We hypothesized that mechanical stimulation with VCPT alters NOx metabolism, which stimulates mucociliary clearance. To test this hypothesis, we measured nasal saccharin transit time and exhaled breath condensate NOx concentrations in healthy normal adult subjects and adults with stable cystic fibrosis before and after VCPT. In this model, we use NSTT as a surrogate of mucociliary clearance.
To our knowledge, this is the first study to associate changes in airway mucociliary clearance with changes in exhaled NOx in human subjects. Our results indicate that VCPT applied to both normal and stable CF subjects significantly increased nasal mucociliary clearance. A single VCPT treatment increased nasal clearance in 18 out of 20 (90%) subjects tested. In aggregate, we found that a vest treatment increased nasal clearance by approximately 35% in both normal and stable CF subjects. Although our study groups were small in number, the effect of percussion on nasal clearance was quite striking with both biologically relevant and statistically significant changes in NSTT.
We originally designed our study to address the mechanism of enhanced clearance by VCPT. We chose to explore the role of NO that might have in enhanced clearance based on in vitro
studies of cilia that date back to 1993 demonstrating that increases of CBF by multiple stimuli require NO production [9
]. Many investigators have corroborated these findings in several different systems [10
]. In this context, we originally hypothesized that VCPT-enhanced clearance would increase exhaled NOx
concentrations. Our results are counterintuitive and exactly the opposite of our expectations regarding the effect of mechanical stimulation via VCPT on exhaled NOx
. In every case, VCPT is associated with dramatic reduction in NOx
concentrations. We speculate that mechanical shaking with the VCPT device enhances mucus clearance by stimulating calcium release from airway epithelial cells, resulting in calmodulin activation of eNOS with resultant production of NO in the airway epithelial cell. If NO production increases with mechanical stimulation, then we have to reconcile our observation that EBC NOx
decreased following VCPT. An alternative explanation for the difference between our findings in this study and those published with NOx
measurements performed in ciliated cells in vitro
is that our study focused on the metabolites of NO that were exhaled into the EBC while the in vitro
studies measured the concentration of NOx
present in the media. It is likely that “off-gassing” of NO is the basis of NOx
in EBC [13
], whereas, in vitro
studies focus on dissolved and non-volatile metabolites of NO present in the media.
The decrease in exhaled NOx may be due to increased utilization of NO within the ciliated epithelial cell. In this scenario, the enhanced binding of NO to guanylyl cyclase would provide the signal for elevated cGMP, activation of PKG, and stimulated cilia beating. It is also possible that the increased mucus flow from more distal airways creates a greater barrier to NO diffusion from the airway epithelium during percussion therapy than that present in the resting state. Alternatively, mechanical stimulation through percussion may alter the metabolism of NO through an unknown mechanism.
The literature has clearly established that mechanical stress alone can stimulate cilia beating in both in vitro
] and in vivo
] models. The mechanism of such mechanical stimulation may involve an increase in cytoplasmic calcium resulting from either extracellular calcium influx or inositol-1′,4′,5-triphosphate-mediated internal calcium release [16
]. The calcium pathway is likely independent of the NO-cGMP-PKG stimulation of cilia beating in an ovine model [18
], but others have shown a cGMP-dependent component to calcium-stimulated cilia beat in rabbit airway [18
]. Using human nasal ciliated cells, Alberty et al. demonstrated that mechanical stimulation of cilia could take place in the presence of an NO inhibitor, suggesting that the mechanical induced increases in CBF were independent of endogenous NO production [19
]. Extrapolating from these observations, mechanical stimulation of mucociliary transport in response to VCPT in our study could be independent of NO. However, the significant decrease
in EBC NOx
levels in response to mechanical stimulation in both study groups suggests a distinct mechanical impact on NO metabolism and release from the lung.
An obvious question that arises from our findings is as follows: how does chest percussion alter nasal clearance? The vest percussion apparatus is applied to the chest with the intent of oscillating the lower airways. We observed, however, that the head and neck clearly oscillated in sync with the chest during VCPT. Furthermore, increased nasal discharge typically occurs during percussion treatment consistent with increased nasal clearance (unpublished results). From these observations we surmise that the nasal cavity, in addition to the lower airways, is vigorously shaken during VCPT and results in stimulated nasal mucociliary clearance.
Another important question is as follows: does nasal clearance correlate with lung airway clearance? Because VCPT is typically directed at patients with lung airway clearance disorders such as bronchiectasis, we can only cautiously extrapolate our findings with nasal clearance to lung clearance. We did not directly measure lung clearance due to the cost and exposure to radionuclides involved in such studies. The NSTT is, however, a direct functional measure of mucociliary clearance of the upper airway. Because the vest is thought to enhance lung airway clearance, it is highly likely that both nasal and lung clearance are stimulated by VCPT.
We also note that EBC NOx
tended to be higher
in CF subjects than in control subjects (), which differs from previous studies [20
]. While we acknowledge that the NOx
difference between normal and CF subjects was not statistically different, the best explanation for this disparity is that the earlier study measured only direct exhaled NO, which does not include nitric oxide breakdown products. In contrast, exhaled breath condensate contains the stable conversion compounds of NO, nitrites, and nitrates. Our findings measuring NOx
concentrations in EBC contrast with other studies that measured only the exhaled free radical NO.