Jean-Louis Charli

Cleavage of substance P to an N-terminal tetrapeptide and a C-terminal heptapeptide by a post-proline cleaving enzyme from bovine brain

A post-proline cleaving enzyme isolated from bovine brain and previously shown to act on the neuropeptides thyrotropin releasing hormone (TRH: pGlu-His-Pro-NH2) and luteinizing hormone releasing hormone (LRH: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) has now been found to cleave the Pro4-Gln5 bond in substance P (SP: H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) to yield the N-terminal tetrapeptide H-Arg-Pro-Lys-Pro-OH and the C-terminal heptapeptide H-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. The site of cleavage of the undecapeptide was confirmed by chemical synthesis of SP peptide fragments, by high performance liquid chromatographic analysis, and by high-voltage paper electrophoresis, thin layer chromatography and amino acid analysis. In addition, SP was shown by enzyme kinetic analysis to act as a potent competitive inhibitor of the enzymatic hydrolysis of the synthetic post-proline cleaving enzyme substrate carbobenzoxy-Gly-Pro-para-nitroanilide. It is known that the heptapeptide is as active as SP itself in a variety of bioassays, whereas the tetrapeptide has been reported to induced cyclic AMP formation and neurite extension in neuroblastoma cells and to enehance the phagocytosis activity of macrophages. These observations suggest that SP may play a dual role, with one activity residing in its C-terminal part, and the other in the N-terminal part of the molecule.

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.

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.

Tanycyte pyroglutamyl peptidase II contributes to regulation of the hypothalamic-pituitary-thyroid axis through glial-axonal associations in the median eminence.

Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound metallopeptidase that inactivates TRH in the extracellular space, is tightly regulated by thyroid hormone in cells of the anterior pituitary. Whether PPII has any role in the region where axons containing hypophysiotropic TRH terminate, the median eminence, is unknown. For this purpose, we analyzed the cellular localization and regulation of PPII mRNA in the mediobasal hypothalamus in adult, male rats. PPII mRNA was localized in cells lining the floor and infralateral walls of the third ventricle and coexpressed with vimentin, establishing these cells as tanycytes. PPII mRNA extended in a linear fashion from the tanycyte cell bodies in the base of the third ventricle to its cytoplasmic and end-feet processes in the external zone of the median eminence in close apposition to pro-TRH-containing axon terminals. Compared with vehicle-treated, euthyroid controls, animals made thyrotoxic by the i.p. administration of 10 microg L-T(4) daily for 1-3 d, showed dramatically increased accumulation of silver grains in the mediobasal hypothalamus and an approximately 80% increase in enzymatic activity. PPII inhibition in mediobasal hypothalamic explants increased TRH secretion, whereas i.p. injection of a specific PPII inhibitor increased cold stress- and TRH-induced TSH levels in plasma. We propose that an increase in circulating thyroid hormone up-regulates PPII activity in tanycytes and enhances degradation of extracellular TRH in the median eminence through glial-axonal associations, contributing to the feedback regulation of thyroid hormone on anterior pituitary TSH secretion.

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.

Regional distribution of the membrane-bound pyroglutamate amino peptidase-degrading thyrotropin-releasing hormone in rat brain☆

The brain regional distribution of membrane-bound pyroglutamate aminopeptidase-degrading thyrotropin-releasing hormone (TRH) in rat was studied using a specific radiometric assay. The distribution was not homogeneous: a 10-fold difference was observed between regions. The highest activity was detected in olfactory bulb while the lowest was in the cervical part of spinal cord. There was no correlation with the regional distribution of enzyme activity vs TRH levels, previously reported TRH receptors or in vitro TRH release. The differential distribution of this enzyme is consistent with the hypothesis that it is responsible for extracellular degradation of neuroactive peptides.

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 10–10M, an increase above control at 10–8–10–6M and a reduction at higher concentrations (10–5– 10–4M). The stimulatory effect of 10–8M 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 10–7M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10–3M 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.

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.

Tissue-specific regulation of pyroglutamate aminopeptidase II activity by thyroid hormones.

Among the enzymes capable of degrading thyrotropin-releasing hormone (TRH) in vitro, two pyroglutamate aminopeptidases (PGA) are specific for TRH: thyroliberinase, a seric enzyme and PGAII, a membrane-bound peptidase. The effect of thyroid hormone status on the activity of these enzymes was evaluated in serum and various tissues. Only in adenohypophysis, triiodothyronine treatment increased PGAII to 376% of control; hypothyroidism produced the reverse effect (decrease to 23% of control). As previously reported, similar changes were observed for thyroliberinase. TRH degradation at the adenohypophysis level may participate in the negative feedback control of thyroid hormones.

Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons

Analysis of gene regulatory sequences in primary cultures of neurons has been hampered by inefficient transfection of post-mitotic neurons with reporter plasmids. We describe detailed conditions that allowed a significant improvement of transfection efficiency in primary cultures of serum-supplemented rat fetal hypothalamic cells. Transfected cells expressed the green fluorescent protein (GFP) under the control of the strong but non-cell-specific cytomegalo virus (CMV) promoter or under the thyrotropin-releasing hormone (TRH) promoter, to direct expression only in TRH neurons. Using the CMV promoter-GFP plasmid, we tested several commercially available transfection reagents; the best results were obtained with polyethylenimine (PEI) and Lipofectamine 2000. We optimized the transfection procedure with PEI because it rendered more reproducible results. Transfection with PEI was optimal when cells were transfected at a cellular density of 2.9 x 10(6) cells in 35-mm dishes, with 10 microg of DNA, a PEI/DNA ratio of 8.8 and PEI pH of 6.9. Using these conditions, we were able to detect GFP positive neurons after transfecting the TRH promoter-GFP plasmid. GFP positive cells were successfully purified by FACS. This opens the possibility to use transfection of mammalian CNS post-mitotic neurons for new applications including the purification of specific neuronal subtypes.