Lack of an acute modulatory effect of melatonin on human nocturnal thyrotropin and cortisol secretion

Using a recently developed model for investigating the neuroendocrine role of melatonin in man, we studied melatonin's effect on the nocturnal secretion of thyrotropin and cortisol in 17 normal male volunteers. The model consists of sleep in the dark and all-night sleep deprivation in conditions of: bright light with and without a melatonin infusion, and dim light. We have improved our infusion paradigm so that levels of melatonin during infusion are now indistinguishable from those occurring during sleep in the dark or dim light sleep deprivation. Sleep deprivation per se raised TSH levels compared to normal sleep. However, the three conditions of sleep deprivation could not be distinguished from each other, which suggests that the suppression of TSH by sleep (or the stimulation of TSH by sleep deprivation) is not mediated by melatonin. Cortisol secretion was unaffected by sleep deprivation regardless of melatonin's presence or absence. However, a difference in the pattern of secretion of cortisol in the sleep condition in the early morning (compared to the sleep deprivation conditions) was noted. These data do not implicate melatonin in the acute regulation of TSH or cortisol in normal man. These data also provide a method of melatonin infusion that replicates the pattern and levels seen in sleep.     

Lack of nocturnal serum thyrotropin surge after surgery

The effects of surgery on TSH secretion, with particular regard to the nocturnal TSH surge, were evaluated in 10 consecutive patients followed for 6 days after surgery. Surgical trauma was associated in all patients with significant decreases in serum total and free T3 and a significant increase in serum rT3 levels, with no variations in serum total and free T4 concentrations. A marked increase in serum cortisol levels was observed, with higher values at night than in the morning. Serum cortisol levels and circadian rhythm normalized on the fifth day. Serum TSH values in the morning significantly decreased on the first day after surgery and returned to normal on the second day. Serum TSH values at night (2400-0200 h) were higher than in the morning preoperatively, but the nocturnal surge was abolished from days 1-5 after surgery and was restored only on the sixth day. Thus, surgery was associated with a prolonged loss of the nocturnal serum TSH surge. This effect on TSH secretion was more marked than predictable on the basis of serum TSH measurements in the morning alone. An inverse relationship was found between serum cortisol and serum TSH values at night, suggesting that the excessive endogenous cortisol secretion might play a role in the derangement of TSH secretion.     

Participation of serotonin in thyrotropin release. I. Evidence for the action of serotonin on thyrotropin releasing hormone release.

Injection of serotonin (5-HT) into the third ventricle of the rat resulted in a rapid increase of serum TSH; a significant effect was observed 5 min after injection, whereas the maximal effect appeared 10 min after the injection of 1 microgram 5-HT. This stimulating effect of 5-HT was completely prevented by pretreatment with cyproheptadine, a blocker of 5-HT receptors, whereas fluphenazine, a dopamine receptor blocker, was unable to block it. Third venticle injection of 5-HT in rats bearing anterior hypothalamic lesions (which did not affect the suprachiasmatic nucleus or the medio-basal hypothalamus) also induced an increase of serum TSH similar to that observed in normal rats despite the fact that these animals show a lower basal TSH. In vitro, the addition of 5-HT to an incubation medium containing one hemi-anterior pituitary did not modify medium TSH, whereas 5-HT addition induced an increase of medium TSH in the system containing one hemi-anterior pituitary and two hypothalami. We conclude that 5-HT acts on TSH function probably through a stimulation of TRH release.     

Participation of serotonin in thyrotropin release. II. Evidence for the action of serotonin on the phasic release of thyrotropin.

