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

We have investigated the relationship between cyclic nucleotides and nerve function in the sciatic nerve of rats made diabetic with streptozotocin. Cyclic AMP (cAMP) content in the sciatic nerves of diabetic rats was significantly (P < 0·05) lower than in those of normal rats, while cyclic GMP content did not differ between the two groups. Administration of the stable prostacyclin analogue iloprost or dibutyryl cyclic AMP (dbcAMP) significantly (P<0·05) restored the cAMP content in the sciatic nerves and motor nerve conduction velocity, which reflects nerve function. There was a positive correlation between cAMP content in the sciatic nerves and motor nerve conduction velocity in both normal and diabetic rats.

Endoneurial preparations of sciatic nerves obtained from normal rats were incubated in Krebs–Ringer bicarbonate buffer containing d-glucose (30 or 5·5 mmol/l). Cyclic AMP accumulation was significantly (P<0·05) suppressed in the buffer containing 30 mmol d-glucose/l compared with that containing 5·5 mmol/l. Iloprost (P<0·05) and dbcAMP (P<0·01) increased cAMP accumulations in the tissues incubated in buffer containing both 5·5 and 30 mmol d-glucose/l. When non-metabolizing hexoses, such as l-glucose or 3-O-methylglucose instead of d-glucose were used, cAMP accumulations at 30 mmol hexose/1 were not significantly different from those at 5·5 mmol/l.

Cyclic AMP phosphodiesterase activity in the sciatic nerves of diabetic rats did not change compared with that in nerves from normal rats. Although not significant, mean ATP content in the sciatic nerves of diabetic rats was about 30% lower than that in nerves of normal rats. Basal, iloprost-stimulated and forskolin-stimulated adenylate cyclase activities in the sciatic nerves of diabetic rats were significantly (P<0·05) reduced when compared with those of control rats.

We therefore conclude that reduction of cAMP content in peripheral nerves may be involved in the pathogenesis of diabetic neuropathy and is mainly caused by the impairment of adenylate cyclase activity in the diabetic state.

Journal of Endocrinology (1993) 136, 431–438

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

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