Figure 1.

Human subcutaneous fibroblasts (HSCF) express ecto-5′-nucleotidase/CD73, the enzyme responsible for AMP dephosphorylation into adenosine, but lack adenosine deaminase (ADA), resulting in extracellular adenosine accumulation that may signal via co-expressed A_2A and A₃ receptor subtypes. Panel A shows the immunoreactivity against fibroblast cell markers, vimentin (red), and type I collagen (green). The panel B shows that HSCF stain positively against A_2A and A₃ receptors (green), with very little amounts of A_2B and A₁ receptor subtypes. The panel C shows that HSCF are ecto-5′-nucleotidase/CD73 positive ADA negative cells. Nuclei are stained in blue with DAPI; scale bar is 60 μm. Micrographs were obtained from at least three different individuals with a laser scanning confocal microscope using the same acquisition settings. Panel D shows the time course of the extracellular catabolism of AMP (30 µM, (i) and adenosine (ADO, 30 μM, (ii) in HSCF cultures allowed to grow for 11 days. AMP and ADO were added to the culture medium at time zero. Samples (75 μL) were collected from each well at indicated times in the abscissa. Each collected sample was analyzed by HPLC to separate and quantify AMP (filled squares), adenosine (open squares), inosine (filled triangles), and hypoxanthine (open triangles). Each point represents pooled data from four individuals; two replicas were performed in each individual experiment. Vertical bars represent SEM and are shown when they exceed the symbols in size. The calculated half-life time (t½, min) for each initial substrate is shown for comparison.