Kinin-B2 Receptor Mediated Neuroprotection after NMDA Excitotoxicity Is Reversed in the Presence of Kinin-B1 Receptor Agonists

Background: Kinins, with bradykinin and des-Arg 9-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg 9-bradykinin as well as Lys-des-Arg 9-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-Daspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. Principal Findings: Bradykinin at 10 nM and 1 mM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg 9-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spik

Kinin-B2 receptor mediated protection against NMDA-induced excitotoxicity and its reversion by Lys-des-Arg⁹-bradykinin.

<p>Peak areas of synaptically elicited population spikes (PSs) recorded in the <i>stratum pyramidale</i> region of hippocampal slices are reported as mean values ± S.E.M. Bradykinin (BK) (10 nM and 1 µM) protected against NMDA (0.5 mM)-mediated cytotoxicity (n = 21, *** p<0.001, compared to control values in the presence of NMDA alone). Neuroprotection induced by 10 nM BK was abolished in the presence of 100 nM HOE-140 (HOE) (n = 21, ### p<0.001, values obtained in the presence of NMDA and BK compared to those collected in the presence of NMDA, BK and HOE-140). The MEK/MAPK inhibitor PD98059 (50 µM) did not interfere with BK-mediated neuroprotection. The PI3-kinase inhibitor LY294002 (10 µM) co-applied with 10 nM BK blocked neuroprotection conferred by BK (n = 21, p<0.05, compared to control values in the presence of BK alone). BK (10 nM)-exerted effects were abolished by 100 nM of the B1BKR agonist Lys-des-Arg⁹-BK (p<0.001, compared to control values in the presence of BK alone). Lys-des-Arg⁹-BK-mediated blockade of neuroprotection was reverted in the presence of PD98059 (50 µM) or the B1BKR antagonist Lys-des-Arg⁹-Leu⁸-bradykinin (1 µM) (p<0.001, compared to control values in the presence of BK and Lys-des-Arg⁹-BK) (n = 21). Statistical analysis was done by one way ANOVA followed by the Dunn's method.</p

Verification of population spike recovery in the presence of kinin-B1 receptor agonists and antagonists.

<p>Synaptically elicited PSs were recorded in the stratum pyramidale region in the absence of NMDA. Control slices were treated only with ACSF and compared with slices treated with 100 nM or 1 µM concentrations of the B1BKR receptor antagonist Lys-des-Arg⁹-Leu⁸-BK or agonist Lys-des-Arg⁹-BK. PSs were measured before and after the application of peptides. Data are presented as mean values ± S.D. (n = 28).</p

Relation of population spike recovery with apoptosis rates.

<p>Initial population spikes (PS) were recorded in <i>stratum pyramidale</i> region of hippocampal slices prior to and following a 10 min application of 0.5 mM NMDA. A) Z-LEHD-FMK, a cell-permeant caspase 9 inhibitor (C.I.), was superfused during 1 h prior to NMDA administration or during 1 h after NMDA. Each lane was superfused for 1 h with ACSF, and the initial PS was recorded from seven slices per lane. For the NMDA lane, the perfusion with ACSF continued for 1 h. Then 0.5 mM NMDA was applied for 10 min; the second lane was superfused with 5 µM of the caspase 9 inhibitor for 1 h after NMDA washout; the third lane was superfused with the inhibitor for 1 h prior to exposure to 0.5 mM NMDA for 10 min. After that, all three lanes were superfused with ACSF for 1 h, and at the end of this time, the final PS was recorded. PS recovery rates (peak areas) obtained in the NMDA alone were compared with those obtained in the presence of 0.5 mM NMDA plus 5 µM Z-LEDH-FMK (C.I.) (n = 21, ***, <i>p</i><0.001, as analyzed by Student's t-test). B) The same protocol describe above was used for GSK-3 inhibition (GSK I) by 25 µM SB-216763 (n = 21, * <i>p</i><0.05). C) NMDA-triggered excitotoxicity induces cytochrome c release from mitochondria and is correlated with the decrease of PS areas. Cytochrome c release was measured after 3 hours. The amount of cytochrome c released after NMDA was more than 2 fold greater (220±20%) than detected in control fraction obtained from slices superfused with ACSF (n = 3, p<0.05). (A) to (C): Data are presented as mean values ± standard deviation (S.D.).</p