRH and thyroid hormone were measured by individual radioimmunoassays. TSH was determined by the enzyme-immunoassay method. The hypothalamic TRH contents increased significantly after orexin A injection, whereas its plasma concentrations tended to decrease, but not significantly. The plasma TSH levels decreased significantly in a dose-related manner with a nadir at 15 min after injection. The plasma thyroid hormone levels showed no changes. TRH release from the rat hypothalamus in vitro was inhibited significantly in a dose-related manner with the addition of orexin A. TSH release from the anterior pituitary in vitro was not affected with the addition of orexin A. The findings suggest that orexin A acts on the hypothalamus to inhibit TRH release.

Gene amplification as a common cause of inherited thyroxine-binding globulin excess: analysis of one familial and two sporadic cases.

T4-binding globulin (TBG) is the major thyroid hormone transport protein in humans. Inherited abnormalities in the level of serum TBG have been classified as partial deficiency, complete deficiency and excess. A single nucleotide deletion or substitution in the TBG gene, located on Xq22, has been detected in partial and complete deficiencies. As for inherited TBG excess, the gene amplification has been recognized in two Japanese families recently. In this study, an additional three Japanese families, one familial (F-I) and two sporadic TBG excess (F-II, F-III), were analyzed. Serum TBG levels in hemizygous males were 73, 47 and 42 microg/ml, three- to two-fold the normal value. The molecule had normal properties in terms of heat stability and isoelectric focussing pattern. The gene dosage of TBG was evaluated by coamplification with autosomal betaGlobin or X-chromosomal Duchenne Muscular Dystrophy (DMD) and subsequent quantitation by HPLC. The TBG/betaGlobin ratios of the affected male and female of F-I were 3.09- and 3.86-times, respectively, compared to that of the normal males. The TBG/DMD ratios were 2.93- and 2.09-times, respectively. These results are compatible with three copies of the TBG gene on the affected X-chromosome. Similarly, a twofold increase in gene dosage was demonstrated in the affected males of sporadic cases. Their mothers with normal TBG values had the same TBG gene dosage as normal females, suggesting that de novo gene duplication arose in gametes probably during meiosis. Amplification of the TBG gene was not recognized in these three families by in situ hybridization of prometaphase chromosomes. Though the mechanism remains unproved, gene amplification of TBG was considered to be a common cause for inherited TBG excess.

Distribution of thyrotropin releasing hormone receptor type 2 in rats: an immunohistochemical study.

OBJECTIVE: To investigate the organ distribution of thyrotropin releasing hormone receptor (TRHR) type 2 in rats by immunohistochemical method. METHODS: TRHR type 2 was identified immunohistochemically in the rat tissues using specific anti-TRHR antiserum raised in New Zealand white rabbits immunized with a conjugate of synthetic TRHR type 2 (5-23) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. RESULTS: TRHR type 2 immunoreactivity was visualized in the central nervous system, anterior pituitary, gastric mucosa, Auerbach's and Meissner's nervous branch of the stomach, small intestine and colon, retina amd testis. Significant stain was detected in neural perikarya, axons and dendrites. When using antiserum preincubated with synthetic TRHR type 2(5-23) or anterior pituitary homogenates, no significant stain of anterior pituitary was detected. CONCLUSIONS: These findings suggest that TRHR type 2 is widely distributed and that the method used is valuable in studying the distribution of TRHR type 2 in rats.

Distribution of calcium sensing receptor in rats: an immunohistochemical study.

OBJECTIVE: To investigate the organ distribution of calcium sensing receptor (CaR) in rats by immunohistochemical method. METHODS: CaR was identified immunohistochemically in the rat tissues using specific anti-CaR antiserum raised in New Zealand white rabbits immunized with a conjugate of synthetic CaR peptide (186-204) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. RESULTS: CaR immunoreactivity was visualized in the central nervous system, anterior pituitary, gastric mucosa, small intestine and colon, Auerbach,s and Meissner,s gastric nervous branch, small intestine and colon, pancreas, adrenal medulla, kidney and testis. When using antiserum preincubated with synthetic CaR peptide (186-206) or kidney homogenates, no significant stain of kidney was detected. CONCLUSIONS: The findings suggest that CaR is widely distributed and that the method used is valuable in studying the distribution of caR in rat.

Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity.

Xeroderma pigmentosum variant (XP-V) represents one of the most common forms of this cancer-prone DNA repair syndrome. Unlike classical XP cells, XP-V cells are normal in nucleotide excision repair but defective in post-replication repair. The precise molecular defect in XP-V is currently unknown, but it appears to be a protein involved in translesion synthesis. Here we established a sensitive assay system using an SV40 origin-based plasmid to detect XP-V complementation activity. Using this system, we isolated a protein from HeLa cells capable of complementing the defects in XP-V cell extracts. The protein displays novel DNA polymerase activity which replicates cyclobutane pyrimidine dimer-containing DNA templates. The XPV polymerase activity was dependent on MgCl2, sensitive to NEM, moderately sensitive to KCl, resistant to both aphidicolin and ddTTP, and not stimulated by PCNA. In glycerol density gradients, the activity co-sedimented with a 54 kDa polypeptide at 3.5S, indicating that the monomeric form of this polypeptide was responsible for the activity. The protein factor corrected the translesion defects of extracts from three XPV cell strains. Bypass DNA synthesis by the XP-V polymerase occurred only in the presence of dATP, indicating that it can incorporate only dATP to bypass a di-thymine lesion.

Nociceptin stimulates thyrotropin secretion in rats.

