Chronic stress inhibits hypothalamus-pituitary-thyroid axis and brown adipose tissue responses to acute cold exposure in male rats.

PURPOSE: Cold exposure activates the hypothalamus-pituitary-thyroid (HPT) axis, response blunted by previous acute stress or corticosterone administration. Chronic stressors can decrease serum T3 concentration, and thyrotropin-releasing hormone (Trh) expression in the paraventricular nucleus (PVN), but impact on the response to cold is unknown; this was studied in rats submitted to daily repeated restraint (rRes) that causes habituation of hypothalamus-pituitary-adrenal (HPA) axis response, or to chronic variable stress (CVS) that causes sensitization and hyperreactivity. METHODS: Wistar male adult rats were submitted to rRes 30 min/day, or to CVS twice a day, for 15 days. On day 16, rats were exposed 1 h to either 5 or 21 °C. Parameters of HPT and HPA axes activity and of brown adipose tissue (BAT) cold response were measured; gene expression in PVN and BAT, by RT-PCR; serum hormone concentration by radioimmunoassay or ELISA. RESULTS: Compared to naïve animals, Crh and corticosterone concentrations were attenuated at the end of rRes, but increased at the end of CVS treatments. Cold exposure increased mRNA levels of Crh, Trh, and serum concentration of thyrotropin in naïve, but not in rRes or CVS rats; corticosterone increased in all groups. Cold induced expression of thermogenic genes in BAT (Dio2 and Ucp1) in naïve but not in stressed rats; Adrb3 expression was differentially regulated. CONCLUSION: Both types of chronic stress blunted HPT and BAT responses to cold. Long-term stress effects on noradrenergic and/or hormonal signaling are likely responsible for HPT dysfunction and not the type of chronic stressor.

An acute injection of corticosterone increases thyrotrophin-releasing hormone expression in the paraventricular nucleus of the hypothalamus but interferes with the rapid hypothalamus pituitary thyroid axis response to cold in male rats.

The activity of the hypothalamic-pituitary-thyroid (HPT) axis is rapidly adjusted by energy balance alterations. Glucocorticoids can interfere with this activity, although the timing of this interaction is unknown. In vitro studies indicate that, albeit incubation with either glucocorticoid receptor (GR) agonists or protein kinase A (PKA) activators enhances pro-thyrotrophin-releasing hormone (pro-TRH) transcription, co-incubation with both stimuli reduces this enhancement. In the present study, we used primary cultures of hypothalamic cells to test whether the order of these stimuli alters the cross-talk. We observed that a simultaneous or 1-h prior (but not later) activation of GR is necessary to inhibit the stimulatory effect of PKA activation on pro-TRH expression. We tested these in vitro results in the context of a physiological stimulus on the HPT axis in adult male rats. Cold exposure for 1 h enhanced pro-TRH mRNA expression in neurones of the hypophysiotrophic and rostral subdivisions of the paraventricular nucleus (PVN) of the hypothalamus, thyrotrophin (TSH) serum levels and deiodinase 2 (D2) activity in brown adipose tissue (BAT). An i.p. injection of corticosterone stimulated pro-TRH expression in the PVN of rats kept at ambient temperature, more pronouncedly in hypophysiotrophic neurones that no longer responded to cold exposure. In corticosterone-pretreated rats, the cold-induced increase in pro-TRH expression was detected only in the rostral PVN. Corticosterone blunted the increase in serum TSH levels and D2 activity in BAT produced by cold in vehicle-injected animals. Thus, increased serum corticosterone levels rapidly restrain cold stress-induced activation of TRH hypophysiotrophic neurones, which may contribute to changing energy expenditure. Interestingly, TRH neurones of the rostral PVN responded to both corticosterone and cold exposure with an amplified expression of pro-TRH mRNA, suggesting that these neurones integrate stress and temperature distinctly from the hypophysiotrophic neurones.

A rapid interference between glucocorticoids and cAMP-activated signalling in hypothalamic neurones prevents binding of phosphorylated cAMP response element binding protein and glucocorticoid receptor at the CRE-Like and composite GRE sites of thyrotrophin-releasing hormone gene promoter.

