Table 1

Effects of pharmacologic ATP transport inhibitors on eATP levels, ATP-metabolizing ecto-enzyme activities, and cell toxicity in hypotonically stressed chondrocyte cultures
Inhibitor Target Dosage Fold change in ATP P N Alk phos NTPPPH 5’NT Toxicity (fold change)
Probenecid ANK, Hemichannels 5 mM 0.59* 0.004 83 103 ± 2.9 108 ± 6.9 122 ± 24.8 1.31
Monensin Vesicular 100 uM 0.74 0.097 50 103 ± 1.8 109 ± 5.3 120 ± 20.2 1.49
GdCl3 Maxianion 50 uM 0.88 0.309 51 106 ± 6.9 104 ± 2.1 126 ± 11.9 2.21
N-ethylmalemide (NEM) Vesicular 100 uM 0.89 0.947 57 101 ± 3.2 110 ± 3.6 116 ± 19.3 1.68
Brefeldin Vesicular 100 uM 0.88 0.739 60 101 ± 3.2 110 ± 3.6 116 ± 19.3 1.68
Carbeneoxolone (CBX) Hemichannels 5 uM 1.45 0.627 40 103 ± 2.9 109 ± 8.4 109 ± 16.9 3.25
Flufenamic acid (FFA) Hemichannels 30 uM 1.14 0.545 40 98 ± 6.5 118 ± 6.0 108 ± 14.4 1.33
10Panx1 Pannexin-1 100 uM 1.3 0.850 43 105 ± 4.7 137 ± 4.6 115 ± 16.8 1.33
10Panx1 Scramble Pannexin-1 100 uM 1.5 0.256 41 104 ± 6.6 142 ± 8.8 83 ± 10.8 1.27
Brilliant Blue G P2X7, P2X4 50 uM 0.367* 0.001 24 147 ± 5.9 142 ± 7.4 155 ± 32 1.26
A438079 P2X7 300 nm 2 0.042 24 131 ± 4.4 136 ± 5.7 92 ± 21.9 1.25
AZ10606120 P2X7 10 nM 1.2 0.806 24 123 ± 6.9 124 ± 4.6 104 ± 23.6 1.12

Chondrocytes were exposed to control media with and without various pharmacologic ATP pathway inhibitors for 30 minutes. Aliquots of media were removed and replaced with 35% H20 as described. After 10 minutes, eATP levels were measured in the media. eATP results are expressed as a mean fold change in osmotically challenged eATP levels over control (no inhibitor) conditions; N = number of replicates pooled from three to five experiments: *statistically significantly inhibition of ATP levels compared to controls. In parallel cultures, specific activities of ATP metabolizing enzymes - alkaline phosphatase (Alk phos), nucleoside triphosphate pyrophosphohydrolase (NTPPPH), and 5′ nucleotidase (5' NT) - were measured in the cell layer using standard colorimetric assays. Results are expressed as percent of specific activity of the control (no inhibitor) conditions (means ± standard error, n=6). Cell toxicity was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are expressed as a fold increase over control (no inhibitor) conditions. There were no statistically significant changes in levels of Alk phos, NTPPPH, 5’NT activity or cell toxicity in the presence of inhibitors. ANK, progressive ankylosis gene product.

Rosenthal et al.

Rosenthal et al. Arthritis Research & Therapy 2013 15:R154   doi:10.1186/ar4337

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