Kynurenic acid is the only known endogenous excitatory amino acid receptor blocker with a broad spectrum of antagonistic properties. It can block, in low micromolar concentrations, the glycine allosteric site of the N-methyl-D-aspartate (NMDA) receptor. Its affinity to the glutamate binding site is at least 10 times lower than to the binding site of glycine, 1 whereas it exhibits a weak antagonistic effect on the α-amino-3-hydroxy-5-methyl-isoxasolpropionate (AMPA) and kainate receptors. 2,3 Kynurenic acid is a metabolic product of the tryptophan pathway (Fig. 1). 4,5 Another product of this pathway is quinolinic acid with action opposite to kynurenic acid (i.e., it is an endogenous excitotoxin and an agonist of the NMDA receptor). 4,6 Alterations in their concentrations in the brain were described in several neurologic diseases, (e.g., Huntington disease, temporal lobe epilepsy, neuroinfection, immunologic disorders, brain trauma, and cerebral ischemia as reviewed by Stone 3).
Kynurenic and quinolinic acid cross the blood-brain barrier slowly. In contrast, kynurenine (KYN), the precursor of kynurenic acid possessing a protein carrier, easily enters the brain. 7,8 Systematically administered KYN is able to reach maximal cerebral concentrations within 60 to 120 minutes, 9 and exerts pharmacological effects. 10,11 Production of kynurenic acid in the brain is determined by the amount of l-KYN. 12,13 Peripherally administered l-KYN is able to increase the concentration of cerebral kynurenic acid dose dependently, and the existence of a functional, inducible KYN pathway in the central nervous system has been suggested. 14–16 It has been proposed that by shifting kynurenine metabolism toward kynurenic acid formation, it is possible to reduce glutamate receptor activation and excitotoxic neuronal damage. 17
Kynurenic acid proved to be neuroprotective in neonatal rats by reducing anoxia- 18 or hypoxia-ischemia–19 induced brain edema and in adult rats 20 and gerbils 21 given prior to ischemia induction. Only high doses of kynurenic acid proved to be neuroprotective in all cases investigated.
The aim of our present study was to examine whether peripherally administered l-KYN can influence the normal and the unilateral carotid artery occlusion induced ischemic corticocerebral blood flow (cCBF) in conscious rabbits.
Altogether 60 New-Zealand white rabbits weighing 2.5 to 3 kg were used. The animals were fed commercial laboratory rabbit food pellets and had free access to water.
Introduction of Electrodes into the Cerebral Cortex
Animals were anesthetized with a solution of intravenously administered diazepam (20 mg/kg) and ketamine (20 mg/kg). Six hydrogen-sensitive electrodes consisting of glass-insulated (O.D., 0.5 mm) platinum-iridium wire (diameter 0.1 mm) with a bare tip length of 1.0 to 1.5 mm were implanted stereotaxically. In control animals and in those with left-carotid occlusion electrodes were placed in the right and left parietal cortex (to the area of the right and left “gyrus suprasylvius anterior and media”) through adequately placed bore holes in the skull and fixed with dental cement in aseptic conditions. Stereotaxic placements were calculated from the atlas of Monnier and Gangloff. 22 Before closing the wound 200,000 IU benzylpenicillin-potassium salt was applied locally.
Occlusion of Carotid Artery
In the same anesthesia (as described previously), the left external and internal carotid artery was occluded by ligation under sterile conditions (n = 6 per group). A further 6 animals in each group that did not undergo carotid occlusion served as controls.
Measurement of Corticocerebral Blood Flow
Rabbits were allowed to recover for three days after surgery and corticocerebral blood flow (cCBF) was determined by means of the hydrogen clearance technique23 as applied by ourselves earlier. 24 During the measurements the rabbits rested quietly in a comfortable wooden stock and 2 to 5% hydrogen gas in air mixture was administered via a funnel. Clearance curves were registered and cCBF was evaluated with the aid of an 80-MHz IBM 486 SX computer. The basal and unilateral carotid occlusion induced impaired cCBF and flow changes following l-KYN treatment were determined in the first minute and in every 30 minutes throughout 4 hours. One of the groups of 6 control conscious rabbits without drug treatment was used to measure the effect of physiological salt solution (in the same volume as used for drug treatments) on cCBF.
Measurement of Arterial Blood Pressure, Heart Rate, and Blood Gas Parameters
In each animal the left or right central ear artery was cannulated for the monitoring of blood pressure and the withdrawal of arterial blood samples. Mean arterial blood pressure was measured in the conscious rabbits through a pressure transducer (Statham P23 Db) attached to a Hellige electromanometer. To follow heart rate, electrocardiograms were continuously recorded by means of a radiotelemetry system (Innopoint, Budapest). Arterial pH, pCO2, pO2, and O2 saturation were measured at intervals utilizing a blood gas analyzer (Model OP-216, Radelkis, Budapest).
