Engagement of the EP₂ prostanoid receptor closes the K⁺ channel K_Ca3.1 in human lung mast cells and attenuates their migration

Abstract

Human lung mast cells (HLMC) express the Ca²⁺-activated K⁺ channel K_Ca3.1, which plays a crucial role in their migration to a variety of diverse chemotactic stimuli. K_Ca3.1 activation is attenuated by the β₂-adrenoceptor and the adenosine A_2A receptor through a G_s-coupled mechanism independent of cyclic AMP. Prostaglandin E₂ promotes degranulation and migration of mouse bone marrow-derived mast cells through the G_i-coupled EP₃ prostanoid receptor, and induces LTC₄ and cytokine secretion from human cord blood-derived mast cells. However, PGE₂ binding to the G_s-coupled EP₂ receptor on HLMC inhibits their degranulation. We show that EP₂ receptor engagement closes K_Ca3.1 in HLMC. The EP₂ receptor-specific agonist butaprost was more potent than PGE₂ in this respect, and the effects of both agonists were reversed by the EP₂ receptor antagonist AH6809. Butaprost markedly inhibited HLMC migration induced by chemokine-rich airway smooth muscle-conditioned media. Interestingly, PGE₂ alone was chemotactic for HLMC at high concentrations (1 µM), but was a more potent chemoattractant for HLMC following EP₂ receptor blockade. Therefore, the G_s-coupled EP₂ receptor closes K_Ca3.1 in HLMC and attenuates both chemokine- and PGE₂-dependent HLMC migration. EP₂ receptor agonists with K_Ca3.1 modulating function may be useful for the treatment of mast cell-mediated disease.

Introduction

Mast cells are tissue-dwelling cells derived from bone marrow progenitors. They are present in all organs throughout the human body, both at mucosal surfaces and within connective tissues. Mast cells play a major role in tissue homeostasis, host defence and the pathophysiology of many diverse diseases ¹. These include pulmonary fibrosis, rheumatoid disease and atherosclerosis, but they are most commonly associated with allergic disease due to their activation by allergen ². In many diseases mast cells re-locate to specific compartments within tissue. This is typified in asthma where mast cells migrate into the airway epithelium ³, airway smooth muscle (ASM) ⁴ and submucosal glands ⁵. This places activated mast cells in direct contact with these dysfunctional airway elements, allowing the specific delivery of detrimental cell–cell signals. Drugs that inhibit this tissue relocation by preventing mast cell migration may prove particularly effective in the treatment of mast cell-mediated disease.

Human mast cells express the intermediate conductance Ca²⁺-activated K⁺ channel K_Ca3.1, which plays a critical role in their migration to diverse chemotactic stimuli ⁶, and to a lesser extent in their degranulation ^{7, 8}. Drugs that directly block this channel or which close it indirectly therefore have potential as novel therapies for mast cell-dependent disease. K_Ca3.1 in human lung mast cells (HLMC) is closed by both salbutamol and adenosine via the β₂-adrenoceptor and A_2A adenosine receptors, respectively ^{9, 10}, which are both G_s-coupled G protein-coupled receptors (GPCR). In keeping with a critical role for K_Ca3.1 in HLMC migration, adenosine also inhibits HLMC chemotaxis via the A_2A receptor ¹⁰.

PGE₂ is a prostanoid with four specific GPCR, designated EP_1–4. EP₂ and EP₄ couple to G_s, EP₃ couples predominantly to G_i although two isoforms also couple to G_s, while EP₁ receptors mobilise intracellular Ca²⁺, probably through G_q ¹¹. PGE₂ therefore has diverse biological activities depending on the receptors it interacts with and the cells expressing them. With respect to mast cells, mouse mast cells express the EP₃ receptor whose activation induces both degranulation and chemotaxis ^{12, 13}. Human cord blood-derived mast cells, express both EP₂ and EP₃ receptors. In these cells, PGE₂ enhances degranulation and PGD₂ production in cells primed by IL-4, an effect mediated through the EP₃ receptor. However, following FcɛRI-dependent activation PGE₂ inhibits human cord blood-derived mast cell LTC₄, PGD₂, IL-5 and TNF-α production via the EP₂ receptor but has no effect on degranulation ¹⁴. In contrast, several studies have shown that PGE₂ consistently inhibits degranulation and eicosanoid production by HLMC, an effect mediated via the EP₂ receptor ^15–17. This receptor also appears to dominate PGE₂-dependent signalling on mast cells within the human asthmatic lung as PGE₂ markedly attenuates both the early and late phase airway response to allergen challenge ¹⁸.

