Increased TRH and TRH-like peptide release in rat brain and peripheral tissues during proestrus/estrus.

Women are at greater risk for major depression, PTSD, and other anxiety disorders. ERß-selective agonists for the treatment of these disorders are the focus of pharmacologic development and clinical testing. Estradiol and its metabolites contribute to the neuroprotective effects of this steroid class, particularly in men, due to local conversion of testosterone to estiradiol in key brain regions which are predisposed to neurodegenerative diseases. We have used young adult female Sprague-Dawley rats to assess the role of TRH and TRH-like peptides, with the general structure pGlu-X-Pro-NH2 where "X" can be any amino acid residue, as mediators of the neurobiochemical effects of estradiol. The neuroprotective TRH and TRH-like peptides are coreleased with excitotoxic glutamate by glutamatergic neurons which contribute importantly to the regulation of the estrus cycle. The levels of TRH and TRH-like peptides during proestrus and/or estrus in the 12 brain regions analyzed were significantly decreased (due to accelerated release) 106 times but increased only 25 times when compared to the corresponding levels during diestrus days 1 and 2. These changes, listed by brain region in the order of decreasing number of significant decreases (↓) and/or increases (↑), were: striatum (20↓,1↑), medulla oblongata (16↓,2↑), amygdala (14↓,1↑), cerebellum (13↓,1↑), hypothalamus (12↓,1↑), entorhinal cortex (6↓,6↑), posterior cingulate (10↓,1↑), frontal cortex (3↓,5↑), nucleus accumbens (5↓,3↑), hippocampus (5↓,2↑), anterior cingulate (2↓,1↑), and piriform cortex (1↑). In peripheral tissues the corresponding changes were: ovaries (23↓), uterus (16↓,1↑), adrenals (11↓,3↑), and pancreas (1↓,6↑). We conclude that these peptides may be downstream mediators of some of the therapeutic effects of estrogen.

Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by leptin.

Leptin is not only a feedback modulator of feeding and energy expenditure but also regulates reproductive functions, CNS development and mood. Obesity and major depression are growing public health concerns which may derive, in part, from disregulation of leptin feedback at the level of the hypothalamic feeding centers and mood regulators within the limbic system. Identifying downstream mediators of leptin action may provide therapeutic opportunities. We and others have previously reported that thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH(2)) and TRH-like peptides (pGlu-X-Pro-NH(2), where "X" can be any amino acid residue) have neuroprotective, antidepressant, anti-epileptic, analeptic, anti-ataxic, and anorectic properties. For this reason, young, adult male Sprague-Dawley rats were injected ip with 1mg/kg rat leptin and peptide and protein levels were measured in brain and peripheral tissues at 0, 0.5, 1 and 2h later. Eleven brain regions: pyriform cortex (PYR), entorhinal cortex (ENT), cerebellum (CBL), nucleus accumbens (NA), frontal cortex (FCX), amygdala (AY), posterior cingulate (PCNG), striatum (STR), hippocampus (HC), medulla oblongata (MED) and anterior cingulate (ACNG) and five peripheral tissues (adrenals, testes, epididymis, pancreas and prostate) were analyzed. TRH and six TRH-like peptide levels in STR fell by 0.5h consistent with leptin-induced release of these peptides: STR (7 downward arrow). Significant changes in TRH and TRH-like peptide levels for other brain regions were: CBL (5 downward arrow), ENT (5 downward arrow), HC (4 downward arrow), AY (4 downward arrow), FCX (3 downward arrow), and ACNG (1 downward arrow). The rapid modulation of TRH and TRH-like peptide release combined with their similarity in behavioral, neuroendocrine, immunomodulatory, metabolic and steroidogenic effects to that of leptin is consistent with these peptides participating in downstream signaling.

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.

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.

Lithium modulates expression of TRH receptors and TRH-related peptides in rat brain.

