ABSTRACT

We have developed a fetal rat hypothalamic cell culture system for the study of factors controlling the acute release of TRH. Release of TRH by the cells has been characterized by reversed-phase high pressure liquid chromatography and about 86% of the total immunoreactivity in the medium co-eluted with synthetic TRH. Release of TRH by the cells in response to 56 mmol K⁺/l increased between days 5 and 9 of culture but reached a plateau thereafter. Cell contents of TRH did not change significantly between days 5 and 14 of culture.

Release of TRH from the cells was stimulated by K⁺ (56 mmol/l), veratridine (100 μmol/l) and ouabain (100 μmol/l) to 550, 480 and 335% of basal release respectively over a 1-h period. Release of TRH was dependent upon calcium in that it was absent when calcium-free medium was used and could be blocked by verapamil (20 μmol/l); however it could not be blocked by nifedipine (50 μmol/l). The calcium ionophore A23187 (1 μmol/l) stimulated TRH release to 340% of basal release. Tetrodotoxin (1 μmol/l) completely abolished the release in response to veratridine but had no effect on the release stimulated by K⁺ (56 mmol/l).

The calmodulin antagonists trifluoperazine and triflupromazine (50 μmol/l) inhibited veratridine-stimulated TRH release. This was at a site after calcium influx as they also inhibited A23187-stimulated TRH release. The highly specific calmodulin antagonist W7 (10 μmol/l) also inhibited both veratridine and A23187-stimulated TRH release whereas, at the same concentration, its inactive analogue W5 did not significantly inhibit TRH release in response to either stimulus.

These results confirm that fetal rat hypothalamic cell cultures release authentic TRH which can be stimulated by a number of depolarizing agents. Calcium is essential for depolarization-induced release which is also dependent on calmodulin. Fetal rat hypothalamic cell cultures are a valid model for the study of factors controlling the release of TRH.

J. Endocr. (1987) 115, 255–262

ABSTRACT

The aim of this study was to investigate the role of thyroid hormones and glucocorticoids on GH secretion. Secretion of GH in response to GH-releasing hormone (GHRH) (5 μg/kg) was markedly (P < 0·001) decreased in hypothyroid rats in vivo (peak GH responses to GHRH, 635 ± 88 μg/l in euthyroid rats vs 46 ±15 μg/l in hypothyroid rats). Following treatment with tri-iodothyronine (T₃; 20 μg/day s.c. daily for 2 weeks) or cortisol (100 pg/day s.c. for 2 weeks) or T₃ plus cortisol, a marked (P <0·01) increase in GH responses to GHRH was observed in hypothyroid rats (peak GH responses, 326 ±29 μg/l after T₃ vs 133+19 μg/l after cortisol vs 283 ± 35 μg/l after cortisol plus T₃). In contrast, none of these treatments modified GH responses to GHRH in euthyroid animals. Hypothyroidism was also associated with impaired GH responses to the GH secretagogue, Hisd-Trp-Ala-Trp-d-Phe-Lys-NH₂ (GHRP-6). Secretion of GH in response to GHRP-6 in vivo was reduced (P <0·01) in hypothyroid rats (peak GH responses, 508 ± 177 μg/l in euthyroid rats vs 203 ± 15 μg/l in hypothyroid rats). In-vitro studies carried out using monolayer cultures of rat anterior pituitary cells derived from euthyroid and hypothyroid rats showed a marked impairment of somatotroph responsiveness to both GHRP-6 and somatostatin in cultures derived from hypothyroid rats. In summary, our data suggest that thyroid hormones and glucocorticoids influence GH secretion by modulating somatotroph responsiveness to different GH secretagogues.

Journal of Endocrinology (1989) 121, 31–36

ABSTRACT

We have studied the effect of dopamine together with agonist and antagonist drugs of different specificities on the release of TRH from the perfused, intact hypothalamus of the adult rat in vitro. Dopamine produced a dose-related stimulatory effect on TRH release with maximal effect being achieved at 1 μmol/l (increase over basal, 118 ±16·5 (s.e.m.) fmol TRH; P <0·001 vs basal). This effect was mimicked by the specific D₂-agonist drugs bromocriptine (0·1 μmol/l) and LY 171555 (0·1 μmol/l) (increase over basal values, 137·5±13·75 fmol and 158·6± 10·7 fmol respectively; P <0·001 vs basal), but not by the D₁-agonist SKF 38393A. The stimulatory effect of dopamine (1 μmol/l) was blocked in a stereospecific manner by the active (d) but not by the inactive (l) isomers of the dopamine antagonist butaclamol. Similar blockade was achieved with the specific D₂-antagonist domperidone (0·01 μmol/l) whereas the D₁-antagonist SCH 23390 was only effective when used at a concentration 100 times greater. Lower concentrations (0·01 μmol/l) of this D₁ -antagonist did not block the stimulatory effect of dopamine. High-performance liquid chromatography characterization of the material secreted within the hypothalamus showed one single peak of immunoreactive material which coeluted with synthetic TRH. These data suggest that dopamine exerts a stimulatory role in the control of hypothalamic TRH release by acting at specific D₂-receptors.

