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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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B1R distribution in thoracic spinal cord of STZ-treated rats was shown by confocal microscopy with [Nα-Bodipy]-des-Arg9-BK. Shown are pictures from low (i) to high magnification (V) of the dorsal horn. Scale bars = 200, 50, 20, 5 and 2.5 μm, respectively from (i) to (V). Pictures are representative of a minimum of 4 sections per rat from 4 different STZ-diabetic rats.
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Figure 1: B1R distribution in thoracic spinal cord of STZ-treated rats was shown by confocal microscopy with [Nα-Bodipy]-des-Arg9-BK. Shown are pictures from low (i) to high magnification (V) of the dorsal horn. Scale bars = 200, 50, 20, 5 and 2.5 μm, respectively from (i) to (V). 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)

B1R distribution in thoracic spinal cord of STZ-treated rats was shown by confocal microscopy with [Nα-Bodipy]-des-Arg9-BK. Shown are pictures from low (i) to high magnification (V) of the dorsal horn. Scale bars = 200, 50, 20, 5 and 2.5 μm, respectively from (i) to (V). 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 1: B1R distribution in thoracic spinal cord of STZ-treated rats was shown by confocal microscopy with [Nα-Bodipy]-des-Arg9-BK. Shown are pictures from low (i) to high magnification (V) of the dorsal horn. Scale bars = 200, 50, 20, 5 and 2.5 μm, respectively from (i) to (V). 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