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We analyzed cultured cells from hyperfunctioning thyroid adenoma and its surrounding thyroid tissue from a Japanese woman and determined the nucleotide sequences of genes encoding the alpha subunit of the stimulatory G-protein 1 (G alphas) and thyrotropin (TSH) receptor in its tumor tissue. Primary culture of cells from hyperfunctioning thyroid adenoma and its surrounding thyroid tissue revealed that cAMP production was constitutively activated while intracellular Ca2+ concentration was suppressed both at the basal level and in the response to TSH stimulation in the cells from tumor tissue compared with those from non-tumor tissue. Nucleotide sequence analysis demonstrated the somatic missense mutation at codon 201 (CGT(Arg)-CAT(His)) of G alphas gene in tumor tissue but not in its surrounding tissue. No mutation was observed in the transmembrane region of TSH receptor. These results suggest that cAMP regulatory cascade is constitutively activated while phospholipase C-Ca2+ signaling cascade is suppressed in hyperfunctioning thyroid adenoma with an activating mutation of G alphas gene in the present case.

Soluble guanylate cyclase is a key enzyme of nitric oxide (NO)-related intracellular signal transduction in platelets. In the present study, we investigated the effects of sex and age on the enzyme activity in human platelets. Soluble guanylate cyclase activity was determined by generation of cyclic GMP in platelet cytosol. No significant differences in the basal activity of soluble guanylate cyclase were observed between in men and women, and between in young and old subjects. However, soluble guanylate cyclase activity in response to sodium nitroprusside, an exogenous NO donor, was higher in young men than in young and old women. Furthermore, the enzyme activity was lower in old than in young men, but there were no differences in female platelets between from young and old subjects. The present data suggest that NO-related signal transduction system in the platelet is affected by sex and age, which, to certain extent, contributes to different sensitivity of human platelets.

To elucidate the underlying mechanisms of platelet dysfunction in diabetes mellitus, we examined the activity of soluble guanylate cyclase (sGC), a key enzyme in the nitric oxide (NO)-related signalling pathway, in platelets from NIDDM (non-insulin dependent diabetes mellitus) patients. The sGC activity was determined by measuring the amount of cyclic GMP produced in platelet cytosol. In the first study, we investigated the platelet sGC activity in untreated NIDDM patients without diabetic complications. In the male NIDDM patients, sodium nitroprusside (SNP) caused a significantly lower sGC response than that in age-matched control male subjects, while the enzyme activity of female diabetics did not differ from that in the controls. Secondly, we investigated effects of diabetic-associated factors on the enzyme activity in the male NIDDM patients. There was no difference in the SNP-stimulated sGC activity in platelets from male diabetics between with and without retinopathy. In the male diabetic patients with retinopathy, however, the platelet sGC activity was slightly increased by treatment with insulin. Interestingly, the changes in enzyme activity did not correlate with plasma glycosylated hemoglobin A1c levels in diabetic patients. The impairment of the NO-related signalling pathway may contribute to the platelet dysfunction observed in patients with diabetes mellitus.

We studied the mechanism by which thyrotropin-releasing hormone (TRH)-Gly stimulated prolactin and thyrotropin (TSH) secretion in pituitary, using a pituitary mammotropic cell line, GH3 cells, and a cell line stably expressing a human TRH receptor (TRH-R). In GH3 cells expressing endogenous TRH-R, an addition of TRH-Gly evoked an immediate rise of intracellular calcium concentration, indicating that TRH-Gly reacted directly without converting from TRH-Gly to TRH. In order to determine whether this reaction might occur through TRH-R, we established a cell line stably expressing a human TRH-R, by transfecting a human TRH-R cDNA into Chinese hamster ovary cells (CHO cells). In this cell line, 10 nM TRH elevated intracellular calcium significantly; the Kd for MeTRH was 1.7 nM. One micromolar and 100 nM TRH-Gly also elevated intracellular concentration of calcium significantly, but not in CHO cells which were not transfected with the TRH-R cDNA. Competition studies further revealed that TRH-Gly displaced MeTRH binding (IC50, 12 microM). These data indicate that at high concentration, TRH-Gly interacts directly with TRH-R to activate signal transduction pathway, and that release of prolactin and TSH induced by TRH-Gly in vitro may be due, at least in part, to the direct effect of TRH-Gly on the TRH-R.

