Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA ß subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates.

Electron beam inactivation of Tulane virus on fresh produce, and mechanism of inactivation of human norovirus surrogates by electron beam irradiation.

Ionizing radiation, whether by electron beams or gamma rays, is a non-thermal processing technique used to improve the microbial safety and shelf-life of many different food products. This technology is highly effective against bacterial pathogens, but data on its effect against foodborne viruses is limited. A mechanism of viral inactivation has been proposed with gamma irradiation, but no published study discloses a mechanism for electron beam (e-beam). This study had three distinct goals: 1) evaluate the sensitivity of a human norovirus surrogate, Tulane virus (TV), to e-beam irradiation in foods, 2) compare the difference in sensitivity of TV and murine norovirus (MNV-1) to e-beam irradiation, and 3) determine the mechanism of inactivation of these two viruses by e-beam irradiation. TV was reduced from 7 log10 units to undetectable levels at target doses of 16 kGy or higher in two food matrices (strawberries and lettuce). MNV-1 was more resistant to e-beam treatment than TV. At target doses of 4 kGy, e-beam provided a 1.6 and 1.2 log reduction of MNV-1 in phosphate buffered saline (PBS) and Dulbecco's Modified Eagle Medium (DMEM), compared to a 1.5 and 1.8 log reduction of TV in PBS and Opti-MEM, respectively. Transmission electron microscopy revealed that increased e-beam doses negatively affected the structure of both viruses. Analysis of viral proteins by SDS-PAGE found that irradiation also degraded viral proteins. Using RT-PCR, irradiation was shown to degrade viral genomic RNA. This suggests that the mechanism of inactivation of e-beam was likely the same as gamma irradiation as the damage to viral constituents led to inactivation.

Food-restricted and dehydrated-induced anorexic rats present differential TRH expression in anterior and caudal PVN. Role of type 2 deiodinase and pyroglutamyl aminopeptidase II.

TRH synthesized in hypothalamic paraventricular nucleus (PVN) regulates thyroid axis function and is also implicated in anorexigenic effects. Under energy deficit, animals present decreased PVN TRH expression and release, low TSH levels, and increased appetite. Dehydration-induced anorexia (DIA) model allows insight into underlying mechanisms of feeding regulation. Animals drinking a 2.5% NaCl solution for 7 d present body weight reduction; despite their negative energy balance, they avoid food and have increased PVN TRH expression and TSH serum levels. These findings support an inhibiting role of PVN TRH in feeding control. We compared TRH expression by in situ hybridization in PVN subdivisions of 7-d dehydrated male rats to those of a pair-fed group (forced food-restricted) with similar metabolic changes than DIA, but motivated to eat, and to controls. We measured peripheral deiodinase activities, and expression and activity of medial basal hypothalamic type 2 deiodinase and pyroglutamyl-aminopeptidase II, to understand their regulating role in PVN TRH changes between food restriction and anorexia. TRH mRNA levels increased in anterior (aPVN) and medial-caudal subdivisions in DIA rats, whereas it decreased in medial PVN in both experimental groups. We confirmed the nonhypophysiotropic nature of aPVN TRHergic cells by injecting ip fluorogold tracer. Findings support a subspecialization of TRHergic hypophysiotrophic cells that responded differently between anorexic and food-restricted animals; also, that aPVN TRH participates in food intake regulation. Increased type 2 deiodinase activity seemed responsible for low medial PVN TRH synthesis, whereas increased medial basal hypothalamic pyroglutamyl-aminopeptidase II activity in DIA rats might counteract their high TRH release.

The gamma ray response of alanine film dosimeters at low temperatures.

The response of alanine film EPR dosimeters was studied for low temperature gamma irradiation conditions (77-293 K) in the dose interval from 6.3 to 80 kGy. It was found that the response of the dosimeter decreases with decreased irradiation temperature and saturates at lower doses for lower irradiation temperatures. The analysis of the EPR signal suggests that the radical species formed at low temperature are the same as those used for dosimetry at room temperature, but with different concentrations. Their concentrations evolve as the temperature of the sample increases until the usual EPR signal used at room temperature is obtained.

Electron beam irradiation dose dependently damages the bacillus spore coat and spore membrane.

