Applicability of microplate assay coupled to Fiske-Subbarow reducer for the determination of phosphorous produced by in vivo human lymphocytes: PKC is probably cross talking with ecto 5′-nucleotidase

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-5′-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 assay−1. 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 cells−1 h−1. The addition of MADP to the incubation media decreased the AMPase activity to 2.43±0.9 nmol Pi million cells−1 h−1 (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.