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Increased Brain Neurotensin and NTSR2 Lead to Weak Nociception in NTSR3/Sortilin Knockout Mice

View Article: PubMed Central - PubMed

ABSTRACT

The neuropeptide neurotensin (NT) elicits numerous pharmacological effects through three different receptors (NTSR1, NTSR2, and NTSR3 also called sortilin). Pharmacological approaches and generation of NTSR1 and NTSR2-deficient mice allowed to determine the NT-induced antipsychotic like behavior, the inhibitory of weak fear memory and the nociceptive signaling in a rat formalin tonic pain model to NTSR1. Conversely, the effects of NT on thermal and tonic nociceptions were mediated by NTSR2. However, the role of NTSR3/sortilin on the neurotensinergic system was not investigated. Here, by using C57Bl/6J mouse model in which the gene coding for NTSR3/sortilin has been inactivated, we observed a modification of the expression of both NTSR2 and NT itself. Quantitative PCR and protein expression using Western blot analyses and AlphaLisa™ technology resulted in the observation that brain NTSR2 as well as brain and blood NT were 2-fold increased in KO mice leading to a resistance of these mice to thermal and chemical pain. These data confirm that NTSR3/sortilin interacts with other NT receptors (i.e., NTSR2) and that its deletion modifies also the affinity of this receptor to NT.

No MeSH data available.


Related in: MedlinePlus

Measurement of brain and blood NT content in WT and NTSR3/sortilin KO mice (A) Quantitative PCR of NT from WT and NTSR3/sortilin KO mouse brains. (B) Competitive inhibition of biotinylated NT by unlabeled NT, the standard curve was the mean ± SEM from 3 independent experiments performed in triplicate, the corresponding IC50 was 0.48 nM. (C) NT concentrations in sera and in brain from WT and NTSR3/sortilin KO mice measured using AlphaLisa™ technique. Each bar in the graphs represents the mean value ± SEM of NT concentrations determination in serum (n = 19) and in brain extracts (n = 3). *p < 0.05.
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Figure 3: Measurement of brain and blood NT content in WT and NTSR3/sortilin KO mice (A) Quantitative PCR of NT from WT and NTSR3/sortilin KO mouse brains. (B) Competitive inhibition of biotinylated NT by unlabeled NT, the standard curve was the mean ± SEM from 3 independent experiments performed in triplicate, the corresponding IC50 was 0.48 nM. (C) NT concentrations in sera and in brain from WT and NTSR3/sortilin KO mice measured using AlphaLisa™ technique. Each bar in the graphs represents the mean value ± SEM of NT concentrations determination in serum (n = 19) and in brain extracts (n = 3). *p < 0.05.

Mentions: As we observed an important increase of NTSR2 expression at the plasma membrane, we wondered whether the expression of its ligand may also be modified in NTSR3/sortilin KO mice by using the dosing method developed for NT. We first observed that the amount of NT mRNA was also significantly enhanced in the brain of KO mice (p < 0.05) (Figure 3A). The higher level of NT mRNA measured in the brain from NTSR3/sortilin KO mice prompted us to quantify the peptide content in brain extracts and serum from both mice. To perform these experiments, we developed tools (specific antibodies and biotinylated NT) to be used according to the AlphaLisa™ method (Perkin). The Figure 3B illustrated the competition curve between biotinylated NT and unlabeled NT. The amount of NT present in the serum or in the brain extracts was determined from this curve (Figure 3C). We observed a significant increase of the peptide in serum (from 12 nM in WT to 18 nM in KO mice) and brain extracts (from 21 nM in WT to 45 nM in KO) (Figure 3C).


Increased Brain Neurotensin and NTSR2 Lead to Weak Nociception in NTSR3/Sortilin Knockout Mice
Measurement of brain and blood NT content in WT and NTSR3/sortilin KO mice (A) Quantitative PCR of NT from WT and NTSR3/sortilin KO mouse brains. (B) Competitive inhibition of biotinylated NT by unlabeled NT, the standard curve was the mean ± SEM from 3 independent experiments performed in triplicate, the corresponding IC50 was 0.48 nM. (C) NT concentrations in sera and in brain from WT and NTSR3/sortilin KO mice measured using AlphaLisa™ technique. Each bar in the graphs represents the mean value ± SEM of NT concentrations determination in serum (n = 19) and in brain extracts (n = 3). *p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121284&req=5

Figure 3: Measurement of brain and blood NT content in WT and NTSR3/sortilin KO mice (A) Quantitative PCR of NT from WT and NTSR3/sortilin KO mouse brains. (B) Competitive inhibition of biotinylated NT by unlabeled NT, the standard curve was the mean ± SEM from 3 independent experiments performed in triplicate, the corresponding IC50 was 0.48 nM. (C) NT concentrations in sera and in brain from WT and NTSR3/sortilin KO mice measured using AlphaLisa™ technique. Each bar in the graphs represents the mean value ± SEM of NT concentrations determination in serum (n = 19) and in brain extracts (n = 3). *p < 0.05.
Mentions: As we observed an important increase of NTSR2 expression at the plasma membrane, we wondered whether the expression of its ligand may also be modified in NTSR3/sortilin KO mice by using the dosing method developed for NT. We first observed that the amount of NT mRNA was also significantly enhanced in the brain of KO mice (p < 0.05) (Figure 3A). The higher level of NT mRNA measured in the brain from NTSR3/sortilin KO mice prompted us to quantify the peptide content in brain extracts and serum from both mice. To perform these experiments, we developed tools (specific antibodies and biotinylated NT) to be used according to the AlphaLisa™ method (Perkin). The Figure 3B illustrated the competition curve between biotinylated NT and unlabeled NT. The amount of NT present in the serum or in the brain extracts was determined from this curve (Figure 3C). We observed a significant increase of the peptide in serum (from 12 nM in WT to 18 nM in KO mice) and brain extracts (from 21 nM in WT to 45 nM in KO) (Figure 3C).

View Article: PubMed Central - PubMed

ABSTRACT

The neuropeptide neurotensin (NT) elicits numerous pharmacological effects through three different receptors (NTSR1, NTSR2, and NTSR3 also called sortilin). Pharmacological approaches and generation of NTSR1 and NTSR2-deficient mice allowed to determine the NT-induced antipsychotic like behavior, the inhibitory of weak fear memory and the nociceptive signaling in a rat formalin tonic pain model to NTSR1. Conversely, the effects of NT on thermal and tonic nociceptions were mediated by NTSR2. However, the role of NTSR3/sortilin on the neurotensinergic system was not investigated. Here, by using C57Bl/6J mouse model in which the gene coding for NTSR3/sortilin has been inactivated, we observed a modification of the expression of both NTSR2 and NT itself. Quantitative PCR and protein expression using Western blot analyses and AlphaLisa&trade; technology resulted in the observation that brain NTSR2 as well as brain and blood NT were 2-fold increased in KO mice leading to a resistance of these mice to thermal and chemical pain. These data confirm that NTSR3/sortilin interacts with other NT receptors (i.e., NTSR2) and that its deletion modifies also the affinity of this receptor to NT.

No MeSH data available.


Related in: MedlinePlus