Pharmacological characterisation of paf-stimulated Ca2+ responses in human lung interstitial macrophages

P. Bahra, T.K. Finney-Hayward, L.E. Donnelly, C.T. Poll, J. Westwick & S. Li. Novartis Institutes for Biomedical Research, UK and National Heart & Lung Institute, London, UK.

Macrophages are reported to play a major role in the inflammation observed in COPD (Tetley, 2002). The inflammatory mediator, platelet-activating factor (PAF), 1-O-Palmityl-2-acetyl-sn-glyceryl-3-phosphorylcholine, stimulates receptor -mediated Ca2+ entry (RMCE) in human alveolar macrophages (Schaber et al., 1991), however to date little is known about Ca2+ entry pathways present in macrophages. The aim of this study was to pharmacologically characterise PAF-stimulated Ca2+ influx in human lung interstitial tissue macrophages (IMs) using the cation channel blockers SK&F 96365, econazole, nifedipine, Gd3+ (Sigma-Aldrich) and LOE908 (QuChem).

IMs were isolated from the human lung tissue of smokers and COPD patients (Smith et al. 2004) and loaded with Fluo-4 (Molecular Probes, 1.5x105 per well in 96 well plates). Changes in intracellular free Ca2+ concentration ([Ca2+]i) in response to PAF stimulation were measured at room temperature using FLIPR (FLuorescence Imaging Plate Reader, Molecular Devices). Experiments were performed in Hanks Buffered Salt Solution (HBSS) except for the studies using Gd3+, where a phosphate-free buffer was used containing in mM concentrations, 140NaCl, 5.4KCl, 1.0MgCl2, 1.2CaCl2, 15HEPES, pH 7.4. Cells were pre-treated with compounds for 2 min prior to stimulation with PAF (1 m M). Data are expressed as mean ±standard error of the mean (sem). Statistical significance was assumed when P<0.05 using a one-way ANOVA with Dunnett’s multiple comparisons test.

PAF concentration-dependently stimulated an increase in IM [Ca2+]i with an EC50 value of 37.4± 5.8nM (n=3, peak increase in [Ca2+]i). The PAF (1 μM) Ca2+ response was inhibited by the PAF receptor antagonist, PCA4248 (Biomol) with an IC50 value of 8.8± 1.9 μM (n=3). As peak increase in [Ca2+]i represents Ca2+ release from intracellular stores, the magnitude of the PAF-evoked Ca2+ influx component was determined from area under the curve (AUC) analysis by measuring the difference between the total PAF-stimulated AUC [Ca2+]i response obtained in the presence and absence of external Ca2+. The Ca2+-chelating agent EGTA (2mM) was used to chelate extracellular Ca2+ and had no effect (102.8± 15.2% of the control, n=4, P>0.05) on PAF induced Ca2+ release from intracellular stores (peak increase in [Ca2+]i). However, EGTA inhibited the PAF AUC [Ca2+]i response by 62.4± 2.5% (n=4, P<0.05) and this represents the Ca2+ influx component. Gd3+ was the most effective Ca2+ influx inhibitor tested. SK&F 96365 and econazole only partially inhibited the PAF Ca2+ influx response whilst the nonselective cation channel blocker LOE908 and the selective L-type voltage operated Ca2+ channel blocker, nifedipine were ineffective (Table 1).

Table 1. Data represent the mean (± sem) % inhibition of the initial peak increase in [Ca2+]i (Peak) and total increase in [Ca2+]i (AUC) following stimulation of IMs with PAF (1μM).

* Indicates P<0.05 versus control PAF response.

These data suggest that RMCE represents a major component of PAF-stimulated increase in [Ca2+]i with differential sensitivity to these cation channel blockers and warrants further investigation .

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