Enzymes secreted onto epithelial surfaces play a vital role in innate mucosal defense, but are believed to be steadily removed from the surface by mechanical actions. Thus, the amount and availability of enzymes on the surface are thought to be maintained by secretion. In contrast to this paradigm, we show here that enzymes are retained at the apical surface of the airway epithelium by binding to surface-associated hyaluronan, providing an apical enzyme pool 'ready for use' and protected from ciliary clearance. We have studied lactoperoxidase, which prevents bacterial colonization of the airway, and kallikrein, which mediates allergic bronchoconstriction that limits the inhalation of noxious substances. Binding to hyaluronan inhibits kallikrein, which is needed only in certain situations, whereas lactoperoxidase, useful at all times, does not change its activity. Hyaluronan itself interacts withthe receptor for hyaluronic acid-mediated motility (RHAMM or CD168) that is expressed at the apex of ciliated airway epithelial cells. Functionally, hyaluronan binding to RHAMM stimulates ciliary beating. Thus, hyaluronan plays a previously unrecognized pivotal role in mucosal host defense by stimulating ciliary clearance of foreign material while simultaneously retaining enzymes important for homeostasis at the apical surface so that they cannot be removed by ciliary action.

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.

We have shown that an inhaled glucocorticosteroid (GS) causes alpha(1)-adrenergic antagonist-blockable, rapid, and transient bronchial vasoconstriction in healthy and asthmatic subjects. Steroids inhibit norepinephrine (NE) uptake by non-neuronal cells, thereby increasing NE concentration at alpha-adrenergic receptor sites. This could explain the GS-induced bronchial vasoconstriction. We therefore studied expression of the steroid-sensitive extraneuronal monoamine transporter (EMT) and steroid sensitivity of NE uptake in human bronchial artery and rabbit aorta (as a substitute for the limited supply of human bronchial artery). NE uptake was measured using a semiquantitative, sucrose-potassium phosphate-glyoxylic acid fluorescence method that we newly adapted for use in single cells. Both human bronchial arteries and rabbit aorta expressed messenger RNA for EMT, and steroids blocked NE uptake into freshly dissociated human bronchial arterial and rabbit aortic smooth-muscle cells (SMCs). In the latter, inhibition of NE uptake by steroids was not altered, either by a protein synthesis inhibitor (cycloheximide) or by a transcription inhibitor (actinomycin D), and corticosterone made membrane-impermeant by conjugation to bovine serum albumin inhibited NE uptake equipotently. These data show that NE uptake into bronchial arterial and rabbit aortic SMCs is sensitive to steroids, possibly mediated by EMT, and suggest a mechanism for GS-induced bronchial vasoconstriction.

Hyaluronic acid (hyaluronan, or HA) is secreted by submucosal glands, but its function in airway secretions other than influencing the rheology of mucus is not fully understood. HA is known to modulate cell behavior and to enhance sperm motility. Because sperm tails and cilia have the same microtubular structure, we studied the effect of HA on ciliary beat frequency (CBF) in vitro. CBF of cultured ovine airway epithelial cells was measured continuously by digital video microscopy. After removal of endogenous HA by hyaluronidase, cells were exposed to 50 to 100 microg/mL of HA at different times in culture. No change in CBF in response to HA was seen in cells cultured less than 7 days. After 7 days, however, 6 of 10 measured cells (from three different sheep) showed a transient CBF increase from a baseline of 6.4 +/- 0.3 Hz (mean +/- SE) to 7.4 +/- 0.4 Hz or 16% above baseline (p < 0.05). At these time points (but not before), cytochemical staining was positive for endogenous HA using a biotinylated HA-binding protein. These data suggest that HA can increase CBF of tracheal epithelial cells only late in culture when HA is able to bind to an unspecified cell surface structure. Because this binding has a physiological effect, we hypothesize that it is an HA-binding receptor, that is either transiently expressed late in culture or initially destroyed by the protease treatment for cell dispersion.

Ciliary beat frequency (CBF) is regulated, at least in part, by the cytoplasmic calcium concentration ([Ca(2+)](i)). Because Ca(2+) can stimulate nitric oxide (NO) production by nitric oxide synthase (NOS) and NO has been implicated in the regulation of CBF in some species, we examined whether NOS is present in cultured ovine ciliated epithelial cells and whether NO plays a role in the Ca(2+)-mediated muscarinic stimulation of CBF. Dissociated ovine tracheal epithelial cells were grown in culture for 2 to 14 days. Frequency from a single cilium was measured by on-line Fourier transform methods using video microscopy. [Ca(2+)](i) was determined with fura-2 using fluorescence ratio imaging from the same single cells. Ciliated cells contained NOS in culture as indicated by NADPH-diaphorase staining. Acetylcholine (ACh) increased CBF and [Ca(2+)](i) transiently as previously shown. Measurements with 2',7'-dichlorofluorescin diacetate indicated that reactive oxygen/nitrogen species were produced in these cells on ACh exposure. NOS inhibitors N(G)-nitro-L-arginine methyl ester (< or =10 mM), N(G)-nitro-L-arginine (< or =10 mM), and 7-nitro indazole (1 microM) were unable to block the CBF or [Ca(2+)](i) response to ACh. Furthermore, the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine (< or =1 mM) did not change CBF or [Ca(2+)](i). Above these concentrations, they both lead to a reversible decrease in CBF. The membrane-permeable cyclic guanosine monophosphate analogue 8-bromo-cyclic guanosine monophosphate had no effect on CBF, whereas 8-bromo-cyclic adenosine monophosphate stimulated CBF. Taken together, these results suggest that NO does not play a role in mediating the ACh-induced increase in CBF through [Ca(2+)](i). The role and targets for NO in ovine ciliated cells remains to be determined.