In vitro effect of quinolinic acid on energy metabolism in brain of young rats
Introduction
Neurotoxic actions have been attributed to quinolinic acid (QA), an important intermediate of the kynurenine pathway of tryptophan metabolism (Wolfensberger et al., 1983). Since QA is an endogenous glutamate agonist with a relative selectivity to the N-methyl-d-aspartate (NMDA) receptor, it has been hypothesized that part of its deleterious effects are due to its ability to induce excessive activation of NMDA receptors, associated with increased cytosolic Ca2+ concentrations, ATP and γ-aminobutyric acid (GABA) depletion and specific GABAergic and cholinergic neuronal death (Foster et al., 1983, Schwarcz et al., 1984, Santamaria and Rios, 1993, Stone, 1993, Moroni et al., 1999, Chiarugi et al., 2001).
Furthermore, a number of studies indicate that intrastriatal injection of 240 nmol of QA induces oxidative stress, giving support to a possible relationship between the excitotoxicity and oxidative processes following QA administration (Santamaria and Rios, 1993, Perez-Severiano et al., 1998, Perez-Severiano et al., 2004, Cabrera et al., 2000, Rodriguez-Martinez et al., 2000, Santamaria et al., 2001, Rossato et al., 2002, Ganzella et al., 2006). More recently it has been shown that micromolar concentrations of QA (20–100 μM) elicits lipid peroxidation and decrease of the brain antioxidant defenses in vitro, indicating its capability to induce oxidative stress itself by a NMDA-independent mechanism (Rios and Santamaria, 1991, Behan et al., 1999, Rodriguez-Martinez et al., 2000, Santamaria et al., 2001, Leipnitz et al., 2005).
On the other hand, it was postulated that QA may be involved in the pathogenesis of various disorders in which it is accumulated, such as infectious, as well as in inflammatory and non-inflammatory human neurological diseases (Stone and Perkins, 1981, Moroni et al., 1986, Schwarcz et al., 1988, Heyes et al., 1990, Heyes et al., 1992, Stone, 1993, Heyes, 1996). Experiments in rodents have demonstrated that a single unilateral intrastriatal injection of 100 nmol of QA mimics many of the neurochemical and histological features of Huntington's disease (HD), including GABA depletion and striatal spiny cell loss (Schwarcz et al., 1983, Beal et al., 1986). Thus, this neurotoxin has been used as a chemically-induced animal model of this disease (Schwarcz et al., 1983, Schwarcz et al., 1984, Beal et al., 1986, Ferrante et al., 1993). Interestingly, abnormal energy metabolism has been demonstrated in postmortem HD brain, most notably reduced activity of succinate-linked oxidation and of the activities of the respiratory chain complexes (Brennan et al., 1985, Mann et al., 1990, Gu et al., 1996). Further evidence has shown that the mitochondrial abnormality in HD brain is similar to that induced by malonate and 3-nitropropionic acid, the complex II inhibitors of the respiratory chain (Ludolph et al., 1992, Beal et al., 1993).
Brain physiological QA concentrations range from 0.03 to 0.1 μM in humans, rats and mice (Stone, 1993), whereas in neurodegenerative and neuroinflammatory diseases QA levels may achieve 10–246-fold higher (10–40 μM), especially due to macrophage invasion and microglia activation (Stone, 2001, Beagles et al., 1998). Furthermore, QA activates NMDA receptors in vitro at 20–50 μM and higher concentrations, as measured by electrophysiological studies (Schurr and Rigor, 1993).
Although QA-induced excitotoxicity and free radical generation have been extensively studied as mechanisms of neural cell damage, very few studies were designed to examine the role of QA on brain energy metabolism despite the fact that increased concentrations of QA and mitochondrial energy dysfunction have been found in the brain of HD patients and of other neurodegenerative disorders. In this scenario, it was demonstrated that intrastriatal injection of 60 nmol of QA provokes a progressive time-dependent mitochondrial dysfunction reflected by a low respiratory chain ratio (decreased respiration) and a reduction of ATP, NAD+, aspartate and glutamate concentrations (Bordelon et al., 1997). Furthermore, disturbances in neuronal activity and ion gradients secondarily to metabolic impairment have been also attributed to QA administered in vivo (Bordelon et al., 1998). Although these studies revealed that electrical activity and cellular respiration are reduced by intrastriatal administration of QA, they did not evaluate the underlying involved mechanisms. Furthermore, these QA-induced effects may be indirect secondary to excitotoxicity and to our mind there is no report in the literature on effects of QA on brain energy metabolism in vitro.
Thus, the present study was undertaken to investigate the in vitro influence of QA on important parameters of energy metabolism, such as glucose uptake, 14CO2 production from [U-14C] glucose, [1-14C] acetate and [1,5-14C] citrate and lactate production, as well as on the activities of the respiratory chain complexes I–V, of the citric acid cycle (CAC) enzymes, creatine kinase (CK), lactate dehydrogenase (LDH) and Na+,K+-ATPase in cerebral cortex of young rats in order to clarify the mechanisms involved in the mitochondrial dysfunction provoked by QA.
Section snippets
Reagents
All chemicals were purchased from Sigma Chemical Co., St. Louis, MO, USA, except for [U-14C] glucose, [1-14C] acetate and [1,5-14C] citrate, which were purchased from Amersham International plc, UK.
Animals
A total of 109 thirty-day-old Wistar rats obtained from the Central Animal House of the Departamento de Bioquímica, ICBS, UFRGS, were used. Rats were kept with dams until weaning at 21 days of age. The animals had free access to water and to a standard commercial chow and were maintained on a 12:12 h
QA stimulates glucose uptake by rat cortical prisms
We initially investigated the in vitro effect of QA on glucose uptake by rat cerebral cortex. For these experiments a total of six rats were used. Fig. 1A shows that 100 μM QA significantly increased glucose uptake (up to 55%) by rat cortical prisms [F(3, 20) = 6.62; P < 0.01] in a dose-dependent manner [β = 0.66; P < 0.001]. These results indicate that QA activated glucose utilization by the brain. Next, we pre-incubated 100 μM QA with the NMDA receptor antagonist MK-801 in an attempt to clarify whether
Discussion
Quinolinic acid (QA) neurotoxicity has been mainly attributed to overstimulation of NMDA receptors and to oxidative stress induction (Perkins and Stone, 1983, Foster and Schwarcz, 1989, Rios and Santamaria, 1991, Winn et al., 1991, Santamaria and Rios, 1993, Stone, 1993, Stipek et al., 1997, Perez-Severiano et al., 1998, Perez-Severiano et al., 2004, Behan et al., 1999, Moroni et al., 1999, Cabrera et al., 2000, Rodriguez-Martinez et al., 2000, Stone et al., 2000, Chiarugi et al., 2001,
Acknowledgements
This work was supported by grants from CNPq, PRONEX II, FAPERGS and PROPESQ/UFRGS.
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