ATP storage and uptake by isolated pancreatic zymogen granules

TY - JOUR

T1 - ATP storage and uptake by isolated pancreatic zymogen granules

AU - Haanes, Kristian Agmund

AU - Novak, Ivana

N1 - .

PY - 2010/7/15

Y1 - 2010/7/15

N2 - ATP is released from pancreatic acini in response to cholinergic and hormonal stimulation. The same stimuli cause exocytosis of ZG (zymogen granules) and release of digestive enzymes. The aim of the present study was to determine whether ZG stored ATP and to characterize the uptake mechanism for ATP transport into the ZG. ZG were isolated and the ATP content was measured using luciferin/luciferase assays and was related to protein in the sample. The estimate of ATP concentration in freshly isolated granules was 40-120 μM. The ATP uptake had an apparent Km value of 4.9±2.1 mM when granules were incubated without Mg2+ and a Km value of 0.47±0.05 mM in the presence of Mg2+, both in pH 6.0 buffers. The uptake of ATP was significantly higher at pH 7.2 compared with pH 6.0 solutions. The anion transport blockers DIDS (4,4′-diisothiocyanostilbene- 2,2′-disulfonate) and Evans Blue inhibited ATP transport. Western blot analysis on the ZG showed the presence of VNUT (vesicular nucleotide transporter). Together, these findings indicate that VNUT may be responsible for the ATP uptake into ZG. Furthermore, the present study shows the presence of ATP together with digestive enzymes in ZG. This indicates that co-released ATP would regulate P2 receptors in pancreatic ducts and, thus, ductal secretion, and this would aid delivery of enzymes to the duodenum.

AB - ATP is released from pancreatic acini in response to cholinergic and hormonal stimulation. The same stimuli cause exocytosis of ZG (zymogen granules) and release of digestive enzymes. The aim of the present study was to determine whether ZG stored ATP and to characterize the uptake mechanism for ATP transport into the ZG. ZG were isolated and the ATP content was measured using luciferin/luciferase assays and was related to protein in the sample. The estimate of ATP concentration in freshly isolated granules was 40-120 μM. The ATP uptake had an apparent Km value of 4.9±2.1 mM when granules were incubated without Mg2+ and a Km value of 0.47±0.05 mM in the presence of Mg2+, both in pH 6.0 buffers. The uptake of ATP was significantly higher at pH 7.2 compared with pH 6.0 solutions. The anion transport blockers DIDS (4,4′-diisothiocyanostilbene- 2,2′-disulfonate) and Evans Blue inhibited ATP transport. Western blot analysis on the ZG showed the presence of VNUT (vesicular nucleotide transporter). Together, these findings indicate that VNUT may be responsible for the ATP uptake into ZG. Furthermore, the present study shows the presence of ATP together with digestive enzymes in ZG. This indicates that co-released ATP would regulate P2 receptors in pancreatic ducts and, thus, ductal secretion, and this would aid delivery of enzymes to the duodenum.

KW - Faculty of Science

KW - Key words: ATP release, ATP uptake, pancreas, purinergic receptor, vesicular nucleotide transporter (VNUT), zymogen granule

U2 - 10.1042/BJ20091337

DO - 10.1042/BJ20091337

M3 - Journal article

C2 - 20441565

SN - 0264-6021

VL - 429

SP - 303

EP - 311

JO - Biochemical Journal

JF - Biochemical Journal

IS - 2

ER -