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Essential role of vesicular nucleotide transporter in vesicular storage and release of nucleotides in platelets.

Hiasa M, Togawa N, Miyaji T, Omote H, Yamamoto A, Moriyama Y - Physiol Rep (2014)

Bottom Line: The release of nucleotides triggers one of the first steps in a series of cascades responsible for blood coagulation.However, the mechanism of how the nucleotides are accumulated in the granules is still far less understood.The transporter protein responsible for storage of nucleotides in the neuroendocrine cells has been identified and characterized.

View Article: PubMed Central - PubMed

Affiliation: Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

No MeSH data available.


Vesicular nucleotide transporter (VNUT) was responsible for release of nucleotides from MEG‐01 cells. (A) Time course of ATP and ADP release in a Ca2+‐dependent manner from MEG‐01 cells. ATP release was initiated by the calcium ionophore A23187 at 5 μmol/L. (B) RNAi against human VNUT decreased VNUT expression. Quantitative analysis for VNUT mRNA levels was performed by real‐time PCR. The amount of hVNUT mRNA was quantified as the ratio to G3PDH and expressed as relative to that of negative control siRNA. (C) Immunoblotting indicated that the concentration of VNUT protein decreases without affecting the expression of other proteins by RNAi. (D) Exocytosis of ATP and ADP were measured after 20 min the calcium ionophore A23187 stimulation at 5 μmol/L from control siRNA‐treated MEG‐01 cells and VNUT siRNA‐treated MEG‐01 cells.
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fig04: Vesicular nucleotide transporter (VNUT) was responsible for release of nucleotides from MEG‐01 cells. (A) Time course of ATP and ADP release in a Ca2+‐dependent manner from MEG‐01 cells. ATP release was initiated by the calcium ionophore A23187 at 5 μmol/L. (B) RNAi against human VNUT decreased VNUT expression. Quantitative analysis for VNUT mRNA levels was performed by real‐time PCR. The amount of hVNUT mRNA was quantified as the ratio to G3PDH and expressed as relative to that of negative control siRNA. (C) Immunoblotting indicated that the concentration of VNUT protein decreases without affecting the expression of other proteins by RNAi. (D) Exocytosis of ATP and ADP were measured after 20 min the calcium ionophore A23187 stimulation at 5 μmol/L from control siRNA‐treated MEG‐01 cells and VNUT siRNA‐treated MEG‐01 cells.

Mentions: Then, we investigated whether VNUT gene expression is linked with the secretion of nucleotides from MEG‐01 cells. Nucleotide secretion was triggered by the addition of A23187, a Ca2+ ionophore, since it causes entry of extracellular Ca2+ into the cell interior; the resultant rapid increase in Ca2+ facilitates exocytosis of secretory vesicles. As shown in Figure 4A, appreciable amounts of ATP and ADP were released from the cells in a time‐dependent fashion. In the absence of extracellular Ca2+ in the medium, no A23187‐dependent release of nucleotides was observed.


Essential role of vesicular nucleotide transporter in vesicular storage and release of nucleotides in platelets.

Hiasa M, Togawa N, Miyaji T, Omote H, Yamamoto A, Moriyama Y - Physiol Rep (2014)

Vesicular nucleotide transporter (VNUT) was responsible for release of nucleotides from MEG‐01 cells. (A) Time course of ATP and ADP release in a Ca2+‐dependent manner from MEG‐01 cells. ATP release was initiated by the calcium ionophore A23187 at 5 μmol/L. (B) RNAi against human VNUT decreased VNUT expression. Quantitative analysis for VNUT mRNA levels was performed by real‐time PCR. The amount of hVNUT mRNA was quantified as the ratio to G3PDH and expressed as relative to that of negative control siRNA. (C) Immunoblotting indicated that the concentration of VNUT protein decreases without affecting the expression of other proteins by RNAi. (D) Exocytosis of ATP and ADP were measured after 20 min the calcium ionophore A23187 stimulation at 5 μmol/L from control siRNA‐treated MEG‐01 cells and VNUT siRNA‐treated MEG‐01 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4208647&req=5

fig04: Vesicular nucleotide transporter (VNUT) was responsible for release of nucleotides from MEG‐01 cells. (A) Time course of ATP and ADP release in a Ca2+‐dependent manner from MEG‐01 cells. ATP release was initiated by the calcium ionophore A23187 at 5 μmol/L. (B) RNAi against human VNUT decreased VNUT expression. Quantitative analysis for VNUT mRNA levels was performed by real‐time PCR. The amount of hVNUT mRNA was quantified as the ratio to G3PDH and expressed as relative to that of negative control siRNA. (C) Immunoblotting indicated that the concentration of VNUT protein decreases without affecting the expression of other proteins by RNAi. (D) Exocytosis of ATP and ADP were measured after 20 min the calcium ionophore A23187 stimulation at 5 μmol/L from control siRNA‐treated MEG‐01 cells and VNUT siRNA‐treated MEG‐01 cells.
Mentions: Then, we investigated whether VNUT gene expression is linked with the secretion of nucleotides from MEG‐01 cells. Nucleotide secretion was triggered by the addition of A23187, a Ca2+ ionophore, since it causes entry of extracellular Ca2+ into the cell interior; the resultant rapid increase in Ca2+ facilitates exocytosis of secretory vesicles. As shown in Figure 4A, appreciable amounts of ATP and ADP were released from the cells in a time‐dependent fashion. In the absence of extracellular Ca2+ in the medium, no A23187‐dependent release of nucleotides was observed.

Bottom Line: The release of nucleotides triggers one of the first steps in a series of cascades responsible for blood coagulation.However, the mechanism of how the nucleotides are accumulated in the granules is still far less understood.The transporter protein responsible for storage of nucleotides in the neuroendocrine cells has been identified and characterized.

View Article: PubMed Central - PubMed

Affiliation: Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

No MeSH data available.