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ß₂-Adrenoceptor regulation of the K⁺ channel iK_Ca1 in human mast cells

Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK

² Correspondence: Department of Respiratory Medicine, Glenfield Hospital, Groby Rd., Leicester LE3 9QP, UK. E-mail: pbradding{at}hotmail.com

SPECIFIC AIMS

Our hypothesis is that drugs that attenuate human mast cell secretion interfere with the ion channel activity required for normal stimulus-secretion coupling by inhibiting prosecretory channel activity or via the opening of antisecretory channels. The aim of this study was to characterize the effects of ß₂-adrenoceptor stimulation on human mast cell ion channel function.

PRINCIPAL FINDINGS

1. Effect of salbutamol on "resting" human mast cells
Electrical currents were measured using the patch clamp technique. Addition of salbutamol to resting human lung mast cells (HLMC) and human peripheral blood-derived mast cells (HPBDMC) did not induce ion channel activity (n=13). In contrast, salbutamol (1–10 µM) produced a dose-dependent and reversible closure of the intermediate conductance Ca²⁺-activated K⁺ channel iK_Ca1 (also known as K_Ca3.1) in one of two cells expressing iK_Ca1 currents at baseline (Fig. 1 A).

View larger version (23K): [in this window] [in a new window]   Figure 1. Current-voltage curves of a HPBDMC demonstrating an iKCa1 current at rest, and its reversible suppression by salbutamol (A), and a HLMC activated by anti-IgE, demonstrating suppression of the iKCa1 current by salbutamol and its reversal by ICI 118551 (B).

2. Salbutamol closes the iK_Ca1 channel after IgE-dependent activation
Salbutamol 10 µM inhibited iK_Ca1 activity after IgE-dependent activation by >50% in 3/3 cells tested (current at +40 mV post-anti-IgE 73.4±27.8 pA, post-salbutamol 30.7±11.0 pA, P=0.13). There was a corresponding positive shift in reversal potential (Vm) (Vm post-anti-IgE –45.3±7.7 mV, post-salbutamol –29.2±4.6 mV, P=0.037). This was reversed by the competitive ß₂-adrenoceptor antagonist and inverse agonist ICI 118551 (1 µM) in 2/2 cells tested (Fig. 1B ), suggesting that the effect of salbutamol was mediated via the ß_2-adrenoceptor.

3. Salbutamol closes iK_Ca1 in the presence of the iK_Ca1 opener 1-EBIO
Because salbutamol might potentially inhibit many cell activation pathways that could reduce cytosolic free Ca²⁺ and thus reduce iK_Ca1 activity indirectly, we studied the effects of salbutamol on iK_Ca1 currents induced by the iK_Ca1 opener 1-EBIO. Addition of salbutamol (1–10 µM) to HLMC and HPBDMC in which the iK_Ca1 channel had been activated by 1-EBIO produced a dose-responsive inhibition of channel activity with an associated positive shift in membrane potential. Salbutamol produced a >20% inhibition of current in 20/27 cells (for all cells tested current at +40 mV post-EBIO 84.3±13.8 pA, post-salbutamol 41.8±6.45 pA, P=0.003). There was a corresponding positive shift in reversal potential (Vm post-EBIO –44.1±3.4 mV, post-salbutamol –27.0±3.6 mV, P<0.0001). The effect of salbutamol was reversed within 1 min by removing salbutamol from the recording solution (current post-salbutamol 39.2±17.5 pA, post-wash 103.1±31.6 pA, P=0.05; Vm post-salbutamol –18.0±6.7 mV, post-wash –41.9±7.7 mV, n=6, P=0.04) (Fig. 2 A), indicating that rundown was not responsible for the effects seen. ICI 118551 (0.1–1.0 µM) reversed the effect of salbutamol within 1 min (current post-salbutamol 39.1±10.4 pA, post-ICI 118551 65.2±16.0 pA, P=0.02; Vm post-salbutamol –27.6±7.4 mV, post-ICI 118551 –45.9±6.5 mV, n=7, P=0.03) (Fig. 2B ).

View larger version (21K): [in this window] [in a new window]   Figure 2. Current voltage curves from an HLMC demonstrating suppression of the 1-EBIO-induced iKCa1 current by salbutamol and its reversal after removal of salbutamol from the recording chamber (A) and an HLMC demonstrating suppression of the 1-EBIO-induced iKCa1 current by salbutamol and its reversal by ICI 118551 (B).

