Thyroid hormone activation in human vascular smooth muscle cells: expression of type II iodothyronine deiodinase.

Thyroid hormone has been reported to have significant effects on the peripheral vascular system, including relaxation of vascular smooth muscle cells and antiatherosclerotic effects. To exert its biological activity, thyroxine, which is a major secretory product of thyroid gland, needs to be converted to 3,5,3'-triiodothyronine (T(3)) by iodothyronine deiodinase. Type I iodothyronine deiodinase (DI) is widely distributed and maintains circulating T(3) level, whereas type II iodothyronine deiodinase (DII) is present in a limited number of tissues to provide local intracellular T(3). In the present study, we have identified iodothyronine deiodinase in cultured human coronary artery smooth muscle cells (hCASMCs) and human aortic smooth muscle cells (hASMCs). All of the characteristics of the deiodinating activity in hCASMCs and hASMCs were compatible with DII. Northern analysis demonstrated that DII mRNA was expressed in both hCASMCs and hASMCs, and DII mRNA levels as well as DII activities were rapidly increased by dibutyryl-cAMP or forskolin. These data demonstrate, for the first time, the expression of DII in human vascular smooth muscle cells, which is regulated by a cAMP-mediated mechanism. The present results suggest a previously unrecognized role of local T(3) production by DII in the pathophysiology of human vascular smooth muscle cells.

Thyrotropin receptors in brown adipose tissue: thyrotropin stimulates type II iodothyronine deiodinase and uncoupling protein-1 in brown adipocytes.

It has been demonstrated that TSH receptors are expressed not only in thyroid gland but also in extrathyroidal tissues. Brown adipose tissue of guinea pig has been reported to express TSH receptor messenger RNA (mRNA), but the physiological roles of TSH receptors in brown adipose tissue have not been understood. We studied the expression and function of TSH receptors in rat brown adipose tissue and cultured rat brown adipocytes. Northern analysis demonstrated the expression of TSH receptor mRNA in rat brown adipose tissue and cultured rat brown adipocytes. TSH receptor mRNA in rat brown adipose tissue was decreased by cold exposure of the rat, and its mRNA in cultured rat brown adipocytes was also decreased by incubation with TSH or (Bu)(2)cAMP. TSH increased the intracellular cAMP concentration in cultured rat brown adipocytes in a dose dependent manner. Type II iodothyronine deiodinase mRNA, its activity, and uncoupling protein-1 mRNA in cultured rat brown adipocytes were significantly increased by incubation with TSH in a dose-dependent manner. These results suggest the expression of functional TSH receptors in brown adipose tissue, which may be involved in regulation of the expression of type II iodothyronine deiodinase and uncoupling protein-1.

Expression and regulation of type II iodothyronine deiodinase in human thyroid gland.

We have studied the expression of type II iodothyronine deiodinase (DII) in human thyroid tumors and cultured human thyroid cells to elucidate the mechanisms involved in the regulation of DII expression in human thyroid gland. Three cases with hyperfunctioning thyroid adenoma, including a case that showed an activating mutation of G(s)alpha with a constitutive activation of cAMP production in cultured cells, and six cases with papillary thyroid carcinoma were analyzed in the present study. Free T(3) was increased, whereas free T(4) was within the normal range in all patients with hyperfunctioning thyroid adenoma. Thyroid tumor tissue and surrounding nontumor tissue were obtained at the time of surgery, and DII expression was compared between tumor tissue and nontumor tissue in each case. Northern analysis demonstrated the presence of DII messenger RNA (mRNA) approximately 7.5 kb in size in all of the tumor and nontumor tissues. DII mRNA and DII activity in hyperfunctioning thyroid adenoma were significantly increased compared with those in nontumor tissue in each case. In contrast, DII mRNA and DII activity in papillary thyroid carcinoma were decreased compared with those in nontumor tissue in each case. DII mRNA and DII activity in cultured human thyroid cells were significantly stimulated by TSH in a dose-dependent manner. The promoter activity of the human DII gene including the complete cAMP response element, transfected to cultured human thyroid cells, was stimulated by (Bu)(2)cAMP. In summary, these results suggest that DII expression in human thyroid gland is regulated at the transcriptional level through the TSH receptor-G(s)alpha-cAMP regulatory cascade, which may be related to the increase in circulating T(3) level in patients with Graves' disease and hyperfunctioning thyroid adenoma.

Pretranslational regulation of rhythmic type II iodothyronine deiodinase expression by beta-adrenergic mechanism in the rat pineal gland.

