Enhancement of bradykinin-induced relaxation by focal brain ischemia in the rat middle cerebral artery: Receptor expression upregulation and activation of multiple pathways

Abstract

Focal brain ischemia markedly affects cerebrovascular reactivity. So far, these changes have mainly been related to alterations in the level of smooth muscle cell function while alterations of the endothelial lining have not yet been studied in detail. We have, therefore, investigated the effects of ischemia/reperfusion injury on bradykinin (BK)-induced relaxation since BK is an important mediator of tissue inflammation and affects vascular function in an endothelium-dependent manner. Focal brain ischemia was induced in rats by endovascular filament occlusion (2h) of the middle cerebral artery (MCA). After 22h reperfusion, both MCAs were harvested and the response to BK studied in organ bath experiments. Expression of the BK receptor subtypes 1 and 2 (B1, B2) was determined by real-time semi-quantitative RT-qPCR methodology, and whole mount immunofluorescence staining was performed to show the B2 receptor protein expression. In control animals, BK did not induce significant vasomotor effects despite a functionally intact endothelium and robust expression of B2 mRNA. After ischemia/reperfusion injury, BK induced a concentration-related sustained relaxation in all arteries studied, more pronounced in the ipsilateral than in the contralateral MCA. The B2 mRNA was significantly upregulated and the B1 mRNA displayed de novo expression, again more pronounced ipsi- than contralaterally. Endothelial cells displaying B2 receptor immunofluorescence were observed scattered or clustered in previously occluded MCAs. Relaxation to BK was mediated by B2 receptor activation, abolished after endothelium denudation, and largely diminished by blocking nitric oxide (NO) release or soluble guanylyl cyclase activity. Relaxation to BK was partially inhibited by charybdotoxin (ChTx), but not apamin or iberiotoxin suggesting activation of an endothelium-dependent hyperpolarization pathway. When the NO-cGMP pathway was blocked, BK induced a transient relaxation which was suppressed by ChTx. After ischemia/reperfusion injury BK elicits endothelium-dependent relaxation which was not detectable in control MCAs. This gain of function is mediated by B2 receptor activation and involves the release of NO and activation of an endothelium-dependent hyperpolarization. It goes along with increased B2 mRNA and protein expression, leaving the functional role of the de novo B1 receptor expression still open.

Fig 1. Concentration-related relaxation induced by bradykinin (BK) of middle cerebral artery (MCA) ring segments precontracted with U46619 (3x10^-7 M).

The ring segments were obtained from rats which had undergone a 2h MCA occlusion followed by 22h of reperfusion. Shown are the results for the right (ischemic) and left (contralateral) MCA. The vasomotor effects were comparable upon the first and second application of BK in both sides. Solvent indicates time matched solvent control measurements to check for the stability of precontraction. Given are mean ±SEM based on ≥7 observations.

Fig 2. Effects of selective bradykinin (BK) receptor antagonists on the relaxation induced by bradykinin in rat middle cerebral artery (MCA) ring segments precontracted with U46619 (3x10^-7 M).

Segments were obtained 24h following transient MCA occlusion from the ischemic side. The BK-induced relaxation was not at all affected by the selective B₁ receptor antagonist lys-(des-arg⁹, leu⁸)-BK (10⁻⁵ M) while it was completely abrogated in the presence of the selective B₂ receptor antagonist Hoe140 (10⁻⁶ M). Indicated are mean ± SEM based on ≥6 observations. *p<0.05, **p<0.01 vs. BK alone.

Fig 3. Whole mount immunofluorescence staining for the expression of B₂ receptors in the middle cerebral artery wall following ischemia/reperfusion injury.

Structures in blue are nuclei, while the B₂ immunofluorescence presents in green. The endothelial cell nuclei run in the length axis of the artery while the smooth muscle cell nuclei are arranged perpendicular to the endothelial cells. Filled arrows mark endothelial cells displaying high level of immunofluorescence while the empty arrow points to an endothelial cell with low level staining. Asterisks mark smooth muscle cell nuclei (randomized choice) which are not in the focus of the image due to the thickness of the vessel wall. The clustering of endothelial cells displaying a high level of B₂ receptor immunofluorescence was a typical finding, and we never found any immunofluorescent signal in smooth msucle cells.

Fig 4. Role of the NO-cGMP axis in bradykinin-induced relaxation in rat middle cerebral arteries obtained 24h after induction of focal brain ischemia.

Preincubation with N^ω-nitro-l-arginine (L-NNA) inhibited BK-induced relaxation in a concentration-related manner. Similarly, inhibition of the soluble guanylyl cyclase by ODQ abrogated the effect of BK. Indicated are mean±SEM based on ≥ 4 observations.*p<0.05 vs control, **p<0.01 vs control.

Fig 5. In the presence of N^ω–nitro-l-arginine (L-NNA, 10⁻⁵ M) bradykinin (BK) elicits a transient relaxation in middle cerebral artery segments after transient focal brain ischemia.

(A) Representative trace from a segment which was precontracted with U46619 (3x10^-7 M). Application of high concentrations of BK (10⁻⁷ M to 10⁻⁵ M) induced transient peaks of relaxation. (B) Mean values of the transient peaks of relaxation induced by high concentrations of BK in segments obtained from the previously occluded middle cerebral artery. Indicated are mean±SEM (n = 5).

Fig 6. Effects of potassium channel blockers on the relaxation induced by bradykinin (BK) in middle cerebral artery segments after ischemia/reperfusion injury.

The response to BK was partially inhibited by charybdotoxin (ChTx, 10⁻⁸ M, n = 8), but did not change in the presence of apamin (10^-7M, n = 7) or iberiotoxin (IbTx, 10⁻⁸ M, n = 6). Indicated are mean±SEM. ** p<0.01 vs. control.