During a 12-h light 12-h dark schedule (lights off at 1900 h), male Sprague-Dawley rats show a circadian rhythm of plasma TSH with a zenith near midday. The participation of serotonin (5HT) in the phasic release of TSH was studied using both pharmacological and surgical-stereotaxical approaches. Animals treated with parachlorophenylalanine methyl ester (pCPA), an inhibitor of 5HT synthesis (one or two injections of 250 mg/kg each) showed a reduction or a disappearance of the diurnal peak of TSH, respectively. Additional treatment by 5-hydroxytryptophan, a precursor of 5HT, completely, restored the diurnal TSH peak. Treatment with 5,6-dihydroxytryptamine creatine sulfate, a neurotoxin which selectively destroys 5HT terminals, also induced alterations of the diurnal peak of TSH. There were no major modifications observed in the low nocturnal levels of TSH in rats treated with pCPA, 5-hydroxytryptophan, or 5,6-dihydroxytryptamine. The major serotoninergic innervation of the hypothalamus originates from the raphe dorsalis or centralis; destruction of these two nuclei caused a quasiabolition of the diurnal TSH peak (only a low amplitude TSH circadian rhythm persisted). Hypothalamic 5HT content was measured in the majority of these experiments; the greatest depletions (near 90%) were observed after two injections of pCPA or in rats bearing raphe lesions. We conclude that the diurnal peak of TSH, observed during the physiological circadian rhythm, is serotoninergic dependent.     

Circadian and pulsatile thyrotropin release in treated acromegalics

We studied the 24-h TSH profiles of 16 treated male acromegalic patients (age range 26-68 yr) in clinical and biochemical remission. Eight had undergone transsphenoidal surgery, the others surgery and pituitary irradiation. Blood samples were taken at 20-min intervals; circadian rhythms were established by cosinor analysis, pulsatile release with the Cluster programme. All patients, except one irradiated subject, were euthyroid. TSH reserve was diminished preoperatively in 7 subjects and at the time of the profile study in 10 subjects, one of whom was biochemically hypothyroid. A significant circadian rhythm was present in 14 subjects and absent in the hypothyroid patient. The acrophase occurred at 2.46 +/- 0.51 h in nonirradiated patients and at 3.37 +/- 0.38 h in irradiated patients (NS). About 10 TSH pulses/24 h (range 6-13) were detected; there was no significant difference between irradiated and non-irradiated patients. With cross-correlation techniques synchronous release of TSH and PRL was demonstrated in 7 out of 8 nonirradiated patients in contrast to only 2 of the irradiated patients. This study demonstrates a qualitatively normal TSH secretion pattern for treated acromegalic patients, but the absolute TSH levels are clearly low compared with published data on normal subjects. The present findings can be explained by a diminished TSH cell mass; in addition radiation therapy causes a disturbance at the hypothalamic level, as indicated by the loss of synchronism between TSH and PRL release.     

Effect of diabetes mellitus on thyrotropin release from isolated rat thyrotrophs.

The effect of streptozotocin-induced diabetes on thyrotropin (TSH) secretion was studied in vitro in male rats, using purified thyrotrophs isolated by centrifugal elutriation. The development of diabetes was associated with a significant fall in serum T4 (4.9 +/- 1.5 vs. 1.2 +/- 0.7 micrograms/dl, control vs. diabetic), T3 (59 +/- 16 vs. 20 +/- 9 ng/dl) and TSH (53 +/- 22 vs. 23 +/- 18 microU/ml). In vitro, basal TSH release was lower in cells from diabetic rats (1570 +/- 248 microU/10(6) cells) than in controls (2612 +/- 765 microU/10(6) cells) after 48 h in culture. Furthermore, thyrotropin-releasing hormone (TRH)-induced TSH release was decreased about 30% and the intracellular TSH content was also reduced 45% in diabetic rat thyrotrophs compared with controls. The addition of 200 microU/ml of insulin in the incubation media did not change the TRH-stimulated TSH release, but it decreased the basal TSH release by 42%. Preincubation of thyrotrophs with glucose (25 mM) did not impair the basal or TRH-stimulated TSH release. In the same experimental conditions, no effect of insulin or glucose was seen on the basal or TRH-stimulated TSH release from thyrotrophs of diabetic rats. Preincubation of thyrotrophs with 10, 100, or 1000 nM of T4 for 48 h decreased basal TSH release by 27%, 45%, and 48%, respectively, and reduced TRH-induced TSH release by 46%, 48%, and 55%; 100 nM T4 also reduced TRH-induced TSH release from diabetic thyrotrophs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of terbutaline on TRH-induced prolactin and thyrotropin release in normal subjects

