Table 2.
Effects of FS and FS analogs for recombinant ACs 1, 2 and 5 in the presence of 7 mM Mn2+.
| Mn2+ | AC1 | AC2 | AC5 | |||
|---|---|---|---|---|---|---|
| Diterpene | EC50 [μM] |
Efficacy [%] |
EC50 [μM] |
Efficacy [%] | EC50 [μM] |
Efficacy [%] |
| FS(+) | 3.3 ± 1.3 | 100 | 38.3 ± 14.9 | 100 | 17.8 ± 6.5 | 100 |
| DMB-FS(c) | 2.8 ± 1.6 | 65.4 ± 8.9 | 50.8 ± 31.2 | 46.0 ± 14.5 | 10.9 ± 1.0 | 81.4 ± 14.3 |
| 6A7DA-FS(aa, bb) | 3.0 ± 1.0 | 110 ± 2.2 | 16.7 ± 7.4 | 69.6 ± 3.5 | 13.4 ± 3.4 | 69.7 ± 7.0 |
| 7DA-FS(aa,cc) | 9.3 ± 1.7 | 108.8 ± 4.6 | 80.5 ± 46.8 | 61.7 ± 7.9 | 38.2 ± 6.4 | 124.2 ± 12.9 |
| 9d-FS(aa,cc) | 17.0 ± 3.3 | 98.3 ± 3.7 | 127 ± 31.8 | 29.5 ± 6.2 | 60.8 ± 12.3 | 108.1 ± 12.9 |
| 1d-FS(a) | Ineffective | 2.9 ± 0.7 | Ineffective | 0.4 ± 0.8 | Ineffective | 0.7 ± 0.3 |
| BODIPY-FS(xx, aaa, ccc) | 1.2 ± 0.2 | 73.1 ± 10.3 | 0.17 ± 0.07 | −18.9 ± 12.3 | 2.7 ± 1.1 | 68.5 ± 9.3 |
AC activities were determined as described in “Materials and Methods”. Reaction mixtures contained 7 mM Mn2+, [α–32P]ATP (0.3 μCi/tube), 10 μM GTPγS, 100 μM cAMP, 0.4 mg/mL creatine kinase, 9 mM phosphocreatine, 100 μM IBMX and diterpenes at concentrations from 100 nM – 300 μM. Data were analyzed by non-linear regression to determine the EC50-values. The efficacy for each analog was determined by dividing the maximal stimulation obtained for the analog by the maximum stimulation obtained by treatment with 300 μM FS expressed in percent. Statistical differences between the examined AC isoforms were analyzed by ANOVA Bonferroni test. Comparison of EC50-values: AC1 vs. AC2 (+) p < 0.05, (++) p < 0.005 and (+++) p < 0.0001; AC1 vs. AC5 (#) p < 0.05, (##) p < 0.005 and (###) p < 0.0001; AC2 vs. AC5 (x) p < 0.05, (xx) p < 0.005 and (xxx) p < 0.0001. Comparison of Emax-values: AC1 vs. AC2 (a) p < 0.05, (aa) p < 0.005 and (aaa) p < 0.0001; AC1 vs. AC5 (b) p < 0.05, (bb) p < 0.005 and (bbb) p < 0.0001; AC2 vs. AC5 (c) p < 0.05, (cc) p < 0.005 and (ccc) p < 0.0001.