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.

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.

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.

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.

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.

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.

Effect of neurotransmitters on the in vitro release of immunoreactive thyrotropin-releasing hormone from rat mediobasal hypothalamus.

The in vitro release of TRH from hypothalamic fragments or purified nerve endings (synaptosomes) has been evaluated after incubation for 10 min in the presence of various concentrations of K+ or neurotransmitters. Release of the hormone from fragments but not from synaptosomes was enhanced in the presence of 56 mM K+ in a Ca++ -dependent manner. Neurotransmitter effects were thus tested on the fragments. Addition of histamine (10 (-7)-10(-5) M) induced a significant increase over the basal release of TRH. A comparable effect was obtained with dimaprit (10(-5) M), a highly specific agonist of histamine H2 receptors; conversely, the response to histamine was blocked by the addition of a H2 (metiamide; 10(-6) M) but not of a H1 (mepyramine; 10(-6) M) antagonist to the incubation medium. Dopamine (10(-7) M) slightly inhibited the release of TRH, but antagonists of dopamine receptors (10(-7)-10(-6) M fluphenazine or 10(-6) M alpha-flupentixol) exhibited an inhibitory effect by themselves, so that specific receptors involved in mediating dopamine actions could not be further characterized. In contrast, noradrenalin, serotonin gamma-aminobutyric acid and acetylcholine (tested at concentration of 10(-7) M) did not alter the basal release of the tripeptide.

Pups removal enhances thyrotropin-releasing hormone mRNA in the hypothalamic paraventricular nucleus.

Previous studies have shown that lactation and suckling alter thyrotropin-releasing hormone (TRH) biosynthesis in hypothalamic paraventricular neurons. The amounts of paraventricular TRH mRNA and mediobasal hypothalamus (MBH) TRH were determined following removal of the pups to examine whether paraventricular TRH neuron activity is altered during the transition from lactation to estrous cycle. Paraventricular TRH mRNA and MBH TRH levels were determined by Northern blot analysis and radioimmunoassay, respectively. We had shown previously that after an 8-h withdrawal of the pups at mid-lactation the MBH TRH and paraventricular TRH mRNA levels are not modified. This condition was compared to one where pups were removed for 56 h, finding a significant decrease (46%, p < 0.005) of MBH TRH and a significant increase (156%, p < 0.02) of paraventricular TRH mRNA. The effect observed in the paraventricular TRH mRNA was correlated negatively with the serum corticosterone levels, a potential negative regulator of paraventricular TRH mRNA. The results were similar if a 1-h suckling period was introduced 8 h after withdrawal of the pups to induce a transient increase of corticosterone levels. The pattern of TRH mRNA was specific to the paraventricular nucleus because there was no enhancement in the preoptic area-anterior hypothalamus. In summary, our data suggest that TRH biosynthesis in paraventricular neurons is slowly adjusted after withdrawal of the pups, possibly to prepare TRH neurons to the new secretory demands of the estrous cycle.

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.

Multiple hypothalamic factors regulate pyroglutamyl peptidase II in cultures of adenohypophyseal cells: role of the cAMP pathway.

