Stimulation of ovine airway epithelial cells with 10 μm ATP for 1 min at 25 °C transiently increased both cytoplasmic calcium (fura‐2 epifluorescence microscopy) and ciliary beat frequency (CBF; differential interference contrast microscopy) with a similar time course. Identical purinergic stimulation of human airway epithelial cells at 25 or 35 °C, however, lead to an increase in CBF that outlasted the calcium transient at least 20 min. While a nitric oxide synthase inhibitor had no effect, pre‐treatment of human cells with inhibitors of cAMP‐dependent kinase (PKA), 10 μm myristoylated PKA‐inhibitory peptide and 1 μm KT‐5720, as well as an inhibitor of adenylyl cyclase, 1 mm SQ22536, blocked the prolonged, but not calcium‐coupled CBF increase. Addition of PKA inhibitors after purinergic stimulation only partially reduced CBF from its elevated plateau. Prolonged CBF increases did not depend on adenosine production as 10 μm UTP had an effect similar to ATP and 8‐sulphophenyl‐theophylline did not block them. After increasing human CBF in a PKA‐dependent manner to a stable plateau with forskolin (10 μm), ATP caused only a transient, calcium‐coupled CBF increase. Calcium transients were necessary for both short‐term and prolonged CBF changes as ATP failed to produce CBF increases after emptying calcium stores with 1 μm thapsigargin. These data suggest that in human, but not ovine airway epithelial cells, ATP‐induced calcium transients activate a signalling cascade including adenylyl cyclase and PKA. The resulting prolonged CBF stimulation does not rely only on PKA activity, suggesting that the decay of CBF is influenced by ciliary phosphatase activity.

Frohock, Jeffrey I., Corrine Wijkstrom-Frei, and Matthias Salathe. Effects of albuterol enantiomers on ciliary beat frequency in ovine tracheal epithelial cells. J Appl Physiol 92: 2396-2402, 2002. First published February 8, 2002 10.1152/japplphysiol.00755.2001.-␤ 2-Adrenergic agonists stimulate ciliary beat frequency (CBF), an integral part of mucociliary clearance. To evaluate the differential effects of albuterol enantiomers and their racemic mixture on ciliary function, CBF and intracellular calcium were measured at room temperature from single ovine airway epithelial cells with use of digital videomicroscopy. Baseline CBF was 7.2 Ϯ 0.2 (SE) Hz (n ϭ 80 measurements). R-albuterol (10 M to 1 mM) stimulated CBF in a dose-dependent manner to maximally 24.4 Ϯ 5.4% above baseline. Racemic albuterol stimulated CBF to maximally 12.8 Ϯ 3.6% above baseline, a significantly lower increase compared with R-albuterol alone, despite identical R-enantiomer amounts in both groups. Simultaneous recordings of intracellular calcium concentration and CBF from single cells indicated that the CBF increase in response to R-albuterol was mediated through ␤-receptors and stimulation of protein kinase A, in a calcium-dependent and -independent fashion. S-albuterol had a negligible effect on CBF and did not change intracellular calcium. Together, these results suggest that R-albuterol is more efficacious than racemic albuterol in stimulating CBF. Thus S-albuterol may interfere with the ability of R-albuterol to increase CBF. airway; calcium; beta-agonists; signaling ␤-AGONISTS HAVE BEEN SHOWN to stimulate the mucociliary transport rate (4, 5, 22), and thus they are used clinically to improve mucociliary clearance (10, 14, 15, 18), a major airway host defense mechanism. Ciliary beating is an integral part of the mucociliary transport apparatus, and it has been well established that ␤ 2 -adrenergic agonists stimulate ciliary beat frequency (CBF) in a variety of airway epithelial cells (11,27,33,34). Therefore, the increase in CBF is believed to be the main contributor to higher mucociliary transport rates seen after ␤ 2 -agonist administration. Albuterol, the most commonly used ␤ 2 -agonist in a clinical setting, possesses two stereoisomers due to an asymmetric carbon adjacent to the aromatic ring. The R-enantiomer binds to the ␤ 2 -adrenoceptor, leading to cAMP production, whereas the S-enantiomer has an over 100-fold lower affinity to the receptor. In addition, some recent data indicate that the S-enantiomer may have properties not usually associated with ␤ 2 -agonists, such as smooth muscle contraction through intracellular calcium concentration ([Ca 2ϩ ] i ) elevation (13) and other side effects (for review, see Ref. 8). Usually, racemic albuterol mixtures are used in a clinical setting. These mixtures bring with them the potential for divergent responses of different cellular functions for each of the enantiomers. We therefore conducted a study to compare the effects of the two enantiomers as well as the racemic mixture of albut...