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.

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.

Effect of intragastric ammonia on gastrin-, somatostatin-and somatostatin receptor subtype 2 positive-cells in rat antral mucosa.

Somatostatin suppresses gastrin and somatostatin secretion via somatostatin receptors (SSTRs). Ammonia produced by Helicobacter pylori has been reported to modify gastric gastrin and somatostatin levels. We investigated the distribution of SSTR-subtype 2 (SSTR-2) in relation to gastrin- and somatostatin-containing cells and the effect of ammonia solution (0.01%-0.1%) administered orally for 2 to 4 weeks on these cells in rat antral mucosa by immunohistochemistry. The majority of SSTR-2 peptide [31-41]-positive cells were located in the basal third of the glands. Double staining experiments revealed that SSTR-2 peptide [31-41]-positive cells are co-localized in 85.0 +/- 2.2% of the gastrin-containing cells and in 34.4 +/- 4.8% of the somatostatin-containing cells. Ammonia solution significantly decreased the number of somatostatin-containing cells and increased the proportion of SSTR-2 peptide [31-41]-labeling in the somatostatin-containing cells in a duration-dependent manner. Maximum changes were observed in rats treated with ammonia solution at the lowest level of 0.01% accompanied by an increase in serum gastrin levels in the portal vein. Sodium hydroxide at the similar pH to 0.01% ammonia solution had no effect. These findings suggest that SSTR-2 are localized in antral endocrine cells and that ammonia solution mainly decreases somatostatin-containing cells without SSTR-2 expression, resulting in an increase in gastrin secretion into the portal vein.

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.

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.

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.

Organ distribution of iodide transporter (symporter) in the rat: immunohistochemical study.

Iodide transporter/symporter (NIS) was identified immunohistochemically in rat tissues using specific antipeptide serum. Anti-NIS serum was raised in New Zealand white rabbits immunized with a conjugate of synthetic NIS peptide (39-53) with bovine serum albumin. Immunohistochemical analysis was performed by avidine-biotin complex method. NIS immunoreactivity was visualized in the thyroid gland, gastric and small intestine mucosa, anterior pituitary, adrenal medulla, pancreatic islets, kidney, chorioid plexus and several brain and spinal cord nuclei. When using antiserum preincubated with synthetic NIS peptide (39-53) or rat thyroid homogenate containing NIS, no significant stain of the thyroid gland was detected. These findings suggest that NIS is widely distributed and that the method used is suitable for studying the distribution of NIS in rats.

DISTRIBUTION OF SOMATOSTATIN RECEPTOR TYPE 3 IN THE RAT: IMMUNOHISTOCHEMICAL STUDY.

Somatostatin receptor type 3 (SSTR-3) was identified immunohistochemically in the rat tissues using specific anti-SSTR-3 serum which was raised in New Zealand white rabbits immunized with a conjugate of synthetic SSTR-3 peptide (28-41) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. SSTR-3 immunoreactivity was visualized in the central nervous system, anterior pituitary, gastric and duodenal mucosa, Auerbach's and Meissner's nervous branch of gastrointestinal tract, adrenal medulla, testis and pancreas. Significant staining was detected in neural perikarya, axons and dendrites. When using antiserum preincubated with synthetic SSTR-3 peptide (28-41) or rat anterior pituitary homogenate which contains SSTR-3 peptide, no significant stain of the anterior pituitary or neurons in the hypothalamus was detected. These findings suggest that SSTR-3 is widely distributed and that this method is valuable in studying the distribution of SSTR-3 in rats.

Effect of anti-TRH-receptor antibody on corticosterone release from rat adrenal gland in vitro.

The effect of anti-thyrotropin-releasing hormone (TRH) receptor antibody on corticosterone release from the rat adrenal gland in vitro was studied. The adrenal glands were incubated in medium 199 with 1.0 mg/ml bacitracin (pH 7.4, medium) for 20 min and the release of corticosterone into the medium was measured by radioimmunoassay. TRH inhibited corticosterone release from the adrenal gland in a dose-related manner. The inhibitory effect of TRH on corticosterone release from the adrenal gland was prevented by the addition of anti-TRH-receptor antibody. The present findings suggest that TRH inhibits corticosterone release from the adrenal gland in vitro and its effect is mediated via TRH receptor.

Effects of serotonin on the release of thyrotropin-releasing hormone from the rat retina in vitro.

Effects of serotonin on the release of thyrotropin-releasing hormone (TRH) from the rat retina were studied in vitro. The retina was incubated in medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (medium) for 20 min. 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 serotonin and enhanced with cyproheptadine. The inhibitory effect of serotonin on TRH release from the retina was blocked with the addition of cyproheptadine. The elution profile of methanol extract of the rat retina was identical to that of synthetic TRH. The findings suggest that the serotonergic system inhibits TRH release from the rat retina in vitro.

Distribution of somatostatin receptor type 1 in the rat. An immunohistochemical study.

Somatostatin receptor type 1 (SSTR-1) was identified immunohistochemically in the rat tissues using anti-peptide antiserum. Anti-somatostatin receptor antiserum was raised in New Zealand white rabbits immunized with a conjugate of synthetic SSTR-1 peptide (41-52) with bovine serum albumin. Immunohistochemical analysis was performed by the ABC method. SSTR-1 immunoreactivity was visualized in the central nervous system, anterior pituitary, mucosa of stomach and duodenum, Auerbach's nervous branch and Mysner's nervous branch of gastrointestinal tract and pancreas. Significant staining was detected in neural perikarya, axon and dendrite. When using antiserum preincubated with synthetic SSTR-1 peptide (41-52) or rat anterior pituitary homogenated which contains SSTR-1, no significantly stained anterior pituitary cells or neurons in the hypothalamus were detected. These findings suggest that SSTR-1 is widely distributed and that this method is valuable in studying the distribution of somatostatin receptor in rats.

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

Thyrotropin releasing hormone (TRH) receptor was identified immunohistochemically in the rat tissues using anti-peptide antiserum. Anti-TRH receptor antiserum was raised in New Zealand white rabbits immunized with a conjugate of synthetic TRH-receptor peptide (15-28) to bovine serum albumin. Immunohistochemical analysis was performed by the avidin-biotin complex method. TRH-receptor immunoreactivity was visualized in the central nervous system and anterior pituitary thus supporting previous investigations of TRH receptor distribution using in vitro autoradiographic ligand binding. Significant stain was detected in neural perikarya, axons and dendrites as well as in many cells of the retina, adrenal medulla, stomach mucosa, Auerbach's nervous branch and Mysner's nervous branch of the stomach, small intestine and colon. When using antiserum preincubated with synthetic TRH-receptor peptide (15-28) or rat anterior pituitary homogenate which contains TRH-receptor peptide, no significant stain of the anterior pituitary cells or neurons in the hypothalamus was detected. These findings suggest that TRH-receptor is widely distributed and that this method is valuable in studying the distribution of TRH-receptor in rats.