Abstract 15858: Phosphodiesterase Isoforms 2–4 Modulate cAMP Signalling to Phospholemman During β-adrenergic Stimulation
There is now considerable evidence showing that phosphodiesterases (PDEs) contribute to the intracellular compartmentalization of cAMP in cardiac myocytes by preventing its free diffusion within the cytosol. We hypothesised that PDEs play a role in modulating cAMP signalling to phospholemman (PLM), the accessory protein to the Na/K ATPase, during β-adrenergic (β-AR) stimulation. Freshly-isolated adult rat ventricular myocytes were voltage-clamped at 35oC using the perforated-patch technique to measure whole-cell Na/K pump current (Ip). Cells were challenged with a submaximal concentration of isoproterenol (ISO: 300nM) alone or were pretreated with selective inhibitors of PDE isoforms; either 100µM IBMX (broad-spectrum inhibitor), or 10µM rolipram (PDE4), cilostamide (PDE3) or EHNA (PDE2) followed by ISO (300nM) in the maintained presence of the inhibitor. Low-dose ISO alone significantly increased Ip (14±1%, p<0.001, n=12). In the absence of ISO, Ip was significantly augmented on addition of IBMX (20±3%, p<0.001, n=9), rolipram (19±2%, p<0.001, n=10), cilostamide (9±2%, p<0.05, n=10) or EHNA (10±2%, p<0.01, n=12). Subsequent addition of 300nM ISO resulted in a significantly greater increase in Ip compared to that induced by ISO alone (IBMX: 23±5%, p<0.001, n=9; rolipram: 21±4%, p<0.001, n=10; cilostamide: 9±5%, p<0.05, n=10; EHNA: 9±5%, p<0.05, n=12). Immunoprecipitations show PLM co-associates with PDE4D and Western blots show rolipram significantly increases both basal and ISO stimulated phosphorylation of PLM Ser 68. Forskolin-induced Na/K pump activation was also blocked by including Ht31 (a competitive inhibitor of AKAP binding) in the pipette implicating AKAP binding in the local control of PLM phosphorylation. Our study demonstrates a determinant contribution of PDE4 activity (and to a lesser extent, PDE2 and PDE3) to cAMP signalling to PLM (and therefore Na/K ATPase activity) during β-AR stimulation. This suggests there is functional cAMP compartmentalization in the vicinity of PLM, which may have important implications for the regulation of Na/K ATPase function.
- © 2010 by American Heart Association, Inc.