Human thyrotropin (TSH) receptors were expressed in Chinese hamster ovary (CHO) cells using eukaryotic expression plasmid pCXN2, which contains beta-actin promoter. We measured cAMP stimulation in CHO cells expressing human TSH receptors (CHO-hTSH-R cells) by immunoglobulin G (IgG) of patients with Graves' disease and Hashimoto's thyroiditis, and compared the results with a conventional thyroid-stimulating antibody (TS-Ab) assay using porcine thyroid cells and a TSH-binding inhibiting immunoglobulin (TBII) assay. Nineteen untreated patients with Graves' disease, including a case who developed hyperthyroidism after interferon -alpha therapy for chronic hepatitis C, and 13 treated patients with Graves' disease, 10 patients with Hashimoto's thyroiditis and 8 control subjects were studied. In 19 untreated patients with Graves' disease, 17 patients showed positive CHO-hTSH-R cell stimulation, 11 patients showed positive porcine thyroid cell stimulation and 15 patients showed positive TBII. All the untreated patients showed positive results in at least one assay. Although significantly positive correlations were observed among CHO-hTSH-R cell stimulation, porcine thyroid cell stimulation and TBII activities, the IgG of several patients showed significant discrepancy in the assay results. In a patient with interferon-induced hyperthyroidism only CHO-hTSH-R cell stimulation was positive, while porcine thyroid cell stimulation and TBII were negative. After the treatment with propylthiouracil for 6 months, CHO-hTSH-R cell stimulation became negative. The IgG of patients with Hashimoto's thyroiditis did not show significant stimulation of CHO-hTSH-R cells. These results suggest that the CHO-hTSH-R cell stimulation assay is clinically useful for the diagnosis and follow-up of patients with Graves' disease.

TRH receptor-related signal transduction mechanism in the pituitary cells and the central nervous system was reviewed. In pituitary cells, TRH binds to its specific receptor on the cell membrane, followed by hydrolysis of inositol phospholipids by activation of phospholipase C leading to an increase in inositol 1,4,5-trisphosphates (IP3) and diacylglycerol (DG). IP3 mobilizes intracellular Ca2+, which activates Ca2+ and Calmodulin dependent protein kinase (Ca-CaM kinase) and DG activates protein kinase C (PKC). Both Ca-CaM kinase and PKC phosphorylates several proteins in the nucleus, plasma membranes, and cytosol resulting in cell responses including hormone secretion and gene expression. Protein dephosphorylation is also involved in TRH action in the pituitary. In the central nervous system, TRH possesses different intracellular signaling systems, which vary with brain regions.

We evaluated whether prolonged oral administration of a novel analog of thyrotropin-releasing hormone (TRH), TA-0910, may change the pituitary-thyroid axis in human subjects with brain stroke. The subjects were given one oral dose of 2.5-20 mg of TA-0910 daily for 8 weeks, and then blood levels of TA-0910, thyroid hormones and thyrotropin (TSH) were measured. Plasma levels of TA-0910 were elevated with increasing doses. After administration of TA-0910, the basal levels of thyroid hormones and TSH had a tendency to increase and decrease, respectively, but those changes remained in the normal range. The secretion of TSH in response to TRH was not significantly affected by TA-0910 administration. No significant changes in other pituitary hormones were observed after TA-0910 administration. The present data indicate that prolonged oral administration of TA-0910 did not significantly alter the pituitary-thyroid axis in subjects with brain stroke.

An antiserum raised against an alignment of amino acid-(32-56), termed TSHRP-1, in the extracellular domain of human thyrotropin (TSH) receptor was used to identify the TSH receptor-like substance in plasma of Graves' disease. The dilution curve of plasma TSHRP-1-like immunoreactivity was observed in a manner parallel to the standard synthetic peptide curve in radioimmunoassay, and its molecular weight estimated approximately 60 kDa. The amounts of TSHRP-1-like immunoreactivity were significantly higher in Graves' plasma than those in plasma of normal and hypothyroid patients due to Hashimoto's thyroiditis. The present results indicate that human peripheral blood possesses a soluble form of the extracellular domain of TSH receptor which may contribute to the pathophysiology of Graves' disease.

