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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

TFLLR-induced glutamate is not by vesicular exocytosis. A) To test whether Best1–shRNA affects the expression of exocytotic protein transcripts, mRNA expression levels of several endogenous vesicular machineries such as Syt4, Munc18-1, Vamp2 were analyzed from cultured astrocytes expressing Best1–shRNA or scrambled shRNA at least for 72 h. PCR cycles for Best1, Syt4 (Synaptotagmin4), Munc18-1, Vamp2 (Vesicle-associated membrane protein 2), and GFAP were 35, 35, 30, 30, and 25, respectively. B) Time-lapse imaging of relative CFP/YFP ratio from GluSFnR-expressing cultured astrcytes plotted as mean ± sem. Arrowhead indicates the time point of 500 μM TFLLR puff. Naïve astrocytes (Control); naïve astrocytes pretreated with 0.5 mM concanamycin (Conc.A; 2 hrs); naïve astrocytes pretreated with 10 nM tetanus toxin (TeNT; ~14 hrs); naïve astrocytes treated with hyperosmotic solution puff (Hyper). Bar graphs represent the averaged relative CFP/YFP ratio from 30 sec to 70 sec. Numbers of cells from at least two independent culture batches are indicated on the bar graph.
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Figure 5: TFLLR-induced glutamate is not by vesicular exocytosis. A) To test whether Best1–shRNA affects the expression of exocytotic protein transcripts, mRNA expression levels of several endogenous vesicular machineries such as Syt4, Munc18-1, Vamp2 were analyzed from cultured astrocytes expressing Best1–shRNA or scrambled shRNA at least for 72 h. PCR cycles for Best1, Syt4 (Synaptotagmin4), Munc18-1, Vamp2 (Vesicle-associated membrane protein 2), and GFAP were 35, 35, 30, 30, and 25, respectively. B) Time-lapse imaging of relative CFP/YFP ratio from GluSFnR-expressing cultured astrcytes plotted as mean ± sem. Arrowhead indicates the time point of 500 μM TFLLR puff. Naïve astrocytes (Control); naïve astrocytes pretreated with 0.5 mM concanamycin (Conc.A; 2 hrs); naïve astrocytes pretreated with 10 nM tetanus toxin (TeNT; ~14 hrs); naïve astrocytes treated with hyperosmotic solution puff (Hyper). Bar graphs represent the averaged relative CFP/YFP ratio from 30 sec to 70 sec. Numbers of cells from at least two independent culture batches are indicated on the bar graph.

Mentions: We noted that the amount of released glutamate was not completely inhibited by gene-silencing of Best1 (Figure 4E and 4F), raising a possibility that astrocytes have other Best1-independent mechanisms for glutamate release such as vesicular exocytosis. It is possible that Best1-shRNA affects the expression level of other genes involved in vesicular release. To address this possibility, we performed semi-quantitative RT-PCR analysis of cultured astrocytes with Best1-shRNA. Best1-shRNA showed a knock down efficiency for Best1 of over 90% while not affecting the expression level of other known genes such as Syt4 (Synaptotagmin4), Munc18-1, Vamp2 (Vesicle-associated membrane protein 2) involved in vesicular release (Figure 5A). We also tested whether inhibiting vesicular release machinery affects the glutamate release by using FRET based glutamate imaging. We found that the pretreatment with Conconomycin A (preventing vesicular glutamate release by inhibiting vesicular hydrogen ATPase, [31]) and Tetanus toxin (interfering all vesicular releases by inhibiting fusion of vesicles, [32]) slightly but not significantly reduced TFLLR-induced glutamate release from cultured astrocytes (Figure 5B and 5C). Consistent with these results, hyperosmotic challenge, which is known to cause a Ca2+ independent exocytosis of glutamate containing releasable vesicles, did not show any significant increase in glutamate release (Figure 5B and 5C). Taken together, PAR1-induced glutamate release is not mediated by vesicular exocytosis in cultured astrocytes.