Since K_Ca3.1 in HLMC is closed by G_s-coupled β₂-adrenoceptors and A_2A adenosine receptors, we hypothesised that activation of the G_s-coupled EP₂ receptor would also close K_Ca3.1 in these cells. Furthermore, if PGE₂ was to close K_Ca3.1, then it should inhibit HLMC chemotaxis. To test this hypothesis we have used the patch-clamp technique to investigate the effects of PGE₂ on HLMC ion channel function, and investigated the effect of PGE₂ on HLMC migration induced by asthmatic ASM-conditioned medium.

Results

PGE₂ alone does not open K_Ca3.1

We initially examined whether PGE₂ opens K_Ca3.1 in HLMC under resting baseline conditions. HLMC are typically electrically silent at rest and it is possible that PGE₂ could open either K_Ca3.1 or another channel as reported previously for adenosine ¹⁰. No significant change in current amplitude was observed in seven cells following the addition of 10⁻⁵ M PGE₂ (current changed from 1.22±0.54 to 1.42±0.85 pA at +40 mV; p=0.678).

PGE₂ closes K_Ca3.1 in the presence of the K_Ca3.1 opener 1-EBIO

We then examined whether PGE₂ closes K_Ca3.1. Because PGE₂ might potentially inhibit many IgE-dependent cell activation pathways that could reduce cytosolic-free Ca²⁺, and thus reduce K_Ca3.1 activity indirectly, we concentrated on studying the effects of PGE₂ on K_Ca3.1 currents that were induced by the K_Ca3.1 opener 1-EBIO ^8–10. This compound opens K_Ca3.1 with a half-maximal value of about 30 µM for heterologously expressed K_Ca3.1, with a maximal effect at about 300 µM ¹⁹. 1-EBIO is specific for K_Ca3.1 in HLMC and opens it by enhancing the channels sensitivity to [Ca²⁺]_i ¹⁹. Thus at 100 µM EBIO, maximal K⁺ currents are achieved in the presence of 100 nM free Ca²⁺, which is below the resting [Ca²⁺]_i of most cell types including HLMC ⁸.

In cells in which K_Ca3.1 had been activated by 1-EBIO, addition of PGE₂ (10⁻⁸–10⁻⁵ M) produced a rapid (within 30 s) dose-responsive inhibition of channel activity with an associated positive shift in membrane potential (Fig. 1A–E). PGE₂ 10⁻⁵ M suppressed the K_Ca3.1 current in >90% of cells (Fig. 1B). Thus, addition of 10⁻⁵ M PGE₂ reduced K_Ca3.1 membrane current at +40 mV from 155.4±20.9 to 92.6±13.7 pA (p=0.0001, n=22 cells)(Fig. 1B), with a corresponding shift in reversal potential (Vm) from −69.5±2.8 to −56.3±3.9 mV (p=0.0004) (Fig. 1C). Half maximal suppression (IC₅₀) of K_Ca3.1 by PGE₂ occurred at approximately 4.0×10⁻⁷ M (calculated from six cells). Importantly, the effect of PGE₂ was partially reversed within 1 min by removing it from the recording solution (current post PGE₂ 49.6±20.8 pA, post wash 103.4±34.5 pA, p=0.046; Vm post PGE₂ −44.1±11.7 mV, post wash −72.1±6.81 mV, p=0.028, n=5) (Fig. 1D and E), indicating that non-specific “rundown” was not responsible for the effects seen.

Figure 1.

