Adenosine-evoked Na+ transport in human airway epithelial cells

Abstract

Background and purpose: Absorptive epithelia express apical receptors that allow nucleotides to inhibit Na(+) transport but ATP unexpectedly stimulated this process in an absorptive cell line derived from human bronchiolar epithelium (H441 cells) whilst UTP consistently caused inhibition. We have therefore examined the pharmacological basis of this anomalous effect of ATP.

Experimental approach: H441 cells were grown on membranes and the short circuit current (I(SC)) measured in Ussing chambers. In some experiments, [Ca(2+)](i) was measured fluorimetrically using Fura -2. mRNAs for adenosine receptors were determined by the polymerase chain reaction (PCR).

Key results: Cross desensitization experiments showed that the inhibitory response to UTP was abolished by prior exposure to ATP whilst the stimulatory response to ATP persisted in UTP-pre-stimulated cells. Apical adenosine evoked an increase in I(SC) and this response resembled the stimulatory component of the response to ATP, and could be mimicked by adenosine receptor agonists. Pre-stimulation with adenosine abolished the stimulatory component of the response to ATP. mRNA encoding A(1), A(2A) and A(2B) receptor subtypes, but not the A(3) subtype, was detected in H441 cells and adenosine receptor antagonists could abolish the ATP-evoked stimulation of Na(+) absorption.

Conclusions and implications: The ATP-induced stimulation of Na(+) absorption seems to be mediated via A(2A/B) receptors activated by adenosine produced from the extracellular hydrolysis of ATP. The present data thus provide the first description of adenosine-evoked Na(+) transport in airway epithelial cells and reveal a previously undocumented aspect of the control of this physiologically important ion transport process.

Figure 1

Effects of ATP and UTP on ion transport (I_SC) measured across cells mounted in standard Ussing chambers either under control conditions (n=4) or in the presence of 10 μM apical amiloride (n=3). (a) Changes in I_SC induced by adding ATP followed by UTP (both 100 μM) to the apical solution. (b) Effect of adding UTP followed by ATP (both 100 μM). Data are mean±s.e.m.

Figure 2

Effects of ATP and UTP on ion transport (I_SC) and Fura-2 fluorescence ratio (F₃₄₀/F₃₈₀) measured simultaneously. (a) Changes in the two parameters evoked by 100 μM ATP (n=33) (b) Effect of 100 μM UTP (n=22). Both signals are presented as mean±s.e.m.

Figure 3

Effect of consecutive doses (100 μM apical) of ATP (a and b; n=5) and UTP (c and d; n=5) upon I_SC and F₃₄₀/F₃₈₀ measured simultaneously. Both the initial peak increase in I_SC (‘Transient') and the increase after 10 min exposure to the nucleotide (‘Sustained') are presented as mean±s.e.m. (a and c). The peak increases in Ca²⁺ (ΔF₃₄₀/F₃₈₀) evoked by the first and second applications of nucleotide were also quantified (‘Peak'; mean±s.e.m.; b and d) Asterisks denote statistically significant differences between the responses evoked by the first and second applications of nucleotide (^*P<0.05, ^**P<0.002).

Figure 4

Effect of ATP followed by UTP (a, c and d) and UTP followed by ATP (b, e and f) on I_SC and F₃₄₀/F₃₈₀ measured simultaneously. (a) and (b) show the time courses of the cross-desensitization experiments. The dashed lines represent a 10 min wash with control saline. Both the initial peak increase in I_SC (‘Transient') and the increase after 10 min exposure to the nucleotide (‘Sustained') are shown (c and e). The peak increases in Ca²⁺ (ΔF₃₄₀/F₃₈₀) evoked by the first and second applications of nucleotide were also quantified (‘Peak'; d and f). All data are mean±s.e.m. (n=5) and asterisks denote statistically significant differences between the data derived from control and prestimulated cells (^*P<0.05, ^**P<0.02).

