Study On The Regulation Mechanism Of Arachidonic Acid On Voltage-gated Sodium Channels And RNav1.2 On Rat Cerebellar Granulosa Cells

Arachidonic acid (AA), which possesses both neurotoxic and neurotrophic activities, has been implicated as a messenger in both physiological and pathophysiological processes. In the present study, we investigated the effects of both extracellular and intracellular application of AA on the activity of voltage-gated Na+ channels (Nav) in rat cerebellar granule cells.The extracellular application of AA inhibited the resultant Nav current (INa), wherein the current-voltage curve shifted to a negative voltage direction. Because this effect could be reproduced by treating the granule cells with eicosatetraynoic acid (ETYA), or a membrane impermeable analogue of AA, arachidonoyl coenzyme A (arachidonoyl-CoA), we inferred that AA itself exerted the observed modulatory effects on INa.In contrast, intracellular AA significantly augmented the elicited INa peak, when the same protocol that was used for extracellular AA was followed. The observed INa3 increase that was induced by intracellular AA was mimicked by the AA cyclooxygenase metabolite prostaglandin E2 (PGE2) but not by ETYA. Furthermore, cyclooxygenase inhibitors decreased INa and quenched AA-induced channel activation, indicating that the effect of intracellular AA on Nav was possibly mediated through AA metabolites. In addition, the PGE2-induced activation of INa was mimicked by cAMP and quenched by a protein kinase A inhibitor, a Gs inhibitor and EP receptor antagonists.Knocking down Nav1.2 genes by small interfering RNA not only decreased the INa amplitude significantly, but also eliminated the effects triggered by extracelluar/intracellular application of AA on INa, suggesting that Nav1.2 encoded INa channels were involved in this complicated process.The full-length cDNA coding sequences for rat brain Nav1.2 a-subunits were cotransfected with eGFP, which was used as the marker to detect transfected CHO cells by lipofectamine2000. Observations on the reconstituted channel, rNav1.2, showed that external AA still exhibited an inhibition on INa, while internal AA could augment INa by a leftward shift for the steady-state activation characteristics of rNav1.2 and a rightward shift for its steady-state inactivation property. Our data suggest that extracellular AA modulates Nav channel activity in rat cerebellar granule cells without metabolic conversion, whereas intracellular AA augments the INa by PGE2-mediated activation of cAMP/PKA pathways. These observations may explain the dual character of AA in neuronal pathogenesis.