It has been demonstrated that type II iodothyronine deiodinase is present in rat pineal gland, and the deiodinase activity markedly increases during the hours of darkness, primarily through beta-adrenergic mechanism. We have studied the relationship between pineal type II iodothyronine deiodinase messenger RNA (mRNA) and the deiodinase activity to elucidate the mechanisms involved in the nocturnal rise in pineal deiodinase activity. Northern analysis has demonstrated that type II iodothyronine deiodinase mRNA is expressed in rat pineal gland, and the mRNA markedly increases during the hours of darkness. The nocturnal increase in pineal type II iodothyronine deiodinase activity is preceded by the increase in its mRNA. Daytime isoproterenol administration resulted in a rapid increase in pineal type II iodothyronine deiodinase mRNA followed by the increase in deiodinase activity. Propranolol treatment, bilateral superior cervical ganglionectomy, or constant light exposure significantly suppressed the nocturnal rise in type II iodothyronine deiodinase mRNA as well as the deiodinase activity. Moreover, isoproterenol or (Bu)2AMP stimulated type II iodothyronine deiodinase mRNA and the deiodinase activity in cultured rat pineal glands. These results suggest that the rhythmic change in pineal type II iodothyronine deiodinase activity is regulated at least in part at the pretranslational level by a beta-adrenergic mechanism transmitted through superior cervical ganglia.

Discrete characteristics of antibodies raised against thyrotropin receptor-related peptides whose sequences are not conserved in the luteinizing hormone/chorionic gonadotropin receptor.

In order to identify the specific regions in the human TSH receptor for TSAb and thyroid stimulation-blocking antibody (TSBAb), we produced rabbit antibodies raised against several peptides of the extracellular domain of the human TSH receptor, where sequences are not conserved in the LH/CG receptor, and measured the TSAb activity and TSBAb activity of those antibodies using Chinese hamster ovary cells expressing human TSH receptors. Only antisera from rabbits that were immunized with a peptide of amino acid 32-56, including the small insertion near the N-terminal end of the extracellular domain, showed apparent TSAb activities and have been shown to be significantly precipitated by IgG of patients with Graves' disease. TSAb activity positively correlated with the antibody titers against the peptide in those rabbits. In contrast, antisera from rabbits immunized with a peptide of amino acid 352-378, including a part of the large insertion near the C-terminal end of the extracellular domain, showed the obvious TSBAb activities. TSBAb activity also positively correlated with the degree of antibody titers against the peptide in those rabbits. Moreover, this peptide was significantly immunoprecipitated by the IgG from hypothyroid patients who had TSBAb, and the immunoprecipitation of this peptide positively correlated with TSBAb activities. These results suggest that the epitope responsible for TSAb is quite different from that for TSBAb in the extracellular domain of the human TSH receptor.

Expression and regulation of type II iodothyronine deiodinase in cultured human skeletal muscle cells.

T4, which is a major secretory product of the thyroid gland, needs to be converted to T3 by iodothyronine deiodinase to exert its biological activity. After the molecular cloning of human type II iodothyronine deiodinase (DII) complementary DNA, DII expression was unexpectedly detected in human skeletal muscle tissue. In the present study, we have identified DII activity and DII messenger ribonucleic acid (mRNA) in cultured human skeletal muscle cells and studied the mechanisms involved in the regulation of DII expression in those cells. All of the characteristics of the deiodinating activity in cultured human skeletal muscle cells were compatible with those of DII. Northern analysis has demonstrated that DII mRNA, approximately 7.5 kb in size, was expressed in cultured human skeletal muscle cells. DII mRNA and DII activity were rapidly increased by (Bu)2cAMP, forskolin, or beta-adrenergic agonists and were negatively regulated by thyroid hormones in cultured human skeletal muscle cells. Although interleukin-1beta and interleukin-6 did not decrease DII expression in cultured human skeletal muscle cells, tumor necrosis factor-alpha decreased DII expression in those cells in a dose-dependent manner. These data have demonstrated, for the first time, that DII activity and DII mRNA are present in cultured human skeletal muscle cells, and that the DII expression is stimulated by beta-adrenergic mechanisms through a cAMP-mediated pathway and is negatively regulated by thyroid hormones and tumor necrosis factor-alpha.

Expression of type II iodothyronine deiodinase in brain tumors.

Type II iodothyronine deiodinase (DII) messenger ribonucleic acid (mRNA) and its activity have been demonstrated in human normal brain. Although DII activity has been demonstrated in brain tumors, expression of DII mRNA has not been studied in these tumors. To investigate the mechanisms involved in the expression of DII activity in brain tumors, we studied DII mRNA and DII activity in astrocytoma (two cases), glioblastoma (three cases), and oligodendroglioma (one case). DII mRNA, the size of which was indistinguishable from that in control cerebral cortical tissue, was demonstrated in all of the brain tumors tested, although the intensity of the hybridization signal showed wide variation among the tumors. DII activity was also detected in all tumors. DII mRNA and DII activity were highest in the tissue from oligodendroglioma. A significantly positive correlation was observed between DII mRNA and DII activity in these tumors (r = 0.94; P < 0.01), suggesting that DII expression in brain tumors is regulated at the pretranslational level. The present results demonstrate, for the first time, that DII mRNA as well as DII activity are expressed in brain tumors, and that DII mRNA is significantly correlated with DII activity in those tissues.

