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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.


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Quantification of NT receptors from WT and NTSR3/sortilin KO mice (A) Quantitative PCR of NTSR1 and NTSR2 from WT and NTSR3/sortilin KO mouse brains, ***p < 0.001. (B,C) Protein expression of NTSR1-like and NTSR2-like in plasma membranes (PM), high and low density vesicles (HLDM) and total homogenates prepared from brains from WT and NTSR3/sortilin KO mice. Each bar in the graphs represents the mean ± SEM of bands intensitiy quantified using the corresponding compartment markers from 5 independent experiments. *p < 0.05 using Mann and Whitney Student t-test. (D) Representative Western blot analysis of NTSR1-like and NTSR2-like proteins expressed in plasma membranes (PM), high and low density vesicles (HLDM) and total homogenates prepared from brains from WT and NTSR3/sortilin KO mice. NaK-ATPase; Sodium Potassium-ATPase, TGN38; Trans-Golgi Network protein of 38 kDa.
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Figure 2: Quantification of NT receptors from WT and NTSR3/sortilin KO mice (A) Quantitative PCR of NTSR1 and NTSR2 from WT and NTSR3/sortilin KO mouse brains, ***p < 0.001. (B,C) Protein expression of NTSR1-like and NTSR2-like in plasma membranes (PM), high and low density vesicles (HLDM) and total homogenates prepared from brains from WT and NTSR3/sortilin KO mice. Each bar in the graphs represents the mean ± SEM of bands intensitiy quantified using the corresponding compartment markers from 5 independent experiments. *p < 0.05 using Mann and Whitney Student t-test. (D) Representative Western blot analysis of NTSR1-like and NTSR2-like proteins expressed in plasma membranes (PM), high and low density vesicles (HLDM) and total homogenates prepared from brains from WT and NTSR3/sortilin KO mice. NaK-ATPase; Sodium Potassium-ATPase, TGN38; Trans-Golgi Network protein of 38 kDa.

Mentions: Intriguingly, quantitative PCR (qPCR) determination indicated that the mRNA of NTSR1 remained unchanged whereas the amount of NTSR2 mRNA was significantly increased in the brain of KO mice (p < 0.001) (Figure 2A). This increase of NTSR2 mRNA was in contradiction with the loss levocabastine-sensitive NT binding sites. The similar mRNA level of NTSR1 between WT and KO mice did not correspond to the increase of levocabastine-sensitive NT binding sites observed in the brain of KO mice. Therefore, we verified the protein expression of both receptors after subcellular fractionation and Western blot analysis. The quantification determined from 5 independent experiments indicated that the protein level of NTSR1-like remained similar at the plasma membranes (PM), in the high and low density vesicles (H/LDM) and in the total extracts from brain from WT and KO mice (Figure 2B). However, the amount of NTSR2-like protein was significantly increased by a factor 2 (p < 0.05) at the plasma membranes prepared from KO mouse brain but was similar between WT and KO mice in H/LDM and total extracts (Figures 2C,D).


Increased Brain Neurotensin and NTSR2 Lead to Weak Nociception in NTSR3/Sortilin Knockout Mice
Quantification of NT receptors from WT and NTSR3/sortilin KO mice (A) Quantitative PCR of NTSR1 and NTSR2 from WT and NTSR3/sortilin KO mouse brains, ***p < 0.001. (B,C) Protein expression of NTSR1-like and NTSR2-like in plasma membranes (PM), high and low density vesicles (HLDM) and total homogenates prepared from brains from WT and NTSR3/sortilin KO mice. Each bar in the graphs represents the mean ± SEM of bands intensitiy quantified using the corresponding compartment markers from 5 independent experiments. *p < 0.05 using Mann and Whitney Student t-test. (D) Representative Western blot analysis of NTSR1-like and NTSR2-like proteins expressed in plasma membranes (PM), high and low density vesicles (HLDM) and total homogenates prepared from brains from WT and NTSR3/sortilin KO mice. NaK-ATPase; Sodium Potassium-ATPase, TGN38; Trans-Golgi Network protein of 38 kDa.
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Related In: Results  -  Collection

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Figure 2: Quantification of NT receptors from WT and NTSR3/sortilin KO mice (A) Quantitative PCR of NTSR1 and NTSR2 from WT and NTSR3/sortilin KO mouse brains, ***p < 0.001. (B,C) Protein expression of NTSR1-like and NTSR2-like in plasma membranes (PM), high and low density vesicles (HLDM) and total homogenates prepared from brains from WT and NTSR3/sortilin KO mice. Each bar in the graphs represents the mean ± SEM of bands intensitiy quantified using the corresponding compartment markers from 5 independent experiments. *p < 0.05 using Mann and Whitney Student t-test. (D) Representative Western blot analysis of NTSR1-like and NTSR2-like proteins expressed in plasma membranes (PM), high and low density vesicles (HLDM) and total homogenates prepared from brains from WT and NTSR3/sortilin KO mice. NaK-ATPase; Sodium Potassium-ATPase, TGN38; Trans-Golgi Network protein of 38 kDa.
Mentions: Intriguingly, quantitative PCR (qPCR) determination indicated that the mRNA of NTSR1 remained unchanged whereas the amount of NTSR2 mRNA was significantly increased in the brain of KO mice (p < 0.001) (Figure 2A). This increase of NTSR2 mRNA was in contradiction with the loss levocabastine-sensitive NT binding sites. The similar mRNA level of NTSR1 between WT and KO mice did not correspond to the increase of levocabastine-sensitive NT binding sites observed in the brain of KO mice. Therefore, we verified the protein expression of both receptors after subcellular fractionation and Western blot analysis. The quantification determined from 5 independent experiments indicated that the protein level of NTSR1-like remained similar at the plasma membranes (PM), in the high and low density vesicles (H/LDM) and in the total extracts from brain from WT and KO mice (Figure 2B). However, the amount of NTSR2-like protein was significantly increased by a factor 2 (p < 0.05) at the plasma membranes prepared from KO mouse brain but was similar between WT and KO mice in H/LDM and total extracts (Figures 2C,D).

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