Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10557224 | Microchemical Journal | 2005 | 6 Pages |
Abstract
In this research, the phorbol ester, phorbol 12-myristate 13-acetate (PMA), was used to assess the effect of protein kinase C (PKC) activation on the specific activity of ecto-5â²-nucleotidase (eNT) in human lymphocytes. PMA mimics the effects of diacylglycerol, a natural compound released by the hydrolysis of the glycosilphosphatidilinositol (GPI) moiety, in activating PKC. In order to evaluate the activity of eNT in living lymphocytes, a micro-assay method was established with a low detection limit for inorganic phosphate (Pi) of 0.94 nmol Pi assayâ1. The dephosphorylation of Adenosine monophosphate (AMP) by functional lymphocytes was evaluated and the contribution of the eNT activity was calculated by its inhibition with the specific eNT inhibitor α,β-methylene ADP (MADP) and the use of the broad spectrum phosphatases inhibitor (but not eNT), levamisole. Under the conditions tested, we obtained an AMPase value of 8.05±4.4 nmol Pi million cellsâ1 hâ1. The addition of MADP to the incubation media decreased the AMPase activity to 2.43±0.9 nmol Pi million cellsâ1 hâ1 (p<0.05). On the other hand, when lymphocytes were incubated with PMA, an increase of 182% in the AMPase activity was observed. However, the addition of levamisole inhibited the AMPase activity by about 17%, while the co-incubation of cells with PMA and levamisole reduced only an 8% of the total PMA-increased AMPase activity. These results show that (1) a non-radioactive micro-method can be used to assess the Pi production in living cells; (2) the obtained data strongly suggest that eNT is the main ecto-enzyme present on the surface of circulating lymphocytes responsible for the hydrolysis of extracellular AMP; and (3) that PKC is cross talking with eNT.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Alejandro MartÃnez-MartÃnez, Laura A. de la Rosa, Claudia A. JÃmenez-Muñoz, Angel Gabriel DÃaz-Sánchez, Jesús A. Araujo-González, José R. Peralta-Videa, Guadalupe de la Rosa, Jorge L. Gardea-Torresdey,