Pierre Le Grevès

Calcitonin gene-related peptide is a potent inhibitor of substance P degradation

Calcitonin gene-related peptide (CGRP) was found to potently inhibit a substance P endopeptidase isolated from human CSF. CGRP potentiated substance P irritant actions; a possible mechanism is interaction for a common metabolic step. Somatostatin is another peptide capable of competing with substance P endopeptidase.

Measurement of Substance P Metabolites in Rat CNS

Abstract: A procedure based on ion‐exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P(SP), the SP(1–7), and C‐terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50‐ to 100‐fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1–7) comigrated with the authentic standard whereas practically all activity isolated as C‐terminal fragments comigrated with SP(5–11). The levels of C‐terminal fragments in rat brain areas rich in SP and in spinal cord were 1–2% of those of parent compound. The levels of SP(1–7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1–7) levels fell more rapidly than those of SP or C‐terminal fragments.

Characterization of substance P(1-7) and (1-8) generating enzyme in human cerebrospinal fluid

A substance P-hydrolyzing endopeptidase has been purified from a large quantity of human cerebrospinal fluid by ion exchange chromatography (DEAE-Sepharose CL-6B) and molecular sieving (Sephadex G-100 and Sephacryl S-200). The purification was monitored by measuring the conversion of synthetic substance P using a radioimmunoassay specific for its (1-7) fragment. The enzyme has an apparent molecular weight of 43,000. It cleaves predominantly at the Phe7-Phe8 and Phe8-Gly9 bonds but gives no or negligible conversion of the other tachykinins, neuromedin K and L (substance K).

Growth hormone induces age-dependent alteration in the expression of hippocampal growth hormone receptor and N-methyl-D-aspartate receptor subunits gene transcripts in male rats

Studies were conducted to evaluate the effects of s.c. injected recombinant human growth hormone (GH) on the expression of the gene transcript of N-methyl-D-aspartate receptor subunits type 1 (NR1), type 2A (NR2A), and type 2B (NR2B) in the male rat hippocampus. The GH-induced effects on the expression of hippocampal gene transcripts of GH receptor (GHR) and GH-binding protein were also examined. Male Sprague–Dawley rats, kept in four groups of two different ages, was treated with the hormone or saline during 10 days before decapitation and tissue dissection. Brain tissues collected were analyzed for mRNA content by using the Northern blot technique. The results indicated that in adult young rats (11 weeks of age) the hormone elicited a decrease in the mRNA expression of NR1 but an increase in that of the NR2B subunit. In elderly adult rats (57–67 weeks of age) GH induced an increase in the expression of the hippocampal message for NR1 and NR2A. Meanwhile, the hormone induced a significant up-regulation of the GHR transcript in hippocampus of adult young rats but not in elderly adult rats. It was further found that a significant positive correlation exists between the level of GHR mRNA and the expression of the NR2B subunit transcript in adult young rats. The GH-induced increase in the expression of hippocampal mRNA for the NR2B subunit is compatible with a previously observed memory promoting effect seen for the hormone, because overexpression of this N-methyl-D-aspartate receptor subunit is shown to enhance cognitive capabilities.

Growth hormone replacement in hypophysectomized rats affects spatial performance and hippocampal levels of NMDA receptor subunit and PSD-95 gene transcript levels

Clinical studies have demonstrated that growth hormone (GH) promotes learning and memory processes in GH-deficient (GHD) patients. In animal studies, GH also influences the N-methyl-D-aspartate (NMDA) receptor system in the hippocampus, an essential component of long-term potentiation (LTP), which is highly involved in memory acquisition. This study was designed to examine the beneficial effects of recombinant human GH (rhGH) on cognitive function in male rats with multiple hormone deficiencies resulting from hypophysectomy (Hx). The performance of an rhGH-treated group and an untreated control group was appraised in the Morris water maze (MWM). The rhGH-treated group performed significantly better in the spatial memory task than the control animals on the second and third trial days. Further training eliminated this difference between the groups. Hippocampal mRNA expression of the NMDA subunits NR1, NR2A and NR2B, insulin-like growth factor type 1 receptor (IGF-1R), and postsynaptic density protein-95 (PSD-95) was then measured in the animals by Northern blot analysis. The results suggest that there may be a relationship between the NMDA receptor subunit mRNA expression levels and learning ability, and that learning is improved by rhGH in Hx rats. Furthermore, a link between MWM performance and PSD-95 was also suggested by this study.

Effects of an anabolic-androgenic steroid on the regulation of the NMDA receptor NR1, NR2A and NR2B subunit mRNAs in brain regions of the male rat

The expression of the N-methyl-D-aspartate (NMDA) receptor subunits NR1, NR2A and NR2B mRNAs was examined in discrete areas of the male rat brain (including hippocampus, hypothalamus, nucleus accumbens and cortex) following 14 days daily intramuscular injections of high doses (5 and 15 mg/kg) of an anabolic-androgenic steroid (AAS) (nandrolone decanoate). The results indicated that the drug produced a significant decrease in the mRNA expression of the NR2A receptor subunit both in the hypothalamus and hippocampus. A decrease in the level of NR2B receptor mRNA was observed in hypothalamus at the lower dose of the AAS but in other areas examined, this receptor subunit mRNA was not affected. Except for a decreased expression in the nucleus accumbens at the higher dose of AAS the NR1 receptor subunit mRNA was not affected by the drug. The three subunit mRNAs in cortex were not significantly altered. The effects of the steroid on the mRNA expression for the NMDA receptor subunits in hippocampus and hypothalamus are suggested to be involved in the mechanism behind aggressive behaviour, a feature previously associated with AAS misuse. The downregulation of the mRNA for the NR1 receptor subunit in nucleus accumbens may relate to a mechanism involved in the recently suggested AAS-induced stimulation of the brain reward system.

