Extracellular metabolism of 3′,5′-cyclic AMP as a source of interstitial adenosine in the rat airways

Abstract

In the respiratory tract, intracellular 3′,5′-cAMP mediates smooth muscle relaxation triggered by the β2-adrenoceptor/Gs protein/adenylyl cyclase axis. More recently, we have shown that β2-adrenoceptor agonists also increase extracellular 3′,5′-cAMP levels in isolated rat trachea, which leads to contraction of airway smooth muscle. In many other tissues, extracellular 3′,5′-cAMP is metabolized by ectoenzymes to extracellular adenosine, a catabolic pathway that has never been addressed in airways. In order to evaluate the possible extracellular degradation of 3′,5′-cAMP into 5′-AMP and adenosine in the airways, isolated rat tracheas were incubated with exogenous 3′,5′-cAMP and the amount of 5′-AMP, adenosine and inosine (adenosine metabolite) produced was evaluated using ultraperformance liquid chromatography–tandem mass spectrometry. Incubation of tracheal tissue with 3′,5′-cAMP induced a time- and concentration-dependent increase in 5′-AMP, adenosine and inosine in the medium. Importantly, IBMX (non-selective phosphodiesterase (PDE) inhibitor) and DPSPX (selective ecto-PDE inhibitor) reduced the extracellular conversion of 3′,5′-cAMP to 5′-AMP. In addition, incubation of 3′,5′-cAMP in the presence of AMPCP (inhibitor of ecto-5′-nucleotidase) increased extracellular levels of 5′-AMP while drastically reducing extracellular levels of adenosine and inosine. These results indicate that airways express an extracellular enzymatic system (ecto-phosphodiesterase, ecto-5′-nucleotidase and adenosine deaminase) that sequentially converts 3′,5′-cAMP into 5′-AMP, adenosine and inosine. The observation that extracellular 3′,5′-cAMP is a source of interstitial adenosine supports the idea that the extrusion and extracellular metabolism of 3′,5′-cAMP has a role in respiratory physiology and pathophysiology.