The marginal ear vein was cannulated and used for administration of l-KYN and other agents.
l-KYN free base (Sigma, St. Louis, MO, USA) was freshly dissolved in physiological salt solution each day and was administered intravenously in a volume of 1.5 to 2 mL. The baseline values were registered, and the effect of l-KYN on cCBF, blood pressure, and heart rate was followed for 240 minutes.
L-NAME hydrochloride (40 mg/kg) (Sigma, St. Louis, MO, USA) was applied intravenously in aqueous solution in a volume of 1 mL after registration of basal cCBF values. Thereafter the measurements were repeated; 45 minutes later l-KYN was administered and the changes in cCBF, blood pressure, and heart rate were determined. The dose of l-NAME was chosen on the basis of the results of Iadecola et al25 according to which 40 mg/kg of the compound was effective in attenuation of hypercapnic cCBF response reaching a maximum at 45 minutes.
Atropine sulfate (1 mg/kg) (EGIS Rt., Hungary) was given intravenously in aqueous solution in a volume of 2 to 3 mL 5 minutes before administration of l-KYN. cCBF, blood pressure, and heart rate values and the l-KYN–induced responses were registered.
The results were expressed as means ± SEM. Data were collected in pairs from the same measuring sites (electrodes) before and after the experimental intervention or administration of agents. The statistical significance of the observed differences was calculated by Student's paired t-test and repeated measures Analysis of Variance (ANOVA). Multiple comparisons of different time points and groups was carried out by means of one-way ANOVA. P values less than 0.05, when obtained with both statistical tests, were considered significant.
Significance of reductions in mean basal cCBF values in the controls and in animals with unilateral carotid occlusions treated with 0.3, 1, and 3 mg/kg l-KYN (“1,” “2,” and “3”) were calculated by unpaired Student's t test respectively. P values less than 0.05 were considered significant.
This work was approved by the Ethics Committee for the Protection of Animals in Research of Albert Szent-Györgyi Medical University, Szeged, Hungary. All experiments involved in this study were conducted in strict compliance with established professional and National Institutes of Health guidelines.
Administration of l-KYN resulted in a significant increase in the normal cCBF (Table 1). This effect was particularly obvious after administration of 1 mg/kg l-KYN. The cCBF was enhanced by all 3 applied doses of l-KYN within 30 minutes, attained its maximum values soon thereafter, and remained high even around minute 240 (i.e., at the end of the recording period).
Physiological salt solution treatment did not change the cCBF significantly. The following cCBF values (ml/min/100 g tissue; mean ± SEM) were measured: baseline = 107 ± 15; 30 min = 123 ± 12; 60 min = 108 ± 14; 90 min = 117 ± 11; 120 min = 103 ± 16; 150 min = 104 ± 15, 180 min = 114 ± 16; 210 min = 106 ± 12; 240 min = 110 ± 11.
Unilateral carotid occlusion caused a significant reduction in cCBF from 117 ± 15 to 57 ± 11 (“1”), from 90 ± 12 to 52 ± 8 (“2”), and from 113 ± 10 to 58 ± 11 (“3”) ml/min/100 g tissue (Fig. 2). Following administration of l-KYN there was an immediate increase in cCBF in animals with carotid occlusion (Table 2). The l-KYN–induced maximal percentage increases in cCBF in the ischemic animals were more pronounced than the cCBF decreases caused by unilateral occlusion (92–94% at 1 mg/kg dose/ vs. 42–51%) (Fig. 2) (Table 2), and in l-KYN–treated animals with carotid occlusion the cCBF became closer to or even exceeded (at 1 mg/kg l-KYN) the normal basal values measured in control animals (Table 1 vs. Table 2). This effect was of long duration and peak values were recorded 60 to 240 minutes after l-KYN injection.
Pretreatment with atropine or l-NAME prevented the cCBF-increasing effect of 1 mg/kg l-KYN in control rabbits and also in those with carotid occlusion (Table 3).
As shown in Table 4, l-KYN did not alter arterial blood pressure or heart rate, whereas l-NAME caused a small increase in blood pressure and atropine in heart rate. The duration of the latter effects was relatively short (< 60 minutes). No significant changes in arterial blood gas parameters and pH were noted after administration of l-KYN, atropine, or l-NAME (Table 5).
