β1‐ and β2‐adrenergic receptors (βARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by β1AR but not β2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that β1AR forms a signaling complex with a cAMP‐specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β‐arrestin/PDE complex reported previously. The β1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the β2AR is a prerequisite for the recruitment of a complex consisting of β‐arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of β1‐ and β2‐adrenoceptor signaling.

Cyclic nucleotide PDE4 (phosphodiesterase 4) inhibitors are being developed as potent anti-inflammatory drugs for use in chronic lung diseases, but the complexity of the PDE4 family has hampered this process. The four genes comprising the PDE4 family, PDE4A, PDE4B, PDE4C and PDE4D, are all expressed as multiple splice variants. The most widely used criterion to identify PDE4 variants expressed endogenously is their migration on SDS/PAGE. However, when a PDE4D3-selective antibody was used for immunoprecipitation, the pattern of expression obtained did not confirm the expression predicted by SDS/PAGE. This observation, together with the recent discovery of additional PDE4D transcripts, prompted us to re-evaluate the pattern of expression of these variants. The nine rat PDE4D splice variants, PDE4D1 to PDE4D9, were cloned, their electrophoretic properties compared, and their in vivo mRNA and protein levels determined. Using this approach, we found that the pattern of distribution of the PDE4D splicing variants is more complex than previously reported. Multiple variants co-migrate in single immunoreactive bands, and variant-selective antibodies were necessary to discriminate between splice variants. Tissues that were thought to express only PDE4D3, express three closely related proteins, with PDE4D8 and PDE4D9 as the predominantly expressed forms. In addition, activation of cAMP signalling produces phosphorylation and activation of variants other than PDE4D3, and expression of PDE4D mRNA does not always correlate with the pattern of protein expression. As PDE4 inhibitors have different affinities for distinct PDE4D splicing variants, our results indicate that a better definition of the pattern of PDE4 expression is required for target validation.

Steady-state activation of cardiac β-adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer–based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca ²⁺ channel current ( I _Ca,L ) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. I _Ca,L amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for ≈5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 μmol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 μmol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-l-methylxanthine (300 μmol/L), cAMP signals and I _Ca,L declined with a time constant >10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to ≈50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct β-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4.

(2013). PDE4 as a target for cognition enhancement. Expert Opinion on Therapeutic Targets: Vol. 17, No. 9, pp. 1011-1027.