Influence of thyroid hormones and transforming growth factor-ß1 on cystatin C concentrations.

Serum cystatin C concentrations are reported to increase in the hyperthyroid state. Serum concentrations of cystatin C and transforming growth factor-ß1 (TGF-ß1) were measured in patients with thyroid dysfunction, and the effects of 3,5,3'-tri-iodothyronine (T(3)) and TGF-ß1 on cystatin C production in human hepatoblastoma (Hep G2) cells were studied. Serum concentrations of cystatin C and TGF-ß1 were significantly higher in patients with Graves' disease compared with control subjects. Significantly positive correlations were observed between thyroid hormones and cystatin C, thyroid hormones and TGF-ß1, and TGF-ß1 and cystatin C in patients with thyroid dysfunction. Serum concentrations of cystatin C and TGF-ß1 decreased after treatment for hyperthyroidism. Cystatin C mRNA levels and cystatin C secretion were increased by T(3) and TGF-ß1 in cultured Hep G2 cells. These results suggest that serum cystatin C concentrations increase in patients with hyperthyroidism. The mechanisms for this may involve elevation of serum TGF-ß1 levels and the stimulatory effects of T(3) and TGF-ß1 on cystatin C production.

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.

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.

In vivo transfer of antisense oligonucleotide against urinary kininase blunts deoxycorticosterone acetate-salt hypertension in rats.

We have previously reported that the renal kallikrein-kinin system suppressed the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Kinins were degraded in the kidney mainly by carboxypeptidase Y (CPY)-like kininase. Blockade of renal kinin degradation may reduce hypertension in the developmental stage. We constructed an antisense oligonucleotide against rat CPY homologue (5'-CAT-CTC-TGC-TTC-CTT-GTG-TC-3', AS) and its randomized control oligonucleotide (5'-TCC-TTC-CTG-CTT-GAG-TTC-CT-3', RC), and prepared an HVJ-liposome complex that prolongs and increases the effectiveness of the antisense oligonucleotide. Antisense oligonucleotide was transfected (25 nmole rat(-1), in terms of nucleotide) into the kidney from the renal artery. Blood pressure was measured through a catheter inserted into the abdominal aorta. Mean blood pressure (MBP) in DOCA-salt treated (for 2 weeks) Sprague Dawley strain rats was 130+/-3 mmHg (n=11), and was reduced significantly (P<0.05) more by AS transfection (122+/-4 mmHg, n=6) than by RC treatment (137+/-6 mmHg, n=5) 4 days after the transfection. This reduction in MBP was accompanied by increased urinary sodium excretion (AS, 8.4+/-1.5 mmole day(-1); RC, 4.6+/-0.5 mmole day(-1), P<0.05) and a reduction in urinary CPY-like kininase activity. Ebelactone B (5 mg kg(-1), twice a day, p.o.), an inhibitor for urinary CPY-like kininase, also reduced MBP and induced natriuresis to the same degree as AS. Lisinopril, an inhibitor for angiotensin converting enzyme (ACE) failed to reduce the elevated MBP. These results suggest that CPY-like kininase may have more contribution than ACE to degrade kinin in the kidney, and that knockdown of CPY-like kininase in the kidney may partly prevent rat DOCA-salt hypertension.

Tyrosine kinase inhibitors enhance GHRH-stimulated cAMP accumulation and GH release in rat anterior pituitary cells.