J. Endocr. (1987) 115, 419–424

ABSTRACT

Effects of thyroid status on brain catecholamine turnover in adult rats were investigated using a steady-state method. Rats were treated for 3 weeks with s.c. injections of l-thyroxine (0·4 mg/kg), aminotriazole in drinking water (0·1%, w/v) or vehicle. After 2 weeks of treatment rats were implanted chronically with lateral intracerebroventricular (i.c.v.) cannulae. They were injected i.c.v. with [³H]tyrosine 1 week later. Catecholamine and tyrosine content and specific activity were measured in mediobasal hypothalamus, anterior hypothalamus and striatum, using high-performance liquid chromatography with electrochemical detection. Thyroxine treatment resulted in a significant increase in noradrenaline and dopamine synthesis localized to the mediobasal hypothalamus. Conversely, aminotriazole treatment resulted in a significant decrease in noradrenaline synthesis localized to the mediobasal hypothalamus. The localization of these changes in catecholamine turnover to the mediobasal hypothalamus suggests that they may be specific functional effects which are of importance in the overall integrated control of thyroid function.

J. Endocr. (1986) 111, 383–389

Abstract

We have studied the effects of glucose on the release of somatostatin (SS), TRH and GHRH from incubated hypothalami of normal and genetically diabetic, Goto-Kakizaki (GK) rats. The active isomer d-glucose caused a dose-related inhibition of SS, TRH and GHRH from normal rat hypothalami over a 20-min incubation period in vitro. In contrast, in GK rats the effects of glucose on TRH and SS were significantly reduced and the effects on GHRH were abolished. These data indicate that the sensitivity of SS-, TRH- and GHRH-producing hypothalamic neurones is reduced in diabetic rats. The effect is most pronounced for GHRH release as there was no change in the release of this peptide with increasing glucose concentrations. In conclusion, it appears that the diabetic state in GK rats causes differential desensitisation (GHRH>TRH and SS) of neuronal responses to subsequent changes in glucose concentrations in vitro. This may be due to alterations in the neurotransmitter control and/or a reduction in number, affinity or function of glucose transporters on these peptidergic neurones or other intermediary neuronal pathways.

Journal of Endocrinology (1996) 151, 13–17

ABSTRACT

We have tested the hypothesis that α-adrenergic drive is involved in the nocturnal increase in TSH in man. Seven mildly hypothyroid women (basal TSH levels 5·0–11·0 mU/1), aged 38–60 years, and nine euthyroid women, aged 27–60 years, were studied. Subjects underwent α-adrenergic blockade by infusion of thymoxamine (210 μg/min from 19.00 to 24.00 h); the same women were used as controls, with saline infused on different nights. Subjects were not allowed to sleep during the study period.

A clear evening rise in basal TSH levels was apparent in both normal subjects and patients. Although overall secretion of TSH was slightly decreased in normal subjects (mean ± s.e.m. area under the curve, 29·93 ± 0·96 vs 30·71 ±mU/1 per h; P<0·05), thymoxamine infusion did not produce any major alteration in the gradual rise in TSH levels during the evening (incremental change above baseline +0·96± 0·21 during control infusion and + 0·97 ± 0·27 mU/1 during thymoxamine infusion). In mildly hypothyroid patients the TSH changes were exaggerated and α-adrenergic blockade caused a reduction in basal TSH levels and a delayed rise in TSH (incremental change above baseline +2·93 ± 1·42 during control infusion and +2·26 ± 0·73 mU/1 during thymoxamine infusion; P < 0·02). Overall TSH secretion was significantly decreased by thymoxamine (mean ± s.e.m. area 106 ± 2·45 mU/1 per h vs 123·32 ± 3·68 in the control study; P<0·0001). As expected, no circadian change was observed in basal prolactin levels in either controls or patients.

