Fig. 3.
MeATP is rapidly metabolized by eNPP1 in VSMC to generate methylene diphosphonate (PCP) that acts as an inhibitor of autocrine PPi accumulation. A: HPLC chromatograms of extracellular media from intact HEK-green fluorescent protein (GFP) cells (left) or HEK-NPP1 cells (right) pulsed with 20 μM exogenous etheno-β,γ-methylene ATP (ɛ-MeATP) and incubated for the indicated times. B: HPLC chromatograms of extracellular media from rat VSMC pulsed with 300 μM exogenous MeATP and incubated for the indicated times. Inset: time course of MeATP (300 μM) hydrolysis based on calibrated absorbance from the HPLC chromatogram in B. C: extracellular PPi generation by VSMC incubated in the absence (▪) or presence of 30 μM (□), 100 μM (•), or 300 μM (○) PCP. Data points represent the average ± range of duplicates from representative experiments (each repeated three times with similar results) using passage 4 VSMC. D: extracellular ATP release by VSMC incubated in the absence (▪) or presence of 300 μM MeATP (□), 300 μM PCP (•), or 300 μM MeATP + 300 μM PCP (○). PCP data points indicated by solid circles overlap with the control data points indicated by solid squares. E: total intracellular ATP was measured in control or PCP-treated VSMC by rapid permeabilization of the cell monolayers with digitonin (50 μg/ml) to release ATP for quantification by the luciferase-based assay. VSMC were incubated in the absence or presence of 300 μM PCP for the indicated times before digitonin permeabilization. Data points in D and E represent means ± SE from 3 separate experiments (each performed in triplicate) with VSMC preparations at passages 2-4 (in D, error bars are smaller than symbols for some data points).