Lithium is an established mood stabilizer and neuroprotective agent frequently used in the treatment of bipolar disorder and as an adjuvant in drug-resistant unipolar depression. The mechanisms underlying both the therapeutic efficacy of lithium and the exacerbation of symptoms following rapid withdrawal are not understood. From previous studies showing antidepressant and neuroprotective activities of thyrotropin releasing hormone (TRH) and TRH-related neuropeptides we hypothesized that lithium may have substantial effects on the expression and secretion of these peptides and/or their receptors in various rat brain regions involved in the regulation of mood. Chronic lithium effect on TRH receptor binding studies: The effect of 1 and 2 weeks of dietary lithium on [(3)H]3-Me-His-TRH binding to plasma membranes of nucleus accumbens, amygdala and pituitary of young adult male Wistar and the endogenously 'depressed' Wistar Kyoto (WKY) rats was measured by the method of Burt and Taylor [Burt, D.R., Taylor, R.L., Endocrinology 106 (1980) 1416-1423]. Acute, chronic and withdrawal effect of lithium on TRH and TRH-like peptide levels in young, adult male Sprague-Dawley rats: Rats were divided into four lithium treatment groups. Control animals received a standard laboratory rodent chow. The acute group received a single i.p. injection of 1.5 milli-equivalents of LiCl 2 h prior to killing. The chronic and withdrawal groups received standard rodent chow containing 1.7 g/kg LiCl for 2 weeks. Withdrawal rats were returned to standard chow 48 h prior to killing while the chronic animals continued on the LiCl diet. TRH, TRH-Gly (pGlu-His-Pro-Gly, a TRH precursor), EEP (pGlu-Glu-Pro-NH(2), a TRH-like peptide with antidepressant activity) and Ps4 (a prepro-TRH-derived TRH-enhancing decapeptide) immunoreactivity (IR) were measured in 13 brain regions. The remaining samples were pooled and fractionated by high-pressure liquid chromatography followed by EEP radioimmunoassay. Chronic lithium treatment increased [(3)H]3Me-TRH binding in the nucleus accumbens and amygdala about two-fold in both Wistar and WKY rats but no change was observed in pituitary binding. The most widespread changes in TRH and TRH-related peptide levels were observed in the withdrawal group compared to the controls. The direction of change for the total IR was consistent for all TRH-IR and TRH-related peptide-IR within a given tissue. For example, withdrawal increased all peptide levels in the pyriform cortex and striatum but decreased these levels in the anterior cingulate and lateral cerebellum. Both acute injection and chronic treatment with LiCl decreased TRH and TRH-related peptide levels in the entorhinal cortex. Acute injection and withdrawal both increased EEP-IR in striatum by more than two-fold. The acute effects are most likely due to changes in the release of these peptides since 2 h is not sufficient time for alterations in peptide biosynthesis. Chronic treatment increased levels of pGlu-Phe-Pro-NH(2) levels in hippocampus, pGlu-Leu-Pro-NH(2), and peak '2' in septum by more than four-fold. The present results are consistent with a component role for TRH and related peptides in the mood-altering effects of lithium administration and withdrawal frequently observed during treatment for depression and bipolar disorder.

Antidepressant effects of thyrotropin-releasing hormone analogues using a rodent model of depression.

The antidepressant potential of two naturally occurring analogues of thyrotropin-releasing hormone (TRH), pGLU-GLU-PRO-NH2 (EEP) and pGLU-PHE-PRO-NH2 (EFP), were examined using a rodent model of antidepressant efficacy. The Porsolt Swim Test was used to assay the antidepressant properties of these two peptides. Both analogues of TRH produced significant antidepressant effects, with EEP producing the stronger response. No effect of EEP upon triiodothyronine (T3) was observed at the dosage used. EFP, which has previously been demonstrated to crossreact with the TRH receptor, significantly increased serum T3. Since an effect upon T3 was only observed in the weaker of the two compounds, these data suggest that the behavioral effect of EEP was not secondary to stimulation of thyroid hormone. Additionally, the differential behavioral response to the two compounds suggests a degree of sequence specificity in the ability of TRH-like tripeptides to produce an antidepressant effect.

Regulation of TRH and TRH-related peptides in rat brain by thyroid and steroid hormones.