In this study, the role of tyrosine phosphorylation in agonist-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. It was found that genistein, a tyrosine kinase inhibitor, while having no effect on its own, potentiated GHRH-stimulated cAMP accumulation in a concentration-dependent manner. In comparison, daidzein, an inactive analogue of genistein, was ineffective and vanadate, a phosphotyrosine phosphatase inhibitor, reduced GHRH-stimulated cAMP accumulation. Additional structurally unrelated tyrosine kinase inhibitors, erbstatin and tyrphostins, also potentiated GHRH-stimulated cAMP accumulation. To determine the site of action of the tyrosine kinase inhibitors, pituitary adenylate cyclase-activating polypeptide (PACAP), cholera toxin and forskolin were used to increase cAMP accumulation. Genistein enhanced the PACAP-, cholera toxin- or forskolin-stimulated cAMP accumulation, suggesting that the site of action is at the post-receptor level. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein did not potentiate and vanadate did not inhibit GHRH-stimulated cAMP accumulation, indicating that phosphodiesterase is a probable site of action for the inhibitor. Genistein and erbstatin also enhanced GHRH-stimulated GH release and the effect of vanadate was inhibitory. These results indicate that tyrosine kinase inhibitors enhance cAMP accumulation through their action on phosphodiesterase activity in rat anterior pituitary cells and the tyrosine kinase pathway appears to be involved in the control of GH release.

Protein kinase C potentiation of the tyrosine kinase inhibitor-stimulated cyclic GMP production in rat pinealocytes.

Inhibition of tyrosine kinase activities elevates cyclic GMP (cGMP) levels in rat pinealocytes. Since protein kinase C (PKC) and intracellular Ca2+ both interact with the agonist-stimulated cGMP accumulation, in this study their interactions with the tyrosine kinase inhibitor-mediated cGMP response were investigated. Two tyrosine kinase inhibitors, genistein and tyrphostin B42, increased basal cGMP accumulation concentration dose-dependently. This increase in cGMP accumulation was potentiated by 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of PKC, and blocked by calphostin C, a specific PKC inhibitor. The tyrosine kinase inhibitors had no effect on the in vitro or PMA-mediated translocation of PKC activity. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine (IBMX), neither the tyrosine kinase inhibitors alone nor in combination with PMA had an effect on cGMP accumulation, suggesting that phosphodiesterase is a probable site of action of the inhibitors. In comparison, elevation of intracellular Ca2+ by BayK 8644, ionomycin, or KCl inhibited the genistein- or tyrphostin B42-mediated increase in cGMP accumulation. This inhibition persisted in the presence of IBMX and was partly reversed by a Ca2+/calmodulin inhibitor. These results suggest that PKC modulates the rate of cGMP degradation through signalling pathways involving tyrosine phosphorylation. However, the inhibitory effect of the Ca(2+)-elevating agents on the tyrosine kinase inhibitor-stimulated cGMP accumulation appears to be independent of phosphodiesterase inhibition.

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.

Enzyme immunoassay of testosterone using the testosterone-glucoamylase complex.

A highly sensitive, reproducible method was established for enzyme-coupled immunoassay of testosterone, involving coupling of testosterone to glucoamylase [EC 3.2.1.3] with a water-soluble coupling reagen carbodimide. No enzyme activity was lost during this coupling procedure. The sensitivity of the method was comparable to that of competitive protein binding assay.

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.

Thyroid hormones regulate the formation of inositol phosphate in response to thyrotropin-releasing hormone in rat anterior pituitaries.

The effects of thyroid hormones on TSH secretion and inositol phospholipid hydrolysis in response to thyrotropin-releasing hormone (TRH) in rat anterior pituitaries were examined. Experimental hypothyroidism caused a significant increase in [3H]inositol phosphate ([3H]IP) formation in response to TRH in rat anterior pituitaries with a concomitant elevation of blood TSH. In contrast, administration of thyroxine (T4) to hypothyroid rats resulted in a complete restoration of blood TSH and TRH-stimulated [3H]IP formation to the euthyroid control value. Furthermore, in vitro pre-treatment of anterior pituitaries with triiodothyronine (T3) produced a dose-dependent decrease in both TSH secretion and the formation of [3H]IP in response to TRH. These results indicate that thyroid hormones regulate TRH receptor-linked inositol phospholipid hydrolysis in the rat anterior pituitary, suggesting that negative feedback action of thyroid hormone occurs at post receptor event in the rat anterior pituitary, which may, to a certain extent, be responsible for the underlying mechanism of T3 inhibition of TSH secretion.

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.