Glucocorticoids or cAMP increase, within minutes, thyrotrophin-releasing hormone (TRH) transcription in hypothalamic primary cultures, although this effect is prevented if cells are simultaneously incubated with both drugs. Rat TRH promoter contains a CRE site at -101/-94 bp and a composite GRE element (cGRE) at -218/-197 bp. Nuclear extracts of hypothalamic cells incubated with 8Br-cAMP or dexamethasone, and not their combination, bind to oligonucleotides containing the CRE or cGRE sequences. Adjacent to CRE are Sp/Krüppel response elements, and flanking the GRE half site, two AP1 binding sites. The present study aimed to identify the hypothalamic transcription factors that bind to these sites. We verified that the effects of glucocorticoid were not mimicked by corticosterone-bovine serum albumin. Footprinting and chromatin immunoprecipitation (ChIP) assays were used to examine the interaction of cAMP- and glucocorticoid-mediated regulation of TRH transcription at the CRE and cGRE regions of the TRH promoter. Nuclear extracts from hypothalamic cells incubated for 1 h with cAMP or glucocorticoids protected CRE. The GRE half site was recognised by nuclear proteins from cells stimulated with glucocorticoids and, for the adjacent AP-1 sites, by nuclear proteins from cells stimulated with cAMP or phorbol esters. Protection of CRE or cGRE was lost if cells were coincubated with dexamethasone and 8Br-cAMP. ChIP assays revealed phospho-CREB, c-Jun, Sp1, c-Fos and GR antibodies bound the TRH promoter of cells treated with cAMP or glucocorticoids; anti:RNA-polymerase II immunoprecipitated TRH promoter in a similar proportion as anti:pCREB or anti:GR. Recruitment of pCREB, SP1 or GR was lost when cells were exposed simultaneously to 8Br-cAMP and glucocorticoids. The data show that while pCREB and Sp1 bind to CRE-2, or GR to cGRE of the TRH promoter, the mutual antagonism between cAMP and glucocorticoid signalling, which prevent their binding to TRH promoter, could serve as a mechanism by which glucocorticoids rapidly suppress cAMP and noradrenaline-stimulated TRH transcription.

Phosphorylated cyclic-AMP-response element-binding protein and thyroid hormone receptor have independent response elements in the rat thyrotropin-releasing hormone promoter: an analysis in hypothalamic cells.

BACKGROUND: Thyrotropin-releasing hormone (TRH) from the hypothalamic paraventricular nucleus (PVN) controls the activity of the hypothalamus-pituitary-thyroid axis. TRH is expressed in other hypothalamic nuclei but is downregulated by 3,3',5-L-triiodothyronine (T(3)) exclusively in the PVN. Thyroid hormone receptors (TRs) bind TRH promoter at Site-4 (-59/-52), also proposed to bind phosphorylated cAMP response element-binding protein (pCREB). However, nuclear extracts from 8Br-cAMP-stimulated hypothalamic cells showed no binding to Site-4 and instead to cAMP response element (CRE)-2 (-101/-94). METHODS: We characterized, by DNA footprinting and chromatin immunoprecipitation, the sites in the rat (-242/+34) TRH promoter that bind to nuclear factors of hypothalamic primary cultures incubated with 8Br-cAMP and/or T(3). RESULTS: In primary cultures of fetal hypothalamic cells, TRH mRNA levels rapidly diminished with 10 nM T(3) while they increased by 1 mM 8Br-cAMP (+/- T(3)). Site-4 was protected from DNase I digestion with nuclear extracts from T(3)-incubated cells but not from controls or from those incubated with 8Br-cAMP, which protected CRE-2; T(3) + 8Br-cAMP coincubation caused no interference. The region protected by nuclear extracts from cAMP-stimulated cells included sequences adjacent to CRE-2-containing response elements of the SP/Krüppel family. A TRbeta2 antibody immunoprecipitated chromatin containing Site-4 but not CRE-2, from cells incubated with T(3). A pCREB antibody immunoprecipitated CRE-2 containing chromatin in controls and more in 8Br-cAMP-stimulated cells but none when cells were incubated only with T(3). Recruitment of the 2 transcription factors was preserved in cells simultaneously exposed to 8Br-cAMP and T(3). DISCUSSION: These results show that pCREB binds to a response element in the TRH promoter (CRE-2) that is independent of Site-4 where TRbeta2 is bound; pCREB and TR do not present mutual interference on their binding sites.

