Rapid modulation of TRH and TRH-like peptide levels in rat brain and peripheral tissues by corticosterone.

Disturbance of glucocorticoid signaling has been implicated in several neuropsychiatric disorders including unipolar and bipolar depression and anxiety induced by maternal deprivation. Antidepressants have been shown to be neuroprotective and able to reverse damage to glia and neurons. Thyrotropin-releasing hormone (TRH) is an endogenous antidepressant that reduces the expression of glycogen synthase kinase-3beta (GSK-3beta), an enzyme that hyperphosphorylates tau and is implicated in bipolar depression, diabetes and Alzheimer's disease. In order to understand the potential role of TRH and TRH-like peptides both as mediators of the depressogenic effects of glucocorticoids and as potential therapeutics for neuropsychiatric disease, 300 g male Sprague-Dawley rats were injected i.p. with 4 mg corticosterone/0.5 ml 50% DMSO+50% ethanol and sacrificed 0, 2, 4 and 8h later. Levels of TRH and TRH-like peptides were measured in various brain regions involved in mood regulation and pancreas and reproductive tissues that mediate the metabolic and reproductive impairments associated with high glucocorticoid levels. Significant increases, ranging from 2- to 12-fold, in TRH or TRH-like peptide levels were observed in almost all brain regions studied at 4h after corticosterone injection. In cerebellum, TRH and TRH-like peptides increased 4-14-fold by 8h. TRH-like peptide levels fell 86-98% at 4h after treatment in testis. TRH, derived only from Leydig cells, was not affected. TRH and TRH-like peptides increased 2-4-fold at 8h in pancreas. TRH and TRH-like peptide concentrations in prostate were not affected by corticosterone up to 8h after injection. The 4h needed to detect a highly significant change in the TRH and TRH-like peptide levels in brain and peripheral tissues is consistent with the mediation of most corticosterone-effects via alterations in gene transcription.

Valproate and copper accelerate TRH-like peptide synthesis in male rat pancreas and reproductive tissues.

Treatment with valproate (Valp) facilitates the synthesis of TRH-like peptides (pGlu-X-Pro-NH(2)) in rat brain where "X" can be any amino acid residue. Because high levels of TRH-like peptides occur in the pancreas and pGlu-Glu-Pro-NH(2) (Glu-TRH) has been shown to be a fertilization promoting peptide, we hypothesized that these peptides mediate some of the metabolic and reproductive side effects of Valp. Male WKY rats were treated with Valp acutely (AC), chronically (CHR) or chronically followed by a 2 day withdrawal (WD). AC, CHR and WD treatments significantly altered TRH and/or TRH-like peptide levels in pancreas and reproductive tissues. Glu-TRH was the predominant TRH-like peptide in epididymis, consistent with its fertilization promoting activity. Glu-TRH levels in the epididymis increased 3-fold with AC Valp. Phe-TRH, the most abundant TRH-like peptide in the pancreas, increased 4-fold with AC Valp. Phe-TRH inhibits both basal and TRH-stimulated insulin release. Large dense core vesicles (LDCV's) contain a copper-dependent enzyme responsible for the post-translational processing of precursors of TRH and TRH-like peptides. Copper (500 microM) increased the in vitro C-terminal amidation of TRH-like peptides by 8- and 4-fold during 24 degrees C incubation of homogenates of pancreas and testis, respectively. Valp (7 microM) accelerated 3-fold the processing of TRH and TRH-like peptide precursors in pancreatic LDCV's incubated at 24 degrees C. We conclude that copper, an essential cofactor for TRH and TRH-like peptide biosynthesis that is chelated by Valp, mediates some of the metabolic and reproductive effects of Valp treatment via acceleration of intravesicular synthesis and altered release of these peptides.

Influence of thyroidal status on pituitary content of thyrotropin beta- and alpha-subunit, growth hormone, and prolactin messenger ribonucleic acids.

