Ins(1,4,5)P{sub 3} facilitates ATP accumulation via phosphocreatine/creatine kinase in the endoplasmic reticulum extracted from MDCK cells
- Medical Research Center, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan)
- Joint Laboratory for Frontier Medical Science, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan)
- Central Laboratory for Pathology and Morphology, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan)
- Research Laboratory of Biodynamics, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan)
- Department of Pediatric Dentistry, School of Dentistry of Shanghai Tongji University, Shanghai 200072 (China)
So far, the content and accumulation of ATP in isolated endoplasmic reticulum (ER) are little understood. First, we confirmed using electron microscopic and Western blotting techniques that the samples extracted from MDCK cells are endoplasmic reticulum (ER). The amounts of ATP in the extracted ER were measured from the filtrate after a spinning down of ultrafiltration spin column packed with ER. When the ER sample (5 {mu}g) after 3 days freezing was suspended in intracellular medium (ICM), 0.1% Triton X and ultrapure water (UPW), ATP amounts from the ER with UPW were the highest and over 10 times compared with that from the control with ICM, indicating that UPW is the most effective tool in destroying the ER membrane. After a 10-min-incubation with ICM containing phosphocreatine (PCr)/creatine kinase (CK) of the fresh ER. ATP amounts in the filtrate obtained by spinning down were not changed from that in the control (no PCr/CK). However, ATP amounts in the filtrate from the second spinning down of the ER (treated with PCr/CK) suspended in UPW became over 10-fold compared with the control. When 1 {mu}M inositol(1,4,5)trisphosphate (Ins(1,4,5)P{sub 3}) was added in the incubation medium (ICM with PCr/CK), ATP amounts from the filtrate after the second spinning down were further enhanced around three times. This enhancement was almost canceled by Ca{sup 2+}-removal from ICM and by adding thapsigargin, a Ca{sup 2+}-ATPase inhibitor, but not by 2-APB and heparin, Ins(1,4,5)P{sub 3} receptor antagonists. Administration of 500 {mu}M adenosine to the incubation medium (with PCr/CK) failed to enhance the accumulation of ATP in the ER. These findings suggest that the ER originally contains ATP and ATP accumulation in the ER is promoted by PCr/CK and Ins(1,4,5)P{sub 3}.
- OSTI ID:
- 22202672
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 397, Issue 3; Other Information: Copyright (c) 2010 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
Similar Records
Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating
Phosphocreatine attenuates endoplasmic reticulum stress-mediated hepatocellular apoptosis ameliorates insulin resistance in diabetes model