17ß-Oestradiol indirectly inhibits thyrotrophin-releasing hormone expression in the hypothalamic paraventricular nucleus of female rats and blunts thyroid axis response to cold exposure.

Energy expenditure and thermogenesis are regultated by thyroid and sex hormones. Several parameters of hypothalamic-pituitary-thyroid (HPT) axis function are modulated by 17ß-oestradiol (E(2)) but its effects on thyrotrophin-releasing hormone (TRH) mRNA levels remain unknown. We evaluated, by in situ hybridisation and Northern bloting, TRH expression in the paraventricular nucleus of the hypothalamus (PVN) of cycling rats, 2 weeks-ovariectomised (OVX) and OVX animals injected s.c. during 1-4 days with E(2) (5, 50, 100 or 200 µg / kg) (OVX-E). Serum levels of E(2), thyroid-stimulating hormone (TSH), prolactin, corticosterone and triiodothyronine (T(3)) were quantified by radioimmunoassay. Increased serum E(2) levels were observed after 4 days injection of 50 µg / kg E(2) (to 68.5 ± 4.8 pg / ml) in OVX rats. PVN-TRH mRNA levels were slightly higher in OVX than in virgin females at dioestrous 1 or pro-oestrous, decreasing proportionally to increased serum E(2) levels. E(2) injections augmented serum T(3), prolactin, and corticosterone levels. Serum TSH levels augmented with 4 days 50 µg / kg E(2), but not with the higher doses that enhanced serum T(3) levels. Exposure to cold for 1 h resulted in marked HPT axis activation in OVX rats, increasing the levels of TRH mRNA along the rostro-caudal PVN areas, as well as serum TSH, T(3), corticosterone and prolactin levels. By contrast, no significant changes in any of these parameters were observed in cold-exposed OVX-E (50 µg / kg E(2)) rats. Very few PVN-TRHergic neurones expressed the oestrogen receptor type-α, suggesting that the effects of E(2) on PVN-TRH expression are indirect, most probably as a result of its multiple modulatory effects on circulating hormones and their receptor sensitivity. The blunted response of OVX-E rats to cold coincides with the effects of E(2) on the autonomic nervous system and increased cold tolerance.

Amygdala kindling differentially regulates the expression of the elements involved in TRH transmission.

Subthreshold electrical stimulation of the amygdala (kindling) activates neuronal pathways increasing the expression of several neuropeptides including thyrotropin releasing-hormone (TRH). Partial kindling enhances TRH expression and the activity or its inactivating ectoenzyme; once kindling is established (stage V), TRH and its mRNA levels are further increased but TRH-binding and pyroglutamyl aminopeptidase II (PPII) activity decreased in epileptogenic areas. To determine whether variations in TRH receptor binding or PPII activity are due to regulation of their synthesis, mRNA levels of TRH receptors (R1, R2) and PPII were semi-quantified by RT-PCR in amygdala, frontal cortex and hippocampus of kindled rats sacrificed at stage II or V. Increased mRNA levels of PPII were found at stage II in amygdala and frontal cortex, and of pro-TRH and TRH-R2, in amygdala and hippocampus. At stage V, pro-TRH mRNA levels increased and those of PPII, decreased in the three regions; TRH-R2 mRNA levels diminished in amygdala and frontal cortex and of TRH-R1 only in amygdala. In situ hybridization analyses revealed, at stage II, enhanced TRH-R1 mRNA levels in dentate gyrus and amygdala while decreased in piriform cortex; those of TRH-R2 increased in amygdala, CA2, dentate gyrus, piriform cortex, thalamus and subiculum and of PPII, in CAs and piriform cortex. In contrast, at stage V decreased expression of TRH-R1 occurred in amygdala, CA2/3, dentate gyrus and piriform cortex; of TRH-R2 in CA2, thalamus and piriform cortex, and of PPII in CA2, and amygdala. The magnitude of changes differed between ipsi and contralateral side. These results support a trans-synaptic modulation of all elements involved in TRH transmission in conditions that stimulate the activity of TRHergic neurons. They show that reported changes in PPII activity or TRH-binding caused by kindling relate to regulation of the expression of TRH receptors and degrading enzyme.