Effective control of spore-forming bacilli begs suitable physical or chemical methods. While many spore inactivation techniques have been proven effective, electron beam (EB) irradiation has been frequently chosen to eradicate Bacillus spores. Despite its widespread use, there are limited data evaluating the effects of EB irradiation on Bacillus spores. To study this, B. atrophaeus spores were purified, suspended in sterile, distilled water, and irradiated with EB (up to 20 kGy). Irradiated spores were found (1) to contain structural damage as observed by electron microscopy, (2) to have spilled cytoplasmic contents as measured by spectroscopy, (3) to have reduced membrane integrity as determined by fluorescence cytometry, and (4) to have fragmented genomic DNA as measured by gel electrophoresis, all in a dose-dependent manner. Additionally, cytometry data reveal decreased spore size, increased surface alterations, and increased uptake of propidium iodide, with increasing EB dose, suggesting spore coat alterations with membrane damage, prior to loss of spore viability. The present study suggests that EB irradiation of spores in water results in substantial structural damage of the spore coat and inner membrane, and that, along with DNA fragmentation, results in dose-dependent spore inactivation.

Electron-beam inactivation of a norovirus surrogate in fresh produce and model systems.

Norovirus remains the leading cause of foodborne illness, but there is no effective intervention to eliminate viral contaminants in fresh produce. Murine norovirus 1 (MNV-1) was inoculated in either 100 ml of liquid or 100 g of food. The inactivation of MNV-1 by electron-beam (e-beam), or high-energy electrons, at varying doses was measured in model systems (phosphate-buffered saline [PBS], Dulbecco's modified Eagle's medium [DMEM]) or from fresh foods (shredded cabbage, diced strawberries). E-beam was applied at a current of 1.5 mA, with doses of 0, 2, 4, 6, 8, 10, and 12 kGy. The surviving viral titer was determined by plaque assays in RAW 264.7 cells. In PBS and DMEM, e-beam at 0 and 2 kGy provided less than a 1-log reduction of virus. At doses of 4, 6, 8, 10, and 12 kGy, viral inactivation in PBS ranged from 2.37 to 6.40 log, while in DMEM inactivation ranged from 1.40 to 3.59 log. Irradiation of inoculated cabbage showed up to a 1-log reduction at 4 kGy, and less than a 3-log reduction at 12 kGy. On strawberries, less than a 1-log reduction occurred at doses up to 6 kGy, with a maximum reduction of 2.21 log at 12 kGy. These results suggest that a food matrix might provide increased survival for viruses. In foods, noroviruses are difficult to inactivate because of the protective effect of the food matrix, their small sizes, and their highly stable viral capsid.

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.

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.

Endotoxin stimulates nitric oxide production in the paraventricular nucleus of the hypothalamus through nitric oxide synthase I: correlation with hypothalamic-pituitary-adrenal axis activation.

Nitric oxide (NO) is an unstable gas that is produced in brain tissues involved in the control of the activity of the hypothalamus-pituitary-adrenal (HPA) axis. Transcripts for constitutive neuronal NO synthase (NOS I), one of the enzymes responsible for NO formation in the brain, is up-regulated by systemic endotoxin [lipopolysaccharide (LPS)] injection. However, this change is delayed compared with LPS induced-ACTH release, which makes it difficult to determine whether it is functionally important for the hormonal response. To obtain a more resolutive time course of the NO response, we first measured NO in microdialysates of the paraventricular (PVN) nucleus of the hypothalamus. The iv injection of 100 microg/kg LPS induced a rapid and short-lived increase in concentrations of this gas, which corresponded to the initiation of the ACTH response. LPS-induced Ca2+-dependent NOS activity in the PVN as well as the number of PVN cells expressing citrulline (a compound produced stoichiometrically with NO) also increased significantly over a time course that corresponded to ACTH and corticosterone release. Finally, blockade of NO production with the arginine derivative Nomega-nitro-L-argininemethylester (L-NAME; 50 mg/kg, sc), which attenuated the ACTH response to LPS, virtually abolished basal NOS activity in the PVN, as well as anterior and neurointermediate lobes of the pituitary, and prevented the appearance of citrulline in the PVN of rats injected with LPS. Collectively, these results show that LPS-induced activation of the HPA axis correlates with the activation of the PVN NOergic system, and supports a stimulatory role for NO in the modulation of the HPA axis in response to immune challenges.

TRH inactivation in the extracellular compartment: role of pyroglutamyl peptidase II.

