ABSTRACT
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.
Subject(s)
Receptors, Somatostatin/analysis , Acetone , Animals , Antibody Specificity , Brain Chemistry , Cerebellum/chemistry , Hypothalamus/chemistry , Immune Sera , Iodine Radioisotopes , Isotope Labeling , Male , Pituitary Gland/chemistry , Quality Control , Rabbits , Rats , Rats, Wistar , Stomach/chemistry , Telencephalon/chemistry , Testis/chemistryABSTRACT
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.
Subject(s)
Neuropeptides , Neurotransmitter Agents/analysis , Radioimmunoassay , Animals , Antibodies , Antibody Specificity , Brain Chemistry , Brain Stem/chemistry , Hypothalamus/chemistry , Intracellular Signaling Peptides and Proteins , Male , Neurotransmitter Agents/immunology , Orexins , Organ Specificity , Quality Control , Rabbits , Rats , Rats, Wistar , Testis/chemistryABSTRACT
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.
Subject(s)
Immunohistochemistry , Receptors, Thyrotropin-Releasing Hormone/analysis , Animals , Avidin , Axons/chemistry , Biotin , Brain Chemistry , Colon/innervation , Dendrites/chemistry , Gastric Mucosa/chemistry , Intestine, Small/innervation , Male , Organ Specificity , Pituitary Gland, Anterior/chemistry , Rats , Rats, Wistar , Retina/chemistry , Spinal Cord/chemistry , Stomach/innervation , Testis/innervationABSTRACT
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.
