Airway smooth muscle relaxation is impaired in mice lacking the p47phox subunit of NAD(P)H oxidase

Abstract

NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species and has a central role in airway smooth muscle (ASM) cell proliferation. Since reactive oxygen species also affect ASM contractile response, we hypothesized a regulatory role of NAD(P)H oxidase in ASM contractility. We therefore studied ASM function in wild-type mice (C57BL/6J) and mice deficient in a component (p47phox) of NAD(P)H oxidase. In histological sections of the trachea, we found that the area occupied by ASM was 17% more in p47(phox-/-) than in wild-type mice. After correcting for the difference in ASM content, we found that force generation did not vary between the two genotypes. Similarly, their ASM shortening velocity, maximal power, and sensitivity to acetylcholine, as well as airway responsiveness to methacholine in vivo, were not significantly different. The main finding of this study was a significantly reduced ASM relaxation in p47phox-/- compared with wild-type mice both during the stimulus and after the end of stimulation. The tension relaxation attained at the 20th second of electric field stimulation was, respectively, 17.6 +/- 2.4 and 9.2 +/- 2.3% in null and wild-type mice (P <0.01 by t-test). Similar significant differences were found in the rate of tension relaxation and the time required to reduce tension by one-half. Our data suggest that NAD(P)H oxidase may have a role in the structural arrangement and mechanical properties of the airway tissue. Most importantly, we report the first evidence that the p47phox subunit of NAD(P)H oxidase plays a role in ASM relaxation.

Fig. 1

Histological images illustrating the cross section of the paries membranaceus in transverse sections of a mouse trachea. Sections shown in panel A, B, and C are respectively from the proximal, median, and distal portion of the trachea and illustrate the progressive changes in structural organization along the trachea.

Fig. 2

Typical recordings of force generation in response to 10s electric field stimulation (EFS) in tracheal strips from wild type (C57BL/6J) and p47^phox−/− mice. An impaired spontaneous relaxation can be seen in the p47^phox−/− strip.

Fig. 3

Maximal tissue stress generated by electric field stimulation in tracheal strips from wild type (C57BL/6J) and p47^phox−/− mice. Panel A shows stress normalized per strip cross sectional area. Panel B shows that the greater stress produced by p47^phox−/− strips was cancelled out when corrected for the percent of airway smooth muscle in the airway wall. Means and standard errors are shown, n=16 for both groups.

Fig. 4

A. Force-Velocity curves calculated as average of the single curves obtained for each strip by fitting experimental data with a modification of the Hill’s equation for airway smooth muscle (see text and reference 41). Dashed and solid lines represent data obtained in tracheal strips from wild type and p47^phox−/− mice, respectively. B. Maximal power in tracheal strips from wild type and p47^phox−/− mice. P_o is the maximal stress generated by electric field stimulation at the moment of the force clamp, l_ref is reference length. Means and standard errors are shown, n=6 for both groups.

Fig. 5

The maximal rate of tension relaxation (RTR_end) and the time required to reduce tension by half (t_1/2) after 10 s electric field stimulation in strips from wild type (C57BL/6J) and p47^phox−/− mice are shown respectively in panel A and B. RTR_end was significantly reduced (p<0.01 by t-test), whereas t_1/2 was significantly increased (p<0.01 by t-test) in p47^phox−/− mice compared with wild type. Means and standard errors are shown, n=16 for both groups.

Fig. 6

Typical recordings of force generation in response to 20 s electric field stimulation (EFS) in tracheal strips from wild type (C57BL/6J) and p47^phox−/− mice. No spontaneous relaxation was present during the electric field stimulation in this p47^phox−/− strip.

Fig. 7

The maximal rate of tension relaxation that took place during a 20 s electric field stimulation (RTR_st) and the amount of tension relaxation at 20 s (TR) in strips from wild type (C57BL/6J) and p47^phox−/− mice are reported respectively in panel A and B. Both parameters were significantly reduced (p<0.01 by t-test) in p47^phox−/− mice compared with wild type. Means and standard errors are shown, n=16 for both groups.

Fig. 8

Cumulative concentration-response curves to acetylcholine (ACh) in tracheal rings from wild type (C57BL/6J, circles) and p47^phox−/− mice (triangles). Means and standard errors are shown, n=5 for both groups.

Fig. 9

Increase in total lung resistance (R_T) induced by aerosolized methacholine (MCh) above baseline R_T (panel A) and baseline pressure-volume loops before MCh challenge (panel B) in wild type (C57BL/6J) and p47^phox−/− mice. Means and standard errors are shown, n=6 for both groups.