ABSTRACT

Using chlorethylclonidine (CEC), an α_lb-adrenergic receptor-selective antagonist, we characterized α₁-adrenoceptor subtypes in rat thyroid gland, and investigated the effect of methimazole (MMI)-induced high TSH levels on α₁ receptor subtypes and noradrenaline-induced iodide organification. The density of thyroid α₁-adrenergic receptors was increased about sixfold in rats treated with MMI for 3 weeks compared with controls. Pretreatment of thyroid membrane preparations with CEC (10 μmol/l) caused an 83% decrease in specific 2-[β-(hydroxy-3-[¹²⁵I]iodophenyl) ethylaminomethyl]tetralone binding sites in MMI-treated rats, but only a 43% decrease in control rats. The density of CEC-insensitive α₁ receptors (α^1a) was similar in MMI-treated and control rats, so MMI was shown to increase CEC-sensitive α₁ receptors (α^1b).

Noradrenaline-stimulated iodide organification was threefold greater in MMI-treated rats than in control rats when values were expressed as a per cent increase over basal levels. Pretreatment of thyroid lobes with 10 μmol CEC/1 for 30 min caused a 66% decrease in maximal noradrenaline-induced iodide organification in MMI-treated rats, but a significantly lower decrease (49%) in control rats.

These results suggest that the rat thyroid gland contains both α_1a and α_1b receptors, both of which mediate noradrenaline-induced iodide organification, and also that TSH enhances noradrenaline-induced iodide organification by increasing α_1b receptor density.

Journal of Endocrinology (1990) 126, 317–322

ABSTRACT

Eight rabbits were immunized with a synthetic peptide corresponding to the unique N-terminal region (termed N peptide; amino acid residues 29–57) in the extracellular domain of the human thyrotrophin (TSH) receptor. After 10 weeks, all of the eight rabbits produced anti-N peptide antibodies. Western blot analysis revealed that the antibodies recognized rabbit TSH receptor as an approximately 100 kDa protein. We compared the level of thyroid hormone in serum taken before immunization (preimmune sera) with that of serum taken after immunization (postimmune sera) in these immunized rabbits. Postimmune sera from the eight rabbits had higher mean (± s.d.) levels of tri-iodothyronine (T₃) and thyroxine (T₄) than did preimmune sera (T₃, preimmune 0·82 ± 0·26 μg/l vs postimmune 1·33 ± 0·35, P < 0·01; T₄, preimmune 33·7 ± 10·0 μg/l vs postimmune 41·0 ± 6·0, P < 0·05). T₃ levels in four rabbits and T₄ levels in four rabbits after immunization were over the normal range obtained from six age-matched control rabbits. Seven rabbits exhibited thyroid-stimulating antibody (TSAb) activity with various degrees (241–545%). The concentration of T₃ and T₄ did not increase over 10 weeks in either non-immunized rabbits (T₃, preimmune 0·89 ± 0·34 μg/l vs postimmune 0·82 ± 0·22; T₄, preimmune 31·1 ± 7·3 μg/l vs postimmune 30·3 ± 5·1) or other peptide-immunized rabbits (T₃, preimmune 0·68 μg/l (n = 2) vs postimmune 0·69; T₄, preimmune 33·1 μg/l vs postimmune 26·4). These results indicate that experimentally produced anti-TSH receptor antibody with TSAb activity induces an increase in thyroid hormone in rabbits.

