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RECEPTORS AND SIGNAL TRANSDUCTION

Cytoplasmic cAMP concentrations in intact cardiac myocytes

¹Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio and ²Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany

Submitted 25 January 2008 ; accepted in final form 7 June 2008

In cardiac myocytes there is evidence that activation of some receptors can regulate protein kinase A (PKA)-dependent responses by stimulating cAMP production that is limited to discrete intracellular domains. We previously developed a computational model of compartmentalized cAMP signaling to investigate the feasibility of this idea. The model was able to reproduce experimental results demonstrating that both β₁-adrenergic and M₂ muscarinic receptor-mediated cAMP changes occur in microdomains associated with PKA signaling. However, the model also suggested that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the present study we tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transfer-based biosensor constructed from the type 2 exchange protein activated by cAMP (Epac2-camps). It was determined that in adult ventricular myocytes the basal cAMP concentration detected by the probe is 1.2 µM, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both β₁ and M₂ receptor activation. Modeling suggests that responses detected by Epac2-camps mainly reflect what is happening in a bulk cytosolic compartment with little contribution from microdomains where PKA signaling occurs. These results support the conclusion that even though β₁ and M₂ receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell. This allows receptor stimulation to regulate cAMP activity over concentration ranges appropriate for modulating both higher (e.g., PKA) and lower affinity (e.g., Epac) effectors.

β-adrenergic receptor signaling; muscarinic receptor signaling; live cell imaging; fluorescence resonance energy transfer; biosensors

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