Acute administration of alcohol modulates pyroglutamyl amino peptidase II activity and mRNA levels in rat limbic regions.

Released TRH is inactivated by an ectopeptidase, pyroglutamyl aminopeptidase II (PPII). PPII expression and activity are stringently regulated in adenohypophysis, and in rat brain, during kindling stimulation that activates TRHergic neurons. To gain further insight into the possible regulation of PPII, we studied the effect of an acute intraperitoneal ethanol administration that affects TRH content and expression. PPII activity was determined by a fluorometric assay and PPII mRNA levels by semi-quantitative RT-PCR. Activity decreased in frontal cortex 1 h after ethanol injection and, after 6 h, in hippocampus, amygdala and n. accumbens. PPII mRNA levels decreased at 30 and 60 min in frontal cortex and n. accumbens while increased at longer times in these regions and, in hippocampus and hypothalamus. NMDA and GABA(A) receptors' agonists and antagonists were tested at 1 h (+/-ethanol) on PPII activity and mRNA levels, as well as on TRH content and its mRNA. In n. accumbens, PPII mRNA levels decreased by ethanol, MK-801, and muscimol while picrotoxin or NMDA reversed ethanol's inhibition. Ethanol decreased TRH content and increased TRH mRNA levels as MK-801 or muscimol did (NMDA or picrotoxin reverted the effect of ethanol). In frontal cortex, PPII activity was inhibited by ethanol, NMDA and MK-801 with ethanol; its mRNA levels were reduced by ethanol, MK-801 and muscimol (NMDA and picrotoxin reverted ethanol's inhibition). These results show that PPII expression and activity can be regulated in conditions where TRHergic neurons are modulated. Effects of ethanol on PPII mRNA levels as well as those of TRH and its mRNA may involve GABA or NMDA receptors in n. accumbens. Changes observed in frontal cortex suggest combined effects with stress. The response was region-specific in magnitude, tendency and kinetics. These results give further support for brain PPII regulation in conditions that modulate the activity of TRHergic neurons.

Dexamethasone represses cAMP rapid upregulation of TRH gene transcription: identification of a composite glucocorticoid response element and a cAMP response element in TRH promoter.

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (-776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (-101/-94 and -59/-52) and glucocorticoid response element (GRE) half-site (-210/-205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (-101/-94) but not to CRE (-59/-52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

Chronic ethanol or glucose consumption alter TRH content and pyroglutamyl aminopeptidase II activity in rat limbic regions.

Thyrotropin-releasing hormone (TRH), its receptors and inactivating enzyme (PPII) are present in limbic regions. Nutritional changes or acute ethanol administration in male rats differentially modulate TRH or PPII expression. Chronic ethanol effect was studied in male (3, 6 and 8 weeks) and female rats (6 weeks) including naive and pair-fed (glucose) groups. Daily solid food and liquid intake, serum TSH and corticosterone, TRH content and PPII activity in limbic regions, were quantified. Gender differences were found in ethanol and total caloric intake and body weight gain, TSH and corticosterone levels. Ethanol consumption decreased TRH content and PPII activity in frontal cortex of male rats after 3-6 weeks. In contrast, glucose ingestion altered, by the third week, TRH content in amygdala, hippocampus, hypothalamus and nucleus accumbens, PPII activity in hippocampus and frontal cortex; by the sixth week, TRH content in amygdala and n. accumbens of male and females. Withdrawal at 24 h after 3-week ethanol ingestion decreased TRH content in amygdala and PPII activity in n. accumbens, while withdrawal from glucose reverted some of the effects produced by chronic glucose ingestion. Variations in TRH content or PPII activity support a region specific involvement of TRH neurons that depend on the treatment.