TRH (pGlu-His-ProNH2) inactivation in the brain and pituitary extracellular fluid is reviewed. While TRH could be eliminated by alternative mechanisms, i.e. uptake or internalization, modification, hydrolysis by broad specificity peptidases such as pyroglutamyl peptidase I and prolyl endopeptidase, evidence accumulates to support a specific neuroectopeptidase as the main mechanism responsible for its extracellular inactivation. Pyroglutamyl peptidase II (PPII; E.C. 3.4.19.6) is a narrow specificity zinc metallopeptidase hydrolyzing the pyroglutamyl-histidyl peptide bond of TRH. PPII is an integral membrane protein with a small intracellular domain, a transmembrane segment and a large extracellular domain that contains the catalytic site. It is therefore idealy situated to degrade TRH present in the extracellular space. PPII is highly enriched in brain, specifically present in neuronal cells. PPII inhibition enhances recovery of TRH released in vitro. In situ hybridization studies demonstrate that PPII mRNA colocalizes with TRH-receptor mRNA in various brain regions. However, the existence of exceptions suggest that alternative inactivation mechanisms for TRH may operate. PPII activity is regulated in various pharmacological or pathophysiological conditions which alter TRH transmission. It is also present in adenohypophysis, preferentially on lactotrophs, where its activity is stringently regulated by hormones and hypothalamic factors. PPII activity regulation may contribute to adjust TRH neural and hormonal transmissions.

Multifactorial modulation of TRH metabolism.

1. Thyrotropin releasing hormone (TRH), synthesized in the paraventricular nucleus of the hypothalamus (PVN), is released in response to physiological stimuli through median eminence nerve terminals to control thyrotropin or prolactin secretion from the pituitary. 2. Several events participate in the metabolism of this neuropeptide: regulation of TRH biosynthesis and release as well as modulation of its inactivation by the target cell. 3. Upon a physiological stimulus such as cold stress or suckling, TRH is released and levels of TRH mRNA increase in a fast and transient manner in the PVN; a concomitant increase in cfos is observed only with cold exposure. 4. Hypothalamic cell cultures incubated with cAMP or phorbol esters show a rise in TRH mRNA levels; dexamethasone produces a further increase at short incubation times. TRH mRNA are thus controlled by transsynaptic and hormonal influences. 5. Once TRH is released, it is inactivated by a narrow specificity ectoenzyme, pyroglutamyl peptidase II (PPII). 6. In adenohypophysis, PPII is subject to stringent control: positive by thyroid hormones and negative by TRH; other hypothalamic factors such as dopamine and somatostatin also influence its activity. 7. These combined approaches suggest that TRH action is modulated in a coordinate fashion.

Expression of the proprotein convertases PC1 and PC2 mRNAs in thyrotropin releasing hormone neurons of the rat paraventricular nucleus of hypothalamus.

PC1 and PC2 are subtilisin-like processing enzymes capable of cleaving thyrotropin releasing hormone (TRH) precursor (pro-TRH) at paired basic residues in vitro. In the paraventricular nucleus of the hypothalamus (PVN), pro-TRH is synthesized to control adenohypophysial thyrotropin and prolactin release. Biochemical and immunological approaches have shown that in the hypothalamus, pro-TRH is extensively cleaved at pairs of basic amino acids. We quantified, by two different approaches, in situ hybridization (ISH) on consecutive cryostat sections or double label ISH, the proportion of PVN TRH neurons containing either PC1 or PC2 mRNAs. Both techniques gave similar results: PC2 mRNA was present in 60-70% of TRH neurons, and PC1 mRNA in 37-46%. Values were similar in the anterior and medial parts of the parvocellular PVN. TRH neurons containing either PC1 or PC2 mRNA were found throughout the areas containing TRH cells without any evidence of anatomical segregation. These results suggest a biochemical heterogeneity in PVN TRH biosynthetic machinery.

Phorbol ester or cAMP enhance thyrotropin-releasing hormone mRNA in primary cultures of hypothalamic cells.

Thyrotropin releasing hormone (TRH) biosynthesis is subject to a multifactorial control. TRH mRNA levels are negatively regulated by thyroid hormones in the paraventricular hypothalamic nucleus, and positively in cold exposure or suckling. Effect of second messenger pathways stimulation, a known response to membrane receptors, was studied in vitro; cultures of rat embryonic hypothalami (18 day gestation) were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) or dibutiryl cAMP (dBcAMP, 1 mM) for various times. Levels of TRH mRNA were raised after the first hour of dBcAMP or 2 h of TPA treatment and were still increased at 24 h. These results suggest a neural regulation of TRH biosynthesis.

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.

In vitro TRH release from hypothalamus slices varies during the diurnal cycle.

