Abstract
Dopaminergic nuclei in the basal ganglia are highly sensitive to damage from oxidative stress, inflammation, and environmental neurotoxins. Disruption of adenosine triphosphate (ATP)-dependent calcium (Ca2+) transients in astrocytes may represent an important target of such stressors that contributes to neuronal injury by disrupting critical Ca2+-dependent trophic functions. We therefore postulated that plasma membrane cation channels might be a common site of inhibition by structurally distinct cationic neurotoxicants that could modulate ATP-induced Ca2+ signals in astrocytes. To test this, we examined the capacity of two dopaminergic neurotoxicants to alter ATP-dependent Ca2+ waves and transients in primary murine striatal astrocytes: MPP+, the active metabolite of 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 6-hydroxydopamine (6-OHDA). Both compounds acutely decreased ATP-induced Ca2+ transients and waves in astrocytes and blocked OAG-induced Ca2+ influx at micromolar concentrations, suggesting the transient receptor potential channel, TRPC3, as an acute target. MPP+ inhibited 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ca2+ transients similarly to the TRPC3 antagonist, pyrazole-3, whereas 6-OHDA only partly suppressed OAG-induced transients. RNAi directed against TRPC3 inhibited the ATP-induced transient as well as entry of extracellular Ca2+, which was augmented by MPP+. Whole-cell patch clamp experiments in primary astrocytes and TRPC3-overexpressing cells demonstrated that acute application of MPP+ completely blocked OAG-induced TRPC3 currents, whereas 6-OHDA only partially inhibited OAG currents. These findings indicate that MPP+ and 6-OHDA inhibit ATP-induced Ca2+ signals in astrocytes in part by interfering with purinergic receptor mediated activation of TRPC3, suggesting a novel pathway in glia that could contribute to neurotoxic injury.
Highlights
Calcium (Ca2+) signaling modulates diverse physiological processes in the nervous system including exocytosis of neurotransmitters, neuronal long-term potentiation [1,2], the propagation of intercellular Ca2+ waves in astrocytes that regulate metabolic and trophic support to neurons, and local regulation of cerebral blood flow [3,4]
The acute effects of 1-methyl-4-phenylpyridinium ion (MPP+) and 6-hydroxydopamine (6-OHDA) on ATPinduced Ca2+ transients were determined by adding increasing concentrations of each neurotoxicant to the medium 30 sec prior to stimulation with adenosine triphosphate (ATP)
Expression of TRPC channels was determined by reverse transcriptase PCR (rtPCR) and indicated that subtypes 1–4 of TRPC channels were expressed in primary cortical astrocytes (Table S1c)
Summary
Calcium (Ca2+) signaling modulates diverse physiological processes in the nervous system including exocytosis of neurotransmitters, neuronal long-term potentiation [1,2], the propagation of intercellular Ca2+ waves in astrocytes that regulate metabolic and trophic support to neurons, and local regulation of cerebral blood flow [3,4]. Disruptions in astrocyte Ca2+ signaling could have serious implications for neuronal function and survival during states of neurological injury and disease. Astrocytes tonically protect against excitotoxic neuronal injury by removing excess synaptic glutamate and by dynamically inhibiting glutamatergic synapses through release of ATP that is degraded to adenosine and suppresses pre-synaptic currents via the A1 adenosine receptor [4]. Because astrocytic regulation of excitatory synapses requires Ca2+-dependent release of ATP, deprecations in Ca2+ signaling in astrocytes could predispose regional populations of neurons to excitotoxic injury. Ca2+ signaling in astrocytes could be dysregulated during neuroinflammation, as indicated by recent studies which demonstrated that ATP release from activated microglia evokes P2Y purinergic receptor-dependent release of neuroactive compounds from astrocytes, including ATP, D-serine and glutamate [7]
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