Cytoplasmic cAMP concentrations in intact cardiac myocytes.
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 beta(1)-adrenergic and M(2) 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 approximately 1.2 muM, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both beta(1) and M(2) 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 beta(1) and M(2) 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.
Dept. of Physiology and Biophysics, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106-4970, USA.
Radu V
RV
Iancu
Gopalakrishnan
G
Ramamurthy
Sunita
S
Warrier
Viacheslav O
VO
Nikolaev
Martin J
MJ
Lohse
Stephen W
SW
Jones
Robert D
RD
Harvey
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000109:Acetylcholine / Q000494:pharmacology; D058671:Adrenergic beta-1 Receptor Antagonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D015374:Biosensing Techniques; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003198:Computer Simulation; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D006168:Guinea Pigs; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D043585:Receptor, Muscarinic M2 / Q000819:agonists; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D058671:Adrenergic beta-1 Receptor Antagonists; D000318:Adrenergic beta-Agonists; D020662:Guanine Nucleotide Exchange Factors; D008164:Luminescent Proteins; D043585:Receptor, Muscarinic M2; D000242:Cyclic AMP; D000109:Acetylcholine; D015056:1-Methyl-3-isobutylxanthine