Thyroidectomized rats were used to study the effects of a single injection of T3 on pituitary mRNA synthesis and hormone secretion. T3 was injected ip at doses of 0, 0.2, 1, or 5 micrograms/100 g body weight, and and animals were killed 24 h later. T3 caused a significant decrease in serum TSH, but caused no significant change in either serum GH or PRL. Pituitary mRNA was quantified by slot blot hybridization with cDNA probes specific for alpha-TSH, beta-TSH, PRL, and GH. We found that both the alpha and beta mRNA subunits decreased, that PRL mRNA remained relatively unchanged, and that GH mRNA increased with increasing T3 dose. The data show that a single dose of T3 can profoundly influence mRNA levels in the anterior pituitary; the lowest dose of T3 caused maximum inhibition of alpha-TSH mRNA while beta-TSH mRNA declined further in a dose-dependent manner.

Thyroid hormone 5'-deiodinase activity, nuclear binding, and effects on mitogenesis in UMR-106 osteoblastic osteosarcoma cells.

The hyperthyroid state in vivo is associated with an increase in osteoblast number and activity, suggesting that thyroid hormone may stimulate osteoblast replication and function. We therefore examined the effects of T3 (16-1170 pM) on replication rate as assessed by cell counts in UMR-106 osteoblastic osteosarcoma cells cultured for 5-10 days in medium supplemented with 10% hormone-stripped fetal calf serum (FCS). Despite the virtual absence of thyroid hormone in the control medium (total T3 concentration, 0.02 ng/ml), the addition of T3 in concentrations to 1000 pM did not increase the cell replication rate. At higher T3 concentrations, a slight decrease in growth rate was observed. No significant 5'-monodeiodinase activity was detected in UMR-106 cell homogenates. However, nuclear binding of T3 was demonstrated in intact cells. A high-affinity nuclear binding component was identified with a Ka of 2.6 x 10(10) M-1 and a maximum binding capacity of 7.7 pg T3 per mg DNA, equivalent to 51 binding sites per cell nucleus. A lower affinity nuclear T3 binding component with a Ka of 1.8 x 10(9) M-1 was also identified. Thus, despite the presence of nuclear T3 receptors, UMR-106 cells do not exhibit a mitogenic response to T3.

Tumor necrosis factor, ceramide, transforming growth factor-beta1, and aging reduce Na+/I- symporter messenger ribonucleic acid levels in FRTL-5 cells.

Iodide uptake, which is necessary for thyroid hormone synthesis, can be inhibited by aging, withdrawal of TSH, or increased tumor necrosis factor (TNF) and transforming growth factor (TGF)-beta1 levels resulting from the nonthyroid illness syndrome. TNF induces receptor-mediated activation of sphingomyelinase, which converts sphingomyelin to ceramide, a mediator of TNF actions. Thyroid follicular cells transport iodide from blood into the follicular lumen against an iodide gradient by means of coupled transport of Na+ ions and I- ions via the Na+/I- symporter (NIS). An inward Na+ gradient is maintained by Na+/K+-ATPase. The recent cloning and sequencing of the rat NIS complementary DNA has made possible studies on the mechanism by which TSH, aging, and cytokines regulate I- uptake by thyroid cells. Young (<20 passages) and aged (>40 passages) FRTL-5 cells grown with or without TSH were treated with various concentrations of TNF, TGF-beta1, sphingomyelinase, or ceramide. NIS messenger RNA (mRNA) levels in aged cells were only 2% of those in young cells. Withdrawal of TSH from young cells reduced NIS mRNA levels by more than 90%. TNF reduced NIS mRNA levels in young cells grown with TSH at t1/2 = 0.62 days, a cycloheximide inhibitable effect. Similar treatments with TGF-beta1, sphingomyelinase, or ceramide reduced NIS mRNA by 70-90%. Ceramide reduced 125I(-)-uptake by 50%. The addition of TNF increased both the sphingomyelin and ceramide levels 3- to 5-fold in young and old cells. We conclude that 1) the decline in iodide uptake due to aging, a fall in serum TSH or an increase in TNF or TGF-beta1 is mediated primarily by a reduction in thyroid NIS expression; and 2) that receptor-mediated activation of sphingomyelinase is an important, protein synthesis-dependent, intracellular pathway for inhibition of NIS expression by TNF.

Thyrotropin-releasing hormone (TRH)-Gly conversion to TRH in rat ventral prostate is inhibited by castration and aging.