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)

TFLLR-induced glutamate is not by vesicular exocytosis. A) To test whether Best1–shRNA affects the expression of exocytotic protein transcripts, mRNA expression levels of several endogenous vesicular machineries such as Syt4, Munc18-1, Vamp2 were analyzed from cultured astrocytes expressing Best1–shRNA or scrambled shRNA at least for 72 h. PCR cycles for Best1, Syt4 (Synaptotagmin4), Munc18-1, Vamp2 (Vesicle-associated membrane protein 2), and GFAP were 35, 35, 30, 30, and 25, respectively. B) Time-lapse imaging of relative CFP/YFP ratio from GluSFnR-expressing cultured astrcytes plotted as mean ± sem. Arrowhead indicates the time point of 500 μM TFLLR puff. Naïve astrocytes (Control); naïve astrocytes pretreated with 0.5 mM concanamycin (Conc.A; 2 hrs); naïve astrocytes pretreated with 10 nM tetanus toxin (TeNT; ~14 hrs); naïve astrocytes treated with hyperosmotic solution puff (Hyper). Bar graphs represent the averaged relative CFP/YFP ratio from 30 sec to 70 sec. Numbers of cells from at least two independent culture batches are indicated on the bar graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: TFLLR-induced glutamate is not by vesicular exocytosis. A) To test whether Best1–shRNA affects the expression of exocytotic protein transcripts, mRNA expression levels of several endogenous vesicular machineries such as Syt4, Munc18-1, Vamp2 were analyzed from cultured astrocytes expressing Best1–shRNA or scrambled shRNA at least for 72 h. PCR cycles for Best1, Syt4 (Synaptotagmin4), Munc18-1, Vamp2 (Vesicle-associated membrane protein 2), and GFAP were 35, 35, 30, 30, and 25, respectively. B) Time-lapse imaging of relative CFP/YFP ratio from GluSFnR-expressing cultured astrcytes plotted as mean ± sem. Arrowhead indicates the time point of 500 μM TFLLR puff. Naïve astrocytes (Control); naïve astrocytes pretreated with 0.5 mM concanamycin (Conc.A; 2 hrs); naïve astrocytes pretreated with 10 nM tetanus toxin (TeNT; ~14 hrs); naïve astrocytes treated with hyperosmotic solution puff (Hyper). Bar graphs represent the averaged relative CFP/YFP ratio from 30 sec to 70 sec. Numbers of cells from at least two independent culture batches are indicated on the bar graph.
Mentions: We noted that the amount of released glutamate was not completely inhibited by gene-silencing of Best1 (Figure 4E and 4F), raising a possibility that astrocytes have other Best1-independent mechanisms for glutamate release such as vesicular exocytosis. It is possible that Best1-shRNA affects the expression level of other genes involved in vesicular release. To address this possibility, we performed semi-quantitative RT-PCR analysis of cultured astrocytes with Best1-shRNA. Best1-shRNA showed a knock down efficiency for Best1 of over 90% while not affecting the expression level of other known genes such as Syt4 (Synaptotagmin4), Munc18-1, Vamp2 (Vesicle-associated membrane protein 2) involved in vesicular release (Figure 5A). We also tested whether inhibiting vesicular release machinery affects the glutamate release by using FRET based glutamate imaging. We found that the pretreatment with Conconomycin A (preventing vesicular glutamate release by inhibiting vesicular hydrogen ATPase, [31]) and Tetanus toxin (interfering all vesicular releases by inhibiting fusion of vesicles, [32]) slightly but not significantly reduced TFLLR-induced glutamate release from cultured astrocytes (Figure 5B and 5C). Consistent with these results, hyperosmotic challenge, which is known to cause a Ca2+ independent exocytosis of glutamate containing releasable vesicles, did not show any significant increase in glutamate release (Figure 5B and 5C). Taken together, PAR1-induced glutamate release is not mediated by vesicular exocytosis in cultured astrocytes.

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