Tsuyoshi Nogimori

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.

Concentrations of Immunoreactive Thyrotropin-Releasing Hormone in Spinal Cord of Patients with Amyotrophic Lateral Sclerosis

Concentrations of immunoreactive thyrotropin-releasing hormone (ir-TRH) were measured by specific radioimmunoassay in the spinal cord of six patients with amyotrophic lateral sclerosis (ALS) and seven with non-neurological diseases. Ir-TRH concentrations were the highest in the anterior horn, compared with other areas of the spinal cord, both in nonneurological diseases and ALS. Ir-TRH concentrations in the anterior horn of ALS were significantly lower than in nonneurological diseases, but were the same in both groups in other parts of the spinal cord (e.g. posterior horn, frontal part, lateral and central part, posterior part). Ir-TRH concentrations in rat spinal cords were stable for up to seven hours when spinal cord was stored after death at 4°C or 22°C. An elution profile of methanol-extracted human spinal cord on Sephandex G-10 column was identical to that of synthetic TRH. The cell population in the anterior horn in ALS was decreased markedly. The findings suggest that TRH is present in the human spinal cord and its decreased concentrations in the anterior horn of ALS may be due to a decrease in the cell population.

Peripheral administration of eel calcitonin inhibits thyrotropin secretion in rats

The effects of peripheral administration of eel calcitonin on thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) secretion were studied in rats. Eel calcitonin (50 U/kg) was injected i.v. The hypothalamic immunoreactive TRH (ir-TRH) contents increased significantly after calcitonin injection. Plasma TSH levels decreased in a dose-related manner with a nadir at 30 min after the injection. The plasma ir-TRH and TSH responses to cold as well as the plasma TSH response to TRH were inhibited by calcitonin. The inhibitory effect of calcitonin on TSH levels was prevented in the haloperidol-, pimozide- or p-chlorophenylalanine-pretreated group, but not in the L-DOPA- or 5-hydroxytryptophan-pretreated group. The findings suggest that calcitonin acts on both the hypothalamus and the pituitary to inhibit TRH and TSH release, and that its effects may be modified by amines of the central nervous system.

Effects of leucine-enkephalin on hypothalamic-pituitary-thyroid axis in rats

Basal thyrotropin (TSH) levels in plasma and the TSH response to thyrotropin-releasing hormone (TRH) were inhibited after Leucine-enkephalin (L-EK) administration iv in rats. TRH and TSH responses to cold were inhibited after L-EK administration. In the L-DOPA, haloperidol or 5-hydoxytryptophan-treated rats, the inhibitory effect of L-EK on TSH release was restored. Findings suggested that L-EK acted both the hypothalamus and pituitary. Its inhibitory effects on TRH and TSH release at least partially mediated by interaction with amines in the central nervous system.

Effects of Substance P, Angiotensin II, Oxotremorine and Prostaglandin D2 on Thyrotropin Secretion in Rats

The effects of substance P (SP), angiotensin II, oxotremorine and prostaglandin D2 (PG D2) on thyrotropin-releasing hormone (TRH) and thyrotropin (TSH) secretion in rats were studied. Either SP (100 micrograms/kg), angiotensin II (500 micrograms/kg), oxotremorine (1.0 mg/kg) or PGD2 (500 micrograms/kg) was injected intravenously or intraperitoneally, and the rats were serially decapitated. TRH, TSH and thyroid hormone were measured by means of a specific radioimmunoassay for each. The hypothalamic immunoreactive TRH (ir-TRH) contents were significantly increased by oxotremorine or SP and significantly decreased by angiotensin II, but no by PG D2. The plasma ir-TRH concentrations were significantly increased by angiotensin II, but not by oxotremorine, SP or PG D2. The plasma TSH levels were significantly increased by angiotensin II and significantly decreased by oxotremorine, SP or PG D2 in a dose-related manner. The plasma ir-TRH and TSH responses to cold were inhibited by oxotremorine, SP or PG D2, but enhanced by angiotensin II. The plasma TSH response to TRH was inhibited by SP, but enhanced by angiotensin II. The plasma TSH response to TRH did not differ from that of the control after PG D2 injection. In the haloperidol- or para-chlorophenylalanine (PCPA)-pretreated group, the inhibitory effect of PG D2 or oxotremorine on TSH release was prevented, while in the L-DOPA- or 5-hydroxytryptophan (5-HTP)-pretreated group, the inhibitory effect of SP on TSH release was prevented.(ABSTRACT TRUNCATED AT 250 WORDS)

