Autoradiography-based cytochemical detection of ecto-ATPase, ecto-ADPase, 5′-nucleotidase, and extracellular adenosine production, employing141Ce3+ as a capturing agent

Abstract

A method for the visualization of the ecto-nucleotidase enzyme activities present on the cell surface, employing 141Ce3+ as a capturing and labelling agent, is described. Phosphate ions precipitated at the cell surface can be detected by coating the cells with an autoradiographic emulsion, followed by light microscopical inspection of the formed silver grains. The activities of ecto-ATPase, ecto-ADPase and 5'-nucleotidase were detected by this approach in four different cell lines. Parallel biochemical measurements of the activities of the corresponding enzymes were carried out in order to validate, evaluate, and optimize the cytochemical detection. The finding that Ce3+ ions are inhibitory to ecto-ATPase provided evidence for the necessity of carefully establishing appropriate reaction conditions for the cytochemical determination of ecto-nucleotidases. The application of this method to the indirect detection of extracellular adenosine production from substrates like ATP has also been documented. It allows a cytochemical determination of adenosine formed through cascade nucleotide dephosphorylation. This newly described method is of high sensitivity and potentially of value for a variety of applications, including not only cytochemistry but also cell biology, and molecular biology studies.

Regulation of proliferation of LLC-MK2 cells by nucleosides and nucleotides: the role of ecto-enzymes

“…From these results it can be calculated that the proliferation caused by 100 µM ATP is inhibited by 71 % by suramin and by 84 % by RB-2. The inhibition of the ecto-ATPase activity by the same concentrations of suramin and RB-2, as measured biochemically, was 73 % and 77 % respectively [35]. As determined by Trypan Blue stain cell counting, suramin and RB-2 were shown not to influence the viability of the cells at the concentrations and time periods used in the experiments (viability 105p21 % and 106p22 % respectively, n l 16).…”

“…We focused our attention on LLC-MK # cells for two reasons. First, these cells showed a high proliferative response to externally added ATP and adenosine, and secondly, they express a high activity of ectoenzymes with nucleotide-dephosphorylating capacities [35]. When adenosine, AMP, ADP and ATP were tested for proliferation effects, ATP was shown to influence the cellular proliferation to the highest extent, followed by ADP, AMP and adenosine.…”

“…agent ( Figure 3). This method indirectly detects the adenosine produced by complexing the radioactive phosphate released from ATP at the cell surface with Ce$ + ions [35].…”