Identification of mAC3 activating fatty acids.

(A and B) Effect of 1 µg/assay of fractions E from vacuum-liquid-chromatography (A) and E2 from RP-HPLC (B) on 300 nM Gsα-stimulated mAC isoforms 3 and 5. Activities are shown as % compared to 300 nM Gsα stimulation (100%). n=2, each with two technical replicates. Basal and Gsα activities of mAC3 in (A) were 0.01 and 0.07 and of mAC5 0.06 and 1.32 nmol cAMP•mg-1•min-1, respectively. In (B), basal and Gsα activities of mAC3 were 0.02 and 0.12 and of mAC5 0.09 and 1.1 nmol cAMP•mg-1•min-1, respectively. (C) GC-MS chromatogram of fraction E2. Mass spectra of the fatty acids are shown. Fatty acids’ identity was confirmed by comparing with corresponding standards (TMS: Trimethylsilyl). (D) Effect of fatty acids identified by GC-MS on 300 nM Gsα-stimulated mAC3 (left) and mAC5 (right). Basal and Gsα activities of mAC3 were 0.023 ± 0.02 and 0.17 ± 0.03 and of mAC5 0.08 ± 0.02 and 0.44 ± 0.09 nmol cAMP•mg-1•min-1, respectively. n= 3-23. EC50 of palmitic and oleic acids for mAC3 were 6.4 and 10.4 μM, respectively. Data represent individual experiments (black dots in A and B) or mean ± SEM (D). One-sample t tests were performed. Significances: **P < 0.01, ***P < 0.001; ****P < 0.0001.

Concentration-response curves for Gsα and mAC3 and 5 activities in presence or absence of 20µM oleic acid.

20 µM oleic acid enhances Gsα stimulation of mAC3 (left) but not of mAC5 (center). (Left) EC50 of Gsα in the absence of oleic acid was 549 nM and in the presence of 20 µM oleic acid, it was 471 nM (not significant). mAC3 basal activity was 30 ± 24 pmol cAMP•mg-1•min-1. (Center) The EC50 of Gsα in the absence of oleic acid was 245 nM and in the presence of oleic acid, it was 277 nM (not significant). mAC5 basal activity was 84 ± 60 cAMP•mg-1•min-1. n=2-3, each with two technical replicates. (Right) Fold stimulation of mAC3 and 5 activities incubated with different concentrations of Gsα + oleic acid (n = 2-3). Data are mean ± SEM, paired t test for left and center, and one-way ANOVA for right. Significances: *P < 0.05.

Effect of 20 µM lipids on 300 nM Gsα-stimulated mAC3.

Basal and Gsα stimulated activities were 0.02 ± 0.001 and 0.17 ± 0.01 nmol cAMP•mg-1•min-1, respectively. EPA: Eicosapentaenoic acid; 9-HSA: 9-hydroxystearic acid. Data are mean ± SEM . One-sample t tests, Significances: *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001.

List of lipids tested against mAC isoforms.

Oleic acid enhances cAMP formation in mAC3-transfected HEK293 cells.

Effect of oleic acid on HEK293 cells permanently transfected with mACs 3 and 5 stimulated by 2.5 µM isoproterenol (set as 100%). Basal and isoproterenol stimulated cAMP levels of HEK-mAC3 were 0.02 ± 0.006 and 1.35 ± 0.24 and of HEK-mAC5 2.13 ± 0.69 and 2.60 ± 0.88 pmol cAMP/10000 cells, respectively. n= 3-9. Data are mean ± SEM . One-sample t tests. Significances: *P < 0.05, **P < 0.01, ***P < 0.001.

Fatty acids enhance mAC isoforms 2, 7 and 9 activities.

(A) Effect of 20 µM oleic acid on 300 nM Gsα-stimulated mAC activities normalized to 100%. Basal and Gsα-stimulated activities for each isoform are in appendix 2 Table 1. n= 2-23. (B) Oleic acid activates mACs 2, 7, and 9 stimulated by 300 nM Gsα. n=7-23. (C) Fatty acids activating mACs 2, 7, and 9 at 20 µM. For basal and Gsα-stimulated activities, see appendix 2, Fig.’s 6-9. n= 5-15. Data are mean ± SEM. One-sample t tests were performed. Significances: *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001.

Arachidonic acid attenuates 300 nM Gsα-stimulated activities of mACs 1 and 4.

(A) Arachidonic acid attenuates Gsα-stimulated mACs 1 and 4. Basal and Gsα stimulated activities of mAC1 were 0.12 ± 0.01 and 0.42 ± 0.03 and of mAC4 0.02 ± 0.002 and 0.14 ± 0.02 nmol cAMP•mg-1•min-1, respectively. IC50 of arachidonic acid for mAC1 and mAC4 were 23 and 36 μM, respectively. n= 3-9. (B) Effect of arachidonic acid on HEK-mAC1 and HEK-mAC4 cells. Cells were stimulated by 10 µM isoproterenol (set as 100 %) in the presence of 0.5 mM IBMX (3-isobutyl-1-methylxanthine). Basal and isoproterenol stimulated cAMP levels in HEK-mAC1 were 1.03 ± 0.15 and 1.66 ± 0.28 and in HEK-mAC4 0.20 ± 0.04 and 0.86 ± 0.24 pmol cAMP/10000 cells, respectively. IC50 for HEK-mAC1 was 250 pM. n= 2-11, each with three replicates.

Data are mean ± SEM. One-sample t tests were performed. Significances: **P < 0.01, ***P < 0.001; †P < 0.0001. For clarity, not all significances are indicated.