In the adenohypophysis, thyrotrophin-releasing hormone (TRH) is inactivated by pyroglutamyl peptidase II (PPII), a TRH-specific ectoenzyme localized in lactotrophs. TRH slowly downregulates surface PPII activity in adenohypophyseal cell cultures. Protein kinase C (PKC) activation mimics this effect. We tested the hypothesis that other hypothalamic factors controlling prolactin secretion could also regulate PPII activity in adenohypophyseal cell cultures. Incubation for 16 h with pituitary adenylate cyclase activator peptide 38 (PACAP; 10(-6) M) decreased PPII activity. Bromocryptine (10(-8) M), a D2 dopamine receptor agonist, or somatostatin (10(-6) M) stimulated enzyme activity and blocked the inhibitory effect of [3-Me-His2]-TRH, a TRH receptor agonist. Bromocryptine and somatostatin actions were suppressed by preincubation with pertussis toxin (400 ng ml(-1)). Because these hypophysiotropic factors transduce some of their effects using the cAMP pathway, we analysed its role on PPII regulation. Cholera toxin (400 ng ml(-1)) inhibited PPII activity. Forskolin (10(-6) M) caused a time-dependent decrease in PPII activity, with maximal inhibition at 12-16 h treatment; ED50 was 10(-7) M. 3-isobutyl-1-methylxanthine or dibutiryl cAMP, caused a dose-dependent inhibition of PPII activity. These data suggest that increased cAMP down-regulates PPII activity. The effect of PACAP was blocked by preincubation with H89 (10(-6) M), a protein kinase A inhibitor, suggesting that the cAMP pathway mediates some of the effects of PACAP. Maximal effects of forskolin and 12-O-tetradecanoylphorbol 13-acetate were additive. PPII activity, therefore, is independently regulated by the cAMP and PKC pathways. Because most treatments inhibited PPII mRNA levels similarly to PPII activity, an important level of control of PPII activity by these factors may be at the mRNA level. We suggest that PPII is subject to 'homologous' and 'heterologous' regulation by elements of the multifactorial system that controls prolactin secretion.

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.

The narrow specificity pyroglutamate amino peptidase degrading TRH in rat brain is an ectoenzyme.

In order to determine the pathway of extracellular metabolism of the thyrotropin releasing hormone (pyroglu-his-proNH(2)) in brain, the topographical organization of pyroglutamate aminopeptidase II on the plasma membrane was investigated. Its activity was only slightly increased when intact brain synaptosomes were lysed by osmotic shock or detergent treatment. Trypsin treatment of intact synaptosomes destroyed 70-80% of enzyme activity without affecting lactate dehydrogenase. Pyroglutamate aminopeptidase II activity was present in primary cultures of foetal mice cortical cells. It was detected in intact cells, was not released by the cells and its activity was not increased by saponin pretreatment. Trypsin treatment of the cells reduced pyroglutamate aminopeptidase II by 70% but did not affect pyroglutamate aminopeptidase I and lactate dehydrogenase. These data support that brain pyroglutamate aminopeptidase II is an ectoenzyme. They suggest that this enzyme could be responsible for thyrotropin releasing hormone extracellular catabolism in brain.

Subcellular distribution of the enzymes degrading thyrotropin releasing hormone and metabolites in rat brain.

In order to further understand the role of enzymes degrading Thyrotropin Releasing Hormone (TRH, pglu-his-proNH(2)) and metabolites, we studied their subcellular distribution in rat brain. Brain tissue was homogenized in 0.32 M sucrose, tris-HCl 0.01 M pH 7.4 and fractionated by differential and discontinuous gradient centrifugation; [(3)H]pro-TRH was incubated with the various subcellular fractions and the extent of degradation of each metabolite was measured after separation by thin layer chromatography. Several markers were simultaneously measured (lactate dehydrogenase, 5?-nucleotidase and hexosaminidase) to determine the pattern of distribution of the subcellular organelles. The post-proline cleaving enzyme responsible for pglu-his-pro formation and pyroglutamate amino-peptidase (which requires sulphydryl compounds for maximal activity) were found in cytosol but were barely detectable in the soluble component of synaptosomes; pyroglutamate aminopeptidase (dependent on metals) and post-proline dipeptidyl amino peptidase were found on the membranes of synaptosomes; imido peptidase was not enriched in any particular fraction. These data are consistent with the hypothesis that membrane-bound pyroglutamate aminopeptidase is responsible for TRH degradation once released into the synaptic cleft and that the post-proline dipeptidylaminopeptidase may participate in the extracellular catabolism of his-proNH(2) before it cyclizes to his-pro-DKP. They also suggest that post-proline cleaving enzyme and soluble pyroglutamate aminopeptidase may not play an important role in the regulation of TRH levels in nerve endings.

Regional distribution of pyroglutamyl peptidase II in rabbit brain, spinal cord, and organs.

