Acute effects of parainfluenza virus on epithelial electrolyte transport

Parainfluenza viruses are important causes of respiratory disease in both children and adults. In particular, they are the major cause of the serious childhood illness croup (laryngotracheobronchitis). The infections produced by parainfluenza viruses are associated with the accumulation of ions and fluid in the respiratory tract. It is not known, however, whether this accumulation is because of a direct effect of the viruses on ion and fluid transport by the respiratory epithelium. Here we show that a model parainfluenza virus (the Sendai virus), in concentrations observed during respiratory infections, activates Cl- secretion and inhibits Na+ absorption across the tracheal epithelium. It does so by binding to a neuraminidase-insensitive glycolipid, possibly asialo-GM1, triggering the release of ATP, which then acts in an autocrine fashion on apical P2Y receptors to produce the observed changes in ion transport. These findings indicate that fluid accumulation in the respiratory tract associated with parainfluenza virus infection is attributable, at least in part, to direct effects of the virus on ion transport by the respiratory epithelium.


Introduction
The volume of fluid in the respiratory tract is determined by the balance between the rate of fluid secretion and the rate of fluid absorption by the respiratory epithelium. The secretion of fluid is due to the movement of Cl − into the lumen through CFTR Cl − channels and Ca 2+ -activated Cl − channels in the apical membranes of the epithelial cells and is driven by the Na + -K + -2Cl − cotransporter in their basolateral membranes (1). The absorption of fluid is due to the movement of Na + across the apical membrane through epithelial Na + channels (1)(2)(3). The Na + is then pumped out of the cytosol across the basolateral membrane by the Na + ,K + -ATPase.
Given the importance of ion transport across the respiratory epithelium in determining the volume of the lung surface fluid, it is not surprising that disturbances in it lead to pathological changes in the volume of lung fluid. Hence excessive activity of epithelial Na + channels, such as occurs in cystic fibrosis (4), leads to dehydration of the respiratory surfaces, whereas reduced activity of epithelial Na + channels, such as occurs in pseudohypoaldosteronism type I, is associated with an increase in lung surface fluid (3). Similarly, reduced activity of the epithelial Na + channels in the respiratory epithelium has been implicated in the development of high altitude pulmonary oedema (5), neonatal respiratory distress syndrome (6), cardiogenic pulmonary oedema (7) and serous otitis associated with fluid accumulation in the respiratory tract, which ranges in severity from rhinitis (9) and serous otitis media (14) to a lifethreatening adult respiratory disease syndrome (15). The question thus arises whether parainfluenza viruses could be directly affecting ion transport by the respiratory epithelium. One common respiratory virus, influenza virus, has been shown to directly inhibit Na + absorption by the respiratory epithelium (16). This inhibition is due to the binding of the hemagglutinin in the viral coat to a neuraminidase-sensitive receptor in the apical membrane of the epithelium and is mediated by activation of phospholipase Cβ and protein kinase C (16).
Despite the similarity of their names, which reflects the similarity of the clinical features they produce, influenza viruses and parainfluenza viruses are unrelated (9): influenza viruses have a segmented genome and replicate in the nucleus, whereas the parainfluenza viruses have a non-segmented genome and replicate in the cytoplasm (9). Thus it is not possible to assume that parainfluenza viruses affect epithelial ion transport in the same way as influenza viruses.
In the present study we performed experiments to determine whether parainfluenza viruses rapidly alter ion transport by respiratory epithelia.
We found that not only do they inhibit absorption of the Na + by these epithelia, they also activate epithelial secretion of Cl − . Furthermore, we found that the mechanism by which parainfluenza viruses alter epithelial ion transport differs markedly from that used by influenza viruses.

Statistics:
Results are presented as means ± SEM (n = number of tissues tested). Statistical significance was assessed using unpaired Student's ttests at a probability level of P < 0.05. Asterisks indicate statistically significant differences from control.