4. The ß₂-adrenoceptor inverse agonist ICI 118551 directly opens iK_Ca1
In initial experiments, ICI 118551 directly opened iK_Ca1 in 12/15 mast cells in a partially reversible manner (for all cells tested, baseline current 12.3±3.5 pA, post-ICI 118551 56.4±18.0 pA at +40 mV, P=0.026; baseline Vm –9.4±3.1 mV, post-ICI 118551 –45.8±6.3 mV, P=0.0003). This suggests there is constitutive ß₂-receptor activity in these cells.

5. cAMP manipulation does not modulate iK_Ca1 activity
The stable cAMP analog 8-bromo-cAMP and the adenyl cyclase activator forskolin did not significantly alter iK_Ca1 channel activity (data not shown), suggesting that ß₂-adrenoceptor regulation of iK_Ca1 is unlikely to occur through regulation of cAMP and protein kinase A.

6. Regulation of iK_Ca1 by ICI 118551 is mediated via the Gs but not the Gi G-protein
The inhibitory effects of ß-agonists on human mast cell secretion are believed to be mediated via Gs G-protein signaling. ICI 118551 is a selective antagonist and inverse agonist at the ß₂-adrenoceptor, but has been shown to signal through Gi in the heart. The response to ICI 118551 was identical in the presence of pertussis toxin (PTX) but significantly attenuated in the presence of cholera toxin (CTX). After addition of ICI 118551, iK_Ca1 currents were present in 10/11 control cells, 12/15 PTX-treated cells, but only 3/10 CTX-treated cells (P=0.005 by Chi-square). Across-group analysis by the Kruskall Wallis test for nonparametric data revealed significant differences for current recorded at +40 mV (P=0.029) and reversal potential (P=0.018). These results suggest that regulation of iK_Ca1 by ß₂-adrenoceptors in HLMC occurs through agonism/inverse agonism of Gs.

DISCUSSION

We tested the novel hypothesis that pharmacological inhibition of human mast cell activation interferes with ion channel function in these cells. We show for the first time that the iK_Ca1 ion channel is coupled to the ß₂-adrenergic G-protein-coupled receptor, an effect mediated by Gs but not cAMP. Thus ß₂-adrenoceptor activation results in closure of the prosecretory iK_Ca1 channel in human mast cells.

We chose the ß₂-adrenergic agonist salbutamol as a model to test our hypothesis. ß₂-adrenergic agonists are relatively potent inhibitors of HLMC activation when applied acutely in vivo and in vitro. Salbutamol closed iK_Ca1 after IgE-dependent activation and after its activation by the iK_Ca1 opener 1-EBIO, thus excluding an indirect effect through the perturbation of IgE-dependent cell signaling. iK_Ca1 suppression by salbutamol was reversed by removing the drug from the recording solution and by the addition of the competitive ß₂-adrenoceptor antagonist and inverse agonist ICI 118551, indicating the effect of salbutamol was ß₂-adrenoreceptor mediated. ICI 118551 opened iK_Ca1 directly in "resting" cells, further indicating that the iK_Ca1 channel is coupled to the ß₂-adrenoceptor.

The mechanism whereby ß₂-adrenergic agonists exert their acute mast cell antisecretory effect is assumed to be mediated via elevation of intracellular concentrations of cAMP, in common with other cAMP-elevating agents such as prostaglandin E₂. However, the mechanism by which elevated cAMP couples to inhibition of secretion from HLMC is not known, and its exclusive role in the inhibition of mast cell degranulation and promotion of smooth muscle relaxation has recently been challenged. iK_Ca1 opening enhances IgE-dependent Ca²⁺ influx and degranulation; its blockade attenuates this. Our current data show that salbutamol closes iK_Ca1, providing for the first time a clearly defined mechanism by which ß₂-adrenoceptor stimulation can be linked to attenuated secretion.

