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

• Published in: Microchemical journal 2005-08, Vol.81 (1), p.92-97
• Main Authors: Martínez-Martínez, Alejandro, de la Rosa, Laura A., Jímenez-Muñoz, Claudia A., Díaz-Sánchez, Angel Gabriel, Araujo-González, Jesús A., Peralta-Videa, José R., de la Rosa, Guadalupe, Gardea-Torresdey, Jorge L.
• Format: Article
• Language: English
• Subjects: 5′NT; CD73; Ecto-5′-nucleotidase; Lymphocyte; Nucleotidase; Pi microassay; PKC; PMA; α-β-MADP
• ISSN: 0026-265X; 1095-9149
• DOI: 10.1016/j.microc.2005.01.013

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