Chronic ingestion of ethanol or glucose solutions affects hypothalamic and limbic TRH metabolism in dams and their pups.

The effect of chronic ethanol consumption during pregnancy and lactation on thyrotropin releasing hormone (TRH) metabolism was investigated in the hypothalamus and limbic areas of female rats and their weaned pups. Pregnant female rats received ethanol or isocaloric glucose solution during pregnancy either alone, or also during the 3 weeks of lactation. Thyrotropin (TSH) and corticosterone levels were measured in serum; TRH and TRH-gly concentrations were determined in hypothalamus, hippocampus, n.accumbens, frontal cortex and amygdala of dams and pups at 21 days after parturition. Ethanol or glucose consumption during pregnancy and lactation produced a decrease in TSH levels compared with control animals fed at libitum; water replacement during lactation normalized TSH levels only in glucose-fed dams. Pups from ethanol or pair-fed dams showed low weight and increased TSH levels compared with normal rats. Variations in TRH metabolism were detected in limbic areas. Chronic ethanol caused a decrease in the levels of TRH in the hippocampus and frontal cortex of dams. In contrast, glucose chronic ingestion increased TRH content specifically in n.accumbens and amygdala of dams. Most of the variations in TRH content of limbic areas of pups were not specific for glucose or ethanol treatment and correlated with the deleterious effect of the mother's thyroid condition, although some differences were observed depending on pup's gender. These results support the involvement of TRHergic neurons in the limbic system of the female rat exposed to alcohol or glucose during pregnancy and lactation.

Thyrotropin-releasing hormone-induced down-regulation of pyroglutamyl aminopeptidase II activity involves L-type calcium channels and cam kinase activities in cultures of adenohypophyseal cells.

Released thyrotropin-releasing hormone (TRH) is inactivated by a narrow specificity ectopeptidase, pyroglutamyl aminopeptidase II (PPII), present in brain and lactotrophs. Various hypothalamic/paracrine factors, including TRH, slowly (in hours) regulate the activity of PPII on the surface of adenohypophyseal cells. TRH-induced down-regulation was mimicked by protein kinase C (PKC) activation but was not affected by inhibition of PKC. Adenylate cyclase activation can also down-regulate PPII. The purpose of this study was to identify elements of the transduction pathway used by TRH to regulate PPII activity. In primary cultures of female adenohypophyseal cells, activation of the stimulatory G protein or adenylate cyclase produced an effect additive to that of TRH; inhibition of protein kinase A activity did not interfere with TRH action. However, regulation of PPII activity by TRH was inhibited by a phospholipase C beta inhibitor or chelation of intracellular calcium. L-type calcium channels (LCC) agonists mimicked TRH action and their effect was not additive with that of TRH. Antagonists of LCC channels and inhibitors of calmodulin or calcium/calmodulin-dependent protein kinase blocked TRH action. Therefore, TRH-induced calcium entry through L-type calcium channels and the activity of calcium/calmodulin-dependent protein kinase are required for TRH effect on PPII activity in primary cultures of adenohypophyseal cells. This pathway may coregulate PPII and prolactin biosynthesis in response to TRH.

Differential responses of thyrotropin-releasing hormone (TRH) neurons to cold exposure or suckling indicate functional heterogeneity of the TRH system in the paraventricular nucleus of the rat hypothalamus.