Results
The baseline electrical properties of mouse tracheal epithelium: The rate at which the tracheal epithelium secretes Cl − ions and absorbs Na + ions can be measured by determining the current flow generated by the movement of these ions across the epithelium. We can do this by measuring the electrical potential difference across the epithelium (the transepithelial potential, V te ) and the change in the transepithelial potential produced by the passage of a standard test pulse of current. We can then use Ohm's Law to estimate the resistance of the epithelium to the flow of the test pulse of current (the transepithelial resistance, R te ), as well as the current that is required to generate the observed transepithelial potential (the so-called short circuit current, I sc = V te / R te ).
To make these measurements, we placed a small piece of trachea in an Ussing chamber, an organ bath that permits the luminal and interstitial surfaces of the tissue to be superfused by separate solutions and the continuous measurement of transepithelial potential and resistance. Under control conditions, we found the potential difference across the tracheal epithelium to be -7.3 ± 0.6 mV (n = 23), with the lumen being negative to the interstitium and the electrical resistance of the epithelium to be 79.4 ± 5.2 Ωcm 2 (n = 23). From these measurements we calculated the short circuit current across the epithelium to be 98.3 ± 7.8 µAcm −2 (n = 23).
This current flow was largely due to transport of Na + through amiloridesensitive Na + channels, as the application of amiloride (10 µM), a selective inhibitor of these channels, to the apical surface of the epithelium reduced the short circuit current by 90.2 ± 6.9 µAcm −2 (n = 23), a 92% reduction.
The effects of Sendai virus: Exposure of the apical membrane of mouse tracheal epithelium to Sendai virus (10 6 pfu/ml) caused the transepithelial potential and the short circuit current to become transiently more negative (∆V te = -1.2 ± 0.2 mV, n = 8; ∆I sc = -43.1 ± 9.0 µAcm −2 ; Fig. 1A). This transient occurred within approximately 1 minute of adding the virus to the epithelium and lasted approximately 5 minutes. It could have been due either to an increase in the rate of Cl − secretion or to an increase in the rate of Na + absorption. We found that it could not be prevented by inhibiting Na + absorption by the addition of 10 µM amiloride to the apical membrane (data not shown), hence could not be attributed to an increase in the rate of Na + absorption. It could be inhibited, however, by blocking the secretion of Cl − by the addition to the basolateral membrane of 100 µM bumetanide, an inhibitor of the Na + -K + -2Cl − cotransporter (data not shown).
Hence the initial transient stimulation of the short circuit current was due to an increase in the rate of Cl − secretion. We further found that this transient stimulation of short circuit current required an increase in the intracellular concentration of Ca 2+ , as it could be prevented by manoeuvres that clamp cytosolic Ca 2+ at a low level, such as the removal of extracellular Ca 2+ or loading the cytosol with the Ca 2+ chelator, BAPTA ( Fig. 4). Hence the initial increase in short circuit current produced by Sendai virus was due to activation of Ca 2+ -activated Cl − channels in the apical membrane of the epithelium.
This decrease in current flow during prolonged exposure to parainfluenza virus was almost entirely due to a reduction in the rate of amiloridesensitive Na + transport (Fig. 1B). In paired control experiments, the ion transport activity of the epithelium did not change over this period (Fig.   1B). The addition of allantoic fluid also did not affect the ion transport activity of the epithelium (data not shown). We further found that, as we had previously observed for influenza virus (16), the action of Sendai virus on the epithelium was not prevented by UV-inactivation of the virus.

UV-inactivated virus produced an initial transient increase in short
circuit current of 23.4 ± 6.0 µAcm −2 (n = 5), and, after an hour of exposure, reduced the rate of amiloride-sensitive Na + absorption from -89.4 To check whether Sendai virus exerted a non-specific toxic effect, we examined other parameters of the function of the tracheal epithelium. In particular, we examined the effect of the virus on: (i) the rate of Cl − secretion by the epithelium in response to an increase in intracellular cyclic AMP produced by exposure to the activator of adenylate cyclase, forskolin (18); (ii) the rate of Cl − secretion in response to an increase in intracellular Ca 2+ produced by muscarinic agonist carbachol (18,19); and (iii) the rate of electrogenic cotransport of Na + and glucose across the epithelium (16,20). We found (Fig. 1C) that although exposure to Sendai virus (10 6 pfu/ml) for 1 hour reduced the response to 100 µM forskolin, it did not affect the response to 100 µM carbachol or the rate of electrogenic glucose transport.
The virus thus appears not to have exerted a nonspecific toxic effect.
Finally, we examined the dependency of the ion transport effects of Sendai virus on the concentration of virus bathing the apical membrane. We found that both the initial transient stimulation of the short circuit current  for 24 hours almost completely inhibited both the initial transient stimulation of short circuit current (Fig. 2F) and the reduction of the amiloride-sensitive current (Fig. 2E) that follows the addition of 10 6 pfu/ml Sendai to the apical bathing solution. In contrast, the inhibition of the amiloride-sensitive current produced by influenza virus in M1 cells was not affected by pre-incubation in PPMP (data not shown).
We further explored the role of glycolipids in mediating the effects of Sendai virus by testing the effect of an antibody directed against the ganglioside asialoGM 1 . This ganglioside has been previously identified as the apical receptor by which many bacterial pathogens evoke mucus and cytokine production by epithelia (28,29). We found that pre-incubation for 20 minutes in an anti-asialoGM1 antibody in a dilution of 1 in 100 completely inhibited the effects of Sendai virus (Figs 2A, 2B and 2D).
Interestingly, as reported in other systems (28,29), when added to tracheal epithelium at the lower dilution of 1 in 20, the antibody acted as an agonist, evoking a transient stimulation of short circuit current followed by a long-term depression of the amiloride-sensitive Na + absorption (Fig.   2C). The findings that the effects of Sendai virus are mediated by ATP acting on purinergic receptors and that they are mediated in part by increasing intracellular Ca 2+ suggested roles also for phospholipase Cβ and protein kinase C. Consistent with this we found that U-73122 (10 µM; Figs 5D and 5F) and edelfosine (10 µM; data not shown), which are blockers of phospholipase C, inhibited the effects of the virus, whereas the inactive isomer of U-73122, U-73343 (20 µM), was without effect (data not shown).