PGE₂ closes K_Ca3.1 in HLMC in a consistent and reversible manner. (A) Current–voltage curve demonstrating suppression of a 1-EBIO-induced K_Ca3.1 current by 10⁻⁵ M PGE₂ and partial reversal of the effect following removal of PGE₂ (wash). (B) Suppression of K_Ca3.1 current measured at +40 mV by 10⁻⁵ M PGE₂ (n=22 cells). (C) Shift in whole-cell current reversal potential (Vm) by 10⁻⁵ M PGE₂ (n=22 cells). (D) K_Ca3.1 current measured at +40 mV after addition of 1-EBIO, suppression by 10⁻⁵ M PGE₂ and then reversibility of suppression following removal of PGE₂ (wash) (n=5 cells). (E) Whole-cell current reversal potential (Vm) after the addition of 1-EBIO, a depolarising positive shift in response to 10⁻⁵ M PGE₂, and then reversibility following removal of PGE₂ (wash) (n=5 cells).

K_Ca3.1 modulation by PGE₂ is mediated via EP₂ receptors

To examine whether the effects of PGE₂ were mediated via EP₂ prostanoid receptors we examined the effects of EP₂ receptor agonists/antagonists. First, we examined the effects of the selective EP₂ receptor agonist butaprost. Butaprost mimicked the effects of native PGE₂ in a dose-dependent manner (Fig. 2A–D). At a concentration of 10⁻⁵ M, butaprost reduced the K_Ca3.1 current from 146.0±18.3 pA to 61.2±6.1 pA (p=0.00006, n=20) (Fig. 2B) with a corresponding shift in reversal from −67.5±1.4 to −54.4±3.2 mV (p=0.00006) (Fig. 2C). Half maximal suppression (IC₅₀) of K_Ca3.1 by butaprost occurred at approximately 2.1×10⁻⁷ M (n=6 cells) (Fig. 2D).

Figure 2.

The EP₂ receptor agonist butaprost closes K_Ca3.1 in HLMC. (A) Current–voltage curve demonstrating suppression of a 1-EBIO-induced K_Ca3.1 current by 10⁻⁵ butaprost. (B) Suppression of K_Ca3.1 current measured at +40 mV by 10⁻⁵ M butaprost (n=20 cells). (C) Shift in whole-cell current reversal potential (Vm) by 10⁻⁵ M butaprost (n=20 cells). (D) Concentration–response curves for K_Ca3.1 suppression by PGE₂ and the EP₂ agonist butaprost (mean±SEM of five cells for each).

The suppression of K_Ca3.1 by 10⁻⁵ M PGE₂ was partially reversed by the competitive EP₁ and EP₂ receptor antagonist AH6809 (Fig. 3A–C). Thus, in experiments studying AH6809 at a concentration of 10⁻⁵ M, current at +40 mV was 112±14.3 pA post PGE₂, increasing to 160.9±23.2 pA post AH6809 (p=0.011, n=10 cells) (Fig. 3B). There was however no significant shift in reversal potential in these experiments explained by the fact that significant K_Ca3.1 currents remained following PGE₂ application (Vm post PGE₂ −60.9±2.1 mV, post AH6809 −63.6±1.8 mV, p=0.17) (Fig. 3C). We also examined the effect of AH6809 on the selective EP₂ agonist butaprost. The suppression of K_Ca3.1 by 10⁻⁵ M butaprost was also partially reversed by 10⁻⁵ M AH6809 (Fig. 3D–F). Current at +40 mV was 58.5±8.3 pA post butaprost, increasing to 111.6±15.9 pA post AH6809 (p=0.0004, n=12) (Fig. 3E). There was also a significant shift in reversal potential (Vm post butaprost −48.1±5.8 mV, post AH6809 −61.0±3.7 mV, p=0.0004) (Fig. 3F). In the presence of AH6809, PGE₂ had no effect on K_Ca3.1 currents that had been induced by 1-EBIO (current at +40 mV 67.0±17.2 pA post 1-EBIO, 66.2±15.7 post AH6809, 62.7±13.7 pA post PGE₂, n=7)(Fig. 4A). Similarly, K_Ca3.1 currents did not appear in resting cells to which AH6809 was added prior to PGE₂ (current 6.9±0.9 pA at baseline, 5.8±0.6 pA post AH6809, 6.3±1.1 pA post PGE₂, n=6) (Fig. 4B). The EP₁ and EP₃ receptor agonist 17-phenyl trinor PGE₂ did not close K_Ca3.1 after activation with 1-EBIO (current at +40 mV post 1-EBIO 128.3±94.9 pA, post 17-phenyl trinor PGE₂ 127.8±90.4 pA, p=0.9567, n=6) and did not open K_Ca3.1 in resting cells (baseline current at +40 mV 5.70±0.88 pA, current post 17-phenyl trinor PGE₂ 5.36±0.949 pA, p=0.202, n=5). These results exclude a role for EP₁, EP₃ or EP₄ receptors in K_Ca3.1 modulation. Taking the data together, it can be concluded that suppression of K_Ca3.1 by PGE₂ is mediated by the EP₂ receptor.