TRH levels in rat prostate are very high in the 2-month-old rat and decline at least 90% during the next 2 yr. This decline in prostatic TRH levels with aging may be a significant factor in benign prostatic hypertrophy in man and other animals. Because prostatic TRH correlates positively with serum testosterone and negatively with serum thyroid hormone levels, we have examined the possibility that the age-related decline in prostatic TRH is hormonally regulated. TRH and a TRH precursor peptide, TRH-Gly (pGlu-His-Pro-Gly), were measured in ventral prostates from 2-, 5-, and 18-month-old Fisher 344 male rats using a combination of HPLC and specific RIAs for TRH and TRH-Gly. Similar measurements were made in a group of 2-month-old Sprague-Dawley rats which were castrated, sham castrated, or left untreated for 3 or 7 days before death. Aging and castration resulted in a significant decrease in the absolute TRH concentration as well as the TRH/TRH-Gly ratio, but no significant change was observed in TRH-Gly levels. The serum testosterone concentration did not change with aging, but serum T4 levels fell significantly. Because testosterone levels do not change during the first 18 months of life in most rat strains, and hypothyroidism in young animals is associated with a significant increase in prostate TRH and TRH-Gly levels, we conclude that the aging-related decline in prostate TRH biosynthesis is not the result of a hypogonadal state.

Aging of FRTL-5 rat thyroid cells causes sensitivity to cytotoxicity induced by tumor necrosis factor-alpha.

While investigating the modulation of the growth and function of the FRTL-5 rat thyroid cell line by recombinant human tumor necrosis factor-alpha (TNF alpha), we noticed that pronounced changes in several response parameters occurred with increasing passage number. For young cells (passage less than 20), TNF alpha by itself slightly increased [3H]thymidine incorporation and DNA content, and had a minimal effect on basal 125I uptake. When combined with TSH, TNF alpha had no influence on TSH-stimulated [3H]thymidine incorporation, but significantly inhibited TSH-stimulated 125I uptake. Compared with young cells, aged cells (passage greater than 40), in contrast, developed a high sensitivity to TNF alpha. TNF alpha markedly stimulated [3H]thymidine incorporation into DNA, inhibited TSH-stimulated 125I uptake per micrograms DNA, but dramatically decreased the total DNA content and cell number. TSH augmented the TNF alpha effect in aged cells, resulting in a further reduction of DNA content. Aphidicolin, a specific inhibitor of DNA polymerase-alpha which is associated with DNA replication, dramatically inhibited TNF alpha-induced [3H]thymidine incorporation in both young and aged cells; this suggested that the effect of TNF alpha on FRTL-5 cell growth is related to DNA replication, rather than DNA repair. 51Cr release from FRTL-5 cells, a measure of cytotoxicity, increased 2-fold over baseline in aged cells at a dose of 400 ng/ml TNF alpha and decreased to 70% of baseline in young cells at this same dose. The protein kinase-A (PKA) and protein kinase-C (PKC) signal transduction mechanisms of TNF alpha in aged cells (passage greater than 40) were also studied. TNF alpha increased cAMP and also increased relative PKA and PKC activity in 1-40 min. Phorbol myristate acetate (PMA), an activator of PKC, increased [3H]thymidine incorporation and DNA content. PMA did not affect the TNF alpha-induced increase in [3H]thymidine incorporation or its reduction of DNA content. When the cells were pretreated with a high concentration of PMA (1 microM/24 h) to down-regulate PKC, the TNF alpha dose-dependent increase in [3H]thymidine incorporation and decrease in DNA content were only slightly inhibited, suggesting that the main effects of TNF alpha are independent of PKC. We conclude that the sensitivity of FRTL-5 cells to the cytotoxic effect of TNF alpha increases with aging.

Thyroid hormones modulate thyrotropin-releasing hormone biosynthesis in tissues outside the hypothalamic-pituitary axis of male rats.