Effects of nociceptin on thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) secretion in rats were studied. Nociceptin (150 microgram/kg) was injected intravenously and rats were serially decapitated after the injection. The effects of nociceptin on TRH release from the hypothalamus and TSH release from the anterior pituitary in vitro were also investigated. TRH and thyroid hormones were measured by individual radioimmunoassays. TSH was determined by enzyme immunoassay. TRH contents in the hypothalamus decreased significantly after nociceptin injection, whereas plasma TRH concentrations showed no changes. Plasma TSH concentrations increased significantly in a dose-related manner. The TRH release from the hypothalamus was enhanced significantly in a dose-related manner with the addition of nociceptin. The TSH release from the anterior pituitary in vitro was not affected by the addition of nociceptin. The plasma thyroxine and 3,3',5-triiodothyronine levels did not change significantly after nociceptin administration. The inactivation of TRH by plasma or hypothalamus in vitro after nociceptin injection did not differ from that of controls. The findings suggest that nociceptin acts on the hypothalamus to stimulate TRH and TSH secretion.

Cat eye syndrome with hypogonadotropic hypogonadism.

A 17-year-old male diagnosed as having Cat Eye Syndrome (CES) with hypogonadotropic hypogonadism showed short stature and no development of secondary sex characteristics. Exogeneous gonadotropin replacement therapy combining human chorionic gonadotropin (hCG) and human menopausal gonadotropin (hMG) was started. As a result, the short stature and androgen deficiency were relieved. The critical region of CES was tetrasomy of 22 pter-->q11. Abnormalities of other chromosomes which cause hypogonadotropic hypogonadism may exist, thus further investigation is needed.

Thyrotropin-releasing hormone and somatostatin inhibit each others release in vitro in the rat retina.

The effect of thyrotropin-releasing hormone (TRH), somatostatin (SS) or octreotide, an analogue of SS, on release of TRH or SS from the rat retina was studied in vitro. The retina was incubated in medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin (medium) for 20 min. The amount of TRH or SS release into the medium was measured by individual radioimmunoassays. The TRH release from the rat retina was inhibited significantly in a dose-related manner by the addition of SS or octreotide. The SS release from the retina was inhibited by TRH, and the inhibitory effect of TRH on SS release from the rat retina was blocked by the addition of anti-TRH receptor antiserum immunoglobulin fraction. The findings suggest an interaction between TRH and SS in the rat retina by which the addition of one inhibits the release of the other.

In vitro effects of endothelin-1 on somatostatin and thyrotropin-releasing hormone release from the rat stomach.

The effects of endothelin (ET) 1 on the release of somatostatin (SS) and thyrotropin-releasing hormone (TRH) from the rat stomach were studied in vitro. The rat stomach was incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) for 20 min. The amounts of SS and TRH released into the medium were measured by individual radioimmunoassays. With the addition of ET-1, the release of SS from the rat stomach was inhibited significantly in a dose-related manner, whereas TRH released from the stomach was enhanced significantly. These effects of ET-1 on SS or TRH release were blocked by BQ-485, a blocker of ETA receptor. These findings suggest that ET-1 inhibits SS and stimulates TRH release from the rat stomach in vitro, and that these effects are mediate via ETA receptor.

Radioimmunoassay for Aquaporin-2.

OBJECTIVE: To develop radioimmunoassay for aquaporin-2 (AQP-2). METHODS: Anti-AQP-2 antiserum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic AQP-2 peptide (257-271) with bovine serum albumin. Radioiodination of synthetic peptide (tyrosine-AQP2 (257-271) was performed by chloramine T method, followed by purification of radioiodinated material on Sephadex G-25 column. RESULTS: The obtained antibody did not crossreact with vasopressin, pituitary hormones, hypothalamic hormones and neuropeptides. The assay was performed with a double antibody system. The values are expressed as an equivalent of synthetic AQP-2 peptide (257-271). The dilution curve of high AQP-2 urine in radioimmunoassay system was parallel to the standard curve. The recovery percentage of AQP-2 added to urine was about 100 % in this assay system. Intra-assay and inter-assay variation was 4.5 % and 7.2 %, respectively. Mean urinary excretion of AQP-2 was 1.16 ng equivalent of AQP-2 (257-271)/mg creatine and was lower in patients with diabetes insipidus. CONCLUSION: These data suggest that his assay system is a suitable to measure AQP-2 in urine.

Distribution of Dopamine Transporter in the Rat: an Immunohistochemical Study.

OBJECTIVE: To investigate the organ distribution of dopamine transporter (DAT) in rats by immunohistochemical method. METHODS: Dopamine transporter (DAT) was identified immunohistochemically in the tissues using specific antipeptide antiserum raised in New Zealand white rabbits immunized with a conjugate of synthetic DAT peptide (29-45) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. RESULTS: DAT immunoreactivity was visualized in the neural perikarya, axons and dendrites of the central nervous system, retina, adrenal medulla, Auerbach's nervous branch and Meissner's nervous branch of the stomach, small intestine and colon, anterior pituitary, and lung. When using antiserum preincubated with synthetic DAT peptide (DAT, 29-45) or hypothalamus homogenate which contains DAT, no significant stain of neurons in the hypothalamus was detected. CONCLUSION: These findings suggest that DAT is widely distributed and that the method used is valuable in studying the distribution of DAT in rats.

Effects of gamma-butyric acid on the release of thyrotropin-releasing hormone from the rat retina in vitro.

Effects of gamma-butyric acid (GABA) on the release of thyrotropin-releasing hormone (TRH) from the rat retina in vitro were studied. The rat retina was incubated in medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (medium). The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat retina was inhibited significantly in a dose-related manner with the addition of GABA, but not with bicuculline. The inhibitory effect of GABA on TRH release from the retina was blocked by adding bicuculline to the medium. The findings suggest that the GABAergic system inhibits TRH release from the rat retina in vitro.