Figure 3.

The effect of the EP₁/₂ receptor antagonist AH6809 on PGE₂- and butaprost -dependent closure of K_Ca3.1. (A) Current–voltage curve demonstrating reversibility of K_Ca3.1 suppression by PGE₂ following administration of the EP_1/2 receptor antagonist AH6809. (B) K_Ca3.1 current measured at +40 mV after addition of 1-EBIO, suppression by 10⁻⁵ M PGE₂ and then reversibility of suppression following addition of AH6809 (n=10 cells). (C) Whole-cell current reversal potential (Vm) after the addition of 1-EBIO, a depolarising positive shift in response to 10⁻⁵ M PGE₂, and then following addition AH6809 (n=10 cells). (D) Current–voltage curve demonstrating reversibility of K_Ca3.1 suppression by butaprost following administration of AH6809. (E) K_Ca3.1 current measured at +40 mV after addition of 1-EBIO, suppression by 10⁻⁵ M butaprost and then reversibility of suppression following addition of AH6809 (n=12 cells). (F) Whole-cell current reversal potential (Vm) after the addition of 1-EBIO, a depolarising positive shift in response to 10⁻⁵ M PGE₂, and then reversibility following addition AH6809 (n=12 cells).

Figure 4.

PGE₂ is ineffective in the presence of the AH6809. (A) K_Ca3.1 current measured at +40 mV after addition of 1-EBIO, stability following addition of AH6809, and failure to suppress following subsequent addition of 10⁻⁵ M PGE₂. (B) K_Ca3.1 current measured at +40 mV in resting cells, after addition of AH6809, and then following subsequent addition of 10⁻⁵ M PGE₂ showing failure of K_Ca3.1 to open.

PGE₂ closes K_Ca3.1 following IgE-dependent activation

Lastly, we confirmed whether PGE₂-dependent regulation of K_Ca3.1 was relevant to K_Ca3.1 channels that had been opened by anti-IgE-dependent activation. Anti-IgE (1:1000 dilution) opened K_Ca3.1 in 5/5 cells tested (baseline current at +40 mV 5.8±1.3 pA, baseline Vm −17.8±3.2 mV; post anti-IgE current 54.2±7.6 pA, Vm −62.4±3.0 mV). PGE₂ (10⁻⁵ M) suppressed the current to 34.3±4.9 pA post PGE₂, p=0.005) (Fig. 5A and B) and produced an associated positive shift in Vm to −52.2±4.1 mV, p=0.051) (Fig. 5C). This suppressive effect of PGE₂ was partially reversed by AH6809 (current 48.3±6.9 pA, p=0.011; Vm −66.6±4.0 mV, p=0.041) (Fig. 5A and C).

Figure 5.