Our data suggest that the effect of salbutamol on HLMC iK_Ca1 is mediated via Gs independent of cAMP, constituting a fundamental shift in our understanding of how ß₂-adrenoceptors contribute to mast cell "stabilization." There are several potential mechanisms independent of cAMP whereby the ß₂-adrenoceptor could modulate iK_Ca1. The "simplest" mechanism would involve a direct membrane-delimited action of Gs or ß subunits on the channel leading to closure, an effect antagonized by the inverse agonist ICI 118551. Such a mechanism has been postulated for the action of the ß₂-receptor on other channels such as voltage-gated Ca²⁺ channels and the large conductance Ca²⁺-activated K⁺ channel bK_Ca, which are closed and opened, respectively, by ß₂-adrenoceptor activation. Whether Gs in HLMC modifies iK_Ca1 affinity for Ca²⁺ or alters its gating properties, and/or involves phosphomodulation via activation of enzymes such as MAP kinases awaits further work.

It is well recognized that ß₂-adrenoceptors and other GPCRs exhibit some degree of intrinsic activity. Evidence for this in native leukocytes is absent, but the ability of ICI 118551 to open iK_Ca1 in "resting" HLMC indicates they exhibit resting ß₂-adrenergic "tone." ICI 118551 has been shown to signal through Gi in the heart, but our data indicate that ICI 118551 was not operating through Gi but through true inverse agonism via Gs. This intrinsic ß₂-adrenoceptor activity in HLMC raises the possibility that this contributes to the variable secretory response to IgE-dependent activation seen with different HLMC preparations and the variable inhibitory response of ß₂-agonists. The exaggerated airway response to bronchial allergen challenge that occurs with chronic dosing of salbutamol can also be explained by a degree of intrinsic ß₂-receptor activity in HLMC, which exerts a small protective effect at baseline but is lost with receptor down-regulation during chronic exposure.

The pharmacological screening of ß₂-agonists has largely revolved around analyzing their effect on cAMP or bronchodilation, but screening alternative outputs such as iK_Ca1 closure could identify compounds with relatively novel activity within the airways. The observation that salbutamol closes iK_Ca1 strengthens the hypothesis that iK_Ca1 is a potential target for the treatment of asthma. Not only does this channel modify mast cell secretion, but its opening promotes T cell proliferation and cytokine secretion. Although blockade is often highly effective in modifying cell behavior, there can be disadvantages. There may be some cases where it may be preferential to inhibit iK_Ca1 in mast cells but perhaps not T cells. Understanding the mechanisms regulating iK_Ca1 gating in different immune and structural cells therefore offers the potential to identify drugs that modulate its activity selectively within certain cell types. Examining whether ß₂-adrenoceptors modify iK_Ca1 function in other cell types and whether iK_Ca1 is modulated by other GPCRs will be of great interest.

Since mast cells recover and regranulate after IgE-dependent activation, we have proposed a hypothetical cycle of electrical activity analogous to that in excitable tissue taking the cell from rest, through activation and back to rest (Fig. 3 ). This is potentially applicable to all nonexcitable cells such as leukocytes. Figure 3 summarizes our evidence based on channels and currents expressed by human mast cells and demonstrates how data regarding the ß₂-adrenoceptor feeds into this.

View larger version (27K): [in this window] [in a new window]   Figure 3. Simplified human mast cell electrical "excitation" cycle based on currents and channels expressed by human mast cells, demonstrating the role of iKCa1 in human lung mast cell activation and its regulation by ß-adrenergic receptors. In disease, the cells may become hyperreactive due to the side cycle indicated by the red arrows, due to positive feedback between iKCa1 and the store-operated Ca2+ channels/currents (SOCC/ICRAC). ß2-Adrenoceptors have the ability to interrupt this cycle through the Gs-mediated closure of iKCa1. The delayed Cl– current might attenuate this process by producing membrane depolarization. SOCC, store-operated Ca2+ channels; ICRAC, Ca2+ release-activated Ca2+ current; TRP, transient receptor potential channels; ELG, extracellular ligand-gated channels (e.g., P2X family).

In summary, we have demonstrated that the ß₂-adrenergic receptor is coupled to the intermediate conductance Ca²⁺-activated K⁺ channel iK_Ca1 in human mast cells through a Gs-mediated mechanism independent of cAMP. This may explain in part how ß₂-agonists attenuate human mast cell secretion; it strengthens the hypothesis that iK_Ca1 is a potential target for the treatment of asthma.

FOOTNOTES

¹ Current address: Food Safety and Quality, Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3439fje; doi: 10.1096/fj.04-3439fje

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