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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: 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.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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Acute exposure to dopaminergic neurotoxicants attenuates the propagation of intercellular Ca2+ waves in primary astrocytes.Calcium waves were initiated in confluent cultures of striatal astrocytes loaded with Fluo-4 AM (2 µM) using a glass micropipette to stimulate a single astrocyte in the center of the field (white arrow). Images were collected every 500 msec for 60 sec. (a) Control wave and kymograph images were generated from the fluorescence intensity of Fluo-4 along a representative line drawn from the point of stimulation to the terminus of the Ca2+ wave across across the field of astrocytes (a, dotted line). Black arrows denote the point of stimulation. (b) The extent and intensity of Ca2+ waves is sharply diminished by acute application of 100 µM MPP+. (c) Similarly to MPP+, 100 µM 6-OHDA diminished the extent and intensity of Ca2+ waves with acute application. (d,e) The mean intensity of intracellular Ca2+ responses in the cells acutely exposed to 100 µM MPP+ and 6-OHDA are significantly decreased compared to control. The vertical bar in (d) denotes 1 relative fluorescent unit; the horizontal bar denotes 10 sec. (f,g) Representative traces of the total distance of the wave front in control and acutely-treated cells. Quantitative analysis of Ca2+ wave propagation indicates that 100 µM MPP+ and 6-OHDA decreases the distance traveled in striatal astrocytes by>50% relative to control, whereas 100 µM DOPAC does not abolish wave distance like MPP+ and 6-OHDA although significantly decreased from control. n = 3 waves analyzed per group for each experiment over 3–4 independent experiments in separate cultures of striatal astrocytes; *p<0.05 relative to control, ** p<0.01 relative to control and treated groups.
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pone-0110996-g002: Acute exposure to dopaminergic neurotoxicants attenuates the propagation of intercellular Ca2+ waves in primary astrocytes.Calcium waves were initiated in confluent cultures of striatal astrocytes loaded with Fluo-4 AM (2 µM) using a glass micropipette to stimulate a single astrocyte in the center of the field (white arrow). Images were collected every 500 msec for 60 sec. (a) Control wave and kymograph images were generated from the fluorescence intensity of Fluo-4 along a representative line drawn from the point of stimulation to the terminus of the Ca2+ wave across across the field of astrocytes (a, dotted line). Black arrows denote the point of stimulation. (b) The extent and intensity of Ca2+ waves is sharply diminished by acute application of 100 µM MPP+. (c) Similarly to MPP+, 100 µM 6-OHDA diminished the extent and intensity of Ca2+ waves with acute application. (d,e) The mean intensity of intracellular Ca2+ responses in the cells acutely exposed to 100 µM MPP+ and 6-OHDA are significantly decreased compared to control. The vertical bar in (d) denotes 1 relative fluorescent unit; the horizontal bar denotes 10 sec. (f,g) Representative traces of the total distance of the wave front in control and acutely-treated cells. Quantitative analysis of Ca2+ wave propagation indicates that 100 µM MPP+ and 6-OHDA decreases the distance traveled in striatal astrocytes by>50% relative to control, whereas 100 µM DOPAC does not abolish wave distance like MPP+ and 6-OHDA although significantly decreased from control. n = 3 waves analyzed per group for each experiment over 3–4 independent experiments in separate cultures of striatal astrocytes; *p<0.05 relative to control, ** p<0.01 relative to control and treated groups.

Mentions: Based upon our previous data that the divalent metal, Mn2+, acutely inhibits mechanically-induced Ca2+ waves in cultured striatal astrocytes [21], we postulated that MPP+ and 6-OHDA might similarly diminish Ca2+ wave activity by targeting membrane cation channels. The data in Figure 2 demonstrate that mechanical stimulation of a single astrocyte in the center of a confluent field using a 0.5 µm glass micropipet initiated Ca2+ waves that propagated outward from the stimulated cell for approximately 500 µm, reaching maximal extent within 60 sec. The amplitude of the wavefront over time is indicated by the kymograph images in Figure 2, representing the fluorescence intensity along the line shown in the image panel at 60 sec following stimulation. Acute application of either MPP+ or 6-OHDA (100 µM each) 30 sec prior to stimulation of Ca2+ waves resulted in a dramatic decrease in the amplitude of the wavefront throughout the field of astrocytes. Quantification of the maximum intensity of the intracellular Ca2+ transient at the wavefront in each group is shown in Figure 2d,e. 100 µM MPP+ was more effective than 6-OHDA at suppressing mechanically-induced Ca2+ waves in striatal astrocytes, although both compounds resulted in greater than 50% inhibition of the maximum amplitude of the wavefront. 100 µM MPP+ and 6-OHDA were equally effective in diminishing the distance of propagation of Ca2+ waves, whereas acute administration of 100 µM DOPAC resulted in only a slight decrease in the extent of wave propagation (Figure 2f,g).