In the present study intravenous administration of l-KYN was found to enhance cCBF in rabbits. The increase in cCBF was apparent not only in control animals but also in rabbits with unilateral carotid occlusion. In the animals without carotid occlusion the highest elevation in cCBF occurred at 1 mg/kg dose of l-KYN (87%). Significant improvement of cCBF was seen in the animals with impaired cerebral blood flow in the presence of all doses investigated and the maximal increases were observed after administration of 1 and 3 mg/kg l-KYN (94 and 78%). When considering these findings, there are several questions to be answered:
- Does l-KYN itself or one of its derivatives (e. g., kynurenic acid) enhance cCBF in rabbits?This question can not be answered on the basis of our present experiment.
- Is the other metabolite of l-KYN, quinolinic acid (an excitotoxin), also involved in the observed cCBF-increasing effect; is it produced in considerable amounts after the administration of the precursor in the above doses?On the basis of the measurements of Guidetti et al 16 it appears that KYN disproportionally favors the production of kynurenic acid over quinolinic acid. So it seems likely that only minimal amount of quinolinic acid might be synthesized after l-KYN administration.
- What might then be suggested as a mechanism of intravenously administered l-KYN in enhancing cCBF in rabbits?
Kynurenic acid is known to possess some NMDA receptor antagonistic properties 2 and the same may apply, at least in directly, to its bioprecursor l-KYN. CGS-19755, a competitive antagonist of NMDA receptor, also enhanced CBF in rats after occlusion of the left middle cerebral and common carotid arteries when given in a dose of 15 mg/kg immediately after the occlusions. 26 MK 801, a non-competitive NMDA receptor antagonist, exhibited different effects depending on the experimental conditions. In normal rats it increased 27 while in another type of experiment performed in halothane anaesthetized rats significantly reduced the local CBF in the majority of the brain regions in both the ischemic and non-ischemic hemispheres. 28 A potential explanation for this latter finding can be that the functional consequences of the blockade of the NMDA receptors are markedly modified by anesthetic agents. 29 A possibility for the increase in cCBF might be the activation of the ascending cholinergic pathways as a response to the NMDA receptor blockade. 30,31 In our study, administration of 1 mg/kg atropine prevented the cCBF-increasing effect of 1 mg/kg l-KYN. Nevertheless, it is questionable, whether the mode of action of l-KYN in increasing cCBF is similar to that of the potent NMDA receptor antagonists, but it would be of interest to examine in future experiments whether the cCBF-enhancing effect of NMDA receptor antagonists can be influenced by l-KYN. Human brain extracellular kynurenic acid concentration is about 100 nM. 32 To exert pharmacological effects (ie, an antagonism at the glycine allosteric site of the NMDA receptor) micromolar concentrations of kynurenic acid are necessary. 2 Intraperitoneal administration of 100 mg/kg l-KYN caused a 37-fold increase in striatal extracellular kynurenic acid level in the rat.13 Respecting the fact that human regional brain-tissue content of kynurenic acid is an order of magnitude higher than that of rats, it was suggested that a peripheral precursor loading could produce a brain kynurenic acid level high enough to interact with the glycine site of the NMDA receptor complex. 12 Indeed, at least in rats, pretreatment with 100 mg/kg l-KYN intraocularly, significantly reduced NMDA-induced retinal ganglion cell degeneration. 33 It is, however, unclear, whether systemic administration of the low l-KYN dose applied in the present study (1 mg/kg) can cause a considerable NMDA receptor antagonism. Until now there is no information obtained from in vivo experiments on the NMDA receptor binding of such a small dose of l-KYN. A direct vasodilatory effect of l-KYN or one of its derivatives on cerebral vessels is in doubt. Glutamate or NMDA failed to affect the tone of isolated cerebral arteries. 34 No direct vasodilatory effect was reported with some glutamate receptor agonists or antagonists. 35 Nevertheless, further studies to establish the possible lack of direct (“non-specific”) effects of l-KYN on cerebral blood vessels would be well worthwhile. In the present study, the cCBF-increasing effect of l-KYN in both groups of animals was blocked by l-NAME, an inhibitor of NO synthase. Taking into account that there may be an increased vasodilator parasympathetic activity as response to the NMDA receptor blockade, 30,31 it might induce NO production. 36 NO may open KATP channels causing relaxation of cerebral arterioles. 37 Inhibition of the production of NO may thus diminish the cCBF-enhancing effect of l-KYN. On the basis of our findings it seems apparent that besides its known neuroprotective action, l-KYN may exhibit an additional beneficial effect by increasing cCBF in cerebral ischemia and thereby providing a potentially useful alternative in the therapy for cerebrovascular disorders. However, further detailed studies are required to clarify the mode of action of l-KYN as a vasodilatory agent.
The authors thank Mr. Zoltán Vezekényi for expert technical assistance.
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