Anandamide attenuates 300 nM Gsα-stimulated activities of mACs 1, 4, 5 and 6.

(A) Effect of anandamide on Gsα-stimulated mAC5 and 6. Basal and Gsα activities of mAC5 were 0.05 ± 0.01 and 0.98 ± 0.12 and of mAC6 0.05 ± 0.01 and 0.78 ± 0.12 nmol cAMP•mg- 1•min-1, respectively. IC50 of anandamide were 42 and 22 μM, respectively. n= 3-32. (B) Anandamide attenuates mAC1 but not mAC4 stimulated by Gsα. Basal and Gsα stimulated activities of mAC1 were 0.12 ± 0.01 and 0.40 ± 0.03 and of mAC4 0.02 ± 0.002 and 0.15 ± 0.02 nmol cAMP•mg-1•min-1, respectively. IC50 for mAC1 was 29 μM. n= 3-4, each with two technical replicates. (C) Effect of anandamide on 2.5 µM isoproterenol stimulated HEK-mAC5. Basal and isoproterenol stimulated cAMP levels of HEK-mAC5 were 1.8 ± 0.22 and 2.4 ± 0.48 pmol cAMP/10000 cells, respectively. The control bar represents 2.5 µM isoproterenol stimulation alone. n=5-6, each with three technical replicates. IC50 of anandamide was 133 µM.

Data are mean ± SEM. One-sample t tests (A-B) and paired t test (C) were performed. Significances: ns: not significant P > 0.05;*P < 0.05, **P < 0.01, ***P < 0.001. For clarity, not all significances are indicated.

Receptor properties are exchangeable between mAC isoforms.

(A, left) Detection of AC5(membr)-AC3(cat) receptor chimeras. AC5(membr)-AC3(cat) (AC5-3) (22) was expressed in HEK293 cells with an N-terminal tag for labeling with the protein ligase Connectase. The membrane preparation was incubated with fluorophore-conjugated Connectase and separated by SDS-PAGE. A fluorescence scan of the gel detects AC5(membr)-AC3(cat) (right), the reagent (fluorophore-conjugated Connectase) is detected when using HEK293 membrane (middle) or a buffer control (left); (A, right) Design of the chimeric AC5-3 construct. Numbers are amino acid positions in mAC3 and 5, respectively. (B) Gsα concentration response curve of mAC5-3. Basal activity for mAC5-3 was 0.02 pmol cAMP•mg-1•min-1. Error bars denote SEM of n=3, each with two technical replicates. (C) Effect of 20 µM oleic acid on 300 nM Gsα-stimulated mACs 3, 5, and 5-3. Basal and Gsα activities of mACs 3, 5, and 5-3 were 0.02 ± 0.003 and 0.11 ± 0.02, 0.05 ± 0.01 and 0.98 ± 0.12, and 0.01 ± 0.004 and 0.2 ± 0.02 nmol cAMP•mg-1•min-1, respectively. n=7-33. (D) Effect of 100 µM Anandamide on 300 nM Gsα-stimulated mACs 3, 5, and 5-3. Basal and Gsα activities of mACs 3, 5, and 5-3 were 0.02 ± 0.002 and 0.19 ± 0.02, 0.05 ± 0.01 and 0.98 ± 0.12 and 0.02 ± 0.003 and 0.23 ± 0.04 nmol cAMP•mg-1•min-1, respectively. n=6-9. IC50 for mAC5 and mAC5-3 were 42 and 29 µM, respectively. (E). Exchange of TM domains transfers anandamide effect on mAC3. Basal and Gsα stimulated activities of mAC3 were 0.02 ± 0.002 and 0.12 ± 0.02 nmol cAMP•mg-1•min- 1, respectively. Basal and Gsα stimulated activities of mAC5 were 0.05 ± 0.005 and 0.98 ± 0.12 nmol cAMP•mg-1•min-1, respectively. Basal and Gsα stimulated activities of mAC5-3 were 0.02 ± 0.002 and 0.22 ± 0.03 nmol cAMP•mg-1•min-1, respectively. Calculated IC50 concentrations of Anandamide for mAC5 and mAC5-3 were 42 and 29 µM, respectively. Data are mean ± SEM. One-sample t tests. Significances: **P < 0.01; ****P < 0.0001.

mAC5 – mAC3 receptor-transfer analyzed in HEK293 cells.

Effect of anandamide on HEK-mAC5 and HEK-mAC5-3 cells stimulated by 2.5 µM isoproterenol (set as 100 %). Basal and isoproterenol stimulated cAMP levels in HEK-mAC5 were 1.80 ± 0.22 and 2.29 ± 0.39 and in HEK-mAC5-3 (+ 0.5 mM IBMX) 0.17 ± 0.02 and 3.11 ± 0.55 pmol cAMP/10000 cells, respectively. n= 4-11. IC50 for HEK-mAC5 and HEK-mAC5-3 were 133 and 60 μM, respectively. Anandamide had no effect on basal activity of HEK-mAC5 and stimulated HEK-mAC3 cells in concentrations up to 100 µM (data not shown).

Data are mean ± SEM. One-sample t tests were performed. Significances: *P < 0.05; ***P < 0.001.

Oleic acid concentration-dependently potentiates mAC activity in brain cortical membranes from mouse.

Basal and 300 nM Gsα activities were 0.4 ± 0.1 and 2.7 ± 0.7 nmol cAMP•mg-1•min-1, respectively. N= 4-6. One-sample t test: *P < 0.05; **P < 0.01 compared to 100% (300 nM Gsα stimulation).