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Cellular localization of kinin B1 receptor in the spinal cord of streptozotocin-diabetic rats with a fluorescent [Nalpha-Bodipy]-des-Arg9-bradykinin.

Talbot S, Théberge-Turmel P, Liazoghli D, Sénécal J, Gaudreau P, Couture R - J Neuroinflammation (2009)

Bottom Line: Four days post-STZ treatment, B1R expression was confirmed by quantitative real-time PCR and autoradiography.BdABK failed to displace the B2R radioligand but displaced the B1R radioligand (IC50 = 5.3 nM).In comparison, IC50 values of B1R selective antagonist R-715 and B1R agonist des-Arg9-BK were 4.3 nM and 19 nM, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, Université de Montréal, Succursale Downtown, Montréal, Québec, Canada. sebastien.talbot@umontreal.ca

ABSTRACT

Background: The kinin B1 receptor (B1R) is upregulated by pro-inflammatory cytokines, bacterial endotoxins and hyperglycaemia-induced oxidative stress. In animal models of diabetes, it contributes to pain polyneuropathy. This study aims at defining the cellular localization of B1R in thoracic spinal cord of type 1 diabetic rats by confocal microscopy with the use of a fluorescent agonist, [Nalpha-Bodipy]-des-Arg9-BK (BdABK) and selective antibodies.

Methods: Diabetes was induced by streptozotocin (STZ; 65 mg/kg, i.p.). Four days post-STZ treatment, B1R expression was confirmed by quantitative real-time PCR and autoradiography. The B1R selectivity of BdABK was determined by assessing its ability to displace B1R [125I]-HPP-desArg10-Hoe140 and B2R [125I]-HPP-Hoe 140 radioligands. The in vivo activity of BdABK was also evaluated on thermal hyperalgesia.

Results: B1R was increased by 18-fold (mRNA) and 2.7-fold (binding sites) in the thoracic spinal cord of STZ-treated rats when compared to control. BdABK failed to displace the B2R radioligand but displaced the B1R radioligand (IC50 = 5.3 nM). In comparison, IC50 values of B1R selective antagonist R-715 and B1R agonist des-Arg9-BK were 4.3 nM and 19 nM, respectively. Intraperitoneal BdABK and des-Arg9-BK elicited dose-dependent thermal hyperalgesia in STZ-treated rats but not in control rats. The B1R fluorescent agonist was co-localized with immunomarkers of microglia, astrocytes and sensory C fibers in the spinal cord of STZ-treated rats.

Conclusion: The induction and up-regulation of B1R in glial and sensory cells of the spinal cord in STZ-diabetic rats reinforce the idea that kinin B1R is an important target for drug development in pain processes.

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[Nα-Bodipy]-des-Arg9-BK (BdABK) selectivity for B1R was evaluated by confocal microscopy. While B1R labeling in the presence of BdABK was absent in thoracic spinal cord of control rats (A), it was shown as green dots in STZ-treated spinal cord (B, and enlarged in C). B1R labeling was absent in STZ-treated spinal cord when BdABK (50 μM) was co-incubated with 10-5M SSR240612 (D) or 10-5M R-715 (E). Background staining represents non specific autofluorescence. Scale bars = 20 μm (A, B, D, E) and 8.2 μm (C). Pictures are representative of a minimum of 4 sections per rat from 4 different STZ-diabetic rats.
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Figure 2: [Nα-Bodipy]-des-Arg9-BK (BdABK) selectivity for B1R was evaluated by confocal microscopy. While B1R labeling in the presence of BdABK was absent in thoracic spinal cord of control rats (A), it was shown as green dots in STZ-treated spinal cord (B, and enlarged in C). B1R labeling was absent in STZ-treated spinal cord when BdABK (50 μM) was co-incubated with 10-5M SSR240612 (D) or 10-5M R-715 (E). Background staining represents non specific autofluorescence. Scale bars = 20 μm (A, B, D, E) and 8.2 μm (C). Pictures are representative of a minimum of 4 sections per rat from 4 different STZ-diabetic rats.

Mentions: Figure 1 illustrates B1R labeling with BdABK from low (i) to high (V) magnification (green dots) in dorsal horn of thoracic spinal cord of STZ-treated rats. As depicted in Figure 2, BdABK showed no labeling in control thoracic spinal cord (A), while the labeling of B1R was apparent in thoracic spinal cord of STZ-treated rats as revealed by green dots (B). Selectivity and specificity of the labeling were demonstrated by the absence of BdABK labeling in STZ-spinal cord sections when the B1R antagonists SSR240612 (D) and R-715 (E) were added at 10-5M.