Calcitonin gene-related peptide is metabolized by an endopeptidase hydrolyzing substance P

Calcitonin gene-related peptide (CGRP) is cleaved by an endopeptidase, also known to hydrolyze substance P (SP). The enzyme which was isolated from human cerebrospinal fluid, converted rCGRP into two products, clearly separable on HPLC. Amino acid analysis showed cleavage to occur at Leu16-Ser17. The carboxy-terminal fragment, rCGRP-(17-37), was weakly active in inhibiting 125I-rCGRP binding to a rat medulla oblongata membrane preparation, but it showed no binding to spinal cord membranes. The N-terminal fragment, rCGRP-(1-16), had very low or no affinity. Autoradiography with 125I-rCGRP showed distinct labelling of rat dorsal spinal cord, while there was no consistent pattern with 125I-rCGRP-(1-16). In the isolated guinea pig ileum preparation, the two fragments showed no CGRP-like activity. The ability of CGRP to interfere with SP degradation is offered as the explanation why CGRP has been reported to potentiate several biologic actions of SP.

Age-related effects of IGF-1 on the NMDA-, GH- and IGF-1-receptor mRNA transcripts in the rat hippocampus

Insulin-like growth factor-1 (IGF-1) and growth hormone (GH) have been suggested to promote memory and cognitive capabilities. In a recent publication we observed that GH increase the proportion of the NR2B subunit mRNA transcript of the NMDA receptor in rat hippocampus. NR2B has been suggested to be essential for spatial learning and long-term potentiation (LTP). This effect of GH might be IGF-1-mediated or a result of a co-ordination with IGF-1. To test this hypothesis further, we examined the effects of 10 daily s.c. injections of IGF-1 on NMDA receptor subunits (NR1, NR2A, and NR2B), GH receptor (GHR), GH binding protein (GHBP) and type 1 IGF receptor (IGF-1R) gene transcripts in the hippocampus. The NR2B subunit mRNA increased in young (11 weeks) but not in older (14-16 months) rats and the expression of the NR2A mRNA was decreased in both groups. The ratio of NR2B to NR2A is suggested to mirror the potential for synaptic plasticity. In both age groups, IGF-1 treatment resulted in a significant increase of this ratio at transcription level. The GHR mRNA increased in young rats, mimicking the effect of GH, while the IGF-1R mRNA was decreased in the older group of rats after IGF-1 treatment. These results suggest that IGF-1 in many aspects may mediate the actions earlier shown for GH.

The effect of dehydroepiandrosterone sulfate and allopregnanolone sulfate on the binding of [3H]ifenprodil to the N-methyl-D-aspartate receptor in rat frontal cortex membrane

Neurosteroids have been shown to modulate the N-methyl-d-aspartate (NMDA) receptor function. Dehydroepiandrosterone sulfate (DHEAS) is shown to participate in memory and learning processes as well as preventing glutamate neurotoxicity in hippocampus. In this study we have focused on the modulatory effect of neurosteroids on ifenprodil binding to the NR2B subunit of the NMDA receptor. We show that DHEAS and allopregnanolone sulfate (ALLOPREGS) exert different effects on the [(3)H]ifenprodil binding at 10, 30 or 100 nM, corresponding to physiological concentrations. The effects include changes in the ifenprodil displacement curve, changing it from a one-site fit into a two-site fit leaving B(max), K(d) and K(off) unaffected. Our results indicate that DHEAS and ALLOPREGS induce an allosteric modulation of the NMDA receptor, an observation that might contribute to the understanding of the effects of these neurosteroids.

Endomorphin-1 and endomorphin-2 differentially interact with specific binding sites for substance P (SP) aminoterminal SP1–7 in the rat spinal cord

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) represent two opioid active tetrapeptides with high affinity and selectivity for the mu-opioid (MOP) receptor. Both EM-1 and EM-2 exhibit strong inhibition of pain signals in the central nervous system (CNS). In contrast to these compounds, the undecapeptide substance P (SP) facilitates pain influx in the CNS. SP has been implicated in a number of functions in the central nervous system, including pain processing and reward. Its aminoterminal fragment SP1-7 has been shown to modulate several actions of SP in the CNS, the nociceptive effect included. Although the actions of SP1-7 have been known for long no specific receptor for the SP fragment has yet been cloned. In this study, we demonstrate the presence of specific binding sites for the heptapeptide in the rat spinal cord. The binding affinity for unlabeled SP1-7 to the specific sites for the labeled heptapeptide highly exceeded those of SP and other C- or N-terminal fragments thereof. The NK-1, NK-2 and NK-3 receptor ligands [Sar9, Met(O2)11]SP, R396 and senktide, respectively, showed no or negligible binding. Moreover, both EM-1 and EM-2 were found to interact with SP1-7 binding. However, a significant difference in binding affinity between the two opioid active tetrapeptides was observed. As recorded from replacement curves the affinity of EM-2 was 10 times weaker than that for SP1-7 but about 100 times higher than that of EM-1. Among other Tyr-Pro-containing peptides Tyr-MIF-1 but not Tyr-W-MIF-1 exhibited affinity of similar potency as EM-2. These results strengthen the previously observed differences between EM-1 and EM-2 in various functional studies. Moreover, using a cell line (C6) expressing the MOP receptor it was shown that the labeled SP1-7 did not interact with binding to this receptor and no functional response was seen for the SP heptapeptide on the MOP receptor by means of stimulation in the GTPgammaS assay. This suggests that the identified SP1-7 binding sites, with high affinity also for EM-2, are not identical to the MOP receptor and apparently not to any of the known tachykinin receptors.