Expression of thyrotropin receptors in rat thymus.

Thymic hyperplasia is associated with Graves' disease. It has been demonstrated that thyrotropin receptors are expressed in human thymus, and thymic thyrotropin receptors are suggested to be involved in the pathophysiology of Graves' disease. We have studied whether thyrotropin receptors are expressed in rat thymic tissue. Thyrotropin receptor mRNA was demonstrated in 5-day-old, 10-day-old, 20-day-old and adult rat thymus by reverse transcription polymerase chain reaction. Thyrotropin receptor mRNA was also demonstrated in cultured rat thymic epithelial cells. Thyrotropin stimulated cyclic AMP production in cultured rat thymic epithelial cells, suggesting the expression of functional thyrotropin receptors. The present results indicate that thyrotropin receptors are expressed in rat thymus.

Northern analysis of type II iodothyronine deiodinase mRNA in rat Harderian gland.

It has been known that type II iodothyronine deiodinase activity is present in rat Harderian gland and the activity is significantly increased by isoproterenol administration. We have performed Northern analyses to study whether the transcript for type II iodothyronine deiodinase is expressed in rat Harderian gland and whether the isoproterenol stimulation of type II iodothyronine deiodinase activity in rat Harderian gland is due to the change in its mRNA level. Northern analyses have demonstrated that type II iodothyronine deiodinase mRNA, approximately 7.5 kb in size, is expressed in rat Harderian gland, and the mRNA levels as well as the deiodinase activities are greater in hypothyroid rats than those in euthyroid rats. Type II iodothyronine deiodinase mRNA levels and the deiodinase activities in Harderian gland were increased by isoproterenol administration, and the increase in the mRNA levels preceded that in the deiodinase activities. These results indicate that 7.5 kb transcript for type II iodothyronine deiodinase is expressed in rat Harderian gland and beta-adrenergic stimulation of type II iodothyronine deiodinase activity is due at least in part to the increase in its mRNA level.

[Studies on the efficacy and safety of panipenem/betamipron in infections in surgical domain and the safety when mixed with lactate containing infusion].

Studies were done on the effectiveness and safety of panipenem/betamipron, a new carbapenem antibiotic, in infections in the surgical domain and its safety when dissolved in infusions containing lactate. The obtained results are summarized as follows. The preparation, 0.5 g/0.5 g or 1.0 g/1.0 g, was administered by intravenous drip infusion 2 to 3 times a day to 31 cases of patients with infections in the surgical domain. A physiological saline solution was used as the solvent in 21 cases (group A) of them and Solita T3, an infusion containing lactate, was used as the solvent in the 10 remaining cases (group B). As for its clinical effects, results were rated as "excellent" in 20, "good" in 7, "fair" in 3 and "no response" in 1 out of the 31 cases, and the efficacy rate was 87.1%. Regarding its bacteriological effects, results were rated as "disappeared" in 22, "decreased" in 2, "unchanged" in 1 and "unknown" in 1 out of 26 cases from which bacteria were isolated, hence the bacteria-clearance rate was 88.0%. As for side effects, skin rash was seen in 1 case and slight increases of GOT and GPT were noted as abnormal changes in laboratory data in 2 cases. These side effects were all observed among the cases in group A but not at all in group B where a lactate containing infusion was used as the solvent.

[Experience with high-dose methotrexate therapy for malignant solid tumors].

Thirteen patients with malignant solid tumors, mainly osteogenic sarcomas, were treated by high-dose MTX therapy, 50-400 mg/kg, in a total of 72 cycles. It has been proved that this therapy was relatively safe provided careful clinical surveillance were assured. Of 8 patients with osteogenic sarcomas, 6 had no metastatic lesions on the chest X-rays before this therapy: three were alive without any evidence of disease for 18-25 months after high-dose MTX therapy. In some cases of other malignant solid tumors, particularly intracranial tumors, clinical efficacy was observed. Dose-time relationships, etc. should further be studied for more therapeutic efficacies of high-dose MTX therapy.