An attempt was made to identify a specific binding of inositol 1,4,5-trisphosphate (IP3) to human chromaffin cell tumors. Crude microsomal fractions of pheochromocytomas possessed significant binding sites for [3H]IP3 with high- and low-affinity constants (Kd = 5.46 and 538 nM, respectively). The binding to these sites was rapid, saturable (reached equilibrium by 1 min at 0 degrees C) and reversible. Competition studies with other inositol phosphate analogs indicated the stereotypic specific binding for IP3. Although no significant difference was observed in the binding densities of IP3 between pheochromocytomas and adrenal nonfunctioning adenomas, the values of high-affinity constants were significantly lower in the former than the latter group (5.77 +/- 1.07, n = 5 vs. 11.30 +/- 1.98 nM, n = 4, p less than 0.05). The present data indicate that characteristics of IP3 binding sites are congruous with their receptor functions and that changes in its binding sites may contribute to the biochemical dissimilitudes in pheochromocytomas.

A 59-year-old woman was admitted in February 1991, because of abdominal distension. On admission, she had splenomegaly, autoimmune hemolytic anemia, and idiopathic thrombocytopenic purpura. Evans syndrome had been diagnosed and daily prednisolone therapy had been performed. After this therapy, a rapid increase of lymphocytes was observed accompanied with contraction of spleen. The monoclonal proliferation of B-lymphocytes and rearrangement of JH and J kappa gene were detected and chronic lymphocytic leukemia was diagnosed. With reduction of prednisolone, the lymphocyte counts decreased and the size of the spleen returned to the previous state. It is suspected that the cause of rapid lymphocytosis in this case was due to the redistribution of lymphocytes from the spleen to the peripheral blood.

We studied the effect of thyroid status on thyrotropin-releasing hormone receptor (TRH-R) mRNA levels both in vivo and in vitro (GH3 cells) using a cloned rat TRH-R cDNA by RT-PCR. Experimental hypothyroid rats were produced by total thyroidectomy and were then killed 7 days after the operation. TRH receptor binding in the anterior pituitary and serum TSH level were elevated approximately 2-fold and 8-fold, respectively, in 7 day thyroidectomized rats. TRH-R mRNA levels in hypothyroid rats were also increased significantly compared with those of normal rats. In GH3 cells, however, no significant change of TRH-R mRNA level was observed between cultures treated with triiodothyronine (T3, 10(-9) and 10(-7) M) and the untreated group. The present data indicate that 1) the in vivo effects of thyroid status on TRH-R mRNA levels differ from the in vitro one, and that 2) the down regulation of TRH-R binding by thyroid hormone in GH3 cells may be mediated by translational or post-translational mechanisms.

The effects of okadaic acid, a non-phorbol-12-tetradecanoate-13-acetate (non-TPA)-type tumor promoter and a potent inhibitor of protein phosphatases, on thyroid-stimulating hormone (TSH) secretion from the rat anterior pituitary were examined. Preincubation of anterior pituitaries with okadaic acid caused a time- and concentration-related decrease in a subsequent thyrotropin-releasing hormone (TRH)-stimulated TSH secretion, whereas it did not cause any changes in basal secretion of TSH. In addition, okadaic acid inhibited a subsequent high K(+)-induced TSH secretion. In contrast, ionomycin-induced TSH secretion was not inhibited by pretreatment with okadaic acid. The present results suggest that okadaic acid may block the release of TSH by inhibition of Ca2+ influx through voltage-sensitive and/or receptor-operated Ca2+ channels.

