Isorhamnetin modulates the γ‐aminobutyric acid transporter GAT‐3 in mesencephalic and cortical astrocytes exposed to neurotoxic manganese

Excessive manganese (Mn) exposure leads to a Parkinson's‐like disorder characterized by neurochemical imbalances. A disruption in γ‐aminobutyric acid (GABA) is an early event of Mn‐toxicity, and evidence suggests that Mn impairs the function of GABA transporters leading to elevated synaptic GABA. We suspect that Mn inhibits GABA uptake by altering the density of GABA transporters on the plasma membrane (PM). The purpose of this study was to characterize the effect of Mn on the cellular localization of the GABA transporter GAT‐3 in astrocytes, and to identify whether the blood‐brain permeable quercetin metabolite, isorhamnetin (ISO), preserves membrane GAT‐3 protein levels and function. Primary astrocytes were isolated from the cortex and mesencephalon of Sprague‐Dawley rats at postnatal day two. Astrocytes were exposed to 500μM Mn for 1, 6, or 24hrs with or without 72hr ISO (10μM) pre‐treatment. Western blot analysis of GAT‐3 and phospho protein kinase C‐δ (PKC‐δ) were conducted on PM and cytosolic fractions. Mn decreased total GAT‐3 protein in astrocytes from both regions after 24hr exposure. ISO pre‐treatment did not improve total GAT‐3 levels, but shifted GAT‐3 from the cytosol to the PM in astrocytes from both regions, which corresponded with elevated phospho‐PKCδ at the PM. ISO also enhanced 3 H‐GABA uptake in astrocytes. These data suggest that ISO may protect against early events in Mn‐toxicity.