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Protease activated receptor 1-induced glutamate release in cultured astrocytes is mediated by Bestrophin-1 channel but not by vesicular exocytosis.

Oh SJ, Han KS, Park H, Woo DH, Kim HY, Traynelis SF, Lee CJ - Mol Brain (2012)

Bottom Line: However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.These results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

View Article: PubMed Central - HTML - PubMed

Affiliation: Korea Institute of Science and Technology, Seoul, South Korea.

ABSTRACT

Background: Glutamate is the major transmitter that mediates the principal form of excitatory synaptic transmission in the brain. It has been well established that glutamate is released via Ca2+-dependent exocytosis of glutamate-containing vesicles in neurons. However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.

Findings: We report a novel form of glutamate release in astrocytes via the recently characterized Ca2+-activated anion channel, Bestrophin-1 (Best1) by Ca2+ dependent mechanism through the channel pore. We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.

Conclusions: These results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

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Related in: MedlinePlus

Best1-mediated glutamate release from cultured astrocytes by using glutamate-sensitive FRET sensor, GluSFnR. A) Schematic diagram of principles of FRET-based glutamate sensor and imaging. Released glutamate from astrocyte binds to glutamate binding motif of GluSnFR (glutamate FRET sensor), resulting in decrease of FRET between CFP and YFP or increased CFP/YFP ratio. B) Representative FRET images from GluSnFR-expressing cultured astrocytes processed by CFP/YFP emission ratio. The white bar shown at 0 s image indicates the puff of TFLLR (500 μM, 100 msec). C) Astrocytic GluSFnR responses (CFP/YFP ratio change) by respective puff-treated extracellular glutamate concentration at the time point indicated by an arrow. D) The graph showing the relationship between glutamate concentration and peak value of relative CFP/YFP ratio. The calculated EC50 was ~ 25.57 μM. E) The graph shows the averaged relative CFP/YFP ratio values from time-lapse imaging using GluSFnR expressing-cultured astrocytes. Scrambled shRNA-expressing astrocytes (Sc-shRNA); Best1-shRNA-expressing astrocytes (B1-shRNA); naïve astrocytes pretreated with BAPTA-AM (BAPTA; 30 μM); naïve astrocytes pretreated with niflumic acid (NFA; 100 μM); naïve astrocytes treated with recording buffer puff (Buffer puff). F) Bar graph representing averaged relative CFP/YFP ratio (analyzed during the period indicated by the gray box in Figure 1F). *p < 0.05, **p < 0.01 vs. Sc-shRNA-expressing group. Numbers on each bar indicate number of cells from at least three independent culture batches.
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Figure 4: Best1-mediated glutamate release from cultured astrocytes by using glutamate-sensitive FRET sensor, GluSFnR. A) Schematic diagram of principles of FRET-based glutamate sensor and imaging. Released glutamate from astrocyte binds to glutamate binding motif of GluSnFR (glutamate FRET sensor), resulting in decrease of FRET between CFP and YFP or increased CFP/YFP ratio. B) Representative FRET images from GluSnFR-expressing cultured astrocytes processed by CFP/YFP emission ratio. The white bar shown at 0 s image indicates the puff of TFLLR (500 μM, 100 msec). C) Astrocytic GluSFnR responses (CFP/YFP ratio change) by respective puff-treated extracellular glutamate concentration at the time point indicated by an arrow. D) The graph showing the relationship between glutamate concentration and peak value of relative CFP/YFP ratio. The calculated EC50 was ~ 25.57 μM. E) The graph shows the averaged relative CFP/YFP ratio values from time-lapse imaging using GluSFnR expressing-cultured astrocytes. Scrambled shRNA-expressing astrocytes (Sc-shRNA); Best1-shRNA-expressing astrocytes (B1-shRNA); naïve astrocytes pretreated with BAPTA-AM (BAPTA; 30 μM); naïve astrocytes pretreated with niflumic acid (NFA; 100 μM); naïve astrocytes treated with recording buffer puff (Buffer puff). F) Bar graph representing averaged relative CFP/YFP ratio (analyzed during the period indicated by the gray box in Figure 1F). *p < 0.05, **p < 0.01 vs. Sc-shRNA-expressing group. Numbers on each bar indicate number of cells from at least three independent culture batches.

Mentions: To independently confirm the glutamate release by HPLC method, we tested the effect of Best1-shRNA on glutamate release using fluorescence resonance energy transfer (FRET) based glutamate sensor. The released glutamate was monitored using a glutamate-sensing fluorescent reporter, GluSnFR, which senses glutamate by changing the ratio of FRET from CFP to YFP upon glutamate binding [30] (Figure 4A). The concentration-response curve for glutamate on the sensor was within the range of estimated glutamate release from a single astrocyte in culture (~10-6 M) [7] (Figure 4C and 4D). The calculated EC50 of glutamate was ~25.57 μM (Figure 4D). We observed that a brief pressure application of TFLLR caused a long lasting glutamate release as indicated by the relative change in FRET ratio (Figure 4B and 4E). The amount of glutamate release was on average significantly lower in the astrocytes expressing Best1-shRNA (Figure 4E, red symbols) than those expressing control scrambled shRNA (Figure 4E, black symbols). We found a similar degree of decrease in glutamate release by Best1-shRNA using the FRET based glutamate imaging, as compared to HPLC method (Figures 3C and 4F). Co-application of TFLLR and niflumic acid or treating the cells with BAPTA-AM, significantly reduced the glutamate release from astrocytes (Figure 4E and 4F).


