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Table 1 Effects of pharmacologic ATP transport inhibitors on eATP levels, ATP-metabolizing ecto-enzyme activities, and cell toxicity in hypotonically stressed chondrocyte cultures

From: The progressive ankylosis gene product ANK regulates extracellular ATP levels in primary articular chondrocytes

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

  1. 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.