To gain further insight into the regulation of hypothalamic TRH by thyroid hormones, we measured TRH concentration in specific hypothalamic nuclei and preproTRH mRNA levels in the anterior hypothalamus. Adult male rats were decapitated 1, 7, 14 days after thyroidectomy. Micropunches by the method of Palkovitz from seven hypothalamic nuclei and median eminence were used for measurement of TRH by radioimmunoassay. As compared with normal levels, TRH concentration significantly decreased in the median eminence and five hypothalamic nuclei including paraventricular nucleus (PVN), posterior nucleus, anterior nucleus, arcuate nucleus, and ventromedial nucleus pars lateralis, by 7 days after thyroidectomy. No significant changes were observed in dorsomedial nucleus or ventromedial nucleus pars medialis until 14 days after thyroidectomy. A rapid and simple method to detect specific mRNA for preproTRH was developed using the polymerase chain reaction and a single anterior hypothalamic section. PreproTRH mRNA levels in the anterior hypothalamus increased approximately twice 7 days after thyroidectomy. These data indicate that thyroidectomy caused a marked increase in preproTRH mRNA levels of the anterior hypothalamus, while it significantly reduced TRH concentrations not only in PVN and median eminence but also in other specific hypothalamic nuclei, suggesting that these nuclei might be involved in the thyrotropin regulation in the hypothalamus.

We have attempted to elucidate the effect of thyroid hormone on phospholipase C-linked inositol phospholipid hydrolysis in the rat hypothalamus. Hypothalamic slices of each animal, euthyroid control, hypothyroid, and thyroxine (T4)-supplemented hypothyroid rats were labeled with [3H]myoinositol in the presence of 5 mM LiCl, and then incubated for 60 min in KHG buffer containing either vehicle or 1 mM ouabain, a Na-K ATPase inhibitor. Hypothyroidism caused a significant increase in both basal and ouabain-stimulated accumulation of [3H]inositol phosphate ([3H]IP) in hypothalamic slices, whereas supplement with T4 to hypothyroid rats resulted in a complete restoration of hypothalamic [3H]IP formation to the value of euthyroid control. The present results indicate that thyroid hormone affects phospholipase C-linked inositol phospholipid hydrolysis in the hypothalamus, suggesting that negative feedback action of thyroid hormone may occur at a post-receptor site in the hypothalamus.

The effects of hypothyroidism on the muscarinic cholinergic receptor-coupled inositol phospholipid hydrolysis in the adult rat brain were examined. Tissue slices of striatum, hippocampus, and cortex from either euthyroid or hypothyroid rats were labeled with [3H]myoinositol and incubated with carbachol, a muscarinic cholinergic agonist. In other experiments, crude plasma membranes of each brain region obtained from either euthyroid or hypothyroid rats were incubated with [3H]N-methylquinuclidinyl benzilate ([3H]NMeQNB), a muscarinic cholinergic antagonist, in the presence or absence of atropine. Carbachol produced a significant increase in [3H]inositol phosphate ([3H]IP) formation in each brain region in a dose dependent manner. Hypothyroidism caused a marked decrease in carbachol-stimulated [3H]IP formation in the striatum, whereas it did not affect the formation of [3H]IP in the cortex or hippocampus. In contrast, the affinity constant and the maximal binding of [3H]NMeQNB to plasma membranes in these regions were not changed by hypothyroidism. The present results suggest that thyroid hormones might participate in regulating the muscarinic cholinergic neurotransmission in the striatum of adult rat.

We have attempted to elucidate the effect of glucose concentrations on the release of thyrotropin-releasing hormone (TRH), a brain neuropeptide possessing glucoregulatory function, from rat hypothalamic slices in vitro. Rat hypothalamic slices were preincubated for 60 min at 37 degrees C in Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) containing varying concentrations of glucose (10, 5, 2.5 and 1 mM), and then tissues were incubated in KRB buffer, followed by stimulation with 60 mM K+ or 1 mM ouabain. In addition, after inducing hypoglycemia by insulin administration in the rat, hypothalamic tissues were dissected out and preincubated in KRB buffer (10 mM glucose) and then incubated in fresh KRB buffer containing 1 mM ouabain. A decrease in the glucose concentration of incubation medium caused a dose-dependent decrease in both K(+)- and ouabain-stimulated TRH release from rat hypothalamic slices. Furthermore, the ouabain-stimulated TRH release from the hypothalamus of rats with insulin-induced hypoglycemia was significantly reduced (45% of control values; p less than 0.01). The present results indicate that in vitro and in vivo hypoglycemia resulted in a significant decrease in the release of TRH from the hypothalamus, suggesting that circulating glucose levels affect TRH release which, in turn, might be responsible for peripheral glucoregulation.

