2552 S. Mark Duffy et al. Eur. J. Immunol. 2008. 38: 2548–2556 Figure 4. PGE2 is ineffective in the presence of the AH6809. (A) KCa3.1 current measured at 140 mV after addition of 1-EBIO, stability following addition of AH6809, and failure to suppress following subsequent addition of 10 5 M PGE2. (B) KCa3.1 current measured at 140 mV in resting cells, after addition of AH6809, and then following subsequent addition of 10 5 M PGE2 showing failure of KCa3.1 to open. of KCa3.1 by channel blockers markedly suppresses this HLMC chemotaxis [6], as do molecules that close KCa3.1 such as adenosine [10]. Migration of HLMC using conditioned medium from asthmatic airway smooth muscle was 2.870.9 fold that of medium control (n 5 4, p 5 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 EP1/2 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. 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 mean7s.e.m. net histamine release 19.873.7% versus 7.573.3% with 10 6 PGE2, n 5 4 donors, p 5 0.046). KCa3.1 is not modulated by Gi or Gq-coupled receptors We have now identiﬁed three distinct Gs-coupled receptors that close KCa3.1 in HLMC when exposed to the relevant ligands, and in the case of the b2-adrenoceptor, the inverse agonist ICI 118551 & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 5. The effect of 10 5 M PGE2 on KCa3.1 currents elicited by anti- IgE-dependent mast cell activation. (A) A representative HLMC demonstrating the development of a KCa3.1 current following anti- IgE-dependent activation, suppression of this by 10 5 M PGE2, and reversal of the PGE2-induced suppression by the EP1/2 receptor antagonist AH6809. (B) KCa3.1 current measured at 140 mV after addition of anti-IgE, suppression by 10 5 M PGE2 and then reversibility of suppression following addition of AH6809 (n 5 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 5 cells). opens the channel [9]. In contrast, the Gi-coupled CXCR3 receptor does not couple directly to KCa3.1 [6]. We have therefore investigated further Gi and Gq agonists known to have biological effects on HLMC. Platelet activating factor (PAF, 10 7 M) (Gi), lysophosphatidic acid (LPA, 10 5 M) (Gq,12/13,i) and UTP (10 4 M)(Gq) did not open KCa3.1 in resting HLMC (n 5 Z6 cells for each agonist) and did not close KCa3.1 that had been opened by 1-EBIO (n 5 Z5 cells for each agonist)(data not shown). Discussion In this study we have examined the effects of EP2 receptor activation on KCa3.1 ion channel function in HLMC. In keeping with the known effects of the b2-adrenoceptor and A2A adenosine receptor [9, 10], the Gs-coupled EP2 receptor also closes this channel reversibly. Consistent with this effect on the channel, EP2 receptor activation attenuates HLMC migration, and in conse- www.eji-journal.eu