Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia

Abstract

Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A/PR/8/34 inhibits the amiloride-sensitive Na(+) current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na(+) channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na(+) channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.

Figure 1

The effects of influenza virus on Na⁺ channel activity in tracheal epithelium. (a) Original recordings showing the response of the tracheal epithelium to 10 μmol/liter amiloride under (i) control conditions, (ii) after 1 h exposure of the apical membrane to UV-inactivated PR8 (10⁶ pfu/ml before inactivation), or (iii) after 1 h apical exposure to active PR8 virus (10⁶ pfu/ml). (b) Concentration–response curve for amiloride. I_ammax is the amiloride-sensitive current determined by using a maximal concentration of amiloride, 100 μmol/liter. (c) The effects on amiloride-sensitive current of 1 h apical treatment with: (i) active PR8 influenza virus (10⁶ pfu/ml); (ii) UV-inactivated PRB (10⁶ pfu/ml); (iii) active WSN33 influenza virus (10⁶ pfu/ml); (iv) replication-deficient adenovirus, MX17 (10⁶ pfu/ml); and (v) allantoic fluid not infected with any virus. (d) Time course of the decline produced by 10⁶ pfu/ml PR8 in the amiloride-sensitive current. (e) Viral concentration–response curve for inhibition of the amiloride-sensitive current after apical exposure to purified PR8 for 1 h. In a–e, I_am is the amiloride-sensitive current, and I_am0 is the amiloride-sensitive current before the addition of virus.

Figure 2

Effects of influenza virus on responses of tracheal epithelium to stimulants. (a) Original recordings showing the effects of 10 μmol/liter forskolin plus 100 μmol/liter IBMX, before and after 1 h apical exposure to 10⁶ pfu/ml PR8. (b) Original recordings showing the effects of 100 μmol/liter carbachol before and after 1 h apical exposure to 10⁶ pfu/ml PR8. (c and d) The effects of 1 h apical exposure to 10⁶ pfu/ml PR8 virus on the current activated by 10 μmol/liter forskolin plus 100 μmol/liter IBMX (c) and by 100 μmol/liter carbachol (d).

Figure 3

Effects of influenza virus on nonrespiratory epithelia. (a) Original recordings showing the effect of 10 μmol/liter amiloride on M1 mouse collecting duct cells before and after 1 h apical exposure to 10⁶ pfu/ml PR8. (b) Original recordings showing the effect of 10 μmol/liter amiloride on mouse colonic epithelium before and after 1 h apical exposure to 10⁶ pfu/ml PR8. (c) Effect of 10⁶ pfu/ml PR8 on the amiloride-sensitive current in M1 cells and mouse colonic epithelium.

Figure 4

Mechanism of Na⁺ channel inhibition by influenza virus. (a) Effect on the PR8-induced inhibition of the amiloride-sensitive current in trachea of cytochalasin D (30 μg/ml; CD); chloroquine (500 μmol/liter; CQ); amantadine (200 μmol/liter; AM) and pretreatment of the apical membrane with neuraminidase (0.1 units/ml) for 30 min at 37°C (NA). (b) Effect of “split virus” (48 μg total protein/milliliter) on the amiloride-sensitive current and its neutralization by antihemagglutinin antibody (1:100 dilution; hemagglutinin antibody). (c) Effects of staurosporine (2 μmol/liter; Stauro), bisindolylmaleimide I (0.1 μmol/liter; BIM), Gö-6983 (20 nmol/liter; Gö) and U-73122 (10 μmol/liter; U-73122) on the inhibition by 10⁶ pfu/ml PR8 of the amiloride-sensitive current. (d) Effect of 1,2-dioctanoyl-sn-glycerol (10 μmol/liter; DOG) and BIM (0.1 μmol/liter; BIM) on the amiloride-sensitive short-circuit current.