Pyroglutamyl peptidase II (PPII) is a narrow specificity ectoenzyme that degrades thyrotropin-releasing hormone (TRH). We detected the enzyme in the brain of various mammals, with highest specific activity in rabbit brain. In this species, activity was heterogeneously distributed in the central nervous system. There was a 28-fold difference between regions of highest and lowest PPII activity. Enzyme activity was highest in the olfactory bulb and posterior cortex. In the spinal cord, activity was low but unevenly distributed, with highest values detected in the thoracic (T) region. Segments T1 and T2 activities were particularly high. Other organs contained low or undetectable levels of activity. The levels of TRH-like immunoreactivity (TRH-LI) in spinal cord segments were greatest in T3-T4 and lumbar L2-L6. Low concentrations were found in T1 and T9-T12. There was a partial correlation between the distribution of PPII activity and TRH receptors but not with TRH-LI levels. These results demonstrate that PPII is predominantly a central nervous system enzyme, and they support the hypothesis that PPII is responsible for degrading TRH released into the synaptic cleft.

Influence of thyroid status on TRH metabolism in rat olfactory bulb.

The effect of thyroid hormones (TH) on the metabolism of thyrotropin-releasing hormone (TRH) in the olfactory bulb (OB) was compared with the hypothalamic response to TRH. Two methods were used to induce hypothyroidism: propylthiouracyl-methimazole (PTU-M) or 131I treatment. Hyperthyroidism was produced by 3,3',5-triiodo-L-thyronine (T3) injections to the hypothyroid animals. With PTU-M treatment, paraventricular TRH mRNA levels increased 57% and returned to the euthyroid level with T3 treatment. In OB, TRH mRNA was not altered. The TRH content was unaffected in the mediobasal hypothalamus of PTU-M-treated animals whereas it was reduced in OB (31%) with no further response upon T3 treatment. 131I-induced hypothyroidism did not modify the OB TRH content but it was decreased (31%) in hyperthyroids. In the median eminence, TRH increased 26% in hypothyroids, and the response was reversed with T3. Our results demonstrate that treatments that change thyroid status can alter TRH levels in the OB, probably at a translational or postranslational level, though the effects may be pharmacological.

Regional distribution of in vitro release of thyrotropin releasing hormone in rat brain.

To increase our knowledge of the TRH functions in brain and the processes of TRH compartmentalization and release, we studied the in vitro release of endogenous TRH in different brain areas. We also determined the correlation between TRH levels and release under both basal and stimulated conditions. TRH concentration was measured in tissues and media by specific radioimmunoassay. TRH-like material detected in olfactory bulb and hypothalamic incubates (basal or K+ stimulated) were shown to be chromatographically identical to synthetic TRH. Different brain regions showed high variability in the basal release of TRH (1-20% of tissue content). This suggests the existence of different pools. The response to depolarizing stimulus (56 mM K+) was significant only in the following regions: median eminence, total hypothalamus, preoptic area, nucleus accumbens-lateral septum, amygdala, mesencephalon, medulla oblongata and the cervical region of the spinal cord. These regions have been shown to contain a high number of receptors, a high concentration of TRH nerve endings and are susceptible to TRH effects. These results support the hypothesis that TRH functions as neuromodulator in these areas.

TRH in the rat cerebellum: I. Distribution and concentration.

We determined the regional distribution and concentration of endogenous TRH in the rat cerebellum. Radioimmunoassay of endogenous TRH extracted and purified from five different regions of the rat cerebellum and whole hypothalamus showed that the cerebellar vermis contained 24 pg/mg, the hemispheres 74 pg/mg, the deep cerebellar nuclei 148 pg/mg, and the flocculo-nodular region 559 pg/mg of TRH. The highest concentration of TRH was in the cerebellar paraflocculi, which contained 786 pg/mg. The hypothalamic concentration of TRH was 465 pg/mg. Assay of the non-purified tissue fractions (crude extracts) resulted in lower TRH values in accordance with data previously reported by other authors. Bioassay analysis of TRH in purified fractions resulted in values similar to those obtained by radioimmunoassay. On the basis of these findings we hypothesize a functional role for TRH in the cerebellum.