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)

Acute exposure to dopaminergic neurotoxicants attenuates the propagation of intercellular Ca2+ waves in primary astrocytes.Calcium waves were initiated in confluent cultures of striatal astrocytes loaded with Fluo-4 AM (2 µM) using a glass micropipette to stimulate a single astrocyte in the center of the field (white arrow). Images were collected every 500 msec for 60 sec. (a) Control wave and kymograph images were generated from the fluorescence intensity of Fluo-4 along a representative line drawn from the point of stimulation to the terminus of the Ca2+ wave across across the field of astrocytes (a, dotted line). Black arrows denote the point of stimulation. (b) The extent and intensity of Ca2+ waves is sharply diminished by acute application of 100 µM MPP+. (c) Similarly to MPP+, 100 µM 6-OHDA diminished the extent and intensity of Ca2+ waves with acute application. (d,e) The mean intensity of intracellular Ca2+ responses in the cells acutely exposed to 100 µM MPP+ and 6-OHDA are significantly decreased compared to control. The vertical bar in (d) denotes 1 relative fluorescent unit; the horizontal bar denotes 10 sec. (f,g) Representative traces of the total distance of the wave front in control and acutely-treated cells. Quantitative analysis of Ca2+ wave propagation indicates that 100 µM MPP+ and 6-OHDA decreases the distance traveled in striatal astrocytes by>50% relative to control, whereas 100 µM DOPAC does not abolish wave distance like MPP+ and 6-OHDA although significantly decreased from control. n = 3 waves analyzed per group for each experiment over 3–4 independent experiments in separate cultures of striatal astrocytes; *p<0.05 relative to control, ** p<0.01 relative to control and treated groups.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110996-g002: Acute exposure to dopaminergic neurotoxicants attenuates the propagation of intercellular Ca2+ waves in primary astrocytes.Calcium waves were initiated in confluent cultures of striatal astrocytes loaded with Fluo-4 AM (2 µM) using a glass micropipette to stimulate a single astrocyte in the center of the field (white arrow). Images were collected every 500 msec for 60 sec. (a) Control wave and kymograph images were generated from the fluorescence intensity of Fluo-4 along a representative line drawn from the point of stimulation to the terminus of the Ca2+ wave across across the field of astrocytes (a, dotted line). Black arrows denote the point of stimulation. (b) The extent and intensity of Ca2+ waves is sharply diminished by acute application of 100 µM MPP+. (c) Similarly to MPP+, 100 µM 6-OHDA diminished the extent and intensity of Ca2+ waves with acute application. (d,e) The mean intensity of intracellular Ca2+ responses in the cells acutely exposed to 100 µM MPP+ and 6-OHDA are significantly decreased compared to control. The vertical bar in (d) denotes 1 relative fluorescent unit; the horizontal bar denotes 10 sec. (f,g) Representative traces of the total distance of the wave front in control and acutely-treated cells. Quantitative analysis of Ca2+ wave propagation indicates that 100 µM MPP+ and 6-OHDA decreases the distance traveled in striatal astrocytes by>50% relative to control, whereas 100 µM DOPAC does not abolish wave distance like MPP+ and 6-OHDA although significantly decreased from control. n = 3 waves analyzed per group for each experiment over 3–4 independent experiments in separate cultures of striatal astrocytes; *p<0.05 relative to control, ** p<0.01 relative to control and treated groups.
Mentions: Based upon our previous data that the divalent metal, Mn2+, acutely inhibits mechanically-induced Ca2+ waves in cultured striatal astrocytes [21], we postulated that MPP+ and 6-OHDA might similarly diminish Ca2+ wave activity by targeting membrane cation channels. The data in Figure 2 demonstrate that mechanical stimulation of a single astrocyte in the center of a confluent field using a 0.5 µm glass micropipet initiated Ca2+ waves that propagated outward from the stimulated cell for approximately 500 µm, reaching maximal extent within 60 sec. The amplitude of the wavefront over time is indicated by the kymograph images in Figure 2, representing the fluorescence intensity along the line shown in the image panel at 60 sec following stimulation. Acute application of either MPP+ or 6-OHDA (100 µM each) 30 sec prior to stimulation of Ca2+ waves resulted in a dramatic decrease in the amplitude of the wavefront throughout the field of astrocytes. Quantification of the maximum intensity of the intracellular Ca2+ transient at the wavefront in each group is shown in Figure 2d,e. 100 µM MPP+ was more effective than 6-OHDA at suppressing mechanically-induced Ca2+ waves in striatal astrocytes, although both compounds resulted in greater than 50% inhibition of the maximum amplitude of the wavefront. 100 µM MPP+ and 6-OHDA were equally effective in diminishing the distance of propagation of Ca2+ waves, whereas acute administration of 100 µM DOPAC resulted in only a slight decrease in the extent of wave propagation (Figure 2f,g).

Bottom Line: 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.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