We have previously described a daily rhythm in thyrotropin releasing hormone (TRH) and TRH mRNA in the rat hypothalamus. To determine whether TRH release fluctuates in a diurnal manner, we have measured basal and potassium stimulated release from hypothalamic slices, and compared it to release from olfactory bulb slices, during the diurnal cycle. Basal TRH release was higher at 7:00 h than at any other time (1:00, 13:00 or 19:00 h) in either hypothalamus or olfactory bulb. The ratio of stimulated over basal release was higher in the hypothalamus at 19:00 h, when TRH content was highest. Potassium stimulated TRH release from olfactory bulb was not different from basal release at any time. TRH release fluctuations were not due to a rhythm of extracellular inactivation: the activity of pyroglutamyl aminopeptidase II, an ectoenzyme responsible for TRH inactivation, was constant throughout the cycle. Our data demonstrate that diurnal variations of TRH release occur in vitro and that the enhanced responsiveness to potassium stimulation in hypothalamus is correlated with increased levels of peptide.

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.

Suckling and cold stress rapidly and transiently increase TRH mRNA in the paraventricular nucleus.

Thyrotropin releasing hormone (TRH) is released from the median eminence in response to neural stimuli evoked by different physiologic conditions (i.e. cold stress or suckling). The paraventricular nucleus (PVN) synthesizes pro-TRH and responds to negative thyroid hormone feedback. With the aim of determining if TRH biosynthesis is regulated in coordination with its release, we quantified TRH mRNA levels in PVN and in preoptic area-anterior hypothalamus (POA-AH) of rats sacrificed at different times during cold (0.5, 1, 2 or 6 h) or suckling (15, 30 and 60 min) stimulus; TRH-like immunoreactivity (TRH-LI) in medial basal hypothalamus (MBH) and in POA-AH as well as corticosterone, triiodothyronine and prolactin levels in serum were also measured. Increases of serum hormones were observed in both paradigms as has been reported. MBH TRH-LI content decreased during suckling by 33% (p < 0.01) after 1 h, but did not change after cold stimulation. At short stimulation times, PVN TRH mRNA levels were 85% (30 min of suckling) and 97% (1 h in the cold) higher than their respective controls, decreasing to normal after 1-2 h. In the POA-AH, another TRH synthesizing region not involved in TRH hypophysiotropic function, a similar transient enhancement of TRH mRNA (146%) was observed only in cold stimulated animals after 30 min, consistent with its suggested role in thermogenesis. These results show a fast and transient response of TRH mRNA in PVN evoked by a neural stimulus.

Ontogenesis of pyroglutamyl peptidase II activity in rat brain, adenohypophysis and pancreas.

Pyroglutamyl peptidase II (PPII; E.C. 3.4.19.-) is a highly specific membrane-bound ectoenzyme degrading thyrotropin releasing hormone (TRH). The ontogenesis of this enzyme was measured in rat brain regions, adenohypophysis and pancreas. In hypothalamus PPII activity was maximal at day 8 postnatal, decreasing to adult values at day 45. The postnatal ontogenic patterns in posterior cerebral cortex and hypothalamus were similar. In olfactory bulb, two peaks of activity were observed (3th and 22nd day) while in adenohypophysis it appeared only at day 8, increased to day 30, decreasing thereafter to adult values.

Some events of thyrotropin-releasing hormone metabolism are regulated in lactating and cycling rats.

Levels of thyrotropin-releasing hormone (TRH), TRH mRNA and pyroglutamyl peptidase II were analyzed in the hypothalamus-adenohypophyseal axis during lactation and estrous cycle. Mediobasal hypothalamic levels of TRH dropped 41% (p less than 0.01) from pregnancy levels (taken as 100%) on the first day of lactation, recovering until day 15 to the values observed at pregnancy. A sharp decrease was also observed during weaning (36%, p less than 0.01 compared to last day of lactation). TRH levels in the neurohypophysis increased during lactation and dropped at weaning. Highest TRH mRNA levels in the paraventricular nucleus were found at the end of pregnancy and beginning of lactation; they decreased 37% (p less than 0.05) at day 5 of lactation and stayed constant thereafter. Pyroglutamyl peptidase II adenohypophyseal activity was not modified during lactation but changed during estrous cycle. Relative to estrous values, activity diminished 58% (p less than 0.05) at 10.00 h (57% at 14.00 h) during diestrus 2 and 27% at 10.00 h (37% at 14.00 h) during proestrus. Hypothalamic TRH mRNA levels fluctuated in an opposite manner to adenohypophyseal pyroglutamyl peptidase II during the estrous cycle with a peak at diestrus 2: 183% of the estrous value (p less than 0.05). These data point to a regulation of TRH metabolism in conditions where prolactin (PRL) secretion fluctuates. They also suggest a sharp release of TRH between the end of pregnancy and the first day of lactation and that translational efficiency or post-translational processing of TRH precursor in the paraventricular neurons (projecting to the median eminence) increases during lactation and drops at weaning, concomitantly with PRL secretion.