Thyrotropin-releasing hormone (TRH) is released from the median eminence upon neural stimulation such as cold or suckling exposure. Concomitant with the cold- or suckling-induced release of TRH is a rapid and transient increase in the expression of proTRH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. We employed two strategies to determine whether TRH neurons responding to cold exposure are different from those responding to suckling. First, we attempted to identify a marker of cellular activation in TRH neurons of the PVN. Cold induced c-fos expression in about 25% of TRH neurons of the PVN, but no induction was observed by suckling. Moreover, we explored the expression of a variety of immediate early genes including NGFI-A, fra-1 and c-jun, or CREB phosphorylation but found none to be induced by suckling. The number of cells expressing high levels of proTRH mRNA was counted and compared to total expressing cells. An increased number of cells expressing high levels of proTRH mRNA was observed when both stimuli were applied to the same animal, suggesting that different cells respond separately to each stimulus. We therefore analyzed the distribution of responsive TRH neurons as defined by the cellular level of proTRH mRNA. The proTRH mRNA signal was analyzed within three rostrocaudal zones of the PVN and within six mediolateral columns. Results showed that in response to cold, all areas of the PVN of the lactating rat present increased proTRH mRNA levels, including the anterior zone where few hypophysiotropic TRHergic cells are believed to reside. The distribution of the proTRH mRNA expressing cells in response to cold was quite comparable in female and in male rats. In contrast, the response after suckling was confined to the middle and caudal zones. Our results provide evidence of a functional specialization of TRH cells in the PVN.

BDNF increases the early expression of TRH mRNA in fetal TrkB+ hypothalamic neurons in primary culture.

Known effects of neurotrophins in the developing central nervous system include induction or regulation of peptide expression. Hypothalamic postmitotic thyrotropin-releasing hormone (TRH)-producing neurons may require neurotrophins for survival and/or differentiation. This issue was investigated using primary cell cultures derived from 17-day-old fetal rat hypothalamus seeded in serum-free medium and analysed up to 4 days in vitro culture. Neurotrophin receptor (TrkB and TrkC) mRNA expression was detected by RT-PCR in fetal hypothalamus and throughout the culture period. Western blots confirmed the expression of the full-length proteins in vitro. Semi-quantitative RT-PCR showed that the addition of brain-derived neurotrophic factor (BDNF) increases TRH mRNA levels while the addition of neurotrophin-3 does not. TRH cell content was not modified. Studies on the effect of cell density or homologous conditioned medium demonstrated that endogenous factors probably contribute to determine TRH mRNA levels. One of these factors was BDNF because basal TRH mRNA levels were reduced by the addition of a Trk inhibitor or anti-BDNF. TrkB mRNA was expressed in 27% of cells and TRH mRNA in 2% of cells. The number of TRH+ cells was not affected by BDNF treatment. Forty-eight per cent of TRH neurons contained TrkB mRNA; these neurons had higher amounts of TRH mRNA than TrkB- neurons. Only TrkB+ cells responded to BDNF by increasing their TRH mRNA levels suggesting that BDNF may directly affect TRH biosynthesis. In conclusion, fetal hypothalamic TRH neurons are probably heterogeneous in regard to the neurotrophic factors enhancing peptide and mRNA levels. BDNF enhances TRH mRNA levels in a population of TrkB+ fetal hypothalamic TRHergic neurons in primary culture. However, additional influences may be necessary for the establishment of peptide phenotype in the TrkB+ neurons.

Development of pro-TRH gene expression in primary cultures of fetal hypothalamic cells.

Little is known about the temporal relationship and the sequential steps for peptide biosynthesis during the terminal differentiation of the peptide phenotype in central nervous system. Analysis of the TRH phenotype in primary cultures of rat fetal day 17 hypothalamic cells has shown that TRH levels start increasing only after a week in culture, in contrast with in vivo data showing a steady increase during late fetal life. The purpose of this study was to compare the developmental patterns of TRH and pro-TRH mRNA levels in vitro to determine whether the initial low and steady levels of TRH are due to deficient transcription. Pro-TRH mRNA levels were detected by semi-quantitative RT-PCR through the development of primary cultures of serum-supplemented hypothalamic fetal cells from 17 day old embryos. Pro-TRH mRNA levels per dish increased steadily since the beginning of the culture. In contrast, TRH levels per dish were low and stable during the first week increasing afterwards, but remaining low compared to equivalent in vivo values. Pro-TRH mRNA levels per hypothalamus increased between fetal day 17 and postnatal 14, suggesting that the in vitro pattern of pro-TRH mRNA development mimics that occurring in vivo. These data show that pro-TRH gene expression does not limit TRH accumulation in vitro suggesting that the transcriptional and post-transcriptional programs leading to peptide accumulation are established independently.

