Cyclic Nucleotide Phosphodiesterase in Human Bronchial Epithelial Cells: Characterization of Isoenzymes and Functional Effects of PDE Inhibitors

Abstract

Cyclic AMP (adenosine 3′:5′-cyclic monophosphate, cAMP) is an intracellular second messenger that mediates the actions of endogenous hormones and neurotransmitters and also of drugs such as β-adrenoceptor agonists. The presence of functional β-adrenoceptors on human airway epithelial cells has been demonstrated but the expression of the cAMP-metabolizing enzyme, cyclic nucleotide phosphodiesterase (PDE) in these cells has not been studied. We investigated the profile of activity of the different PDE isoenzymes in lysates of a pulmonary epithelial cell line, A549, and of human bronchial epithelial (HBE) cells grown in primary culture. The effects of non-selective and isoenzyme-selective PDE inhibitors on β-agonist-induced elevations in intracellular cAMP concentrations and the production of interleukin (IL) 8 and prostaglandin (PG) E2was also investigated. A549 cells expressed a high level of PDE4, lower levels of PDE1 and PDE3, and minor PDE5 activity. Primary HBE cultures expressed PDE4 and PDE1 activity at approximately equal levels with small additional PDE3 and PDE5 activities. The total PDE activity of the HBE cells was approximately nine-fold lower than that of A549 cells. The β-adrenoceptor agonist salbutamol, caused a slow, concentration-dependent increase in intracellular cAMP levels in HBE cells which was not affected by a non-selective PDE inhibitor, IBMX (100 μm), or by a selective PDE4 inhibitor, rolipram (100 μm). Zardaverine, a dual-selective PDE3/PDE4 inhibitor, had no effect on cAMP levels at 10 μmbut did cause a significant enhancement of salbutamol-induced elevations at 100 μm(150±36 pmol/105cells at 10 μmsalbutamol vs. 64±25 pmol/105cells in the absence of zardaverine; n=3,P<0.01). Neither basal nor tumour necrosis factor α (10 ng/ml)-induced IL8 secretion was affected by salbutamol (10 μm) in the absence or presence of IBMX (100 μm). Salbutamol (10 μm), alone or in the presence of IBMX (100 μm) or rolipram (100 μm), also failed to affect basal or bradykinin (1 μm)-induced PGE2release. Zardaverine (100 μm) caused a significant increase in basal PGE2release but this was not enhanced in the presence of salbutamol (10 μm) and was not related to changes in cAMP levels. We conclude that HBE cells express a low total PDE activity, made up predominantly of PDE1 and PDE4 isoenzymes, and that intracellular cAMP levels in HBE cells are not related to the production of IL8 or PGE2.