Discussion
Strong evidence supports the idea that NO plays a pivotal role in the pathogenesis of migraine [27, 28], a disorder characterized by pain sensitization associated with cranial vascular changes [29–31], but mechanisms and modalities of NO activity are still largely unknown. Systemic GTN activates neuronal groups in selected areas of the rat brain involved in nociception [21, 32, 33] and induces spontaneous-like attacks in migraineurs via multimodal mechanisms that include GTN- induced vasodilation, peripheral sensitization induced by the increased availability of NO at the trigeminovascular level, and possibly also central sensitization [34–37].
GTN administration induces an increase in nNOS that is simultaneous with a hyperalgesic condition and neuronal activation in brain areas involved in migraine pain [38, 39], thus suggesting that NOS inhibition may be a potential therapeutic target for migraine. Experimental and clinical studies suggest that NOS inhibition influences the activation of the trigeminal vascular system and that nonselective NOS inhibition is associated to antimigraine activity [40, 41]. Clinical application of non selelctive NOS inhibition is however hindered by the cardiovascular effects, i.e., increase of mean arterial pressure and a decrease of heart rate for its pharmacokinetic profile [41].
ADMA, is a methylated arginine found in plasma, urine and different tissues [2], which is released when methylated proteins are degraded into their amino acid components during hydrolytic protein turnover [8]. ADMA blocks NO synthesis and can induce endothelial dysfunction, both in vivo and in vitro [2, 3], and cause oxidative stress [42], two potential factors involved in migraine pathogenesis [13, 14]. DDAH regulates ADMA levels and NO signalling in vivo and ADMA/DDAH system is considered as a novel pathway for modulating NO production [43]. DDAH-1 predominates in tissues that express nNOS, whereas DDAH-2 predominates in tissues expressing eNOS [44]. Since large amounts of ADMA and DDAH-1 have been detected in the brain and spinal cord, probably ADMA/DDAH-1 pathway may have a role also in neuronal, inflammatory and other non-cardiovascular pathologies, as migraine pain, where NO has pivotal role [15]. Uzar et al., [15] found elevated plasma levels of ADMA and NO in migraine patients as compared to control subjects, suggesting that an increase in ADMA levels in migraine might represent a compensatory mechanism for blocking NO production and NO-induced excessive vasodilatation [15]. However, differences in ADMA and NO levels when comparing ictal and interictal levels in migraineurs yielded inconclusive findings [15–17]. To the best of our knowledge, no information is available on cerebral ADMA and DDAH-1 expression in experimental animal models of migraine.
In this study, we evaluated the simultaneous changes in ADMA levels and DDAH-1 mRNA expression in brain areas in an animal model specific for migraine in order to evaluate whether ADMA-DDAH-pathway may be involved in migraine. We also evaluated nNOS and eNOS expression in the same brain areas, and ADMA levels in the venous blood, drawn from the vena cava. Our findings show that AMDA levels significantly increased in the hypothalamus and medulla 4 h after GTN administration, the timing where we observe neuronal activation and hyperlagesia. This increase was associated to the inhibition of DDAH-1 expression and to the increase in nNOS expression in the same areas. eNOS expression instead was not affected. Taken together, these results suggest that the increase in brain NO availability, secondary to GTN exposure [45], may have interfered with DDAH-1 expression, possibly via S-nitrosylation of DDAH-1 active site [46, 47]. Indeed, deletion of DDAH-1 gene, or the inhibition of its transcription, is associated with an increase of ADMA levels [48]. Alternatively, DDAH-1 expression may have been inhibited via GTN-induced oxidative stress [49] or GTN-induced activation of inflammatory pathway [50, 51]. Previous reports have indeed shown that DDAH activity and protein expression may be markedly reduced during oxidative stress and/or inflammation [52–54].
Circulating levels of ADMA were not affected by GTN treatment to suggest that GTN interferes with DDAH-1 expression only at cerebral level, but not at peripheral level such as the liver, where high net hepatic uptake of ADMA occurs [55]. In agreement with a selective ‘neuronal’ activity of AMDA in this experimental paradigm is the absence of changes observed in eNOS.