Cellular localization of kinin B1 receptor in the spinal cord of streptozotocin-diabetic rats with a fluorescent [Nalpha-Bodipy]-des-Arg9-bradykinin.

Talbot S, Théberge-Turmel P, Liazoghli D, Sénécal J, Gaudreau P, Couture R - J Neuroinflammation (2009)

[Nα-Bodipy]-des-Arg9-BK (BdABK) selectivity for B1R was evaluated by confocal microscopy. While B1R labeling in the presence of BdABK was absent in thoracic spinal cord of control rats (A), it was shown as green dots in STZ-treated spinal cord (B, and enlarged in C). B1R labeling was absent in STZ-treated spinal cord when BdABK (50 μM) was co-incubated with 10-5M SSR240612 (D) or 10-5M R-715 (E). Background staining represents non specific autofluorescence. Scale bars = 20 μm (A, B, D, E) and 8.2 μm (C). Pictures are representative of a minimum of 4 sections per rat from 4 different STZ-diabetic rats.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667487&req=5

Figure 2: [Nα-Bodipy]-des-Arg9-BK (BdABK) selectivity for B1R was evaluated by confocal microscopy. While B1R labeling in the presence of BdABK was absent in thoracic spinal cord of control rats (A), it was shown as green dots in STZ-treated spinal cord (B, and enlarged in C). B1R labeling was absent in STZ-treated spinal cord when BdABK (50 μM) was co-incubated with 10-5M SSR240612 (D) or 10-5M R-715 (E). Background staining represents non specific autofluorescence. Scale bars = 20 μm (A, B, D, E) and 8.2 μm (C). Pictures are representative of a minimum of 4 sections per rat from 4 different STZ-diabetic rats.
Mentions: Figure 1 illustrates B1R labeling with BdABK from low (i) to high (V) magnification (green dots) in dorsal horn of thoracic spinal cord of STZ-treated rats. As depicted in Figure 2, BdABK showed no labeling in control thoracic spinal cord (A), while the labeling of B1R was apparent in thoracic spinal cord of STZ-treated rats as revealed by green dots (B). Selectivity and specificity of the labeling were demonstrated by the absence of BdABK labeling in STZ-spinal cord sections when the B1R antagonists SSR240612 (D) and R-715 (E) were added at 10-5M.

Bottom Line: Four days post-STZ treatment, B1R expression was confirmed by quantitative real-time PCR and autoradiography.BdABK failed to displace the B2R radioligand but displaced the B1R radioligand (IC50 = 5.3 nM).In comparison, IC50 values of B1R selective antagonist R-715 and B1R agonist des-Arg9-BK were 4.3 nM and 19 nM, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, Université de Montréal, Succursale Downtown, Montréal, Québec, Canada. sebastien.talbot@umontreal.ca

ABSTRACT

Background: The kinin B1 receptor (B1R) is upregulated by pro-inflammatory cytokines, bacterial endotoxins and hyperglycaemia-induced oxidative stress. In animal models of diabetes, it contributes to pain polyneuropathy. This study aims at defining the cellular localization of B1R in thoracic spinal cord of type 1 diabetic rats by confocal microscopy with the use of a fluorescent agonist, [Nalpha-Bodipy]-des-Arg9-BK (BdABK) and selective antibodies.

Methods: Diabetes was induced by streptozotocin (STZ; 65 mg/kg, i.p.). Four days post-STZ treatment, B1R expression was confirmed by quantitative real-time PCR and autoradiography. The B1R selectivity of BdABK was determined by assessing its ability to displace B1R [125I]-HPP-desArg10-Hoe140 and B2R [125I]-HPP-Hoe 140 radioligands. The in vivo activity of BdABK was also evaluated on thermal hyperalgesia.

Results: B1R was increased by 18-fold (mRNA) and 2.7-fold (binding sites) in the thoracic spinal cord of STZ-treated rats when compared to control. BdABK failed to displace the B2R radioligand but displaced the B1R radioligand (IC50 = 5.3 nM). In comparison, IC50 values of B1R selective antagonist R-715 and B1R agonist des-Arg9-BK were 4.3 nM and 19 nM, respectively. Intraperitoneal BdABK and des-Arg9-BK elicited dose-dependent thermal hyperalgesia in STZ-treated rats but not in control rats. The B1R fluorescent agonist was co-localized with immunomarkers of microglia, astrocytes and sensory C fibers in the spinal cord of STZ-treated rats.

Conclusion: The induction and up-regulation of B1R in glial and sensory cells of the spinal cord in STZ-diabetic rats reinforce the idea that kinin B1R is an important target for drug development in pain processes.

Show MeSH
Related in: MedlinePlus