Profiling of cAMP and cGMP phosphodiesterases in isolated ventricular cardiomyocytes from human hearts: comparison with rat and guinea pig.

AIMS: Phosphodiesterases (PDEs) are key enzymes controlling cAMP and cGMP levels and spatial distribution within cardiomyocytes. Despite the clinical importance of several classes of PDE inhibitor there has not been a complete characterization of the PDE profile within the human cardiomyocyte, and no attempt to assess which species might best be used to model this for drug evaluation in heart disease. MAIN METHODS: Ventricular cardiomyocytes were isolated from failing human hearts of patients with various etiologies of disease, and from rat and guinea pig hearts. Expression of PDE isoforms was determined using RT-PCR. cAMP- and cGMP-PDE hydrolytic activity was determined by scintillation proximity assay, before and after treatment with PDE inhibitors for PDEs 1, 2, 3, 4, 5 and 7. Functional effects of cAMP PDEi were determined on the contraction of single human, rat and guinea pig cardiomyocytes. KEY FINDINGS: The presence and activity of PDE5 were confirmed in ventricular cardiomyocytes from failing and hypertrophied human heart, as well as PDE3, with ventricle-specific results for PDE4 and a surprisingly large contribution from PDE1 for hydrolysis of both cAMP and cGMP. The total PDE activity of human cardiomyocytes, and the profile of inhibition by PDE1, 3, 4, and 5 inhibitors, was modelled well in guinea pig but not rat cardiomyocytes. SIGNIFICANCE: Our results provide the first full characterisation of human cardiomyocyte PDE isoforms, and suggest that guinea pig myocytes provide a better model than rat for PDE levels and activity.

Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat.

BACKGROUND AND PURPOSE: The P2X7 receptor is implicated in inflammation and pain and is therefore a potential target for therapeutic intervention. Here, the development of a native tissue radioligand binding, localization and ex vivo occupancy assay for centrally penetrant P2X7 receptor antagonists is described. EXPERIMENTAL APPROACH: Autoradiography studies using the P2X7 antagonist radioligand [³H]-A-804598 were carried out in rat brain and spinal cord. Subsequent in vitro binding and ex vivo occupancy assays were performed using rat cortex homogenate. KEY RESULTS: P2X7 expression was shown to be widespread throughout the rat brain, and in the grey matter of the spinal cord. In binding assays in rat cortex homogenate, ∼60% specific binding was achieved at equilibrium. In kinetic binding assays, k(on) and k(off) values of 0.0021·min⁻¹·nM⁻¹ and 0.0070·min⁻¹ were determined, and the K(d) derived from kinetic measurements was consistent with that derived from saturation analysis. Novel P2X7 antagonists inhibited the binding of [³H]-A-804598 to rat cortex P2X7 receptors with K(i) values of <40 nM. In an ex vivo occupancy assay, a P2X7 antagonist dosed orally to rats caused a concentration-dependent inhibition of the specific binding of [³H]-A-804598 to rat cortex. CONCLUSIONS AND IMPLICATIONS: The present study describes the development of an assay that allows localization of P2X7 receptors, the measurement of the binding affinity of P2X7 receptor antagonists in native tissue, and provides a means of determining central P2X7 receptor occupancy. These assays could form an important part of a P2X7 drug discovery programme.