Previously, we have shown that ethanol (EtOH) stimulates a rapid increase in the ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBECs) via the activation of PKA. We have also shown that inhibitors of nitric oxide synthase block EtOH-stimulated increases in CBF. We hypothesize that EtOH acutely stimulates CBF via the activation of both PKA and PKG pathways. Using chemiluminescence detection of nitric oxide (NO), we directly measured increases in NO production in BBECs treated with 100 mmol/L of EtOH beginning at 25 minutes. Pretreatment of BBECs with guanylyl cyclase inhibitors, ODQ or LY83583, resulted in the inhibition of EtOH-stimulated CBF. Low concentrations (1 nmol/L) of cyclic nucleotide analogues do not stimulate CBF increases. However, a combination of both 1 nmol/L of 8Br-cAMP and 8Br-cGMP stimulates a significant increase over baseline CBF. This effect could be blocked by pretreating BBECs with inhibitors of either PKA or PKG. Very high concentrations of either 8Br-cAMP or 8Br-cGMP (> or =100 micromol/L) were required to cross-activate both PKA and PKG. This suggests that cross-activation of PKA by cGMP is not occurring at the concentrations (1 nmol/L) capable of stimulating CBF. 8-pCPT-cGMPS, an antagonist analogue to cGMP, blocked EtOH-stimulated PKA activity increases. These data support that EtOH-stimulated increases in CBF require the dual activation of both PKA (via cAMP) and PKG (via NO).

Previously, we have shown that the ATPase-dependent motion of cilia in bovine bronchial epithelial cells (BBEC) can be regulated through the cyclic nucleotides, cAMP via the cAMP-dependent protein kinase (PKA) and cGMP via the cGMP-dependent protein kinase (PKG). Both cyclic nucleotides cause an increase in cilia beat frequency (CBF). We hypothesized that cAMP and cGMP may act directly at the level of the ciliary axoneme in BBEC. To examine this, we employed a novel cell-free system utilizing detergent-extracted axonemes. Axoneme movement was whole-field analyzed digitally with the Sisson-Ammons video analysis system. A suspension of extracted axonemes remains motionless until the addition of 1 mM ATP that establishes a baseline CBF similar to that seen when analyzing intact ciliated BBEC. Adding 10 microM cAMP or 10 microM cGMP increases CBF beyond the established ATP baseline. However, the cyclic nucleotides did not stimulate CBF in the absence of ATP. Therefore, the combination of cAMP and cGMP augments ATP-driven CBF increases at the level of isolated axoneme.

Mucociliary clearance is a critical host defense that protects the lung. The mechanisms by which mucociliary function is altered by inflammation are poorly defined. Chronic exposure to cigarette smoke decreases ciliary beating and interferes with proper airway clearance. Bronchoalveolar lavage (BAL) fluid from smokers and ex-smokers has increased amounts of IL-8, which has played a critical role in airway inflammation. We hypothesized that IL-8 might interfere with stimulated ciliary beating in airway epithelium. To test this hypothesis, we stimulated bovine ciliated bronchial epithelial cells (BBECs) with a known activator of ciliary beat frequency (CBF), isoproterenol (ISO; 100 microM), in the presence or absence of IL-8 (100 pg/mL). We measured CBF digitally using the Sisson-Ammons Video Analysis (SAVA) system. CBF increased in untreated cells exposed to ISO (approximately 3 Hz) over baseline. In contrast, cells pre-incubated with IL-8 failed to respond to ISO. Pretreatment with IL-8 also blocked ISO-stimulated cAMP-dependent kinase (PKA) activation, which is known to control ISO-stimulated CBF. In addition, IL-8 pretreated cells revealed a marked decrease in PKA activity when cells were stimulated with forskolin (FSK; 10 microM). Cells were assayed specifically for cAMP-phosphodiesterase (PDE) activity. ISO-stimulated cells demonstrated an increase in PDE activity as compared to control. Pretreatment with IL-8 had no effect on ISO-stimulated PDE activity. Collectively, these data suggest that IL-8 appears to mediate its effect at the level of adenylyl cyclase. It is also possible that IL-8 may not only act as a chemotactic agent, but also as a potential autocrine/paracrine inhibitor of PKA-mediated stimulation of ciliary motility. In conclusion, IL-8 inhibits beta-agonist dependent ciliostimulation and such inhibition of stimulated ciliary activity may contribute to the impaired mucociliary clearance seen in airway diseases. Furthermore, since IL-8 levels are increased in the airway of cigarette smokers, it is likely they may be more resistant to the cilio and muco-ciliostimulating effects of beta-agonists.

No abstract

Relaxin is an insulin-like serum protein secreted during pregnancy and found in many tissues, including the lung. Relaxin is reported to stimulate epithelial cell proliferation, but the effects of relaxin on airway epithelium are unknown. We tested the hypothesis that relaxin would stimulate the increased migration of bronchial epithelial cells (BEC) in response to wounding. Using monolayers of BEC in a wound-healing model, relaxin augmented wound closure with maximal closure occurring at 12.hr (1 μM). Unlike cytokines, relaxin did not stimulate increased BEC interleukin-8 (IL-8) release. Relaxin caused a significant stimulation of ciliary beat frequency (CBF) in BEC. Because protein kinase (PKA) activation increases CBF and relaxin can elevate intracellular cAMP levels, we measured PKA activity in BEC treated with relaxin. Relaxin increased PKA activity 3–4 fold by approximately 4 hr, with a return to baseline levels by 8–10 hr. Relaxin-stimulated PKA activity differs temporally from the rapid (1 hr) β-adrenergic activation of PKA in BEC. These data suggest that relaxin augments epithelial repair by increasing airway cell migration and CBF via PKA-dependent mechanisms.