In the present study we have examined the in vivo effects of thyroid hormone and TRH on secretory tissue concentrations of TRH and TRH-Gly (pGlu-His-Pro-Gly), a TRH precursor. Within secretory granules, TRH-Gly is converted to TRH through alpha-amidation of the C-terminal proline residue, using Gly as the NH2 donor. Using specific RIA, we measured the TRH-Gly immunoreactivity (TRH-Gly-IR) and TRH-IR concentrations in tissues from the reproductive and gastrointestinal systems, adrenals, and other internal organs in euthyroid, hypothyroid, and T4-treated 250-g Sprague-Dawley male rats. TRH-Gly-IR concentrations were more than 2-fold higher than TRH-IR concentrations within the adrenal, pancreas, bowel, and stomach at the time of death. Untreated hypothyroidism and exogenous TRH significantly increased adrenal TRH-Gly-IR levels. Pancreatic TRH-Gly levels increased about 2-fold in hypothyroid rats. Incubation at 60 C significantly increased TRH-Gly-IR levels in the pancreas, adrenal, bowel, stomach, and epididymis by 14-, 3-, 6-, 6-, and 6-fold, respectively. Also after 60 C incubation increases in the TRH-Gly-IR/TRH-IR ratio of 2.7-, 4-, and 1.7-fold were observed in the pancreas, epididymis, and bowel, respectively. Pooled tissue extracts were fractionated by cation exchange and reverse phase HPLC for characterization of TRH-Gly-IR. Both chromatographic methods revealed a major peak of TRH-Gly-IR coeluting with synthetic TRH-Gly. Incubation at 60 C caused 13.5-, 4.1-, 1.5-, and 5-fold increments in the TRH-Gly-IR for adrenal, pancreas, prostate, and thyroid, respectively, compared to the immediately extracted control aliquots. Cation exchange and reverse phase HPLC also revealed production of higher mol wt TRH precursor peptides after incubation at 60 C for 4 or 20 h. Only the TRH-Gly-IR peak coeluting with pGlu-His-Pro-Gly was converted into TRH by rat brain alpha-amidating enzyme. The data suggest that biosynthesis of TRH occurs in rat extrahypothalamic tissues and may be modulated by thyroid status, iv TRH, and selective thermal inactivation of enzymes that convert prepro-TRH to TRH.

Characterization of tumor necrosis factor-alpha receptors in human and rat thyroid cells and regulation of the receptors by thyrotropin.

Administration of recombinant human tumor necrosis factor-alpha (TNF) to rats and mice produces a model of nonthyroid illness in which there is impairment of hypothalamic-pituitary thyroid function, including reduced serum concentrations of T4 and T3, reduced thyroid radioiodine uptake, and reduced response to TSH. In this study, we tested the binding and effects of TNF on FRTL-5 cells and on four human thyroid carcinoma cell lines. The TSH-stimulated [125I]iodide uptake by FRTL-5 cells was inhibited by TNF in a dose-dependent manner. The four human thyroid carcinoma cell lines (NPA, MRO, ARO, WRO) have TSH receptors but did not respond to TSH in regard to iodide uptake and thymidine incorporation. Both human thyroid carcinoma cells and FRTL-5 cells contain specific receptors for TNF. Scatchard analysis showed that the receptor numbers and dissociation constants in human thyroid carcinoma cells and FRTL-5 cells were, respectively; 2.4 x 10(4), 5.4 nM (WRO); 8 x 10(3), 3.4 nM (MRO); 4 x 10(3), 1 nM (ARO); 7 x 10(3), 1 nM (NPA); 3 x 10(3), 1 nM (FRTL-5), and 9 x 10(3), 1 nM (FRTL-5 cells treated with TSH). The results indicate that TNF affects thyroid cell function through binding to the TNF receptor and that the number of TNF receptors is regulated by TSH.

Impairment of hypothalamic-pituitary-thyroid function in rats treated with human recombinant tumor necrosis factor-alpha (cachectin).

