The role of nitric oxide in the anticonvulsant effects of pyridoxine on penicillin-induced epileptiform activity in rats
Introduction
Pyridoxine (Vitamin B6) is an essential dietary element in man, and a precursor of the enzyme cofactors pyridoxal 5′-phosphate (PLP) and pyridoxamine 5′-phosphate, which form the active site of a wide range of essential enzymes in living tissues (Bates et al., 1999). Pyridoxine consists of six vitamers: the alcohol pyridoxine (pyridoxol), the aldehyde pyridoxal, the amine pyridoxamine, and their 5′-phosphorylated esters (Gospe, 2006). Pyridoxine is used to treat seizures in patients with pyridoxine dependency, an autosomal recessive inborn error of metabolism, which presents in infants and young children as well as other forms of childhood seizures that are pyridoxine-responsive (Gospe, 1998, Gospe, 2002, Baxter, 2001). Moreover, the intravenous administration of pyridoxine, at the doses of 50–100 mg, results in a dramatic cessation of clinical and electrographic features of seizures (Clarke et al., 1979, Haenggeli et al., 1991). Shouse (1982) reported that pyridoxine hydrochloride, in a dose of 20 mg/kg completely blocked convulsions in seven of eight animals exposed to monomethylhydrazine. Pyridoxine also yielded a modest but statistically significant elevation in kindled seizure threshold compared with initial and final baselines in cats (Shouse, 1982). However, pyridoxal phosphate and its synthetic analogues, pyridoxal 5′-sulphate and 5-phosphonoethyl analogue of pyridoxal were found to cause epileptic seizures characterized by running fits, vocalization, muscular fasciculation and tonic–clonic convulsions (Ebadi et al., 1983, Ebadi et al., 1985). The most important member of the pyridoxine group is the active coenzyme for more than 100 enzymes, including glutamic acid decarboxylase (GAD), an enzyme involved in gamma-amino butyric acid (GABA) synthesis (Gospe et al., 1994). It was suggested that abnormal binding of the cofactor pyridoxal phosphate, the active form of pyridoxine, to GAD resulted in low GABA levels and consequently to a decrease in central nervous system inhibition (Gospe, 2002). Experimental and clinical study evidence indicates that GABA also has an important role in the mechanism and treatment of epilepsy (Snodgrass, 1992, Treiman, 2001). Experimental studies highlighted that nitric oxide also increased the potentiation of GABAergic transmission due to NMDA-receptor activation and acts an intermediate molecule in the functional balance between excitatory and inhibitory neurotransmitter systems in the regulation of neuronal excitability (Bains and Ferguson, 1997, Tsuda et al., 1997, Lu et al., 1998).
Nitric oxide is produced by the family of NOS in which there are three distinct isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) (Yoshida et al., 1995). Type of nNOS has been detected in the several parts of the brain (Torreilles et al., 1999). Cortical areas are also known to contain the highest packing densities of nNOS-positive interneurons such as the piriform and entorhinal cortices (Bidmon et al., 1999). Contradictory roles for NO in the development and pathogenesis of seizures have been suggested (Garthwaite, 1991, Manzoni et al., 1992, Mulsch et al., 1994, Ayyildiz et al., 2007). l-NAME, the nonspecific inhibitor of NOS potentiated convulsions induced by N-methyl-d-aspartic acid (NMDA) in mice, suggesting an anticonvulsant role for NO (Buisson et al., 1993). On the other hand, l-NAME and NG-nitro-l-arginine (l-NNA), the non-specific inhibitors of NOS inhibited pentylenetetrazol (PTZ) and strychnine-induced convulsions in rats, suggesting a proconvulsant role for NO (Kaputlu and Uzbay, 1997). 7-NI has been described as a selective inhibitor of neuronal NOS in vivo (Moore et al., 1993). 7-NI inhibited kainic acid, pilocarpin, and sound-induced convulsions in animals (Jones et al., 1998, Van Leeuwen et al., 1995, Smith et al., 1996). It was also reported that 7-NI potentiated the antiseizure activity of some anticonvulsant and antiepileptic drugs in different animal models (De Sarro et al., 2000, Luszczki et al., 2006). However, there are no reports of the effects of 7-NI on penicillin-induced epileptiform activity in rats.
In view of the diverse effects of both pyridoxine and NO synthase inhibitors upon susceptibility to different types of seizures, we decided for the first time, to investigate the involvement of NO in the effects of pyridoxine hydrochloride, at the doses of 20, 40, 80 and 160 mg/kg, on the penicillin-induced epileptiform ECoG activity in rats, using nonspecific NOS inhibitor l-NAME and selective neuronal NOS inhibitor 7-NI as well as NO substrate, l-arginine.
Section snippets
Animals
Adult male rats obtained from University of Ondokuz Mayis Experimental Research Centre. Breeders of rat were obtained from DETAM (Istanbul, Turkey) and the animals used were bred at the University of Ondokuz Mayis Experimental Research Centre. The animal studies were conducted with governmental approval according to local guidelines for the care and use of laboratory animals. One hundred and forty male Wistar rats (230–270 g) were used throughout the experiments after at least 1 week of
The effects of pyridoxine on penicillin-induced epileptiform activity
Intracortical injection of penicillin (500 units) induced an epileptiform ECoG activity characterized by bilateral spikes and spike-wave complexes (Figure 1A). This ECoG activity began within 2–4 min after penicillin application and lasted for 3–5 h. It reached a constant level as to frequency and amplitude in the 30 min. The mean of spike frequency and amplitude were 29 ± 2 spike/min, 1.007 ± 193 μV, respectively (Figure 1A).
Pyridoxine was administered 30 min after penicillin injection. Pyridoxine, at
Discussion
In the present study, we used penicillin to induce epileptiform activity in rats which is a widely used method for inducing epileptiform activity by applying penicillin to the cerebral cortex (Holmes et al., 1987). Application of penicillin to the neocortex results in synchronous discharge of neurons, which bears an electrophysiological resemblance to human focal interictal epileptic discharges (Purpura et al., 1972).
Urethane has been widely used as an anesthetic in in vivo experiments. Many
Acknowledgement
The authors would like to thank Dr. Leslie Scarth (UK) for help in polishing the English.
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