Similarly potent inhibition of adenylyl cyclase by P-site inhibitors in hearts from wild type and AC5 knockout mice

Abstract

Adenylyl cyclase type 5 (AC5) was described as major cardiac AC isoform. The knockout of AC5 (AC5KO) exerted cardioprotective effects in heart failure. Our study explored the impact of AC5KO on mouse heart AC activities and evaluated putative AC5-selective inhibitors. In cardiac membranes from AC5KO mice, basal AC activity was decreased, while AC stimulation was intact. The putative AC5-selective P-site inhibitors SQ22,536 [9-(tetra-hydro-2-furanyl)-9H-purin-6-amine], vidarabine (9-β-D-arabinosyladenine) and NKY80 [2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone] inhibited recombinant AC5 more potently than AC2 and AC1, but selectivity was only modest (∼4-40-fold). These compounds inhibited cardiac AC from WT and AC5KO mice with similar potencies. In conclusion, AC regulation in AC5KO hearts was unimpaired, questioning the supposed dominant role of AC5 in the heart. Moreover, the AC inhibitors SQ22,536, NKY80 and vidarabine lack adequate selectivity for AC5 and, therefore, do not present suitable tools to study AC5-specific functions.

Figure 1. mRNA expression analysis of the β-AR-G-protein-AC signaling cascade in mouse hearts.

Box and whisker plots show the gene expression ratios in left ventricles (LVs) from AC5KO- relative to that in WT mice obtained from qRT-PCR experiments described under Methods. (A) Relative mRNA expression of membranous ACs 1–9. AC5 mRNA was not detected in LVs from AC5KO hearts. (B) Relative mRNA expression of β-ARs 1 and 2, G_sα and G_iα 1–3. The lower and upper boundaries of the boxes represent the 25^th percentile and the 75^th percentile. The line within the boxes represents the median. The whiskers show the lower and upper 25% of observations. Data of each target (A and B) was obtained from qRT-PCR experiments with seven WT- and five AC5KO heart LVs performed in duplicates. There were no significant differences in the gene expressions of investigated targets between LVs from WT vs. AC5KO mice except for AC5.

Figure 2. Stimulations of mouse heart AC and recombinant AC1, 2 and 5.

AC activities were determined in membrane preparations of mouse hearts from wild type (WT) and AC5 knockout (AC5KO) mice or in membrane preparations of Sf9-cells expressing recombinant AC1, 2 and 5 respectively. (A–C) Mouse heart AC activities in WT vs. AC5KO mice. (A) Basal and G_sα-stimulated AC activities. G_sα was stimulated GTP (10 µM) or GTPγS (10 µM). (B) AC activities under β-AR-mediated stimulation with isoproterenol. Baseline AC activity represents AC activity under stimulation with GTP (10 µM). (C) Direct activation of mouse heart with forskolin (FS). (D) Comparison of FS mediated AC activation of mouse heart AC (WT) vs. recombinant AC1, 2 and 5. Each experiment assessing mouse heart AC activities was performed with five WT and AC5KO mice respectively. FS stimulated AC activities of recombinant AC isoforms were determined using three independent Sf9-cell membrane preparations. Data represents the means ± S.D. of experiments performed in triplicates (A) or duplicates in (B–D). Not significant (ns) = p>0.05, **p<0.01, ***p<0.001.

Figure 3. Inhibition of mouse heart AC with putative AC5-selective inhibitors.

Concentration response curves of SQ22,536 (A), vidarabine (B) and NKY80 (C) on mouse heart AC from wild type (WT) vs. AC5 knockout mice (AC5KO). MANT-ITPγS served as a potent reference AC inhibitor (D). Maximal AC activities represent AC activity under stimulation with FS (100 µM) and GTPγS (10 µM) in the absence of inhibitor. Absolute activites in WT vs. AC5KO hearts (pmol cAMP · mg⁻¹ · min⁻¹, ± SD): 136.7±46.51 vs. 120.1±31.4; p = 0.64. Data are the means ± S.D. of experiments with cardiac membrane preparations from three WT or AC5KO mice respectively performed in duplicates.

Figure 4. Inhibition of recombinant ACs with putative AC5-selective inhibitors.

Comparison of concentration response curves of putative of AC5-selective inhibitors on recombinant AC1, 2 and 5. (A) SQ22,536, (B) vidarabine, (C) NKY80. MANT-ITPγS served as a potent reference AC inhibitor (D). Maximal AC activities represent AC activity under stimulation with forskolin (100 µM) and GTPγS (10 µM). Data are the means ± S.D. of experiments with three independent membrane preparations of Sf9-cells expressing AC1, 2 or 5 performed in duplicates.