The effect of 10⁻⁵ M PGE₂ on K_Ca3.1 currents elicited by anti-IgE-dependent mast cell activation. (A) A representative HLMC demonstrating the development of a K_Ca3.1 current following anti-IgE-dependent activation, suppression of this by 10⁻⁵ M PGE₂, and reversal of the PGE₂-induced suppression by the EP_1/2 receptor antagonist AH6809. (B) K_Ca3.1 current measured at +40 mV after addition of anti-IgE, suppression by 10⁻⁵ M PGE₂ and then reversibility of suppression following addition of AH6809 (n=5 cells). (C) Whole-cell current reversal potential (Vm) after the addition of anti-IgE, a depolarising positive shift in response to 10⁻⁵ M PGE₂, and then reversibility following addition AH6809 (n=5 cells).

PGE₂ suppresses HLMC migration through the EP₂ receptor

Conditioned medium from asthmatic ASM, which has been activated with TNFα, IFNγ and IL-1β, mediates HLMC chemotaxis predominantly via the CXCL10/CXCR3 pathway with additional contributions from ligands for CXCR1 and CXCR3 ²⁰. Inhibition of K_Ca3.1 by channel blockers markedly suppresses this HLMC chemotaxis ⁶, as do molecules that close K_Ca3.1 such as adenosine ¹⁰. Migration of HLMC using conditioned medium from asthmatic airway smooth muscle was 2.8±0.9 fold that of medium control (n=4, p=0.046) (Fig. 6A) and this was not inhibited significantly by PGE₂ (Fig. 6B). However, the selective EP₂ agonist butaprost produced marked inhibition of HLMC migration to ASM-conditioned medium (Fig. 6B). Interestingly, PGE₂ was chemotactic on its own, but much less potent than in mouse bone marrow-derived mast cells ¹³ (Fig. 6C). This HLMC chemotactic activity of PGE₂ was markedly increased in the presence of EP_1/2 blockade by AH6809 10⁻⁵ M (Fig. 6D). HLMC migration to both ASM-conditioned medium and PGE₂ itself is therefore attenuated by the EP₂ receptor.

Figure 6.

Inhibition of HLMC chemotaxis through EP₂ receptor activation. (A) HLMC migration using conditioned medium from asthmatic airway smooth muscle as the chemotactic stimulus. (B) HLMC migration is attenuated significantly by butaprost (n=4) but not PGE₂ (n=3) in the presence of ASM-conditioned medium. ^*p<0.05 compared with control (no PGE₂ or butaprost). (C) PGE₂ is chemotactic when present in isolation. ^*p<0.05 compared to control (no PGE₂), n=4. (D) PGE₂-dependent chemotaxis is enhanced in the presence of the EP_1/2 receptor antagonist AH6809 (10⁻⁵ M). ^*p<0.05 compared to control (no AH6809), n=4.

PGE₂ attenuates histamine release from cultured HLMC

PGE₂ inhibits the degranulation of HLMC freshly isolated from lung tissue. Because the cells used in this study were cultured in stem cell factor (SCF), IL-6 and IL-10, we investigated whether PGE₂ also inhibits release from these HLMC. In keeping with observations in freshly isolated HLMC, PGE₂ inhibited histamine release dose dependently over the concentration range 10⁻⁹–10⁻⁶ M (control mean±s.e.m. net histamine release 19.8±3.7% versus 7.5±3.3% with 10⁻⁶ PGE₂, n=4 donors, p=0.046).

K_Ca3.1 is not modulated by G_i or G_q-coupled receptors

We have now identified three distinct G_s-coupled receptors that close K_Ca3.1 in HLMC when exposed to the relevant ligands, and in the case of the β₂-adrenoceptor, the inverse agonist ICI 118551 opens the channel ⁹. In contrast, the G_i-coupled CXCR3 receptor does not couple directly to K_Ca3.1 ⁶. We have therefore investigated further G_i and G_q agonists known to have biological effects on HLMC. Platelet activating factor (PAF, 10⁻⁷ M) (G_i), lysophosphatidic acid (LPA, 10⁻⁵ M) (G_q,12/13,i) and UTP (10⁻⁴ M)(G_q) did not open K_Ca3.1 in resting HLMC (n=≥6 cells for each agonist) and did not close K_Ca3.1 that had been opened by 1-EBIO (n=≥5 cells for each agonist)(data not shown).

