Document Type
Article
Publication Date
4-12-2024
Abstract
Background and Purpose
In human airway smooth muscle (hASM) cells, not all receptors stimulating cAMP production elicit the same effects. This can only be explained if cAMP movement throughout the cell is restricted, yet the mechanisms involved are not fully understood. Phosphodiesterases (PDEs) contribute to compartmentation of many cAMP responses, but PDE activity alone is predicted to be insufficient if cAMP is otherwise freely diffusible. We tested the hypothesis that buffering of cAMP by protein kinase A (PKA) associated with A kinase anchoring proteins (AKAPs) slows cAMP diffusion and that this contributes to receptor-mediated, compartmentalized responses.
Experimental Approach
Raster image correlation spectroscopy (RICS) was used to measure intracellular cAMP diffusion coefficients and evaluate the contribution of PKA–AKAP interactions. Western blotting and immunocytochemistry were used to identify the AKAPs involved. RNA interference was used to down-regulate AKAP expression and determine its effects on cAMP diffusion. Compartmentalized cAMP responses were measured using fluorescence resonance energy transfer (FRET) based biosensors.
Key Results
Cyclic AMP movement was significantly slower than that of free-diffusion in hASM cells, and disrupting PKA–AKAP interactions significantly increased the diffusion coefficient. PKA associated with the outer mitochondrial membrane appears to play a prominent role in this effect. Consistent with this idea, knocking down expression of D-AKAP2, the primary mitochondrial AKAP, increased cAMP diffusion and disrupted compartmentation of receptor-mediated responses.
Conclusion and Implications
Our results confirm that AKAP-anchored PKA contributes to the buffering of cAMP and is consequential in the compartmentation of cAMP responses in hASM cells.
Recommended Citation
Sherpa, R. T., Moshal, K. S., Agarwal, S. R., Ostrom, R. S., & Harvey, R. D. (2024). Role of protein kinase A and A kinase anchoring proteins in buffering and compartmentation of cAMP signalling in human airway smooth muscle cells. British Journal of Pharmacology, 181(15), 2622–2635. https://doi.org/10.1111/bph.16357
Copyright
British Pharmacological Society
Included in
Amino Acids, Peptides, and Proteins Commons, Cell Biology Commons, Other Pharmacy and Pharmaceutical Sciences Commons
Comments
This is the accepted version of the article, which has been published in final form at https://doi.org/10.1111/bph.16357. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.