Presence of a membrane bound pyroglutamyl amino peptidase degrading thyrotropin releasing hormone in rat brain.

In the present work we studied the pattern of degradation of [3H-Pro]-TRH by soluble and membrane fractions from rat brain. Demonstration of the membrane bound or soluble nature of the activities was obtained by comparing their distribution to that of lactate dehydrogenase and by looking at the effect of NaCl washes on the membrane fractions. We observed that the pyroglutamyl amino peptidase activity detected in brain homogenates is a result of two different enzymes. One of them is a soluble enzyme previously characterized, that needs DTT and EDTA for its expression, is inhibited by SH-blocking agents such as iodoacetamide and utilizes p-glu-beta-naphtylamide as a substrate. The other one, a membrane enzyme, is inhibited by chelating agents such as EDTA and DTT, is not affected by iodoacetamide and does not degrade p-glu-beta-naphtylamide. The later presents some specificity towards TRH as shown by competition experiments with TRH analogs. We were able to corroborate that the post proline cleaving enzyme acting on TRH is a soluble enzyme. In membranes we demonstrated also the presence of a post-proline dipeptidyl aminopeptidase. The membrane bound pyroglutamidase activity is a potential new source of L-his-L-pro-diketopiperazine in brain. The presence of a TRH degrading enzyme in membrane fractions is of particular importance in searching an inactivation mechanism of this peptide once it is released into the synaptic cleft.

Specific inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase do not change the in vitro release of TRH or its content in rodent brain.

Pyroglutamyl diazomethyl ketone and N-benzyloxycarbonyl prolyl prolinal, specific inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase respectively, were used to study the possible role of these enzymes in the regulation of thyrotropin releasing hormone turnover. In vitro thyrotropin releasing hormone release by male rat hypothalamic slices was studied. Combined in vitro treatment with 10(-5)M of both inhibitors totally inhibited both enzymatic activities. The treatment did not affect basal or 56 mM K+ induced thyrotropin releasing hormone release or thyrotropin releasing hormone levels in slices. Repeated combined intraperitoneal injections of the two inhibitors for up to 12 hours produced a 70%-95% reduction in mouse brain pyroglutamyl peptidase I specific activity and a 65%-85% reduction in prolyl endopeptidase specific activity. Thyrotropin releasing hormone levels were unaffected by this treatment in all regions tested. The data suggest that these two enzymes are not involved in the intra- or extracellular control of thyrotropin releasing hormone levels in brain or hypophysis.

Evaluation of the role of prolyl endopeptidase and pyroglutamyl peptidase I in the metabolism of LHRH and TRH in brain.

Intraneuronal peptide regulatory mechanisms are still poorly understood. The cytosolic enzymes prolyl endopeptidase (EC 3.4.21.26) and pyroglutamyl peptidase I (E.C.3.4.19.3) degrade both TRH and LHRH. Previous studies from this laboratory have not supported a role for these enzymes in the control of TRH levels. These studies have now been extended to cell and organ cultures and examine the effects of enzyme inhibition on LHRH. Exposure of dispersed hypothalamic cells or median eminences in culture to Z-Pro-Prolinal and pyroglutamyl diazomethyl ketone, specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I respectively, did not change TRH content or recovery of released TRH. In vivo and in vitro treatment with these inhibitors did not modify the content of LHRH or recovery of this peptide upon release from several brain regions except in the olfactory bulb where an unexpected decrease in levels was observed. Olfactory bulb levels of TRH also decreased but only after prolonged in vivo inhibitor treatment. The decrease in olfactory bulb LHRH and TRH could not be accounted for by enzyme induction and is likely due to a non-specific or indirect effect of the inhibitors on the processing of these peptides. These studies demonstrate that levels of LHRH and TRH in brain are not controlled by cytosolic peptidases.