Although α-adrenergic pathways may play a role in modulating the nocturnal increase in basal TSH levels, our data suggest that the evening rise in TSH is not a consequence of a primary increase in α-adrenergic drive. The increased TSH changes of mildly hypothyroid patients may, however, be sustained by increased central α-adrenergic stimulation of the TSH secretion.

J. Endocr. (1987) 115, 187–191

ABSTRACT

Recent data suggest that the response of GH to GH-releasing factor (GRF) is reduced following prior exposure to high concentrations of GRF. However, it is unknown whether this is due to alterations in GRF receptors, adenylate cyclase activity or the size of a GRF-releasable storage pool of GH. In order to clarify these questions we have compared the effects of pretreatment with GRF (10 nmol/l every 2 h for 12 h) with those of pretreatment with somatostatin (SRIF; 1 μmol/l), forskolin (10 μmol/l) and GRF plus SRIF (10 nmol/l and 1 μmol/l added together) on the subsequent responses of GH to GRF (1 pmol/l–10 nmol/l), cholera toxin (10 nmol/l), 3-isobutyl-1-methylxanthine (IBMX) (100 μmol/l) and forskolin (10 μmol/l). Experiments were performed on 4-day monolayer cultures of rat anterior pituitary cells. The cells were pretreated with test substances every 2 h for 12 h and incubated with GRF or forskolin for 3 h.

Per cent maximal (B_max) GH responses to GRF (10 nmol/l) were reduced after pretreatment with both GRF (control, 173% of basal; GRF, 25% of basal; P < 0·001) and forskolin (98% of basal; P < 0·001), but were increased after pretreatment with SRIF (246% of basal; P < 0·02). However, GH responses after pretreatment with GRF plus SRIF were not significantly different from those of the control. The dose giving 50% maximum stimulation (ED₅₀) of GRF action was unaltered by SRIF pretreatment (control 48·5 ± 9·2 (s.e.m.) pmol/l vs 140 ± 38 pmol/l; P > 0·05) or by forskolin pretreatment (140 ± 70 pmol/l; P > 0·05), but was increased by GRF pretreatment (900 ± 230 pmol/l; P < 0·05) and GRF plus SRIF pretreatment (1010 ± 730 pmol/l; P < 0·01). Growth hormone responses to forskolin (32% of basal after GRF pretreatment vs 121% of basal in the control saline-treated cells; P < 0·01), cholera toxin (40% of basal after GRF pretreatment vs 106% of basal in the control cells; P < 0·025) and IBMX (195% of basal in the GRF-pretreated cells vs 133% of basal in the control cells; P < 0·025) were also decreased after GRF pretreatment in comparison with controls. Responses of cAMP to 10 μmol forskolin/l were not different from controls after pretreatment with either GRF, SRIF or GRF plus SRIF. However, cAMP responses to 10 nmol GRF/l were reduced after pretreatment with GRF, SRIF, GRF plus SRIF or forskolin.

Reduction of B_max GH responses to GRF, cholera toxin, IBMX and forskolin by GRF pretreatment, and the reversal of this action by combined SRIF plus GRF pretreatment, suggests that depletion of the GRF-sensitive releasable pool of GH may contribute to the desensitization phenomenon. In addition, however, the increase in ED₅₀ of GRF following GRF or GRF plus SRIF pretreatment, as compared with control or SRIF pretreatment alone, together with decreased cAMP responses to GRF after GRF plus SRIF pretreatment, supports the view that uncoupling of the regulatory protein of adenylate cyclase, N_s, also plays a role in this phenomenon.

J. Endocr. (1988) 116, 185–190

ABSTRACT

In order to investigate whether the impaired GH secretion associated with hypothyroidism and hyperthyroidism is due to a hypothalamic or a pituitary disorder, we have studied plasma GH responses to GH-releasing factor (1–29) (GRF) in euthyroid, hypothyroid and hyperthyroid rats. Hypothyroid rats showed a significant (P< 0·001) reduction in GH responses to GRF (5 μg/kg) at 5 min (350 ± 35 vs 1950 ±260 μg/l), 10 min (366±66 vs 2320 ± 270 μg/l) and 15 min after GRF injection (395 ± 72 vs 1420 ± 183 μg/l; means ± s.e.m.) compared with euthyroid rats. Hyperthyroid rats showed a significant (P<0·05) decrease in GH responses to 5 μg GRF/kg after 30 min (200±14 vs 325 ± 35 μg/l) but not at other time-points, or after the administration of 1 μg GRF/kg. These data indicate that in hypothyroidism and perhaps hyperthyroidism there is an alteration in the responsiveness of the somatotroph to GRF administration.

J. Endocr (1986) 109, 53–56