Figure 5

Effect on I_SC (mean±s.e.m.) of apical ATP (100 μM) under control conditions (a; n=7) and in cells that had been preincubated (20 min) in ZM 241385 (b; 1 μM, n=4), MRS 1706 (c; 1 μM, n=4), or DPCPX (d; 1 μM, n=4).

Figure 6

Effect of apical ATPγS (100 μM) on I_SC. (a) Effect under control conditions and (b) Effect on cells that had been preincubated (20 min) in ZM 241385 (1 μM). Data are mean±s.e.m., n=4.

Figure 7

Effects of apical adenosine (100 μM) upon I_SC (upper panel) and F₃₄₀/F₃₈₀ (lower panel). Data are mean±s.e.m. (n=5).

Figure 8

Effect of adenosine on response to ATP. (a) Effect of ATP on I_SC and F₃₄₀/F₃₈₀. (b) Effect of ATP on cells that had been stimulated with 100 μM apical adenosine and then washed with control saline for 10 min. (c) The initial peak increase in I_SC (‘Transient') and the increase after 10 min exposure to ATP (‘Sustained'). (d) The peak increases in Ca²⁺ (ΔF₃₄₀/F₃₈₀) evoked by ATP (‘Peak'; d and f). All data mean±s.e.m. (n=5) and asterisks denote statistically significant differences between control and adenosine prestimulated cells (^**P<0.02).

Figure 9

Effects of adenosine and adenosine receptor agonists upon I_SC. (a) I_SC was recorded during stimulation with apical adenosine: 0.063 μM, n=3, basal I_SC=27.2±3.9 μA cm⁻²; 0.63 μM, n=7, basal I_SC=33.4±3.8 μA cm⁻²; 6.3 μM n=7, basal I_SC=25.7±5.4 μA cm⁻²; or 63 μM n=6, basal I_SC=27.2±3.9 μA cm⁻². All data are shown as mean±s.e.m. and the basal I_SC (i.e. that measured before the addition of adenosine) was subtracted from all records in order to illustrate the adenosine-evoked changes in I_SC. (b) The increases in I_SC measured after ∼20 min exposure to apical adenosine (0.021–200 μM) were measured and plotted (mean±s.e.m.) against the concentration of adenosine used. (c) Cells were exposed to different concentrations of apical SPA, CGS 21680 or IB-MECA and the resultant increases in I_SC measured and normalized to the response to a maximally effective concentration (200 μM) of apical adenosine measured in age-matched cells at identical passage. The results of this analysis are plotted against the concentration of agonist used; all data points are the mean values and error bars have been omitted in the interests of clarity. In all experiments, the cultured epithelial cell layers were each exposed to only a single concentration of agonist, and mean values derived from at least three independent experiments. The solid curves were fitted (least-squares regression) to the experimental data by assuming that the tested drugs bind to a single site as effectively as adenosine. Dashed lines denote extrapolation beyond the range of observed values.

Figure 10

Effects of adenosine receptor antagonists on I_SC. (a) Time course showing the effects of apical adenosine (200 μM) upon I_SC (mean±s.e.m., n=20). Curved bracket indicates the adenosine-evoked increase in I_SC, which was determined for each individual experiment. (b)The effects of DMSO solvent vehicle (control, n=5), DPCPX (1 μM, n=5), ZM 241385 (1 μM, n=5) and MRS 1706 (1 μM, n=5) on the adenosine-evoked increase in I_SC. The bar indicates the period during which data were used to analyse inhibitory effects of the compounds. (c) The inhibitory effects of these compounds, presented as mean±s.e.m.; asterisks denote values that differ significantly from control (^***P<0.0001, ^**P<0.002). (d) Results of experiments in which this protocol was used to quantify the inhibitory actions of different concentrations (0.1–1 μM) of ZM 1706 and of MRS 241385. The inhibitory effect of the solvent vehicle was monitored in each experiment so that could be corrected for the spontaneous/solvent-evoked fall in I_SC.