Discussion

In this study we have examined the effects of EP₂ receptor activation on K_Ca3.1 ion channel function in HLMC. In keeping with the known effects of the β₂-adrenoceptor and A_2A adenosine receptor ^{9, 10}, the G_s-coupled EP₂ receptor also closes this channel reversibly. Consistent with this effect on the channel, EP₂ receptor activation attenuates HLMC migration, and in consequence masks the potential chemotactic activity of PGE₂ on HLMC, which is particularly evident when the EP₂ receptor is blocked pharmacologically.

PGE₂ closed K_Ca3.1 reversibly following IgE-dependent mast cell activation demonstrating physiological relevance. PGE₂ also closed K_Ca3.1 channels that had been activated by the K_Ca3.1 opener 1-EBIO. The inhibition of K_Ca3.1 by PGE₂ was reversed both by removing it from the recording solution, and by the addition of the competitive EP₁ and EP₂ receptor antagonist AH6809 indicating a receptor-mediated mechanism. Since the effects of PGE₂ were mimicked by the specific EP₂ receptor agonist butaprost but not the EP₁ agonist 17-phenyl trinor PGE₂, and no effects of native PGE₂ were seen in the presence of the EP_1/2 receptor antagonist AH6809, we have firm evidence that the suppression of K_Ca3.1 by PGE₂ is mediated via the EP₂ receptor.

Because 1-EBIO opens K_Ca3.1 directly in resting cells by increasing its affinity for Ca^{2+ 19}, it suggests that there is tight coupling between the EP₂ receptor and the channel rather than modulation of intracellular signalling pathways. The ability of PGE₂ to close K_Ca3.1 is in keeping with our previous observations that this channel is closed by G_s-coupled β₂-adrenoceptors ⁹ and G_s-coupled adenosine A_2A receptors ¹⁰. These effects on K_Ca3.1 are not mimicked by cAMP analogues or the activator of adenylate cyclase forskolin ⁹, and considering they are seen in whole-cell configuration of the patch-clamp technique, indicate that the most likely mechanism of action is membrane-delimited involving the G_αs or βγ subunits of these GPCR. This view is further supported by the observation that the β₂-adrenoceptor inverse agonist ICI-118551 actually opens K_Ca3.1 ⁹, but G_i agonists such as CXCL10 and PAF, which lower intracellular cAMP, do not directly activate this channel ⁶.

The ability of GPCR to modify K_Ca3.1 appears to be limited to G_s-coupled receptors as we have not found any evidence that G_i, G_q, or G_12/13-coupled receptors modify K_Ca3.1 activity. In particular blockade of EP₁ and EP₂ in this study did not uncover any G_i-coupled EP₃ effects, and GPCR agonists active on human mast cells such as CXCL10 (G_αi) ⁶, PAF (G_i), LPA (G_{q, −12/13 and i}), and UTP (G_αq P2Y2 receptor) do not open or close K_Ca3.1 in HLMC. It is well established using cell attached, inside-out and outside patch-clamp recording that most classes of GPCR including G_αs couple directly to ion channels through membrane-delimited mechanisms ^{21, 22}. This may lead to either channel opening or channel closing depending on the channel and receptor, and may utilise either the G_α component or specific combinations of βγ subunits ^{21, 22}. A further level of specificity between receptor and channel is likely to be achieved by the close approximation of these proteins in tight membrane-restricted signalling complexes. For example, the β₂ adrenoceptor that can modify both K_Ca1.1 and voltage-gated Ca²⁺ channel gating is associated with these two channels in a macromolecular complex held together by A-kinase-anchoring proteins ²³. It is therefore interesting to speculate that K_Ca3.1 also localises with the various G_αs receptors that modify its function. This and the exact mechanism by which G_αs modifies K_Ca3.1 function will be an important area for future research.

