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.