To investigate whether terbutaline (T) influences the release of prolactin (Prl) and/or thyrotropin (TSH) from the anterior pituitary, 25 micrograms thyrotropin-releasing hormone (TRH) was injected iv in 7 normal subjects who were pre-treated orally with either T or placebo. The TRH-induced Prl response, as reflected by the Prl incremental area, was more pronounced after priming with placebo (2071 +/- 606) than after T (1391 +/- 434; P less than 0.05). In contrast, the TRH-elicited TSH responses did not differ significantly after the two pre-treatments. When TRH was given to 8 additional individuals on iv background infusion of either T or saline, the Prl response was significantly larger during saline (2123 +/- 354) than during T infusion (1540 +/- 235; P less than 0.01), whereas the TSH responses were of similar magnitudes. Six subjects, given 25 micrograms TRH iv on background infusion of T or saline, were also given propranolol orally before commencement of the T infusion and placebo before the saline infusion. This resulted in similar Prl responses and also in similar TSH responses, during the two background treatments. The results imply that oral as well as iv administration of T has inhibitory influence on human lactotrophs, but lacks measurable effect on thyrotrophs.     

Norepinephrine and thyrotropin effects on the thyroid in vitro: simultaneous stimulation of iodide organification and antagonism of thyroxine release

Norepinephrine (NE), which has previously been shown to inhibit TSH-induced T4 release by mouse thyroids in vitro, was found to stimulate iodide organification. The concentration of NE (6 X 10(-7) M) necessary to stimulate organification of iodide was 10 times less than the concentration (6 X 10(-6) M) required for inhibition of TSH-induced T4 release. Both actions of NE were exerted through an alpha-adrenergic receptor, since they were inhibited by phentolamine but not by l-propranolol. One milliunit of TSH maximally stimulated T4 release only, but larger amounts (100 mU) also stimulated organification. TSH stimulation of T4 release and organification was not affected by adrenergic antagonists and therefore was not mediated by adrenergic receptors. N6, O2-Dibutyryl cAMP and isobutylmethylxanthine, like TSH, stimulated T4 release. Their actions were inhibited by NE. However, both compounds, unlike TSH, failed to enhance organification in mouse thyroids. The effects of TSH and NE on the cAMP content of incubated mouse thyroids were also studied. TSH induced a prolonged increase in thyroidal cAMP during the 90-min incubation; this increase was unaffected by alpha- or beta-adrenergic antagonists. In contrast, NE (6 X 10(-5) M) produced a transient but significant increase in cAMP only within the first 5 min. Unlike the action of NE on organification, this short term stimulatory effect on cAMP production was mediated by a beta-adrenergic receptor, since it was blocked by l-propranolol but not by phentolamine. The following conclusions were reached: 1) stimulation of iodide organification and thyroid hormone release involves different sensitivity thresholds for TSH and NE; 2) TSH stimulation of iodide organification, hormone release, and cAMP formation is not exerted through adrenergic receptors; 3) NE stimulates organification and inhibits TSH-stimulated T4 release through alpha-adrenergic receptors, but stimulates cAMP production through beta-receptors; and 4) cAMP may not be the mediator of all TSH actions on the thyroid.     