To investigate the possibility that TRH (pGlu-His-Pro-NH(2)) and EEP (pGlu-Glu-Pro-NH(2)) contribute to the behavioral and mood changes attending hypothyroidism, hyperthyroidism and hypogonadism, we have treated young, adult, male Sprague-Dawley rats (5/group, 250 g bw at time of sacrifice) for one week with either daily ip injections of saline, 5 microg T(4), 3 mg PTU or castration. Immunoreactivity for TRH (TRH-IR), TRH-Gly (pGlu-His-Pro-Gly, a TRH precursor), EEP and Ps4 (prepro-TRH-derived TRH-enhancing peptide) was measured in 8 brain regions by RIA. Castration reduced the Ps4-IR levels in hippocampus by 80%. High pressure liquid chromatography revealed that in many brain regions EEP-IR and TRH-IR consisted of a mixture of TRH and other TRH-like peptides including EEP, Val(2)-TRH, Tyr(2)-TRH, Leu(2)-TRH and Phe(2)-TRH. Transition from the hyperthyroid to the hypothyroid state increased the Val(2)-TRH and Tyr(2)-TRH levels in the accumbens by 10-fold and 15-fold, respectively, and the corresponding ratios for the pyriform cortex increased 9-fold and 12-fold, respectively. Hypothyroidism and castration reduced the levels of TRH and the majority of other TRH-like peptides in the entorhinal cortex. This is the first report that thyroid and steroid hormones alter the levels of TRH, prepro-TRH-derived peptides, and a newly discovered array of TRH-like neuropeptides in limbic brain regions.

Electroconvulsive seizures modulate levels of thyrotropin releasing hormone and related peptides in rat hypothalamus, cingulate and lateral cerebellum.

We have studied the neuroanatomic extent of electroconvulsive (ECS)-responsive prepro-TRH and TRH-related gene expression and its possible interaction with forced swimming. Young adult male Wistar rats were treated in a 2x2 Latin square protocol of swimming, no swimming, three daily ECS or sham ECS. Sixteen different brain regions were dissected and immunoreactivity measured for TRH (pGlu-His-Pro-NH(2)); TRH-Gly, a TRH precursor; Ps4, a prepro-TRH-derived TRH-enhancing decapeptide, and EEP (pGlu-Glu-Pro-NH(2)). ECS, in addition to elevating TRH-immunoreactivity (TRH-IR), TRH-Gly-IR, Ps4-IR and EEP-IR levels in the limbic regions, as we have previously reported, also significantly increased Ps4-IR levels in hypothalamus, posterior cingulate and lateral cerebellum, and increased TRH-Gly-IR levels in hypothalamus. Interestingly, the combination of ECS and swimming significantly reduced the levels of TRH-Gly-IR in the anterior cingulate compared to the sham ECS-no swim group. The combined use of high-pressure liquid chromatography and the EEP radioimmunoassay (RIA) revealed that pGlu-Tyr-Pro-NH(2) and/or pGlu-Phe-Pro-NH(2) occur in amygdala, anterior cingulate, frontal cortex, entorhinal cortex, lateral cerebellum and striatum and make a substantial contribution to the EEP-IR and TRH-IR. We conclude that ECS can alter the expression and secretion of TRH-related peptides in the hypothalamus, cingulate and lateral cerebellum. Such effects have not previously been reported in these limbic and extra-limbic regions which are increasingly implicated in the autonomic, behavioral and volitional changes which accompany severe depression and its treatment.

TRH in therapeutic vs. nontherapeutic seizures: affective and motor functions.

We have modeled some aspects of electroconvulsive therapy (ECT) in rats. In addition to sham-treated controls, one group received two electroconvulsive (ECS) current-doses at grand mal seizure threshold. Two more groups received three additional ECSs at two higher current-doses. Only the two suprathreshold groups showed significant antidepressant (AD) effects in the forced-swim test, but all three seizure groups showed significant increases in TRH and related peptides in anterior cortex (AC), pyriform cortex (PYR), amygdala/entorhinal cortex (AY), and hippocampus (HC). In motor cortex (MC), TRH appeared to be increased only in the lower dose suprathreshold ECS condition. No condition increased TRH in striatum (STR). These results fell short of directly implicating limbic TRH in AD effects, but in HC, MC, and STR, correlations of peptide levels with individual swim scores raise the possibility that this peptidergic system might be involved in motor as well as affective functions. Other peptides related to TRH might also be implicated in affective regulation and antidepressant effects.

Electroconvulsive seizures increase levels of pGlu-Glu-Pro-NH2 (EEP) in rat brain.