Tumor necrosis factor-alpha (TNF; cachectin), a peptide secreted from stimulated macrophages, mediates some of the metabolic derangements in inflammatory and neoplastic disorders. To determine whether TNF is responsible for the changes in hypothalamic-pituitary-thyroid (HPT) function in nonthyroid illnesses, we administered synthetic human TNF to male Sprague-Dawley rats. The rats were given TNF or saline (control; both pair fed and nonpair fed) iv (six to eight per group). HPT function was tested 8 h after administration of 200 micrograms TNF/kg BW, 8 h after 5 days of 150 micrograms TNF/kg BW, and 8 h after a 3-day series of 50, 200, and 800 micrograms TNF/kg BW. The single injection of 200 micrograms TNF/kg significantly reduced (all P less than 0.05) serum TSH, T4, free T4, T3, and hypothalamic TRH compared to the corresponding hormone levels in saline-injected control rats. Serum TSH and hypothalamic TRH recovered to normal levels after 5 days of 150 micrograms/kg TNF treatment. With the increasing daily doses of TNF, serum TSH and hypothalamic TRH fell significantly. Hepatic 5'-deiodinase activity was reduced after 1 day of TNF treatment, but increased after the 3-day series of injections. TNF treatment reduced pituitary TSH beta mRNA, but did not affect alpha-subunit mRNA. TNF treatment also reduced thyroid 125I uptake and reduced thyroidal release of T4 and T3 in response to bovine TSH, but did not change the TSH response to TRH. TNF treatment reduced the binding of pituitary TSH to Concanavalin-A, indicating that it alters the glycosylation of TSH. The TSH with reduced affinity for this lectin had reduced biological activity when tested in cultured FRTL-5 rat thyroid cells. In vitro, TNF inhibited 125I uptake by cultured FRTL-5 rat thyroid cells and blocked the stimulation of [3H]thymidine uptake by these cells. The data indicate that TNF acts on the HPT axis at multiple levels and suggest that TNF is one of the mediators responsible for alterations in thyroid function tests in patients with nonthyroidal illnesses.

Hormonal control of prostatic thyrotropin-releasing hormone (TRH) testosterone modulates prostatic TRH concentrations.

The presence of extremely high concentrations of authentic TRH in the rat prostate prompted us to examine whether prostatic TRH concentrations were under hormonal control. Both the immunoreactive TRH content per prostate and TRH immunoreactivity expressed per 100 mg prostatic protein were lower in animals 2 weeks after hypophysectomy than in sham-operated and calorically restricted weight-matched controls. Since many of the prostatic functions are testosterone dependent, we assessed the possibility that testosterone modulated prostatic TRH concentrations. We measured prostatic TRH concentrations in the following four groups of sexually mature male Sprague-Dawley rats: group I, sham operated; group II, castrated; group III, castrated animals with 5-mm Silastic testosterone implants; and group IV, castrated animals with 20-mm testosterone implants. Prostatic TRH concentrations in these four groups 2 weeks after surgery were 600.5 +/- 33.3, 65.1 +/- 22.6, 169.4 +/- 55.3, and 609.5 +/- 144.3 (+/-SE) ng/100 mg protein. There was good linear correlation between prostatic TRH concentrations and serum testosterone concentrations (r = 0.65; P less than 0.01). By subjecting the pooled prostatic extracts from each group to ion exchange chromatography on a SP-Sephadex C-25 column and measuring the proportion of immunoreactivity coeluting with authentic TRH, it was shown that the fall in prostatic TRH immunoreactivity after castration and hypophysectomy was indeed due to a loss of authentic TRH. We conclude that the prostatic TRH concentrations are under hormonal control and appear to be modulated by serum testosterone concentrations. This is the first demonstration of hormonal regulation of a neuropeptide in a mammalian extrahypothalamic site and suggests a physiological role for this neuropeptide at this site.

The effects of thyrotropin-releasing hormone on the endocrine pancreas.

TRH has been shown to be present in the pancreas. To examine a possible role for TRH in the control of endocrine pancreatic function, we have studied the effects of TRH on the isolated perfused rat pancreas preparation. Arginine caused release of TRH from the preparation. The mean maximum TRH peak was 85 +/- 12 pg/ml and occurred later than the first phase of glucagon release. Glucagon (2000 pg/ml) did not release TRH from the preparation. There was no detectable basal release of TRH. Glucose did not stimulate release of TRH from the pancreas preparation. TRH (10 ng/ml) by itself had no effect on insulin or glucagon release. TRH enhanced arginine-induced glucagon release; mean summated glucagon was 8228 +/- 1138 (SE) pg/ml compared to controls (4530 +/- 447 pg/ml; P less than 0.01). There was a tendency for TRH to enhance second phase glucose-induced insulin release. Pancreatic physiology is in part regulated by locally acting hormones and TRH may be one of these hormones.

beta-Endorphin 61-91 and other beta-endorphin-immunoreactive peptides in human semen.