Neuroendocrine regulation of thyrotropin release in cultured human pituitary cells

The regulation of TSH release in man was investigated using cell cultures derived from human pituitaries obtained within 24 h of accidental death. TRH stimulated TSH release in a dose-dependent manner. The ED50 was 2.9 +/- 0.6 (+/- SEM) nmol/L, similar to that reported for rat pituitary cell cultures. The release of TSH was calcium dependent, since the calcium channel antagonist verapamil inhibited TRH-stimulated TSH release, and the calcium ionophore A23187 stimulated TSH release. 12-O-Tetradecanoyl-phorbol-13-acetate stimulated TSH secretion, while dibuytryl cAMP had no effect. Epinephrine and serotonin stimulated TSH release, and dopamine and somatostatin inhibited TRH-stimulated TSH release. These findings have directly demonstrated that the regulation of TSH secretion by hypothalamic neuropeptides and biogenic amines in the human pituitary is similar to that in the rat. The development of a tissue culture system to study thyrotrophs from postmortem human pituitaries provides the means for detailed studies of the regulation of TSH secretion in man.     

Acute effects of estradiol pretreatment on the response to d-amphetamine in women

Little is known about the interactions between ovarian hormones and responses to psychoactive drugs in humans. Preclinical studies suggest that ovarian hormones such as estrogen and progesterone have direct and indirect central nervous system actions and that these hormones can influence behavioral responses to psychoactive drugs. In the present study, we assessed the subjective and physiological effects of d-amphetamine (AMPH; 10 mg p.o.) after pretreatment with estradiol. Two groups of healthy, regularly cycling women participated in two sessions scheduled during the early follicular phases of two menstrual cycles. One group received estradiol patches (Estraderm TTS; 0.8 mg) which elevated plasma estradiol levels to approximately 750 pg/ml on both sessions; the other group received placebo patches on both sessions. Both groups received AMPH (10.0 mg) and placebo in a randomized and counterbalanced order on the two sessions. Dependent measures included self-report questionnaires, physiological measures, and plasma hormone levels. Most of the subjective and physiological effects of AMPH were not affected by acute estradiol treatment. Nevertheless, estradiol pretreatment increased the magnitude of the effects of AMPH on subjective ratings of 'pleasant stimulation' and decreased ratings of 'want more'. Also, estradiol produced some subjective effects when administered alone: It increased subjective ratings of 'feel drug', 'energy and intellectual efficiency', and 'pleasant stimulation'. These results provide limited evidence that the stimulating effects of AMPH are increased by acute estradiol pretreatment.     

Lack of association between thyrotropin receptor gene polymorphisms and subclinical hypothyroidism in children

Subclinical hypothyroidism is defined as a serum TSH level above the statistically set reference range, associated to normal free thyroid hormone concentrations. Genetic and environmental factors contribute to the inter- and intra-individual biological variations of TSH levels, sometimes leading to uncertainty of treatment in the clinical practice, especially when moderate elevations above the upper limit of the reference range are considered (5< TSH <10 mIU/l). In this view, the study of association between subclinical hypothyroidism and possible molecular effectors, such as polymorphisms in the TSH receptor (TSHR) gene, could be interesting. In this paper, we analyzed the TSHR gene polymorphisms in 103 hyperthyrotropinemic infants. A control group of 120 newborns of the same ethnic background was used to evaluate the frequencies of each polymorphism in the population. We found a statistically significant difference in the allelic frequency of the P52T polymorphism, being that the T variant was more represented in the control group (p=0.03). However, no significant results have been obtained in the analysis of the association between genotypes and serum TSH levels. In conclusion, we analyzed 7 polymorphic variants of TSHR gene in subclinical hypothyroidism. The only significant result refers to the allelic frequency of A in the P52T polymorphism, which is statistically reduced when compared with that of a control group.     

Comparison of acute effects of dynorphin and beta-endorphin on prolactin release in the rat

The ability of dynorphin and beta h-endorphin (beta h-EP) to stimulate prolactin (PRL) release in male rats was examined. Rats were injected intraventricularly with either 1 or 10 microgram dynorphin, 1 or 10 microgram beta h-EP, 10 microgram dynorphin together with 20 microgram naloxone (NAL), or an equivalent volume (13 microliter) of saline. Dynorphin caused a dose-dependent release of PRL which was significant 10 min after injection but not at later sampling times. beta h-EP also increased serum PRL levels and maintained elevated PRL levels for at least 60 min. NAL, a specific opiate antagonist, completely blocked dynorphin-induced PRL release. These results indicate that dynorphin is a potent stimulator of PRL release, but its stimulatory activity is transient relative to the activity exhibited by beta h-EP. Furthermore, dynorphin's action is specific since NAL completely blocked dynorphin induced PRL release.     