Several molecules that attenuate HLMC secretion including PGE₂, adenosine and β₂-adrenoceptor agonists increase intracellular cAMP ²⁴. The generally held view is that this increase in intracellular cAMP couples to inhibition of secretion, supported by the observation that cAMP analogues and non-specific inhibitors of adenylate cyclase can also attenuate secretion from HLMC ²⁴. However, no mechanism has been identified in mast cells that explains how increases in cAMP inhibit the secretory pathway, and the exclusive role of cAMP in the inhibition of other systems such as smooth muscle relaxation has been challenged ²⁵. Opening of K_Ca3.1 enhances IgE-dependent Ca²⁺ influx and degranulation to a submaximal stimulus ⁸, and its blockade by charybdotoxin attenuates this ⁷. Thus, while cAMP plays some role in the inhibition of mast cell mediator release, the demonstration that PGE₂ also closes K_Ca3.1 supports the view that cAMP-independent, K_Ca3.1-dependent mechanisms also contribute in part to EP₂-dependent inhibition of HLMC mediator release.

In many diseases the recruitment of mast cells to key tissue structures appears critical for their pathophysiological effects ^{4, 26, 27}. For example, the contribution of mast cells to the disordered airway physiology of asthma is undoubtedly facilitated by their migration into the airway epithelium ³, submucosal glands ⁵ and ASM ⁴. Inhibition of their migration and subsequent microlocalisation within these structures might therefore offer a novel approach to therapy. Blockade of K_Ca3.1 markedly inhibits HLMC migration in response to a number of diverse chemotactic stimuli including conditioned medium from activated asthmatic ASM ⁶. The ability of PGE₂ to close K_Ca3.1 suggested that it should also inhibit HLMC migration. However, its ability to inhibit HLMC migration in response to ASM-conditioned medium was variable and did not reach statistical significance. In contrast, the selective EP₂ receptor agonist butaprost, which was more potent at closing K_Ca3.1 than PGE₂, produced a marked and consistent inhibition of HLMC migration. PGE₂ was also chemotactic at high concentrations when used alone, but was more potent as a chemoattractant when EP₂ receptors were blocked. This indicates that there are competing pro-migratory and anti-migratory signals when PGE₂ is present, mediated by the EP₂ receptor (inhibitory) and most probably the EP₃ receptor (pro-migratory). These findings are in marked contrast to those in mouse bone marrow-derived mast cells in which PGE₂ in isolation is a potent chemoattractant. This chemotactic activity is mediated through the EP₃ receptor, and the lack of inhibition can be attributed to the absence of EP₂ receptors in mouse bone marrow-derived mast cells ¹³. In human cord blood-derived mast cells, which express both EP₂ and EP₃ receptors, PGE₂ is not a chemoattractant ¹³, compatible with an inhibitory role for the EP₂ receptor. In human lung therefore, PGE₂ is not likely to act as a HLMC chemoattractant, in keeping with its anti-inflammatory activities in this tissue ¹⁸.

In humans PGE₂ inhibits the early and late asthmatic airway response to allergen challenge ¹⁸. The ability of PGE₂ to close K_Ca3.1 provides a mechanism through which it is able to achieve these effects. It will be of great interest to investigate whether PGE₂ has similar effects on lung T-cell K_Ca3.1 function, cytokine secretion and migration, and whether it can inhibit K_Ca3.1-dependent ASM proliferation ²⁸. Drugs that block K_Ca3.1 are also in development as anti-inflammatory treatments ²⁹. Our demonstration that the EP₂ receptor closes K_Ca3.1 in HLMC provides further support for the development of specific EP₂ receptor agonists for the treatment of mast cell-mediated pulmonary disease.

Materials and methods

Reagents

We used the following reagents: SCF, IL-6 and IL-10 (R&D, Abingdon, UK); goat polyclonal anti-human IgE, PGE₂, AH6809, butaprost, PAF, LPA, UTP (Sigma, Poole, Dorset, UK); 17-phenyl trinor PGE₂ (Cayman Chemical Company, Ann Arbor, Michigan, US); mouse IgG₁ mAb YB5.B8 (anti-CD117) (Cambridge Bioscience, Cambridge, UK); sheep anti-mouse IgG₁ Dynabeads (Dynal, Wirral, UK); Dulbecco's Modified Essential Medium (DMEM)/glutamax/HEPES, antibiotic/antimycotic solution, MEM non-essential aminoacids, and fetal calf serum (Life Technologies, Paisley, Scotland, UK).