Deregulated production of interleukin-4 (IL4) in autoimmune thyroid disease assayed with a new radioimmunoassay

A sensitive, reproducible and specific radioimmunoassay for human interleukin-4 (IL4) has been developed. Using 125I-labeled IL4 and polyclonal rabbit antisera raised against recombinant human IL4, a competitive inhibition assay was developed which could detect 5 pg/ml of human IL4. Other interleukins, growth factors, hormones, peptides and lectins did not affect the assay. IL4 was measured in supernatants of culture media of stimulated human peripheral blood mononuclear cells (PBMC). Kinetics of IL4 production in PHA-stimulated PBMC from seven normal subjects revealed that the peak levels of IL4 were seen at 24 h and then declined. Peak IL4 levels in PHA stimulation of PBMC from untreated patients with autoimmune thyroid diseases (Graves disease and chronic thyroiditis) were significantly higher than normal controls. However, after treatment, IL4 production decreased to normal. The present study demonstrates the usefulness of quantitating human IL4 produced by PBMC and that there exists a deregulated production of IL4 in autoimmune thyroid diseases.

Effects of Immunoneutralization of Endogenous Opioid Peptides on the Hypothalamic-Pituitary-Thyroid Axis in Rats

Effects of opioid peptide antisera treatment on the secretion of thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) in rats were studied. Anti-beta-endorphin antiserum, anti-methionine-enkephalin antiserum, or antidynorphin antiserum was injected intraperitoneally and the rats were serially decapitated. TRH levels in the hypothalamus along with plasma TRH, TSH and thyroid hormone levels were measured by individual radioimmunoassay. TRH contents in the hypothalamus decreased significantly after opioid peptide antisera treatment, while its plasma levels tended to decrease, but not significantly. Plasma TSH levels increased significantly after opioid peptide antisera injection. Plasma TRH and TSH level responses to cold as well as plasma TSH level response to TRH were enhanced with treatment of antisera to these peptides. Plasma 3,3,5-triiodothyronine levels increased significantly after treatment of antisera to these peptides. From these findings it is concluded that the treatment of opioid peptide antisera stimulates TRH and TSH secretion in rats.

Effects of histamine and related compounds on thyrotropin secretion in rats.

The effects of histamine (HA) and related compounds on thyrotropin-releasing hormone (TRH) and thyrotropin (TSH) secretion in rats were studied. Histidine (1.0 g/kg), HA (5.0 mg/kg) or histamine antagonists mepyramine (MP) (100 mg/kg) or famotidine (FA) (5.0 mg/kg) were injected intraperitoneally, and the rats were decapitated at various intervals after the injection. The hypothalamic immunoreactive TRH (ir-TRH) content increased significantly after histidine or HA injection, decreased significantly after FA injection, but was not changed by MP. The plasma ir-TRH concentration did not change significantly after injection of these drugs. The plasma TSH levels decreased significantly in a dose-related manner after histidine or HA injection and increased significantly in a dose-related manner after FA injection. The plasma thyroid hormone levels showed no changes. In the FA-pretreated group, the inhibitory effect of histidine or HA on TSH levels was prevented, but not in the MP-pretreated group. The plasma ir-TRH and TSH responses to cold were inhibited by histidine or HA and enhanced by FA. The plasma TSH response to TRH was inhibited by histidine or HA and enhanced by FA. The inactivation of TRH immunoreactivity by hypothalamus or plasma in vitro after histidine, HA, MP or FA was not different from that of the control. These findings suggest that histamine may act both on the hypothalamus and the pituitary to inhibit TRH and TSH release, and that its effects may be mediated via H2-receptor.

Effects of histamine and related compounds on the release of immunoreactive thyrotropin-releasing hormone from the rat stomach in vitro

Effects of histamine and related compounds on the release of immunoreactive thyrotropin-releasing hormone (ir-TRH) from the rat stomach in vitro were studied. The rat stomach was incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The ir-TRH release from the rat stomach was enhanced significantly in a dose-related manner with the addition of histamine and inhibited with the addition of famotidine, but not with mepyramine. The stimulatory effect of histamine on ir-TRH release from the stomach was partially blocked with the addition of famotidine, but not with mepyramine. The elution profile of acid-methanol-extracted rat stomach on Sephadex G-10 was identical to that of synthetic TRH. These findings suggest that histamine stimulated ir-TRH release from the rat stomach in vitro, and that histamines effects may be mediated via a H2-receptor.

Tuftsin stimulates thyrotropin secretion in rats

Tuftsin acts at the hypothalamus level to stimulate thyrotropin-releasing hormone and thyrotropin secretion in rats.