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Dopaminergic neurotoxicants cause biphasic inhibition of purinergic calcium signaling in astrocytes.

Streifel KM, Gonzales AL, De Miranda B, Mouneimne R, Earley S, Tjalkens R - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Environmental Medicine, Colorado State University, Fort Collins, Colorado, United States of America.

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.

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OAG- and ATP-induced currents in striatal astrocytes.(a) Representative time course of OAG (100 µM) -induced whole-cell currents at +80, 0, and −80 mV and in response to TRPC3 inhibitor, pyrazole-3 (Pyr3, 10 µM) in striatal astrocytes. (b) Current (I) vs. voltage (V) relationship of baseline and OAG-induced currents in absence and presence of Pyr3. (c) Summary data of average current density at +80 and −80 mV before and following OAG stimulation and after administration of Pyr3. n  =  5. (*p <0.05). (c) Current (I) vs. voltage (V) relationship of baseline and ATP (1 µM) -induced currents in striatal astrocytes in absence and presence of MPP+ (100 µM). (d) Summary data of average current density at +80 and −80 mV before and following ATP (1 µM) stimulation and after administration of MPP+ (100 µM). n  =  3. (*p <0.05).
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pone-0110996-g007: OAG- and ATP-induced currents in striatal astrocytes.(a) Representative time course of OAG (100 µM) -induced whole-cell currents at +80, 0, and −80 mV and in response to TRPC3 inhibitor, pyrazole-3 (Pyr3, 10 µM) in striatal astrocytes. (b) Current (I) vs. voltage (V) relationship of baseline and OAG-induced currents in absence and presence of Pyr3. (c) Summary data of average current density at +80 and −80 mV before and following OAG stimulation and after administration of Pyr3. n  =  5. (*p <0.05). (c) Current (I) vs. voltage (V) relationship of baseline and ATP (1 µM) -induced currents in striatal astrocytes in absence and presence of MPP+ (100 µM). (d) Summary data of average current density at +80 and −80 mV before and following ATP (1 µM) stimulation and after administration of MPP+ (100 µM). n  =  3. (*p <0.05).

Mentions: To determine whether MPP+-dependent inhibition of Ca2+ signaling correlated to a loss of TRPC3 current, we performed whole-cell patch clamp electrophysiology experiments to isolate native TRPC3 current in primary striatal astrocytes. Large transient outward currents were evoked following the administration of OAG (100 µM), and were blocked with the TRPC3 inhibitor, Pyr3 (10 µM, Figure 7a–c), supporting the presence of native TRPC3 currents in isolated astrocytes. Due to a possible global effect of OAG on counteracting K+ currents, we tested whether local generation of DAG through P2Y receptors could specifically activate TRPC3-like currents in astrocytes. Consistent with Ca2+ imaging experiments, administration of extracellular ATP generated large inward (and outward) currents that were blocked by MPP+ (Figure 7d–e).


Dopaminergic neurotoxicants cause biphasic inhibition of purinergic calcium signaling in astrocytes.

Streifel KM, Gonzales AL, De Miranda B, Mouneimne R, Earley S, Tjalkens R - PLoS ONE (2014)

OAG- and ATP-induced currents in striatal astrocytes.(a) Representative time course of OAG (100 µM) -induced whole-cell currents at +80, 0, and −80 mV and in response to TRPC3 inhibitor, pyrazole-3 (Pyr3, 10 µM) in striatal astrocytes. (b) Current (I) vs. voltage (V) relationship of baseline and OAG-induced currents in absence and presence of Pyr3. (c) Summary data of average current density at +80 and −80 mV before and following OAG stimulation and after administration of Pyr3. n  =  5. (*p <0.05). (c) Current (I) vs. voltage (V) relationship of baseline and ATP (1 µM) -induced currents in striatal astrocytes in absence and presence of MPP+ (100 µM). (d) Summary data of average current density at +80 and −80 mV before and following ATP (1 µM) stimulation and after administration of MPP+ (100 µM). n  =  3. (*p <0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4217743&req=5

pone-0110996-g007: OAG- and ATP-induced currents in striatal astrocytes.(a) Representative time course of OAG (100 µM) -induced whole-cell currents at +80, 0, and −80 mV and in response to TRPC3 inhibitor, pyrazole-3 (Pyr3, 10 µM) in striatal astrocytes. (b) Current (I) vs. voltage (V) relationship of baseline and OAG-induced currents in absence and presence of Pyr3. (c) Summary data of average current density at +80 and −80 mV before and following OAG stimulation and after administration of Pyr3. n  =  5. (*p <0.05). (c) Current (I) vs. voltage (V) relationship of baseline and ATP (1 µM) -induced currents in striatal astrocytes in absence and presence of MPP+ (100 µM). (d) Summary data of average current density at +80 and −80 mV before and following ATP (1 µM) stimulation and after administration of MPP+ (100 µM). n  =  3. (*p <0.05).
Mentions: To determine whether MPP+-dependent inhibition of Ca2+ signaling correlated to a loss of TRPC3 current, we performed whole-cell patch clamp electrophysiology experiments to isolate native TRPC3 current in primary striatal astrocytes. Large transient outward currents were evoked following the administration of OAG (100 µM), and were blocked with the TRPC3 inhibitor, Pyr3 (10 µM, Figure 7a–c), supporting the presence of native TRPC3 currents in isolated astrocytes. Due to a possible global effect of OAG on counteracting K+ currents, we tested whether local generation of DAG through P2Y receptors could specifically activate TRPC3-like currents in astrocytes. Consistent with Ca2+ imaging experiments, administration of extracellular ATP generated large inward (and outward) currents that were blocked by MPP+ (Figure 7d–e).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Environmental Medicine, Colorado State University, Fort Collins, Colorado, United States of America.

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.

Show MeSH
Related in: MedlinePlus