Comparing the Effects of Inhaled Pulmonary Vasodilators and Their Diluents on Airway Inflammation and Ciliary Function

Abstract

Background:

Nitric oxide (NO) and epoprostenol (EPO) are inhaled therapies for pulmonary hypertension. The most common diluents for EPO, glycine (GLY) and arginine (ARG), have a pH of 11-13. pH > 10 has been shown to cause cessation of ciliary beating, cell sloughing, and inhibition of airway drug transport. The effects of EPO-GLY and EPO-ARG on airway epithelia are unknown. NO has been reported to be conditionally pro- or anti-inflammatory. NO donor compounds have disparate effects upon inflammatory mediators in airway cells, but this has not been evaluated in response to NO gas. We hypothesized that EPO-GLY and EPO-ARG would increase inflammatory markers in airway epithelia and that NO exposure would not increase the production of inflammatory markers.

Methods:

Pseudostratified ciliated columnar epithelium (Figure 1) were exposed to NO (20 ppm), EPO-GLY (30 ng/mL), EPO-ARG (30 ng/mL), GLY, ARG, or phosphate-buffered saline (PBS, control). pH of each solution was measured immediately prior to exposure; PBS pH was 7.0, EPO-GLY and GLY pH was 12, EPO-ARG and ARG pH was 12.9. We harvested cell media and lysate for measurement of inflammatory markers. Ciliary beat was observed at each time interval using light microscopy. High-definition video images were recorded. Post-processing included noise reduction and point-feature matching to enhance visualization of cell movement. Ciliary cell clusters were directly observed and ciliary beat frequency (CBF) calculated for each experiment.

Results:

In all cells exposed to GLY, EPO-GLY, ARG, and EPO-ARG ciliary beat decreased immediately (p < 0.05, Figure 1) and ceased within five min of exposure. In control groups and NO group, ciliary beat remained throughout exposure. All cells exposed to GLY, EPO-GLY, ARG, and EPO-ARG died within 30 min of exposure. Lactate dehydrogenase (LDH), an established indicator of cell-stress/cell death, was evaluated via colorimetric assay in all of these groups. LDH was significantly elevated in GLY and EPO-GLY (Figure 1). LDH was not detectable in the ARG or EPO-ARG groups in the (Figure 1) ; to verify cell death, total RNA was evaluated in the mixture of culture media and cellular debris remaining after exposure. No RNA was detectable, indicating total cell death (Figure 1). LDH was no different between the cells exposed to NO and the control group (Figure 1).

Conclusions:

Apical exposure of EPO-GLY, EPO-ARG, GLY, or ARG causes ciliary beat cessation and cell death in NHBE cells.