Human semen contains large amounts of authentic beta-endorphin and other beta-endorphin immunoreactive peptides. To eliminate the non-specific effects of semen in the beta-endorphin RIA, the beta-endorphin immunoreactive peptides were extracted with octadecylsilane-C18 reverse phase beads. beta-Endorphin immunoreactivity was parallel to the RIA standard curve. Sephadex G-50 gel chromatography showed that 93% of the immunoreactive material coeluted with beta-endorphin. Reversed phase high pressure liquid chromatography of semen extract demonstrated several peaks of beta-endorphin immunoreactivity; one of these was coincident with synthetic h-beta-endorphin61-91.

Secretion of thyrotropin with reduced concanavalin-A-binding activity in patients with severe nonthyroid illness.

Patients with nonthyroid illness (NTI) often have reduced serum T3, free T3, T4, and free T4 concentrations. Paradoxically, serum TSH is usually in the normal range. The data suggest a diagnosis of hypothalamic hypothyroidism, in which TSH may have reduced biological activity because TRH, which is necessary for key steps in the glycosylation of TSH, is deficient. To study the glycosylation of TSH in patients with NTI, we measured the serum TSH concentration in 36 such patients hospitalized on our intensive care units and compared the results with those from a group of 18 normal subjects. Serum TSH was measured in 2 assays: 1) a sensitive TSH RIA of unextracted serum (TSH-RIA) and 2) a RIA of serum TSH after its extraction with Concanavalin-A (Con-A), a lectin which binds glycoproteins containing mannose residues in their oligosaccharide side-chains (TSH-Con-A). The ratio of TSH-Con-A to TSH-RIA was significantly reduced in the NTI patients [0.61 +/- 0.03 (+/- SE) vs. 0.89 +/- 0.05 in the normal subjects] due to reduced binding of the TSH to the Con-A. This change was not dependent on the extent of the abnormalities of thyroid hormone levels. The data suggest that the TSH secreted in NTI has altered glycosylation which is associated with reduced biological activity. This finding may explain in part the low serum T4 level in NTI patients in the face of an apparently normal immunoreactive TSH level.

Plasma thyrotropin, thyroxine, and triiodothyronine relationships in man.

The physiologic relationships of plasma TSH, T4 and T3 levels measured every 20 min in seven healthy young men and one healthy young woman have been investigated. A nocturnal TSH surge was observed in all subjects on both nights of the 36-48 h baseline observation period. In males the maximum plasma TSH value occurred at 2300 h. The mean peak TSH level was 2.0 +/- 0.3 (se) muU/ml compared with a mean of 1.3 +/- 0.9 muU/ml for the entire baseline records of the 8 subjects. The effect of iv infusion of 32-1000 mug of somatostatin (SRIF) for 1 1/2-3 h was investigated in four of the male subjects during 2 or 4 consecutive nights following the control period. Temporal relationships between the hormonal fluctuations observed throughout the control period and during the nights of SRIF infusion were investigated using time series analysis and Student's t test. Rapid fluctuations of plasma T4 and T3 concentration were noted, even when corrected for changes in total protein concentration, with an average coefficient of variation of 10% for T3 and 12% for T4. No increment of plasma T4 or T3 followed the nocturnal TSH surge nor were the rapid fluctuations of the thyroid hormones altered by the TSH surge. SRIF infusion commencing at 2300 h suppressed the elevated TSH levels (P is less than 0.01) while similar infusions begun at 2100 h blocked the expected nocturnal TSH rise observed during control periods in male subjects. Plasma T4 and T3 levels were not significantly affected by the administration of SRIF. The relationship of the rapid plasma T4 and T3 variations to postural changes was investigated in four euthyroid male subjects. Serum levels of TSH, T4 and T3 and total protein were determined at 15 min intervals while postural changes were carefully monitored. The ratios of T4 and T3 to total protein were relatively stable (3-4% coefficient of variation) when the subjects were kept in a supine and motionless position. A 50 mug bolus infusion of T4 raised the basal T4 level by only 1-2 mug/dl. The data suggest that short-term fluctuation of plasma T4 and T3 result from changes in protein concentration due to hemodynamic responses to alteration of posture and physical activity and not to pulsatile secretion of T4 and T3.

Effect of normal and reversed sleep-wake cycles upon nyctohemeral rhythmicity of plasma thyrotropin: evidence suggestive of an inhibitory influence in sleep.