Primary culture of cells from hyperfunctioning thyroid adenoma with an activating mutation of G alphas.

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.

Corticosterone facilitation of inhibition of fat intake by enterostatin (Val-Pro-Asp-Pro-Arg).

Enterostatin or Val-Pro-Asp-Pro-Arg (VPDPR) is the amino-terminal pentapeptide of procolipase; VPDPR is generated during tryptic activation of procolipase to lipase. In rodents, exogenous VPDPR has been shown to cause a selective decrement in fat appetite. To understand the mechanism(s) underlying the action of this peptide, we have studied the effects of corticosterone, an adrenal hormone known to modulate caloric intake, on VPDPR-mediated inhibition of appetite. The results of this study show a significant increase in the inhibition of total caloric intake by 250 micrograms/kg VPDPR following corticosterone treatment (control, 2.3%; corticosterone treated, 22.7%). Furthermore, the decrement in the caloric intake in corticosterone-treated rats was exclusively due to the loss of fat intake.

Role of endogenous cyclo(His-Pro) in cold-induced hypothermia in the desert rat (Mastomys natalensis).

Central administration of exogenous cyclo(His-Pro) (CHP) is known to produce hypothermia in rodents. In the present study, we examined the role of endogenous CHP in cold-induced hypothermia in the desert rat, Mastomys natalensis. The results of these studies show that a rise in hypothalamic CHP content accompanied a decrease in rectal temperature during cold exposure. Immunoneutralization of endogenous CHP resulted in a significant decline in cold-induced hypothermia. In addition, central administration of cyclo(Ala-Gly), a structural analogue of CHP, also led to a decrease in cold-induced hypothermia. The results of these studies show that changes in endogenous CHP levels may affect body temperature regulation.

Inhibitory effects of okadaic acid on thyrotropin and prolactin secretion from rat anterior pituitaries.

The present study was undertaken to elucidate the effects of okadaic acid, a potent inhibitor of protein phosphatases, on thyrotropin (TSH) and prolactin (PRL) secretion, and on the hydrolysis of inositol phospholipids in rat anterior pituitaries. Preincubation of anterior pituitaries with okadaic acid caused a dose dependent decrease in TRH- and K(+)-induced TSH secretion, whereas basal secretion of TSH was not affected by pretreatment with okadaic acid. In contrast, okadaic acid resulted in a marked inhibition in both basal, and TRH- and K(+)-stimulated PRL release from anterior pituitaries. In addition, pretreatment with okadaic acid caused a slight, but significant decrease in the formation of [3H]inositol phosphate ([3H]IP) in rat anterior pituitaries. The present study suggests that okadaic acid blocks the release of TSH and PRL by inhibiting Ca2+ influx and that inhibitory effects of okadaic acid on PRL release are, at least in part, due to the inhibition of inositol phospholipid hydrolysis.

Attenuation of alcohol-induced hypothermia by cyclo (His-Pro) and its analogs.

Acute administration of cyclo (His-Pro) to rats cause a dose-dependent decrease in ethanol-induced hypothermia. Bromination of the imidazole moiety of histidine in cyclo (His-Pro) resulted in a significant increase in its potency to attenuate ethanol hypothermia. In contrast, benzylation of the imidazole moiety of histidine or the substitution of one or both of the amino acids in cyclo(His-Pro) led to a total loss of its thermomodulatory activity. In conclusion, it appears from these preliminary data that it may be possible to design analogs of CHP that may be effective antagonists for ethanol hypothermia.

Potential use of serotherapy in the prevention and treatment of infection with the human immunodeficiency virus.

While prevention of infection with the human immunodeficiency virus (HIV) using prophylactic immunization and treatment with anti-viral drugs would appear to be the methods of choice for the prevention and treatment of this infection, neither safe and effective vaccines nor anti-viral agents have yet been developed. A third approach should thus be considered which could be employed both for prophylaxis and treatment of this disease. This approach utilizes specific, anti-HIV antibodies, passively administered, to prevent and/or slow the infectious process. The disadvantages of using xenogeneic antibodies and the advantages of using human antibodies are discussed. The need for large quantities of human antibodies to HIV necessitates the production of cell lines producing these antibodies. The various techniques of producing these lines are summarized. Finally, preliminary data supporting the feasibility of producing human cell lines producing antibody to HIV are presented.

A paradoxical elevation of brain cyclo(His-Pro) levels in hyperphagic obese Zucker rats.