Journal of Endocrinology (1992) 135, 479–484

Abstract

Inositol 1,4,5-trisphosphate (InsP₃) 3-kinase phosphorylates the Ca²⁺-mobilizing second messenger InsP₃ to inositol 1,3,4,5-tetrakisphosphate (InsP₄). InsP₃ 5-phosphatase dephosphorylates InsP₃ to inositol 1,4-bisphosphate (InsP₂). We compared the effects of TSH added to a culture of FRTL-5 thyroid cells on the activity of InsP₃ 5-phosphatase and InsP₃ 3-kinase. InsP₃ 3-kinase activity was decreased at a physiological concentration of TSH. Inhibition of this activity started after 3 h of incubation with TSH and was maximal after 24 h. In contrast, InsP₃ 5-phosphatase activity was not affected by TSH under the same conditions. The inhibitory effect of TSH on InsP₃ 3-kinase was characterized as follows: a) inhibition of activity was mimicked by both dibutyryl cyclic AMP and forskolin; b) activity obtained by mixing lysates of TSH-stimulated and non-stimulated cells was the sum of each activity measured separately; c) inhibition persisted after a crude lysate of TSH-stimulated cells had been subjected to SDS/polyacrylamide gel electrophoresis and the extraction of InsP₃ 3-kinase activity. The data suggest that TSH reduced the activity of InsP₃ 3-kinase in FRTL-5 cells either by a phosphorylation/dephosphorylation mechanism, or by affecting expression of the enzyme.

Journal of Endocrinology (1995) 144, 527–532

ABSTRACT

While prostaglandin F_2α (PGF_2α) has been thought to be a natural luteolysin in non-primates, a luteolytic effect in the human corpus luteum is less evident. We therefore investigated the action of PGF_2α on monolayer cultures of human luteal cells obtained from mid-luteal phase corpora lutea.

PGF_2α increased basal and human chorionic gonadotrophin (hCG)-stimulated progesterone production by human cultured luteal cells. A potent tumour-promoting phorbol ester, phorbol 12-myristate-13-acetate (PMA), also stimulated progesterone production by cultured human luteal cells.

Although human luteal cells were incubated for 24 h with PMA, hCG was still able to stimulate the production of progesterone by PMA-pretreated cells. However, PMA pretreatment blocked the ability of PGF_2α to stimulate progesterone production. It is possible that the luteotrophic effect of PGF_2α may be mediated, in part, by the activation of protein kinase C.

Addition of PGF_2α to suspensions of human luteal cells preincubated with myo-[2-³H]inositol promoted an increase in labelled inositol phosphates. PGF_2α also rapidly increased intracellular free Ca²⁺ in human luteal cells loaded with the fluorescent Ca²⁺ probe, fura-2.

We conclude that PGF_2α and PMA stimulate progesterone production and that PGF_2α increases the intracellular free calcium and inositol phosphates of human cultured luteal cells in the mid-luteal phase.

Journal of Endocrinology (1992) 133, 451–458

ABSTRACT

We have characterized α₁-adrenergic receptor subtypes in functional rat thyroid cells, FRTL, with relation to iodide efflux, and have also examined the effect of TSH on α₁ receptor subtypes. FRTL cells grown in a medium containing 5 mU TSH/ml (6H cells) had five times the number of α₁ receptors of those maintained in TSH-free medium (5H cells) (11·2 fmol/10⁶ cells compared with 2·0 fmol/10⁶ cells). Pretreatment with chlorethylclonidine (CEC; 10 μmol/l), which inactivates only α_1b receptors, caused 98·8% and 97·0% decreases in the density of specific [³H]prazosin-binding sites in 5H and 6H cells respectively. LIGAND computer program analysis of the displacement curves for 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane (WB4101) showed that FRTL cells contained mostly low-affinity WB4101 sites. Using the phenoxybenzamine inactivation method, we found a linear relationship between α₁ receptor density and the cytosolic free Ca²⁺ concentration response in FRTL cells. Pre-exposure of intact FRTL cells to CEC caused a 98·7% decrease in noradrenaline-stimulated maximal increase in cytosolic free Ca²⁺. Also, CEC and 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8), but not nicardipine, inhibited noradrenaline-stimulated iodine efflux. The results suggest that FRTL cells contain mostly the α_1b-adrenergic receptor subtype; that the α_1b receptors mediate cytosolic free Ca²⁺ and iodide efflux responses, and that TSH enhances these responses by increasing the α_1b receptor density without affecting the post-receptor mechanism.

