Adenosine activation of A_2B receptor(s) is essential for stimulated epithelial ciliary motility and clearance

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A₁, A_2A, A_2B, or A₃). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A₁, A_2A, A_2B, or A₃, respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 μM) and/or procaterol (10 μM), in the presence or absence of 5=-(Nethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A₁, A_2A, A₃); 10 μM (A2B)], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 μM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 μM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ∼3 Hz. Likewise, CBF significantly increased ∼2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A1, A2A, and A3 knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A_2B knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A2B adenosine receptor activation, likely via a PKA-dependent pathway.