We have previously reported that electroconvulsive seizures (ECS) increases the level of prepro-TRH-derived peptides in hippocampus, amygdala and pyriform cortex but not the striatum of male rats and that this increase is significantly correlated with reduced immobility (increased swimming) in the Porsolt forced swim test. An abstract by Mabrouk and Bennett published in 1993 described increased locomotor activity in rats following IP injection of TRH (pGlu-His-Pro-NH2) and EEP (pGlu-Glu-Pro-NH2). We have examined the effect of three daily transcorneal ECS on the levels of EEP in various brain regions and their correlation with results from the Porsolt forced swim test. The EEP level (ng/g wet weight) was measured by RIA in 6 brain regions: amygdala (AY), hippocampus (HC), pyriform cortex (PYR), anterior cortex (AC), striatum (STR) and motor cortex (MC). ECS significantly increased EEP levels in AY, HC and PYR. The increased swim behavior following ECS, as measured in the Porsolt test, correlated significantly with the EEP levels in HC and MC within individual subjects. Intraperitoneal (IP) injection of EEP (1.0 mg/kg) resulted in a rapid and sustained rise in EEP levels throughout the brain and a clearance half-time from blood of 2.0 h. Intracardiac injection of 0.5 mg EEP resulted in a peak EEP level in CSF at 2 h followed by a t1/2 of 0.35 h. A 3 compartment model for EEP transport from blood into CSF and then brain was developed. This model revealed a 1.75 h delay in the transit time of EEP from blood to CSF followed by rapid clearance from the CSF but long retention time within various brain tissues. We conclude that (1) ECS significantly increases EEP levels in limbic regions, but not in striatum, of the rat brain, (2) EEP, like TRH, is a potential mediator of the antidepressant effect of ECS and (3) EEP, after IP or IV administration, is readily taken up by, and has a long residence time in, brain tissue.

Is Ps4 (prepro-TRH [160-169]) more than an enhancer for thyrotropin-releasing hormone?

Ps4 (thyrotropin-releasing hormone [TRH]-enhancing peptide), one of the cryptic peptides resulting from the proteolytic processing of prepro-TRH to produce TRH, has a growing list of functions in addition to its well-established ability to enhance the TRH-induced release of thyrotropin (TSH) and prolactin from the pituitary. Intramedullary coadministration of Ps4 and TRH increased gastric acid secretion above the level produced by TRH alone and intracisternal infusion of Ps4 resulted in a substantial reduction in the levels of prepro-TRH-derived peptide levels in the rat pituitary, including Ps4. High-affinity receptors for Ps4 are widely distributed. In addition to the very high Ps4 binding capacity of the folliculo-stellate cells of the anterior pituitary, abundant Ps4 receptors are found in the urinary bladder, vas deferens, central nervous system, reproductive tissues, and pancreas. Targeted prepro-TRH gene disruption results in hyperglycemia as well as the expected hypothyroidism. The observed disregulation of thyroid and glucose homeostasis in the TRH "knockout" mouse clearly demonstrates that prepro-TRH-derived peptides and their cognate receptors within the pituitary, pancreas, and other neural and endocrine systems are of fundamental importance to a variety of physiological systems and merit structural and functional characterization.

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.

Sphingomyelinase and phospholipase A2 regulate type I deiodinase expression in FRTL-5 cells.

We have previously reported (Mol. Cell. Endocrinol. (1994) 101, R31-R35) that the proinflammatory cytokines, tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN-gamma), have a marked inhibitory effect on the expression and activity of type I iodothyronine deiodinase (D1) in FRTL-5 rat thyroid cells, while the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta 1) had no effect. These three proinflammatory cytokines utilize a number of intracellular second messenger systems including the pathways beginning with activation of sphingomyelinase and phospholipase A2. We have studied the time-dependent and dose-dependent effects of sphingomyelinase, ceramide, phospholipase A2 (PLA2), and arachidonic acid on the expression and activity of D1 in FRTL-5 cells. Sphingomyelinase (0.3 U/mL) inhibited D1 activity 55% and reduced D1 mRNA levels 70% to 90% by 8 hours. Similar treatment with 10 U/mL PLA2 inhibited D1 activity 54%. Treatment with 15 microM 5, 8, 11-eicosatriynoic acid (ETI), a nonmetabolizable analog of arachidonic acid, or 15 microM ceramide for 3 hours reduced D1 activity with a half-time of disappearance (t1/2) of 4.2 hours and 3.7 hours, respectively, but ETI and ceramide did not alter the D1 immunoreactivity or mRNA levels. Treatment for 8 hours with cycloheximide (5 or 10 micrograms/mL) had no effect on the D1 mRNA level, but blocked the TNF-induced reduction of this mRNA. We conclude that proinflammatory cytokines inhibit D1 expression and activity in FRTL-5 cells, in part, by activation of sphingomyelinase and PLA2 that results in (1) competitive inhibition of D1 activity by the enzymatic products ceramide and arachidonic acid and (2) reduction of D1 mRNA stability by protein synthesis-dependent mechanisms.

