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

Date Published: June 18, 2018

Publisher: Public Library of Science

Author(s): Youhai Li, Natalia Lapina, Nina Weinzierl, Lothar Schilling, Vardan Karamyan.


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.

Partial Text

The presence of kinins, as well as kinin-synthesizing and–destroying enzymes in the brain, was first described by Hori [1] in rabbits. Based on these and subsequent studies supporting and extending the initial findings, brain tissue is considered to express a full kallikrein-kinin system (KKS). The major biologically active component of the KKS, the nonapeptide bradykinin (BK), acts upon two receptor types termed subtype 1 (B1) and subtype 2 (B2) receptor. In the brain vasculature, BK exerts endothelium-dependent vasodilatation of arteries and arterioles and an increase in capillary permeability eventually leading to (enhancement of) vasogenic brain edema. In healthy conditions, these effects are mediated by activating the B2 receptor [2] which is constitutively expressed on the endothelial cells while the expression of the B1 receptor is typically below detection levels.

Male Sprague Dawley rats (body weight 300–500 g) from Janvier (Isle St. Genest, France) were used throughout. All animals were allowed to accommodate for at least one week before experiments were done. Approval of the experimental protocol was obtained from the Federal Animal Ethics Committee (Regierungspraesidium Karlsruhe). The experiments were performed in compliance with the relevant laws and institutional guidelines for the care and use of animals in research according to the Directive 2010/63/EU. This includes all efforts to minimize pain and stress to the animals and limits to the number of animals used.

A total of 42 rats underwent MCA occlusion. Among these 5 were excluded because of a premature death. Occlusion of the MCA origin resulted in development of focal ischemic damage throughout all animals included in the analysis. Hemispheric swelling was 36±2.5% indicating the presence of marked vasogenic edema. The volume of ischemic damage was 340±16 mm3 (n = 28) after appropriate correction for brain swelling.

The present study describes a significantly enhanced relaxant action of BK in the isolated rat MCA following transient focal brain ischemia. Enhanced relaxation was observed in MCAs from the ischemic as well as from the contralateral hemisphere with the response being more pronounced ipsilaterally. Relaxation was accompanied by a significant upregulation of the B2 and a de novo expression of the B1 mRNA in the MCA wall, again more pronounced ipsi- than contralaterally. Whole mount immunofluorescence staining revealed the presence of B2 receptor protein in previously occluded MCAs but not in arteries taken from non-occluded rats. Relaxation was mediated by B2 activation exclusively as shown in experiments using selective receptor antagonists. The relaxation was endothelium-dependent with release of NO playing a major role. In addition, an EDHF-related pathway, apparently involving intermediate conductance KCa channel activation appeared to be involved as well.




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