Purine-Metabolizing Ectoenzymes Control IL-8 Production in Human Colon HT-29 Cells
Adenylate kinase and ecto-5′-nucleotidase are expressed by HT-29 cells. (a) HT-29 cells were incubated with ADP for 90 min and their supernatants were subjected to RP-HPLC for the determination of purines and ADK activity (). Inset shows the effect of the ADK inhibitor, Ap5A, on ATP production by HT-29 cells after 1 h (). (b) Supernatants from HT-29 cells incubated for 30 min with various concentrations of ADP were subjected to RP-HPLC to determine ATP production (). ((c), (d)) Supernatants from HT-29 cells incubated for 30 min with either 100 µM ATP ((c) ) or ADP ((d) ), in the presence of 10 µM Ap5A or control vehicle, were subjected to RP-HPLC for the determination of adenine nucleotide and adenosine byproducts. (e) Semiquantitative RT-PCR analysis on HT-29 cell line showing the presence of the membrane associated adenylate kinase. A representative experiment of is presented. (f) The expression of ecto-5′-nucleotidase protein was detected by immunoblotting with the rabbit anti-human ecto-5′-nucleotidase h5′NT-I5 ((A) on the left side) and with the commercial antibody clone 4G4 ((B) on right side). For panels (A) and (B), lane 1 represents the positive control of protein extract (6 µg) from HEK 293 cells transiently transfected with a human ecto-5′-Nucleotidase expression vector and lane 2 protein extract (20 µg) from HT-29.