Table 2.
Modulation of 1-MeA-induced inhibition of GJIC by various inhibitors of phospholipases and protein kinase signaling proteins
| † Inhibitor | Target signaling protein | ‡ Effect | §% inhibition of GJIC |
|---|---|---|---|
| None (+1-MeA) | Gap Junction | ↓↓↓ | 100 |
| +D609 (50 μM, 15 min) | PC-PLC | ↑↑↑ | *10 |
| +H-89 (40 μM, 30 min) | PKA, GPCR | ↑↑ | ¶28 |
| +PKI (50 μM, 2 h) | PKA | ↑↑↑ | ¶16 |
| +BEL (2.5 μM, 30 min) | iPLA2 | ↑ | ¶74 |
| +BPB (5.0 μM, 20 min) | sPLA2 | ↓↓↓ | 97 |
| +AACOF3 (10 μM, 30 min) | cPLA2, iPLA2 | ↓↓↓ | 115 |
| +MAFP (1.25 μM, 30 min) | cPLA2, iPLA2 | ↓↓↓ | 101 |
| +MJ33 (10 μM, 30 min) | acidic iPLA2 | ↓↓↓ | 104 |
| +ET-18-OCH3 (30 μM, 30 min) | PI-PLC | ↓↓↓ | 90 |
| +1-butanol (0.5% v/v, 15 min) | PLD | ↓↓↓ | 101 |
| +RHC80267 (50 μM, 20 min) | DAG lipase | ↓↓↓ | 97 |
| +GF109203X (20 μM) | PKC (pan-specific) | ↓↓↓ | 100 |
| +PP2 (40–100 μM) | Src | ↓↓↓ | 102 |
| +Genistein (100 μM) | Protein Tyr kinases (pan-specific) | ↓↓↓ | 102 |
Inhibitors were added before the addition of 1-MeA, which was incubated for 15 min at 70 μM, except for the BEL experiment where the time of 1-MeA treatment was 30 min. The inhibitors were added at concentrations and times indicated in the parentheses, and remained in the medium for the duration of the experiment. D609, tricyclodecan-9-yl-xanthate; H-89, N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; BPB, p-bromophenacyl bromide; AACOCF3, arachidonyl trifluoromethylketone; PKI, myristoylated PKI (14–22) amide; BEL, bromoenol lactone; MAFP, methyl arachidonyl fluorophosphates; MJ33, 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol; ET-18-OCH3, 1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine; RHC80267, 1,6-bis-(cyclohexyloximinocarbonylamino)hexane; GF109203X, bisindoylmaleimide I; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine).
↓↓↓ – complete inhibition of GJIC; ↑, ↑↑ – partial reversal of 1-MeA-induced inhibition of GJIC; ↑↑↑ – complete reversal of 1-MeA-induced inhibition of GJIC.
An average with n = 3, % Inhibition = [(1 − FOC of Inhibitor)/(1 − FOC of vehicle)] × 100.
Significant as determined by a two-tailed paired t-test at the 95% confidence interval, P < 0.001 between the (1-MeA) versus (1-MeA + inhibitor) treated cells.
1-MeA, 1-methylanthracene; DAG, diacylglycerol; GJIC, gap junctional intercellular communication; GPCR, G-protein coupled receptor; PC-PLC, phosphatidylcholine specific phospholipase C; PI-PLC, phosphatidylinositol specific phospholipase C; PKA, protein kinase A; PLD, phospholipase D.