Sendai virus acts by triggering
We also found that an inhibitor of protein kinase C, BIM I (100 nM), partially inhibited the transient response to Sendai virus (Fig. 5F), although it did not prevent the inhibition of amiloride-sensitive Na + absorption ( Fig 5E).
The increased intracellular Ca 2+ concentration that accompanies binding of asialoGM1 has been reported to activate p38 and p42/44 MAP kinases (29,31).
We found, however, that neither inhibition of p38 MAP kinase with 25 µM SB-203580 nor inhibition of p42/44 MAP kinase with 25 µM U-0126 interfered with the actions of Sendai virus on ion transport (Figs 5A, 5B and 5F).
Finally, we examined whether a pertussis toxin-sensitive G protein mediates the effects of Sendai virus. We performed these studies in the presence of BIM I, an inhibitor of protein kinase C, because we have previously found that the B-oligomer of the toxin, which is enzymatically inactive acts as a hemagglutinin, inhibits amiloride-sensitive Na + absorption in mouse tracheal epithelium as a result of activating protein kinase C (32). When we added Sendai virus to tracheal epithelium that had been pre-treated with pertussis toxin in the presence of BIM I (100 nM), we found that the magnitude of the transient stimulation of short circuit current was not different from that observed in the presence of BIM I alone (Fig. 5F).
This treatment, however, abolished completely the effects of Sendai virus on the amiloride-sensitive Na + absorption (Fig. 5C) Furthermore, the P2Y antagonist, suramin, inhibits both the activation of Cl − secretion (data not shown) and the inhibition of the amiloridesensitive Na + absorption (Fig. 6B) produced by UTP. Both actions of UTP were also blocked by the inhibitor of phospholipase C, U-73122 (32) (Fig.   6B). Finally, UTP shows a similar divergence in the mechanisms by which it controls Cl − secretion and Na + absorption to that we had observed with Sendai virus: treatment of the epithelium with the protein kinase C inhibitor BIM I inhibits the effect of UTP on Cl − secretion, but leaves the inhibitory effect of UTP on the amiloride-sensitive Na + absorption unchanged (32) (Fig. 6B).

Discussion
We have found that mouse parainfluenza virus I (Sendai virus) produces rapid changes in ion transport across mouse tracheal epithelium. We observed these effects at concentrations comparable to those observed in the nasal mucosa and lungs of animals during experimentally induced infections, which reach 7 × 10 6 pfu/g wet weight of lung tissue and higher within 3 days of inoculation with the virus (21,23,33). In addition, the nature of the changes in ion transport observed, viz. an increase in Cl − secretion together with an inhibition of Na + absorption, suggests that they may play a significant role in the fluid accumulation in the respiratory tract that accompanies parainfluenza infections (9,23). Since these effects of Sendai virus were also observed in the M1 collecting duct cell line, it would seem that they are not mediated by immune cells in the tracheal mucosa.
The mechanism by which Sendai produces its effects is summarised in Figure   7. It first binds to a glycolipid, which may be asialoGM 1  These bacteria trigger mucus and interleukin production by binding apical asialoglycolipids, leading to release of ATP, autocrine activation of purinergic receptors and increased intracellular Ca 2+ (28,29). Taking these by guest on  http://www.jbc.org/ Downloaded from reports together with our present finding that Sendai virus modulates epithelial ion transport we propose that altered epithelial ion transport and the production of mucus and cytokines are all part of a stereotypic response of airway epithelia to contact with pathogens. In this response, an increase in the volume of fluid bathing the surface of the epithelium hydrates the increased amounts of mucus being secreted and leads to an increase in the rate of mucus clearance so as to facilitate transport of the pathogens out of the lung (39). The possibility that the acute changes in electrolyte transport we have observed form part of a stereotyped epithelial response to pathogens is supported by reports that both Pseudomonas aeruginosa and Klebsiella pneumoniae inhibit Na + transport by the respiratory epithelium (40)(41)(42)(43).
A further implication of our findings is that the release of ATP from epithelia, which has been considered to be an epithelial response to mechanical stimuli (44), may also play a critical role in co-ordinating epithelial responses to pathogens.  Initial ∆I sc produced by Sendai virus (10 6 pfu/ml). Initial ∆I sc was measured as the difference between the peak I sc observed following the addition of Sendai and the I sc observed immediately prior to its addition.

Figure Legends
Statistically significant effects are marked with an asterisk. and PPADS (100 µM; Panels B and E), respectively. I sc-Amil was measured 1 h after the addition of Sendai virus (10 6 pfu/ml). Initial ∆I sc was measured as the difference between the peak I sc observed following the addition of Sendai and the I sc observed immediately prior to its addition.  observed following the addition of Sendai virus (10 6 pfu/ml). Initial ∆I sc was measured as the difference between the peak I sc observed following the addition of Sendai and the I sc observed immediately prior to its addition.
Statistically significant effects are marked with an asterisk.