Limits...
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

Binding of 125I-NT to brain homogenates from WT and NTSR3/sortilin KO mice. (A,B) Brain homogenates from WT (A) or from NTSR3/sortilin KO mice (B) (60 μg of proteins) were incubated with increasing concentrations of 125I-NT alone or isotopically diluted with unlabeled NT in the absence (closed symbols) or in the presence (open symbols) of 1 μM levocabastine. Saturation curves were made from specific binding using GraphPad analysis. (C) Representation of the mean ± SEM of total binding and levocabastine-sensitive and -insensitive binding sites calculated from 5 independent experiments performed in triplicate. *p < 0.05 using Student t-Test.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5121284&req=5

Figure 1: Binding of 125I-NT to brain homogenates from WT and NTSR3/sortilin KO mice. (A,B) Brain homogenates from WT (A) or from NTSR3/sortilin KO mice (B) (60 μg of proteins) were incubated with increasing concentrations of 125I-NT alone or isotopically diluted with unlabeled NT in the absence (closed symbols) or in the presence (open symbols) of 1 μM levocabastine. Saturation curves were made from specific binding using GraphPad analysis. (C) Representation of the mean ± SEM of total binding and levocabastine-sensitive and -insensitive binding sites calculated from 5 independent experiments performed in triplicate. *p < 0.05 using Student t-Test.

Mentions: In order to quantify the amount of NT binding sites corresponding to NTSR1 and NTSR2 in the brain of wild type (WT) and NTSR3/sortilin deficient mice (KO-NTSR3), we first performed saturation binding experiments of iodinated NT on homogenates prepared from the indicated brains in the absence or in the presence of the NTSR2 selective blocker levocabastine (1 μM) (Kitabgi et al., 1987). In brain homogenates from WT mice, in the absence of levocabastine, the saturation curve obtained from a typical experiment indicated a maximal binding capacity (Bmax) of about 200 fmol/mg (Figure 1A). In the presence of levocabastine, the Bmax decreased to 65–70 fmol/mg (Figure 1A), a binding capacity corresponding to the levocabastine insensitive NT binding sites attributed to NTSR1. Interestingly, in brain homogenates from KO-NTSR3 mice, saturation experiments performed in the absence or in the presence of levocabastine revealed the same Bmax (Figure 1B), demonstrating that in KO-NTSR3 mice, the binding of NT is insensitive to the drug. Figure 1C which summarized the Bmax mean values obtained from 5 independent experiments, clearly indicated that the amount of levocabastine-insensitive NT binding sites increased in KO mice (from 63 ± 12 fmol/mg in WT mice to 124 ± 30 fmol/mg in KO mice, p = 0.029). As expected, the amount of levocabastine-sensitive NT binding sites was decreased in brain from KO mice from 88 ± 19 fmol/mg in WT to 14 ± 9 fmol/mg in KO (p = 0.028).


Increased Brain Neurotensin and NTSR2 Lead to Weak Nociception in NTSR3/Sortilin Knockout Mice
Binding of 125I-NT to brain homogenates from WT and NTSR3/sortilin KO mice. (A,B) Brain homogenates from WT (A) or from NTSR3/sortilin KO mice (B) (60 μg of proteins) were incubated with increasing concentrations of 125I-NT alone or isotopically diluted with unlabeled NT in the absence (closed symbols) or in the presence (open symbols) of 1 μM levocabastine. Saturation curves were made from specific binding using GraphPad analysis. (C) Representation of the mean ± SEM of total binding and levocabastine-sensitive and -insensitive binding sites calculated from 5 independent experiments performed in triplicate. *p < 0.05 using Student t-Test.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Binding of 125I-NT to brain homogenates from WT and NTSR3/sortilin KO mice. (A,B) Brain homogenates from WT (A) or from NTSR3/sortilin KO mice (B) (60 μg of proteins) were incubated with increasing concentrations of 125I-NT alone or isotopically diluted with unlabeled NT in the absence (closed symbols) or in the presence (open symbols) of 1 μM levocabastine. Saturation curves were made from specific binding using GraphPad analysis. (C) Representation of the mean ± SEM of total binding and levocabastine-sensitive and -insensitive binding sites calculated from 5 independent experiments performed in triplicate. *p < 0.05 using Student t-Test.
Mentions: In order to quantify the amount of NT binding sites corresponding to NTSR1 and NTSR2 in the brain of wild type (WT) and NTSR3/sortilin deficient mice (KO-NTSR3), we first performed saturation binding experiments of iodinated NT on homogenates prepared from the indicated brains in the absence or in the presence of the NTSR2 selective blocker levocabastine (1 μM) (Kitabgi et al., 1987). In brain homogenates from WT mice, in the absence of levocabastine, the saturation curve obtained from a typical experiment indicated a maximal binding capacity (Bmax) of about 200 fmol/mg (Figure 1A). In the presence of levocabastine, the Bmax decreased to 65–70 fmol/mg (Figure 1A), a binding capacity corresponding to the levocabastine insensitive NT binding sites attributed to NTSR1. Interestingly, in brain homogenates from KO-NTSR3 mice, saturation experiments performed in the absence or in the presence of levocabastine revealed the same Bmax (Figure 1B), demonstrating that in KO-NTSR3 mice, the binding of NT is insensitive to the drug. Figure 1C which summarized the Bmax mean values obtained from 5 independent experiments, clearly indicated that the amount of levocabastine-insensitive NT binding sites increased in KO mice (from 63 ± 12 fmol/mg in WT mice to 124 ± 30 fmol/mg in KO mice, p = 0.029). As expected, the amount of levocabastine-sensitive NT binding sites was decreased in brain from KO mice from 88 ± 19 fmol/mg in WT to 14 ± 9 fmol/mg in KO (p = 0.028).

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