Several studies suggest a role for endogenous cyclo(His-Pro) or CHP in appetite regulation. In the present study, we have examined the regional brain distribution of CHP in hyperphagic obese Zucker rats and their lean littermates. The data show a significant elevation in the levels of CHP in many brain regions, including hypothalamus of the obese rat. Within the hypothalamus, the lateral hypothalamic (LH) nucleus of obese rats had significantly higher levels of CHP when compared to that of the lean littermates. Administration of dehydroepiandrosterone, a steroid hormone known to decrease food intake and body weight gain, to obese rats led to decrease in the levels of CHP in the LH. These data further suggest a role for the endogenous CHP in attenuating food intake.

Thyroid hormone affects the hydrolysis of inositol phospholipids in the rat 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.

Expression and nocturnal increase of type II iodothyronine deiodinase mRNA in rat pineal gland.

It has been demonstrated that thyroxine deiodinating activity is present in rat pineal gland, and its activity increases significantly during the night time. We have studied whether mRNA for type II iodothyronine deiodinase is expressed in rat pineal gland and whether the nocturnal rise of pineal T4 deiodinating activity is due to the change in type II iodothyronine deiodinase mRNA level. Reverse transcription-polymerase chain reaction amplification and Northern blot analyses have demonstrated that type II iodothyronine deiodinase mRNA is expressed in rat pineal gland and its mRNA level increases markedly at midnight. These results suggest that the nocturnal rise in pineal T4 deiodinating activity is due to the change in type II iodothyronine deiodinase mRNA level.

Identification of cyclo(His-Pro)-like immunoreactivity in human follicular fluid: correlation with steroid and peptide hormones.

OBJECTIVE: The purpose of this study was to evaluate human follicular fluid (FF) for the presence of cyclo(His-Pro)-like immunoreactivity (CHP-LI). After verifying its presence, we quantitated the levels and investigated correlations with other follicular parameters, including hormone levels. METHODS: Follicular fluid was collected from female volunteers undergoing controlled ovarian hyperstimulation. Fluid was collected by follicular puncture, either transvaginally (in vitro fertilization) or laparoscopically (gamete intrafallopian transfer) at the time of oocyte retrieval (N = 137). Follicular size was determined ultrasonographically. Assays for steroid and peptide hormones were determined with commercially available radioimmunoassay kits. CHP-LI was measured using a previously reported assay; parallel dilution curves and column chromatography aided in immunoidentity. RESULTS: The mean FF CHP-LI concentration (13.10 +/- 1.83 nmol/L, N = 137) was greater than the corresponding serum values (9.42 +/- 2.45 nmol/L; N = 21) (P < .05). Large follicles (20 mm or greater; 14.45 +/- 1.74 nmol/L) contained significantly more CHP-LI than either medium follicles (16-19 mm; 11.51 +/- 1.88 nmol/L) or small follicles (15 mm or smaller; 10.83 +/- 2.12 nmol/L) (P < .05). Positive correlations were found between FF CHP-LI values and corresponding FF progesterone and prolactin concentrations (r = 0.67 and 0.62, respectively; P < .05). CONCLUSION: Mean CHP-LI levels in the FF are greater than those in the corresponding serum. We suggest that the neuropeptide may be originating from either peptidase cleavage of precursor peptides or from granulosa cell production.

Does cyclo(His-Pro) act like amphetamine?

In many pharmacologic tests, cyclo(His-Pro) (CHP) appears to act like a dopaminergic agonist and augments the actions of amphetamine (AMP). Therefore, to determine whether CHP is an AMP-like peptide, a comparison between CHP and AMP was made using four separate tests known to be AMP-responsive. These include, food intake, locomotor activity, dopamine uptake and modulation of binding sites for amphetamine and mazindol. A decrease in food intake and increase in spontaneous locomotor activity and stereotypy was observed after peripheral administration of amphetamine but not CHP. Chronic CHP administration resulted into a decrease in striatal amphetamine - and increase in mazindol-binding sites; in contrast, chronic amphetamine decreased both amphetamine - and mazindol-binding sites. These results show a clear dissociation between CHP and AMP suggesting that CHP is not an amphetamine-like peptide.

Augmentation of dietary fat preference by chronic, but not acute, hypercorticosteronemia.

Numerous studies have documented a role for corticosterone in appetitive behavior, including caloric intake and dietary fat preference. In the present study, we have examined the mechanism(s) underlying modulation of dietary fat preference by corticosterone. The results of these studies show a) an increased fat preference with increased basal urinary output, or decreased stimulation of corticosterone output on fasting, b) elevation of fat preference following chronic, but not acute, hypercorticosteronemia produced by exogenous corticosterone administration, and c) emergence of hypercorticosteronemia prior to the development of increased fat preference in developing rats. These observations have led us to suggest that increased fat preference after chronic hypercorticosteronemia may be secondary to changes in the levels or actions of agents known to affect fat intake.

Sex and age differences in soluble guanylate cyclase activity in human platelets.

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.