Rapid down regulation of pyroglutamyl peptidase II activity by arachidonic acid in primary cultures of adenohypophyseal cells.

Thyrotropin releasing hormone (TRH; pglu-his-proNH2) is inactivated, in the extracellular space, by pyroglutamyl aminopeptidase II (PPII), a narrow specificity ectopeptidase. In adenohypophysis, multiple hormones regulate PPII surface activity. The intracellular pathways of regulation are still poorly understood. Since some of the neurohormones which regulate PPII activity, including TRH and dopamine, transduce in part their effect through modulation of arachidonic acid (AA) mobilization, we have tested its role in regulation of PPII activity in primary cultures of rat adenohypophyseal cells. Melittin concentrations from 0.25 to 1 ug/ml induced a rapid decrease of PPII activity; 0.5 ug/ml caused a maximum effect (38-45% inhibition) at 20-30 min. AA (0.5 or 5 uM) also inhibited PPII activity (42-72%, maximum at 20 min); AA effect was reversible, with values approaching control at 1 h. The inhibitory effect of AA was blocked by lipoxygenase (10 uM nordihidroguaiaretic acid) but not ciclooxygenase inhibitors (10 uM indomethacin) suggesting the involvement of the lipoxygenase pathway. These data show that production of arachidonic acid by adenohypophyseal cells can rapidly but transiently down regulate surface PPII activity. This is the first evidence that AA mobilization can regulate the activity of an ectopeptidase.

[3-Me-His(2)]-TRH combined with dopamine withdrawal rapidly and transiently increases pyroglutamyl aminopeptidase II activity in primary cultures of adenohypophyseal cells.

TRH is hydrolyzed by pyroglutamyl aminopeptidase II (PP II), a highly specific ecto-enzyme which is localized on the surface of lactotrophs. To study whether PP II activity may be rapidly regulated during a burst of prolactin secretion, we used an in vitro model in which primary cultures of adenohypophyseal cells were incubated with 500 nM dopamine (DA) for 24 h prior to treatments. We observed a rapid increase of PP II activity when 100 nM [3-Me-His(2)]-TRH, a TRH agonist, was added at removal of DA. PPII activity was maximal after 20 min of treatment and reduced to time 0 activity at 30 min. Dopamine withdrawal alone, slightly and transiently, modified the enzyme activity: an initial activation at 15 min was followed by a transient inhibition at 20 min. The specific contribution of [3-Me-His(2)]-TRH in this paradigm was a transient enhancement of PP II activity. If DA was not removed, [3-Me-His(2)]-TRH was ineffective. These data demonstrate that during in vitro conditions that mimic a suckling episode, adenohypophyseal PP II activity is rapidly and reversibly adjusted.

Extracellular matrix proteins increase the expression of pro-TRH and pro-protein convertase PC1 in fetal hypothalamic neurons in vitro.

External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 microgram/ml. Fetal day 17 cells plated at a density of 1250/mm(2) were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus.

Regulation of adenohypophyseal pyroglutamyl aminopeptidase II activity by thyrotropin-releasing hormone and phorbol esters.

Thyrotropin-releasing hormone (TRH) is inactivated by a narrow specificity ectopeptidase, pyroglutamyl aminopeptidase II (PPII), in the proximity of target cells. In adenohypophysis, PPII is present on lactotrophs. Its activity is regulated by thyroid hormones and 17beta-estradiol. Studies with female rat adenohypophyseal cell cultures treated with 3,3',5'-triiodo-L-thyronine (T3) showed that hypothalamic/paracrine factors, including TRH, can also regulate PPII activity. Some of the transduction pathways involve protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP). The purpose of this study was to determine whether T3 levels or gender of animals used to propagate the culture determine the effects of TRH or PKC. PPII activity was lower in cultures from male rats. In cultures from both sexes, T3 induced the activity. The percentages of decrease due to TRH or PKC were independent of T3 or gender; the percentage of decrease due to cAMP may also be independent of gender. These results suggest that T3 and hypothalamic/paracrine factors may independently control PPII activity in adenohypophysis, in either male or female animals.