The relation of nyctohemeral variation in plasma TSH to sleep-wake cycles was examined in 10 normal young men who had their sleep polygraphically monitored and their blood sampled every 20 min for 24,36, or 48 h periods. Studies of normal sleepwake cycles in which sleep was allowed from the usual bedtime to 0630 h totalled 21 nights (night = 1840-0620 h) and their corresponding 16 days. TSH was measured by a sensitive RIA. On 17 nights, the mean nightly TSH significantly exceeded that of the day's and, on 18 nights, clear nyctohemerally maximal peaks in TSH were seen in the 2100-0100 h interval. Greater amplitude, duration and rhythmic repetition over several nights distinguished 2100-0100 h maxima from a background of persistent briefly episodic release. These nyctohemeral peaks were pre-sleep maxima, as rises uniformly began, and on 15 nights, the peaks occurred prior to the onset of sleep. The peaks clustered within the 30 min just before (12 nights) or after (3 nights) entry into sleep. TSH release then declined across sleep. Other evidence suggestive of an inhibitory influence in sleep upon TSH release was that sleep began early on the 3 nights without clear 2100-0100 h TSH maxima and that the mean 2100-0100 h TSH peak was significantly reduced when sleep began prior to the usual 2300-0000 h interval and significantly increased when the onset of sleep was delayed or postponed. After a 24 h baseline, 4 men underwent phase-reversal of their sleep-wake cycles for 48 h, in which sleep was shifted to the 1100-1830 h interval. On the first wakeful night of reversal, the 2100-0100 h peak began normally, but, in the absence of sleep, the enhanced TSH release then simply continued across this night, delaying achievement of the nyctohemeral maxima. On the second wakeful night of reversal, the maximum in mean TSH lay in the same 0400-0600 h interval as that of first reversal night, and the mean 2100-0100 h peak was no longer evident. The TSH of the second 24 h of reversal also was significantly reduced, suggestive of a negative feedback effect of enhanced release of the first reversal day. No shift of basal pre-sleep TSH peaks to the 0900-1300 h interval or of sleep-enhanced TSH release was seen during reversal. Thus, despite the persistence of TSH's nyctohemeral rhythmicity across acute sleep-wake reversal, its pattern changed significantly in relation to shifts in sleep. We currently view these results as consistent with the origin of TSH's nyctohemeral rhythmicity in a circadian mechanism whose expression is subject to modulation by the inhibitory influences of feedback and sleep.

A sensitive and precise radioimmunoassay for human thyroid-stimulating hormone.

A high-sensitivity radioimmunoassay for human thyroid-stimulating hormone (hTSH) has been developed utilizing a highly specific rabbit antibody (developed by AFP) to hTSH with an affinity constant of 4.4 x 10(11) M-1. Optimal conditions which minimized both 125I-hTSH radiation damage and 125I dissociation included storage at -60 C in 0.25% albumin. Repetitive freezing and thawing did not cause loss of structural integrity or of full immunochemical reactivity of 125I-hTSH. Effects of human and calf sera on the precipitation of first antibody-bound 125I-hTSH by second antibody have been tabulated; the addition of TSH-free human serum to the standard curve is necessary to compensate for the serum effect. The minimum detectable amount of TSH in the new assay at a 1:1,000,000 final dilution of the hTSH antibody is 0.02 muU/tube. The mean normal hTSH value of 1.5 +/- 1.0 muU/ml (mean +/- SD) fell within the central one-third of the logit (B/Bo) plot where antigen concentrations are measured with highest precision. This assay gives good precision and reproducibility of measurements throughout the normal range of serum TSH concentrations.

The role of chorionic gonadotropin in transient hyperthyroidism of hyperemesis gravidarum.