Amiodarone effects on thyrotropin receptors and responses stimulated by thyrotropin and carbachol in cultured dog thyroid cells

Amiodarone is an antiarrhythmic drug that often induces thyroid disorders. Its effects on several aspects of thyroid function were studied using cultured dog thyroid cells. Within 5-60 min of incubation of cell membranes with amiodarone, there were profound changes in adenylate cyclase activity and TSH receptor binding. Amiodarone specifically decreased TSH-stimulated adenylate cyclase activity, but not the basal or forskolin-stimulated activities, while it increased the binding of 125I-labeled TSH to its receptors. Significant effects were seen with 5-10 microM amiodarone, with maximal effects at 50-100 microM, when TSH-stimulated adenylate cyclase activity was completely blocked and the labeled TSH binding increased 4- to 5-fold over control. These effects of amiodarone were reversible, since membranes exposed to 50 microM amiodarone for 1 h exhibited normal binding and cyclase activities, when amiodarone was removed by washing before the assay. The above effects of amiodarone were also observed when cells, instead of membranes, were treated with the drug, although the magnitude of changes was less than in membranes. Lower concentrations of amiodarone (10-25 microM) caused significant inhibition of iodide organification, without affecting iodide uptake, while higher concentrations (50-100 microM) inhibited organification by nearly 75% and uptake by about 20%. Amiodarone (10-100 microM) also inhibited [3H]2-deoxy-glucose uptake and the increase in intracellular calcium concentration in response to TSH and carbachol. In contrast to membranes, treatment of cells with amiodarone caused persistent inhibition of TSH-stimulated cAMP formation and iodide organification even 24-48 h after removal of the drug. However, amiodarone had no effect on cell viability, as judged by trypan blue exclusion and ability to remain attached to the culture dishes. These results suggest that amiodarone has specific inhibitory effects on agonist-stimulated functions in thyroid cells, possibly by interfering with TSH-receptor interactions and also at the level of cholinergic receptors.     

Studies on the mechanism of 3,5,3'-triiodothyronine-induced suppression of secretagogue-induced thyrotropin release in vitro

A double column perifusion procedure was used to study the feedback inhibition of L-T3 on TSH secretion from rat anterior pituitary fragments. Matching pituitary halves were pretreated with T3 (10(-7) M) for 2 h before exposure to 10(-8) M TRH, 59 mM K+, or 5 mM Ba2+ . TRH, high K+, and Ba2+ resulted in a 2-fold or greater stimulation of TSH release. T3 significantly inhibited the stimulation by these secretagogues to 0.77, 0.78, and 0.52 of control for TRH, high K+, and Ba2+, respectively. Neither rT3 (10(-7) M) nor T3 added together with TRH had an effect on TSH release by this secretagogue. Perifusion with 3.5 x 10(-5) M cycloheximide or 10(-6) M actinomycin D 1 h before and during T3 administration led to greater TSH release with TRH than in the presence of T3 alone. Neither protein synthetic inhibitor increased TRH responsiveness of pituitary fragments when perifused alone. When cycloheximide was perifused in a similar protocol before high K+ or Ba2+, there again was a significant decrease in the T3-induced inhibition of TSH release by these secretagogues. Cycloheximide alone did not increase TSH release in response to high K+ or Ba2+, eliminating this as a possible explanation for the enhanced TSH response when antibiotic was present with T3. These results indicate that the in vitro effect of T3 on secretagogue-induced TSH release can be blocked by an inhibitor of protein synthesis. The inhibitory effect of T3 on high K+- and Ba2+-induced TSH release suggests that the site of the acute T3 inhibition of TSH release may be subsequent to TRH interaction with its receptor.