Lubiprostone targets prostanoid EP₄ receptors in ovine airways

Abstract

Background and purpose: Lubiprostone, a prostaglandin E₁ derivative, is reported to activate ClC-2 chloride channels located in the apical membranes of a number of transporting epithelia. Lack of functioning CFTR chloride channels in epithelia is responsible for the genetic disease cystic fibrosis, therefore, surrogate channels that can operate independently of CFTR are of interest. This study explores the target receptor(s) for lubiprostone in airway epithelium.

Experimental approach: All experiments were performed on the ventral tracheal epithelium of sheep. Epithelia were used to measure anion secretion from the apical surface as short circuit current or as fluid secretion from individual airway submucosal glands, using an optical method.

Key results: The EP₄ antagonists L-161982 and GW627368 inhibited short circuit current responses to lubiprostone, while EP₁(,)₂(&)₃ receptor antagonists were without effect. Similarly, lubiprostone induced secretion in airway submucosal glands was inhibited by L-161982. L-161982 effectively competed with lubiprostone with a K(d) value of 0.058 µM, close to its value for binding to human EP₄ receptors (0.024 µM). The selective EP₄ agonist L-902688 and lubiprostone behaved similarly with respect to EP₄ receptor antagonists. Results of experiments with H89, a protein kinase A inhibitor, were consistent with lubiprostone acting through a G(s) -protein coupled EP₄ receptor/cAMP cascade.

Conclusions and implications: Lubiprostone-induced short-circuit currents and submucosal gland secretions were inhibited by selective EP₄ receptor antagonists. The results suggest EP₄ receptor activation by lubiprostone triggers cAMP production necessary for CFTR activation and the secretory responses, a possibility precluded in CF tissues.

Figure 1

Effects of the EP₄ receptor antagonist L-161982 on SCC responses to lubiprostone in sheep tracheal epithelium. Record showing response to lubiprostone, 200 nM, applied apically, after amiloride and before addition of L-161982, 0.5 µM, also applied apically (A). Cumulative data (B) show that responses to lubiprostone (lubi) were significantly reduced after L-161982 was added. Data are from seven preparations from four tracheas. The values of the plateau responses were measured at the moment the antagonist was added.

Figure 2

Sidedness of action of lubiprostone and L-161982. (A) shows response to lubiprostone, 500 nM, applied basolaterally (bl) after amiloride and subsequent addition of L-161982, 2.5 µM, initially basolaterally and then apically (ap). Cumulative data from five identical experiments from two tracheas are given in (B).

Figure 3

Effects of the EP₄ receptor antagonist GW627368X on SCC responses to lubiprostone on tracheal epithelium (A). Responses to lubiprostone (L; 200 nM) applied apically after amiloride (A; 10 µM), are shown, followed by GW627368X (GW; 10 µM) also added apically. Plateau values are the SCC increases at the time the GW compound was added. Note the variation in response to the GW compound from no inhibition, incomplete inhibition to complete inhibition. The number at the beginning of each record gives the basal SSC (µA·cm⁻²) before amiloride (A) was added. Cumulative data from seven preparations from three sheep (i and ii) (iii, iv and v) and (vi and vii) are shown at the right (B).

Figure 4

Partial concentration response curves to PGE₂ are shown in the presence and absence of prostanoid receptor antagonists and after amiloride inhibition of sodium absorption. In (A), the EP₄ receptor antagonist L-161982, the mixed EP receptor antagonist AH6809 and the EP₂ receptor antagonist SC-19220 were added in sequence after the plateau response to PGE₂, 1 µM was achieved. In (B) the antagonists were added first. Both the experiments were repeated a further three times, but as SC-19220 produced no additional inhibition to that of L-161982 and AH6809 combined, only the former two were used (mean data given in C). All agents were added apically to the tracheal epithelia (four pairs from three sheep).

Figure 5

Effects of prostanoid receptor antagonists on responses to the selective EP₄ receptor agonist L-902688. The example shows the effects of addition of amiloride, followed by SC-19220 (EP₂ receptor antagonist), AH 6809 (the non-selective EP_1-3 receptor antagonist) and L-161982 on the SCC response to L-902688, all agents added apically (A). Data from five identical experiments are shown in (B).

Figure 6

Concentration-response records to lubiprostone in two tracheal preparations in the absence (A) and presence of L-161982, 1 µM (B). Cumulative concentrations of lubiprostone were added until the maximal response was achieved. All agents, including amiloride, were added to the apical bathing solutions. Responses were converted to percentage of the maximal response and the data plotted on a semi-log scale (C). A second identical experiment gave similar data (D).

Figure 7

Concentration-response records to L-902688 in two tracheal preparations in the absence (A) and presence of L-161982, 1 µM (B). Cumulative concentrations of L-902688 were added until the maximal response was achieved. All agents, including amiloride, were added to the apical bathing solutions. Responses were converted to percentage of the maximal response and the data plotted on a semi-log scale (C). Note lack of effect of DMSO and change in SCC upon addition of L-161982.

Figure 8

Concentration-response records to L-902688 in two tracheal preparations in the absence (A) and presence of L-161982, 2 µM (B). In (A) when the maximal response had been achieved L-161982, 2 µM was added. All agents, including amiloride, were added to the apical bathing solutions.

Figure 10

Persistence of blockade of lubiprostone by L-161982 after its removal. In (A) amiloride, 10 µM and L-161982, 10 µM were added apically (ap) to a short circuited tracheal epithelium for 10 min, after which recording was halted and the apical solution replaced, now containing only amiloride. Subsequent addition of lubiprostone, 500 nM basolaterally (bl), gave only a small increase in current. Data from six experiments confirmed responses to basolaterally applied lubiprostone are significantly smaller after pre-exposure to L-161982 (B).

Figure 9

Effects of the protein kinase A inhibitor (H89) on responses to lubiprostone. One each of three matched pairs of epithelia from two sheep was exposed to H89 (40 µM, both sides) for 30 min. All tissues were then exposed to lubiprostone (200 nM ap) (A). The basal SCC, before amiloride was added is given at the beginning of each record. Peak and plateau responses, together with charge transfer during 500 s, were all significantly inhibited by about 50% in the presence of H89, using a paired t-test (B).

Figure 11

Mean secretion rates of tracheal submucosal glands in response to lubiprostone in the presence and absence of L-161982. Mean rates of secretion (as nl·min⁻¹ per gland) are given for 6 glands from both control and test tissues, measured during a basal period (15 min), following addition of lubiprostone (500 nM bl) (30 min) and following addition of carbachol (CCh, 100 µM bl) (15 min). Note that pre-exposure to L-161982 significantly reduces the basal, as well as the lubiprostone-induced, secretion rate, compared with controls.

Figure 12

Equilibrium binding constants (µM) for L-161982 with recombinant human prostanoid receptors (from Machwate et al., 2001) (A), plus values from functional studies using lubiprostone and L-902688 as agonists (B).