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A Major Functional Role for Phosphodiesterase 4D5 in Human Airway Smooth Muscle Cells

¹ Division of Therapeutics and Molecular Medicine, University of Nottingham, Queens Medical Centre, Nottingham; and ² Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, Leicester, United Kingdom

Correspondence and requests for reprints should be addressed to Ian P. Hall, MRCP, DM, FRCP, Division of Therapeutics and Molecular Medicine, D Floor, South Block, University Hospital, Nottingham NG7 2UH, UK. E-mail: ian.hall{at}nottingham.ac.uk

Relaxation of airway smooth muscle is dependent predominantly upon elevation of cell cAMP content. Although the processes involved in elevation of cell cAMP content are reasonably well established, the mechanisms governing subsequent control of cAMP turnover are less clear. Breakdown of cAMP is solely regulated by phosphodiesterase (PDE) isoenzymes. We have previously reported that PDE4 family members are likely to be important in this process, and that expression of PDE4D variants is actively regulated at the transcriptional level. Here, we demonstrate a key role for PDE4D5 in the control of β₂-adrenoceptor (β₂AR)–stimulated cAMP activity in human airway smooth muscle cells using splice variant–specific small interfering RNA knockdown. Furthermore, we show, using an Epac (exchange protein directly activated by cAMP)-based, cAMP-sensitive fluorescent probe, that these intracellular cAMP gradients are controlled both temporally and dynamically by PDE4D5. Elevation of cAMP within the cytoplasm after β₂AR stimulation is rapid and shows no distinct spatial compartmentalization in these cells. These data suggest that PDE4D5, despite being a minor component of the tissue PDE pool, is the key physiological regulator of β₂AR-induced cAMP turnover within human airway smooth muscle.

Key Words: β₂-adrenoceptor • cAMP • exchange protein directly activated by cAMP • fluorescence resonance energy transfer • small interfering RNA

CLINICAL RELEVANCE As the major relaxant pathway and target for asthma therapy, the β2-adrenoceptor pathway in airway smooth muscle is of significant clinical and scientific interest. By using novel techniques, we find that a specific phosphodiesterase isoform is a key regulator in this pathway.

 

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