Biochemical evidence of hyperthyroidism is frequently encountered in hyperemesis gravidarum, but its relationship to the cause of hyperemesis is unknown. We studied the relationship of serum hCG, thyroid function, and severity of vomiting among 57 hyperemesis patients and 57 controls matched for gestational age. TSH was suppressed in 60% of hyperemesis patients and 9% of controls. hCG correlated directly with free T4(r = 0.45, P < 0.001) and inversely with TSH (r = -0.48, P < 0.001). Hyperemesis patients had significantly greater mean serum hCG, free T4, total T3, and estradiol, and lesser serum TSH compared to controls. Hyperemesis patients with suppressed TSH had significantly greater free T4 and hCG compared to those with TSH in the normal range. Control and hyperemesis subjects were divided into four groups based on the severity of vomiting. The degree of biochemical hyperthyroidism and hCG concentration varied directly with the severity of vomiting. Unextracted serum was tested for thyrotropic activity by measuring its effect on iodide uptake in cultured FRTL-5 rat thyroid cells. Thyrotropic activity correlated with serum hCG (r = 0.50, P < 0.001). These data show that biochemical hyperthyroidism is a common finding in patients with hyperemesis gravidarum and suggest that hCG is the thyroid stimulator in this state. The increased estradiol concentration in patients with hyperemesis gravidarum may be attributed to the effects of hCG on steroidogenesis.

Activation of the thyrotropin (TSH) receptor by human chorionic gonadotropin and luteinizing hormone in Chinese hamster ovary cells expressing functional human TSH receptors.

It is well known that peptide heterogeneity exists in the hCG-beta subunit in pregnancy and in patients with trophoblastic diseases. To elucidate the differences in thyrotropic activity of hCG molecules, we examined cAMP accumulation and TSH receptor binding of intact hCG, hLH, and a recombinant hCG that lacked the C-terminal extension on the beta-subunit, hCG (alpha wt/beta delta T), using Chinese hamster ovary (CHO) cells transfected with hTSH receptors. hLH, which shares 85% sequence identity with the hCG-beta molecule except for the C-terminal amino acid residue extension of the hCG-beta subunit, bound to the TSH receptor and stimulated adenylate cyclase about 10 times more potently than hCG on a molar basis. This was consistent with the result that cAMP stimulation by mutant hCG (alpha wt/beta delta T) was greater than intact hCG. hLH also increased iodide uptake and thymidine incorporation in FRTL-5 rat thyroid cells more potently than intact hCG. These results demonstrate that hLH is a more potent TSH than hCG and that the C-terminal extension of the hCG beta-subunit can interfere with hCG interaction with the hTSH receptor. hCG lacking the C-terminal extension of the beta-subunit occurs in the mixture of heterogeneous hCG molecular forms of pregnancy and trophoblastic diseases and may contribute to the hyperthyroidism in patients with hydatidiform mole, choriocarcinoma, and hyperemesis gravidarum.

Effect of 64-hour sleep deprivation on the circadian waveform of thyrotropin (TSH): further evidence of sleep-related inhibition of TSH release.

Half-hourly sampling of plasma TSH was done across 3 days in four normal young men. Sleep was denied for 64 h from 0700 h on awakening from accommodation sleep until polygraphic sleep was resumed at 7100 h of the third day (D3) such that 2 consecutive nights of usual 2300-0700 h sleep were missed. This protocol allowed examination of any modulatory effects on the daily patterns in TSH concentrations during sleep deprivation on D1-2 (1100-3500, 3500-5900 h) or during resumption of usual nightly sleep on D3 (5900-8300) compared to that of a previously studied group of normal young men. The circadian nature of the daily TSH waveform was evidenced by its daily repetition within a subject both basally and during D1-2 sleep deprivation and by its synchronization within the basal, deprived, or resumed sleep days. The peaks in each subject's daily TSH patterns on D1-2 were consistently longer, and the daily maxima and cosine acrophases on D1-2 were consistently later than those on D3 when basal sleep was resumed. About half the daily TSH concentration maxima and daily cosinor amplitudes on D1-2 were greater than those of the respective sleep-resumed TSH patterns of D3. Both the group mean TSH patterns and the cosinor 95% confidence ellipses also indicated the daily peak in the TSH waveform to be significantly longer, later, and larger during D1-2 sleep deprivation than during the basal or D3 periods. These results indicate that significant alteration of the daily TSH waveform can occur in response to absence of sleep and are compatible with the existence of an inhibitory effect in early nightly sleep on TSH release. The TSH patterns during the 1700-2300 h intervals of rising TSH levels were congruent in the basal, deprived, and resumed sleep periods. Prompt reversion to the basal TSH pattern also occurred when sleep was resumed on D3. Both of these observations suggest the alteration in TSH waveform during sleep deprivation to have arisen from an inhibitory effect in sleep rather than from a change in period or phase of a generating oscillator.