YM14673, a novel analog of TRH, substituting the (oxo-azetidinyl)carbonyl moiety for the pyroglutamyl moiety of TRH, was synthesized and its brain action was evaluated using rat brain slices in vitro. The addition of YM14673 engendered significant and dose-dependent increases in cyclic AMP formation in the hypothalamus and inositol phosphates (IP) accumulation in the cerebellum. The actions of YM14673 did not statistically differ from those of TRH on brain tissues. However, YM14673 did not show a direct interference with TRH binding in the brain membranes. [3H]YM14673 underwent to the same extent the enzymatic degradation by type II pyroglutamyl aminopeptidase as did TRH. As compared with TRH, YM14673 had a lower activity with respect to stimulation of thyrotropin secretion and accumulation of IP in the anterior pituitaries. The data imply that YM14673 possessed potent stimulatory actions on brain intracellular signaling systems with weak hypophyseal activities. These actions of YM14673 were not due to a direct interference with brain TRH binding or a nature impervious to a TRH-degrading enzyme. The present study indicates that the larger doses of YM14673 than TRH can be used for medical treatment of neurological and neuromuscular disorders without strong influences on pituitary functions.

TRH is produced from its possible direct precursor, pGlu-His-Pro-Gly (TRH-Gly), by alpha-amidating enzyme. The quantitative response of TRH-Gly-stimulated PRL, TSH, and GH was evaluated in nine patients with anorexia nervosa, six age-matched normal women, eight patients with uremia, five patients with acromegaly, and two patients with prolactinoma. Intravenous injection (500 micrograms) of TRH-Gly caused a 2.6-fold increase in PRL secretion in patients with anorexia nervosa (basal level, 10.0 +/- 1.4 vs. 25.9 +/- 2.5 micrograms/L 15 min after injection; P less than 0.01). In contrast, no significant change was observed in TRH-Gly-stimulated PRL secretion in normal women (basal level, 13.5 +/- 2.3 vs. 15.3 +/- 2.5 micrograms/L 15 min after injection; P greater than 0.05). TRH-Gly did not alter PRL levels in patients with uremia, acromegaly, or prolactinoma. Secretion of TSH, but not GH, was slightly increased by TRH-Gly injection in patients with anorexia nervosa (basal level, 1.41 +/- 0.13 vs. 2.86 +/- 0.22 min/L 30 min after injection; P less than 0.01), whereas no significant secretory response was observed in normal women. These data provide evidence that PRL secretion in anorectic patients is quantitatively different from that in normal persons.

We explored whether thyrotropin-releasing hormone may affect opioid receptors in the rat brain. Adult male rats were intraperitoneally injected twice a day with varying doses of DN1417, a potent analogue of thyrotropin-releasing hormone, for 2 days, and opioid receptors of the brain (hypothalamus, striatum, hippocampus, midbrain, ponsmedulla, and cortex) homogenates were determined using [3H]naloxone. Intraperitoneal injection of DN1417 in a dose of 0.3 mg/100 g body wt resulted in a significant reduction in naloxone binding of the striatum as compared with the saline-injected group, whereas naloxone binding of other brain regions was not affected by DN1417. DN1417 produced a dose-dependent decrease in naloxone binding of the striatum. The affinity constant of naloxone binding was similar between the saline- and DN1417-injected groups. In vitro addition of DN1417 did not interfere with the brain naloxone binding. The distribution of 3H-labeled DN1417 injected peritoneally did not differ among the brain regions. The present data imply that the opioid antagonistic action of thyrotropin-releasing hormone may be due, at least in part, to the significant decrease in the striatal opioid binding in the rat brain.