Protease activated receptor 1-induced glutamate release in cultured astrocytes is mediated by Bestrophin-1 channel but not by vesicular exocytosis.

Oh SJ, Han KS, Park H, Woo DH, Kim HY, Traynelis SF, Lee CJ - Mol Brain (2012)

Best1-mediated glutamate release from cultured astrocytes by using glutamate-sensitive FRET sensor, GluSFnR. A) Schematic diagram of principles of FRET-based glutamate sensor and imaging. Released glutamate from astrocyte binds to glutamate binding motif of GluSnFR (glutamate FRET sensor), resulting in decrease of FRET between CFP and YFP or increased CFP/YFP ratio. B) Representative FRET images from GluSnFR-expressing cultured astrocytes processed by CFP/YFP emission ratio. The white bar shown at 0 s image indicates the puff of TFLLR (500 μM, 100 msec). C) Astrocytic GluSFnR responses (CFP/YFP ratio change) by respective puff-treated extracellular glutamate concentration at the time point indicated by an arrow. D) The graph showing the relationship between glutamate concentration and peak value of relative CFP/YFP ratio. The calculated EC50 was ~ 25.57 μM. E) The graph shows the averaged relative CFP/YFP ratio values from time-lapse imaging using GluSFnR expressing-cultured astrocytes. Scrambled shRNA-expressing astrocytes (Sc-shRNA); Best1-shRNA-expressing astrocytes (B1-shRNA); naïve astrocytes pretreated with BAPTA-AM (BAPTA; 30 μM); naïve astrocytes pretreated with niflumic acid (NFA; 100 μM); naïve astrocytes treated with recording buffer puff (Buffer puff). F) Bar graph representing averaged relative CFP/YFP ratio (analyzed during the period indicated by the gray box in Figure 1F). *p < 0.05, **p < 0.01 vs. Sc-shRNA-expressing group. Numbers on each bar indicate number of cells from at least three independent culture batches.
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Figure 4: Best1-mediated glutamate release from cultured astrocytes by using glutamate-sensitive FRET sensor, GluSFnR. A) Schematic diagram of principles of FRET-based glutamate sensor and imaging. Released glutamate from astrocyte binds to glutamate binding motif of GluSnFR (glutamate FRET sensor), resulting in decrease of FRET between CFP and YFP or increased CFP/YFP ratio. B) Representative FRET images from GluSnFR-expressing cultured astrocytes processed by CFP/YFP emission ratio. The white bar shown at 0 s image indicates the puff of TFLLR (500 μM, 100 msec). C) Astrocytic GluSFnR responses (CFP/YFP ratio change) by respective puff-treated extracellular glutamate concentration at the time point indicated by an arrow. D) The graph showing the relationship between glutamate concentration and peak value of relative CFP/YFP ratio. The calculated EC50 was ~ 25.57 μM. E) The graph shows the averaged relative CFP/YFP ratio values from time-lapse imaging using GluSFnR expressing-cultured astrocytes. Scrambled shRNA-expressing astrocytes (Sc-shRNA); Best1-shRNA-expressing astrocytes (B1-shRNA); naïve astrocytes pretreated with BAPTA-AM (BAPTA; 30 μM); naïve astrocytes pretreated with niflumic acid (NFA; 100 μM); naïve astrocytes treated with recording buffer puff (Buffer puff). F) Bar graph representing averaged relative CFP/YFP ratio (analyzed during the period indicated by the gray box in Figure 1F). *p < 0.05, **p < 0.01 vs. Sc-shRNA-expressing group. Numbers on each bar indicate number of cells from at least three independent culture batches.
Mentions: To independently confirm the glutamate release by HPLC method, we tested the effect of Best1-shRNA on glutamate release using fluorescence resonance energy transfer (FRET) based glutamate sensor. The released glutamate was monitored using a glutamate-sensing fluorescent reporter, GluSnFR, which senses glutamate by changing the ratio of FRET from CFP to YFP upon glutamate binding [30] (Figure 4A). The concentration-response curve for glutamate on the sensor was within the range of estimated glutamate release from a single astrocyte in culture (~10-6 M) [7] (Figure 4C and 4D). The calculated EC50 of glutamate was ~25.57 μM (Figure 4D). We observed that a brief pressure application of TFLLR caused a long lasting glutamate release as indicated by the relative change in FRET ratio (Figure 4B and 4E). The amount of glutamate release was on average significantly lower in the astrocytes expressing Best1-shRNA (Figure 4E, red symbols) than those expressing control scrambled shRNA (Figure 4E, black symbols). We found a similar degree of decrease in glutamate release by Best1-shRNA using the FRET based glutamate imaging, as compared to HPLC method (Figures 3C and 4F). Co-application of TFLLR and niflumic acid or treating the cells with BAPTA-AM, significantly reduced the glutamate release from astrocytes (Figure 4E and 4F).

Bottom Line: However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.These results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

View Article: PubMed Central - HTML - PubMed

Affiliation: Korea Institute of Science and Technology, Seoul, South Korea.

ABSTRACT

Background: Glutamate is the major transmitter that mediates the principal form of excitatory synaptic transmission in the brain. It has been well established that glutamate is released via Ca2+-dependent exocytosis of glutamate-containing vesicles in neurons. However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.

Findings: We report a novel form of glutamate release in astrocytes via the recently characterized Ca2+-activated anion channel, Bestrophin-1 (Best1) by Ca2+ dependent mechanism through the channel pore. We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.

Conclusions: These results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

Show MeSH
Related in: MedlinePlus