Electroconvulsive seizures increase levels of PS4, the TRH-enhancing peptide [prepro-TRH(160-169)], in rat brain.

We report the development of a radioimmunoassay for prepro-TRH(160-169) (PS4), a thyrotropin-releasing hormone (TRH) enhancing peptide, and its use in characterizing the effect of electroconvulsive seizures on the levels of this peptide in various brain regions of male Wistar rats. We found that electroconvulsive seizures significantly elevated the PS4 levels in hippocampus, amygdala, pyriform (olfactory) cortex, and anterior cortex but not in striatum, motor cortex, locus ceruleus, or ventral lateral medulla. The levels of PS4 were highly correlated with the corresponding TRH (p-Glu-His-Pro-NH2) and TRH-Gly (p-Glu-His-Pro-Gly) levels in hippocampus, amygdala, and pyriform cortex, consistent with the prepro-TRH source of all of these peptides. The PS4 levels in hippocampus and amygdala were significantly correlated with the immobility time in the Porsolt forced swim test, an established animal model for antidepressant effects. The PS4 levels in peripheral blood, hypothalamus, anterior cortex, amygdala, and eyes increased severalfold at 20 min following intracisternal injection of 228 microg of this peptide, suggesting that it readily crosses the blood-brain barrier. The pituitary levels of PS4 and TRH-Gly, on the other hand, were decreased within 20 min by intracisternal PS4, suggesting PS4 stimulated the release of prepro-TRH peptides from the pituitary. Fresh rat and human serum rapidly degraded PS4, indicating that it may act primarily as a paracrine modulator of TRH effects in pituitary, brain, and reproductive system.

Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta 1) inhibit the expression and activity of Na+/K(+)-ATPase in FRTL-5 rat thyroid cells.

We recently reported that tumor necrosis factor-alpha (TNF-alpha) induction of the synthesis and secretion of transforming growth factor (TGF)-beta 1 by FRTL-5 cells is a thyroid-stimulating hormone (TSH)-dependent and age-dependent process. TNF-alpha is only cytotoxic to aged (> 40 passages) FRTL-5 cells grown in TSH-containing medium, whereas TGF-beta induces programmed cell death (apoptosis) in epithelial cells but not in FRTL-5 cells, which otherwise retain many properties of normal thyroid follicular cells. This cell line is, therefore, a convenient model for studies on the TSH-dependent and age-dependent inhibitory effects of these cytokines on epithelial cell growth, viability, and function. One prominent effect of TNF-alpha (and TGF-beta 1) on FRTL-5 cell function is suppression of iodide uptake, which is markedly stimulated by TSH. In aged FRTL-5 cells, iodide uptake is only about 10% that of young control cells. Na+/K(+)-ATPase activity, which drives iodide uptake by thyroid cells, is inhibited by TNF-alpha and TGF-beta. The following experiments quantitate the effects of TSH, aging, TNF-alpha, and TGF-beta 1 on the expression and activity of Na+/K(+)-ATPase activity in FRTL-5 cells. Young (< 20 passages) and aged (> 40 passages) FRTL-5 cells were treated with various doses (0-100 ng/ml) of recombinant human TNF-alpha or TGF-beta 1 for various times (0-3 days) with and without 2 U/liter TSH. These treatments reduced the rate-limiting Na+/K(+)-ATPase beta 1 mRNA level and Na+/K(+)-ATPase activity in parallel in a dose-dependent and time-dependent fashion. Aged FRTL-5 cells were more sensitive to the inhibitory effects of TNF-alpha, whereas young cells were more sensitive to the suppressive effects of TGF-beta 1 on the expression and activity of Na+/K(+)-ATPase. We conclude that inhibition of Na+/K(+)-ATPase activity by TNF-alpha and TGF-beta in FRTL-5 cells is differentially affected by aging and that this inhibitory effect can be dissociated from effects on cell viability.