Human mast cell purification and culture

All human subjects gave written informed consent, and the study was approved by the Leicestershire Research Ethics Committee. HLMC were dispersed and purified from macroscopically normal lung (n=14 donors) obtained within 1 h of resection for lung cancer using immunomagnetic affinity selection as described previously ³⁰. Final mast cell purity determined by Kimura stain was >99% and viability determined by Trypan blue was >97%. HLMC were cultured in DMEM/glutamax/HEPES containing antibiotic/antimycotic solution, non-essential amino acids, 10% fetal calf serum, 100 ng/mL SCF, 50 ng/mL IL-6, and 10 ng/mL IL-10 for up to 10 wk as described previously ^{8, 31}.

Electrophysiology

The whole-cell variant of the patch-clamp technique was used ^{7, 32}. Patch pipettes were made from borosilicate fibre-containing glass (Clark Electromedical Instruments, Reading, UK), and their tips were heat polished, typically resulting in resistances of 4–6 MΩ. The standard pipette solution contained (in mM) KCl, 140; MgCl₂, 2; HEPES, 10; Na⁺-ATP, 2; GTP, 0.1 (pH 7.3). The standard external solution contained (in mM) NaCl, 140; KCl, 5, CaCl₂, 2; MgCl₂,1; HEPES, 10 (pH 7.3). For recording, mast cells were placed in 35-mm dishes containing standard external solution. Whole-cell currents were recorded using an Axoclamp 200A amplifier (Axon Instruments, Foster City, CA, USA), and currents were evoked by applying voltage commands to a range of potentials in 10 mV steps from a holding potential of −20 mV. The currents were digitised (sampled at a frequency of 10 kHz), stored on computer, and subsequently analysed using pClamp software (Axon Instruments). Capacitance transients were minimised using the capacitance neutralisation circuits on the amplifier. Correction for series resistance was not routinely applied. Experiments were performed at 27°C, and the temperature controlled by a Peltier device. Experiments were performed with a perfusion system (Automate Scientific, San Francisco, CA) to allow solution changes, although drugs were added directly to the recording chamber.

PGE₂ was dissolved in ethanol to give a stock solution of 10⁻² M. Thus, at the maximal concentration PGE₂ used (10⁻⁵ M), the concentration of ethanol in the recording chamber was 0.1%. This concentration of ethanol did not affect K_Ca3.1 currents when tested in isolation.

HLMC chemotaxis

HLMC chemotaxis assays were performed using the Transwell system (BD Biosciences, Oxford, UK) with 24-well plates as described previously ^{6, 20}. Conditioned medium from asthmatic ASM that had been activated with TNFα, IL-1β and IFNγ was placed in the lower wells as described previously ²⁰, with appropriate cytokine containing medium in the negative control. PGE₂ was added to the bottom wells in the concentration range 10⁻⁶–10⁻³ M. A total of 1×10⁵ HLMC in 100 µL were added to the top well. After incubating the cells for 3 h at 37°C, we counted the number of HLMC in the bottom well using Kimura stain in a haemocytometer. HLMC migration was calculated as the fold increase of migrated cells in the test wells compared with the negative control (no chemoattractant in the lower well) as described previously ^{6, 20}.

Data presentation and statistical analysis

Data are expressed as mean±SEM. unless otherwise stated. Differences between groups of data were explored using Student's paired or unpaired t-test (two-tailed) as appropriate.

Glossary

Abbreviations

ASM

airway smooth muscle

EP

E prostanoid receptor

1-EBIO

1-ethyl-2-benzimidazolinone

GPCR

G protein-coupled receptor

HLMC

human lung mast cell

LPA

lysophosphatidic acid

PAF

platelet activating factor

PG

prostaglandin

Conflict of interest:

The authors declare no financial or commercial conflict of interest.