Characterisation of receptor-operated calcium influx pathways in human airway smooth muscle cells R. L. Corteling1, S. Li2, J. Westwick2, C. Poll2 and I. P Hall1. 1Division of Therapeutics and Molecular Medicine, University Hospital, Queens Medical Centre, Nottingham, NG7 2UH, UK. 2Novartis Respiratory Research Centre, Wimblehurst Road, Horsham, RH12 5AB, UK.

Print abstract Search PubMed for: Corteling RL Li SW Westwick J Poll CP Hall IP

Many bronchoconstrictors activate G protein-coupled receptors on the surface of airway smooth muscle cells. The coupling of histamine and bradykinin to their specific receptors leads to the activation of phospholipase C and the production of inositol-1,4,5-trisphosphate and diacylglycerol. This in turn induces a biphasic increase in intracellular Ca²⁺ concentration, characterised by the rapid release of Ca²⁺ from the intracellular stores and a sustained influx of Ca²⁺ by an unknown mechanism from the extracellular environment. We therefore set out to investigate the basic pharmacological characteristics of the receptor activated Ca²⁺ influx pathway in primary human airway smooth muscle (HASM) cells when stimulated with histamine or bradykinin.

HASM cells, (passage 3-6) were plated (1x10⁴ per well, 100µl), in black walled clear bottom 96 well tissue culture treated plates and loaded with Fluo-4AM (DMEM, 10% FCS, 10% pluronic acid, 2.5mM probenecid) for 1hour at room temperature. The cells were then washed twice with HBSS (Hanks balanced salt solution) containing 0.1mM CaCl₂and 1mM MgCl₂ and allowed to stand for 30 minutes prior to stimulation. Fluorescence intensity was then continuously recorded at 485nm excitation, and 520nm emission with programmed drug additions using a Flexstation (Molecular Devices). Using a Ca²⁺ re-addition protocol a baseline was established for the first 15 seconds after which test inhibitor (SK&F 96365, LOE 908, La³⁺, Gd³⁺) or vehicle was incubated with the cells for 3 minutes, prior to stimulation with a maximal effective concentration of histamine (100µM) or bradykinin (1µM). 2mM CaCl₂ (final concentration) was then added back to the buffer 4 minutes after agonist addition, to identify any influx from extracellular sources. Ca²⁺ influx was estimated by measuring the area under the curve (AUC) after Ca²⁺ re-addition. For experiments with the lanthanides (La³⁺ and Gd³⁺) the HBSS buffer was replaced with a phosphate free buffer. SK&F 96365 (Calbiochem, UK), lanthanides (Sigma, UK) and LOE 908 synthesised by Quchem, Northern Ireland.

Both La³⁺ and Gd³⁺ inhibited the influx of Ca²⁺ into HASM cells stimulated by either histamine or bradykinin to the same degree. SK&F 96365 however completely abolished histamine-induced Ca²⁺ influx at 10mM, but only partially blocked bradykinin-induced Ca²⁺ influx at the same concentration (Table 1).

Table 1- IC50 values generated, when possible, against histamine and bradykinin-induced Ca²⁺ influx in HASM cells. Data expressed as mean±s.e.m (n=4). * Indicates partial inhibition only, expressed as a percentage of maximum.

The apparent differential sensitivity to SK&F 96365 may reflect that histamine and bradykinin activate distinct Ca²⁺ influx pathways in HASM cells, which is currently being investigated further.