Journal of Endocrinology (1990) 124, 433–441

ABSTRACT

To determine whether thyrotrophin (TSH)-induced desensitization requires a thyroid-specific factor(s), the human TSH (hTSH) receptor was expressed in Chinese hamster ovary cells. The first incubation of the cells with TSH decreased the subsequent response of adenosine 3′,5′-cyclic monophosphate to freshly added TSH in the second incubation. This homologous desensitization was observed as early as after 3 h of the first incubation. The lowest dose of TSH that elicited desensitization was 0·1 nmol/l. The desensitization was not overcome by adding higher doses of TSH in the second incubation. A ¹²⁵I-labelled TSH-binding study revealed a decrease in the number of high-affinity binding sites but not in that of low-affinity binding sites. The data suggest that TSH-induced desensitization in hTSH receptor-transfected cells is caused, at least in part, by a decrease in the number of TSH receptors on the cell surface. The evidence demonstrates, contrary to an earlier report, that a thyroid-specific factor(s) is not required for hTSH receptor desensitization.

Journal of Endocrinology (1993) 139, 425–429

ABSTRACT

The present studies were carried out to investigate the effect of several growth factors on human endometrial stromal cells.

In human endometrial stromal cells, bombesin and bradykinin provoked an increase in intracellular free Ca²⁺ and in labelled inositol phosphates when pre-incubated with [³H]myoinositol. Some or possibly all of the initial increase in intracellular free Ca²⁺ represented a mobilization of Ca²⁺ from intracellular stores and the second phase of the response depended on Ca²⁺ influx from the extracellular medium.

[³H]Thymidine was added to human cultured endometrial stromal cells with bombesin, bradykinin, epidermal growth factor (EGF), prostaglandin F_2α, vasopressin and platelet-derived growth factor. Bombesin, bradykinin and EGF stimulated the incorporation of [³H]thymidine into DNA in quiescent cells.

In conclusion, bombesin and bradykinin are growth factors which activate phospholipase C in human endometrial stromal cells, while EGF stimulates DNA synthesis without the activation of phospholipase C.

Journal of Endocrinology (1991) 131, 313–318

Gonadotropin-releasing hormone (GnRH) and its agonist analog (GnRHa) are well known to have luteolytic effects. We previously reported that prolactin (PRL) stimulated matrix metalloproteinase (MMP)-2 activity to degrade collagen type IV as a mechanism of structural luteolysis. The effects of GnRHa treatment on developed corpora lutea are unknown. In this study we assessed the effect of GnRH on MMP expression and induction of structural involution of developed corpora lutea of superovulated rats using GnRHa. Pregnant mare serum gonadotropin-human chorionic gonadotropin (hCG)-synchronized ovulation and luteinization were induced in immature female rats, followed by daily treatment with GnRHa from 5 days after hCG treatment. GnRHa-induced involution of corpora lutea was evident 3 days after the treatment, as shown by their markedly smaller size (60% of the control weight). Nine days after hCG injection, serum progesterone and 20alpha-dihydroprogesterone concentrations were as low as those associated with structural luteolysis. These findings revealed that GnRHa has the ability to induce structural luteolysis in superovulated rats in the same way that PRL does. To gain information on mechanisms of luteal involution induced by GnRHa, we performed gelatin zymography. This showed a significant increase in the active form of MMP-2 in the luteal extract of GnRHa-treated rats (more than twofold that of the control). Activation of pro-MMP-2 by membrane type-MMP (MT-MMP) is reported to be a rate-limiting step for catalytic function. Another function of MT-MMP is to degrade collagen types I and III. The plasma membrane fraction of corpora lutea of GnRHa-treated rats activated pro-MMP-2 of fetal calf serum, resulting in a marked shift of the 68-kDa band to the 62-kDa band in the zymogram. A Northern hybridization study also revealed simultaneous significant increases in expression of MMP-2 mRNA and MT1-MMP mRNA in corpora lutea of GnRHa-treated rats (more than threefold the control level). In summary, hormonal and histological features of corpora lutea of GnRHa-treated superovulated rats correspond to those of structural luteolysis. GnRHa stimulated the expression of MMP-2 and MT1-MMP in developed corpora lutea associated with involution. These findings support the conclusion that MMP-2, activated by MT1-MMP, and MT1-MMP itself, remodel the extracellular matrix during structural luteolysis induced by GnRHa.