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.

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.

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.