Proton secretion in freshly excised sinonasal mucosa from asthma and sinusitis patients

Abstract

Background: Proton (H+) secretion and the HVCN1 H+ channel are part of the innate host defense mechanism of the airways. The objective of this study was to determine H+ secretion in asthmatic and nonasthmatic patients with chronic rhinosinusitis (CRS) in freshly excised human sinonasal tissue.

Methods: Nasal or sinus mucosa from subjects with three different conditions (normal, CRS, and CRS with asthma) was harvested during sinus surgery. The equilibrium pH and the rate of H+ secretion were measured in an Ussing chamber using the pH-stat titration technique.

Results: Nasal epithelia isolated from subjects with CRS and asthma had a mucosal equilibrium pH = 6.95 (n = 5), which was significantly lower than in normal subjects (7.35 +/- 0.21; n = 5) or from subjects with CRS without asthma (7.33 +/- 0.15 In = 5). Nasal epithelia from CRS with asthma (n = 5) secreted H+ at a rate of 135 +/- 46 nmol x min(-1) x cm(-2). This rate was significantly higher compared with normal (73 +/- 39 nmol x min(-1) x cm(-2); n = 8) or CRS without asthma (51 +/- 28 nmol x min(-1) x cm(-2); n = 7). Mucosal addition of the HVCN1 blocker ZnCl2 blocked H+ secretion by 70% in normal, 53% in CRS without asthma, and by 51% in CRS with asthma. In contrast, measures in sinus tissues were unaffected by the disease condition.

Conclusion: Freshly excised human nasal and sinus epithelia secrete acid. Nasal (but not sinus) tissues from asthmatic CRS patients showed lower mucosal pH values and higher rates of H+ secretion than CRS and normal subjects. The increased acid secretion might contribute to epithelial injury in CRS patients with asthma.

Figure 1

Schematic drawing of pH stat titration method in an Ussing chamber. A thin layer of epithelial tissue, dissected from the surgical specimen, was bathed serosally with HEPES-buffered solution and mucosally with buffer-free solution (5 ml each). Solutions were gassed with oxygen (serosal) and nitrogen (mucosal). The pH of the mucosal solution was continuously measured, maintained, and recorded by a continuous pH stat titration apparatus (TitraLab 856, Radiometer Analytical SAS, Lyon, France).

Figure 2

Distribution of equilibrium pH of freshly excised sinonasal epithelia. Individual equilibrium pH values are plotted and average values are shown as horizontal bars. Nasal epithelia from CRS with asthma had significantly lower equilibrium pH of 6.95, compared to nasal epithelia from normal and CRS without asthma (p = 0.037). Sinus epithelia had a tendency for more alkaline equilibrium pH values compared to nasal epithelia in both groups of CRS with and without asthma (p > 0.05). *: denotes a significant difference (p < 0.05). Solid line: mean. ●: Nasal epithelia. ○: Sinus epithelia.

Figure 3

Proton secretion across freshly excised human nasal epithelia. Rates of proton secretions were plotted from individual experiments and average values are shown as horizontal bars. At a mucosal pH of 8.0, all tested epithelia acidified the mucosal medium. Nasal epithelia from CRS with asthma secreted protons at an average rate of 135.1 nmol·min-1·cm-2. This rate was significantly higher compared to normal and CRS without asthma (p = 0.005). Nasal epithelia from CRS without asthma secreted less protons than normal controls (p > 0.05). Solid line: mean. *: denotes a significant difference (p < 0.05).

Figure 4

Inhibition of proton secretion by using the proton channel inhibitor ZnCl₂. Rates of proton secretions were plotted from individual experiments and average values are shown as horizontal bars. Mucosal addition of ZnCl₂ blocked 71.0% (normal), 52.6%, (CRS without asthma) and 50.8% (CRS with asthma) indicating a role of mucosal Zn²⁺-sensitive proton channels (HVCN1) in proton secretion by these tissues. Magnitude of ZnCl₂ sensitive proton secretion was higher in normal nasal epithelia, compared to nasal epithelia from CRS with and without asthma (p < 0.05). Solid line: mean.

Figure 5

Model of acid release in the mucosal membrane of airway surface epithelial cells. In this model, a Duox-based NADPH oxidase releases intracellular H⁺ from NADPH and acidifies intracellular pH. Intracellular protons exit across a Zn²⁺-sensitive proton channel (HVCN1). In addition, airway cells release acid by way of a bafilomycin-sensitive H⁺ ATPase and an ouabain-sensitive K⁺/H⁺ ATPase. The source of H⁺ for the H⁺ channel is NADPH, whereas the ATPases release H⁺ from water resulting in an alkalinization of intracellular pH in this model.