TNF-alpha, TSH, and aging regulate TGF-beta synthesis and secretion in FRTL-5 rat thyroid cells.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine produced by macrophages in response to a variety of pathological conditions, can inhibit thyroid cell function in vitro and in vivo. TNF-alpha induction of transforming growth factor-beta 1 (TGF-beta 1) in rat endothelial cells suggested that TGF-beta, a known mediator of inflammatory effects of TNF-alpha, may be involved in the sensitivity of aged thyroid cells to TNF-alpha (G. Chen, A. E. Pekary, and J. M. Hershman. Endocrinology 131: 863-870, 1992). To determine whether TNF-alpha induces TGF-beta production in FRTL-5 cells, young (< 20 passages) and aged (> 40 passages) FRTL-5 cells were grown to near confluency in medium containing 2 U/l of bovine thyroid-stimulating hormone [TSH; 6-hormone (6H) medium] or no TSH [5-hormone (5H) medium]. TNF-alpha (0-100 ng/ml) was added 0-48 h before total RNA was extracted. Northern blots were hybridized with 32P-TGF-beta 1, -beta 2, and -beta 3 cDNAs. In aged cells TNF-alpha increased their TGF-beta 1 (and -beta 2 and -beta 3) mRNA levels 5.4-fold (and > 10-fold), respectively, while in young cells all TGF-beta mRNAs remained almost undetectable during incubation with TNF-alpha. In contrast, TNF-alpha and TSH had a highly significant stimulatory effect on the secretion rate of TGF-beta precursors in both young and old cells as measured in the mink lung cell bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

TRH gene products are implicated in the antidepressant mechanisms of seizures.

1. After a series of electroconvulsive seizures, levels of TRH-Gly (the immediate precursor of TRH) in four limbic regions correlate significantly and highly with increased swimming in the forced-swim test model of antidepressant efficacy. Only in hippocampus did TRH itself correlate with swimming. 2. After ECS, limbic forebrain regions differ in the relationship of TRH to its precursor peptides. This probably results from differences in the coordination of induction of TRH-processing enzymes, as well as differences in the level of prepro-TRH following seizures. 3. Sprague-Dawley rats that are partially kindled with corneal stimulation swim less in the forced-swim test, opposite to the effect seen with antidepressant agents. 4. Pyriform cortex is unique among the four limbic regions examined in showing decreased amounts of the TRH precursor following swim/stress. 5. Combining ECS with the forced-swim test of antidepressant effects creates a useful model for studying the involvement of TRH and its precursor peptides in both the antidepressant and anticonvulsant effects of controlled therapeutic seizures in the treatment of major depressive disorders. Regional differences between the effects of pinnate and corneal ECS on peptides and behavior support the idea that corneal ECS is a better model than pinnate ECS for human bitemporal ECT. 6. Together with recent results in other laboratories, our results suggest that a series of generalized seizures results in prolonged and increased release and action of TRH in limbic forebrain.

Cytokines modulate type I iodothyronine deiodinase mRNA levels and enzyme activity in FRTL-5 rat thyroid cells.

Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interferon-gamma (INF-gamma) have inhibitory effects on thyroid function both in vivo and in vitro. We have studied the effects of these cytokines on type I 5'-deiodinase (5'-DI) mRNA expression and enzyme activity in FRTL-5 cells maintained in standard cell culture medium containing 0 (5H) or 2 mIU/ml bovine TSH (6H). Northern blots were hybridized with 5'-DI cDNA. 5'-DI mRNA levels were reduced to 20% of control values after treating cells with 100 ng/ml TNF-alpha in 6H for 2 days while the corresponding enzyme activity was reduced 50%. Other cytokines, including IL-1beta and interferon-gamma, also significantly inhibited expression of 5'-DI in FRTL-5 cells grown in 6H medium. Because the majority of circulating T3 in the rat is secreted by the thyroid gland, the highly significant decline in the serum T3/T4 ratio following in vivo administration of cytokines may be due to their direct inhibitory effect on thyroidal 5'-DI expression.