Limits...
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.

Show MeSH

Related in: MedlinePlus

[Nα-Bodipy]-des-Arg9-BK affinity for B2R was evaluated by quantitative autoradiography. Increasing concentration (10-10 to 10-6 M) of Hoe 140 (selective B2R antagonist) displaced total binding of 200 pM [125I]-HPP-HOE-140 to B2R. In contrast, same concentrations of [Nα-Bodipy]-des-Arg9-BK (fluorescent B1R agonist) did not displace the B2R radioligand. Data are means ± SEM of 4 sections per rat from 7 different rats for each compound.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2667487&req=5

Figure 4: [Nα-Bodipy]-des-Arg9-BK affinity for B2R was evaluated by quantitative autoradiography. Increasing concentration (10-10 to 10-6 M) of Hoe 140 (selective B2R antagonist) displaced total binding of 200 pM [125I]-HPP-HOE-140 to B2R. In contrast, same concentrations of [Nα-Bodipy]-des-Arg9-BK (fluorescent B1R agonist) did not displace the B2R radioligand. Data are means ± SEM of 4 sections per rat from 7 different rats for each compound.

Mentions: Competition experiments using 200 pM [125I]-HPP-desArg10-Hoe 140 and 10-10 to 10-6 M of des-Arg9-BK, R-715, or BdABK revealed that kinin analogues decreased in a concentration-dependent manner the binding of [125I]-HPP-desArg10-Hoe 140 in the thoracic spinal cord of STZ-treated rats (Fig. 3). The rank order of potency to inhibit total B1R binding sites was R-715 = BdABK > des-Arg9-BK with IC50 values of 4.3 ± 0.2 nM, 5.3 ± 0.1 nM and 19 ± 0.2 nM, respectively. In contrast, BdABK (10-10 to 10-6 M) failed to inhibit the binding of 200 pM [125I]-HPP-Hoe 140 to B2R in the thoracic spinal cord of STZ-treated rats (Fig. 4). In comparison, same concentrations of Hoe 140 displaced B2R binding sites with IC50value of 1.33 ± 0.1 nM.


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 affinity for B2R was evaluated by quantitative autoradiography. Increasing concentration (10-10 to 10-6 M) of Hoe 140 (selective B2R antagonist) displaced total binding of 200 pM [125I]-HPP-HOE-140 to B2R. In contrast, same concentrations of [Nα-Bodipy]-des-Arg9-BK (fluorescent B1R agonist) did not displace the B2R radioligand. Data are means ± SEM of 4 sections per rat from 7 different rats for each compound.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: [Nα-Bodipy]-des-Arg9-BK affinity for B2R was evaluated by quantitative autoradiography. Increasing concentration (10-10 to 10-6 M) of Hoe 140 (selective B2R antagonist) displaced total binding of 200 pM [125I]-HPP-HOE-140 to B2R. In contrast, same concentrations of [Nα-Bodipy]-des-Arg9-BK (fluorescent B1R agonist) did not displace the B2R radioligand. Data are means ± SEM of 4 sections per rat from 7 different rats for each compound.
Mentions: Competition experiments using 200 pM [125I]-HPP-desArg10-Hoe 140 and 10-10 to 10-6 M of des-Arg9-BK, R-715, or BdABK revealed that kinin analogues decreased in a concentration-dependent manner the binding of [125I]-HPP-desArg10-Hoe 140 in the thoracic spinal cord of STZ-treated rats (Fig. 3). The rank order of potency to inhibit total B1R binding sites was R-715 = BdABK > des-Arg9-BK with IC50 values of 4.3 ± 0.2 nM, 5.3 ± 0.1 nM and 19 ± 0.2 nM, respectively. In contrast, BdABK (10-10 to 10-6 M) failed to inhibit the binding of 200 pM [125I]-HPP-Hoe 140 to B2R in the thoracic spinal cord of STZ-treated rats (Fig. 4). In comparison, same concentrations of Hoe 140 displaced B2R binding sites with IC50value of 1.33 ± 0.1 nM.

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