Three TRH-like molecules are released from rat hypothalamus in vitro.

TRH-like immunoreactivity distinct from TRH is present in various tissues and fluids. In order to determine whether TRH-like molecules are secreted by the hypothalamus, we analyzed tissues and media from hypothalamic slices incubated in Krebs Ringer bicarbonate. Media from basal or high KCl conditions contained 3 TRH-like molecules evidenced by reverse phase high performance liquid chromatography followed by TRH radioimmunoassay. Peak I corresponded to authentic TRH (73% of total immunoreactivity) and peaks II and III had a higher retention time. These additional TRH-like forms were neither detected in hypothalamic tissue nor in tissue or medium from olfactory bulb. Gel filtration analysis of hypothalamic media revealed only one TRH-like peak eluting as TRH, suggesting that the molecular weights of peaks II and III are similar to that of TRH. Peak II retention time was similar to that of pglu-phe-proNH2. We analysed if they could be produced by post secretory metabolism of TRH. Incubation of hypothalamic slices with [3H-Pro]-TRH did not produce radioactive species comigrating with peaks II or III. However, it induced rapid degradation to [3H-Pro]-his-prodiketopiperazine ([3H]-HPDKP). Inhibitor profile suggested that pyroglutamyl aminopeptidase II, but not pyroglutamyl aminopeptidase I, is responsible for [3H]-HPDKP production. These data are consistent with the hypothesis that pyroglutamyl aminopeptidase II is the main aminopeptidase degrading TRH in hypothalamic extracellular fluid. Furthermore, we suggest that the hypothalamus releases additional TRH-like molecules, one of them possibly pglu-phe-proNH2, which may participate in control of adenohypophyseal secretions.

Dexamethasone rapidly regulates TRH mRNA levels in hypothalamic cell cultures: interaction with the cAMP pathway.

The biosynthesis of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) is subject to neural and hormonal regulations. To identify some of the potential effectors of this modulation, we incubated hypothalamic dispersed cells with dexamethasone for short periods of time (1-3 h) and studied the interaction of this hormone with protein kinase C (PKC) and PKA signaling pathways. TRH mRNA relative changes were determined by the RT-PCR technique. One hour incubation with 10(-10)-10(-4) M dexamethasone produced a concentration-dependent biphasic effect: an inhibition was observed on TRH mRNA levels at 10(-10) M, an increase above control at 10(-8)-10(-6) M and a reduction at higher concentrations (10(-5)- 10(-4) M). The stimulatory effect of 10(-8) M dexamethasone on TRH mRNA was essentially independent of new protein synthesis, as evidenced by cycloheximide pretreatment. Changes in TRH mRNA levels were reflected by enhanced TRH cell content. Incubation with a cAMP analogue (8-bromo-cAMP, 8Br-cAMP) or with a PKC activator (12-O-tetradecanoylphorbol-13-acetate, TPA) increased TRH mRNA levels after 1 and 2 h, respectively. An increase in TRH mRNA expression was observed by in situ hybridization of dexamethasone or 8Br-cAMP-treated cells. The interaction of dexamethasone, PKA and PKC signaling pathways was studied by combined treatment. The stimulatory effect of 10(-7) M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10(-3) M 8-Br-cAMP and dexamethasone diminished the stimulatory effect of both drugs. An inhibition was observed when the cAMP analogue was coincubated with TPA or TPA and dexamethasone. These results demonstrate that dexamethasone can rapidly regulate TRH biosynthesis and suggest a cross talk between cAMP, glucocorticoid receptors and PKC transducing pathways.