We have studied the mechanisms of extracellular calcium entry in cultured human airway smooth muscle cells (HASMCs) using the fluorimetric dye fura PE-3 as an intracellular calcium indicator. Our results suggest that three distinct pathways are involved in calcium entry in HASMCs. This study was approved by the Guy’s and St Thomas’ Hospitals’ Research Ethics Committee and informed consent was obtained. The studies were performed on tissue derived from patients undergoing surgery. Results are expressed as means ± S.E.M., and n is the number of cell lines/patients.

Both basal Ca²⁺ influx in HASMCs from early passages, and influx induced by agonists or store mobilisation was insensitive to nifedipine and Ni²⁺, but was blocked by 100 µM SK&F 96, 365 (n = 5-12). The basal Ca²⁺ influx pathway was permeable to Mn²⁺ and Sr²⁺, completely blocked by 1 µM La³⁺, and partially blocked (51.1 ± 11.1 %, n = 4) by 2-APB (the cell-permeant IP₃R function inhibitor/non-store-operated Ca²⁺ entry blocker). SOC could be induced in all cells from all passages by the SERCA inhibitors thapsigargin and cyclopiazonic acid (CPA). The SOC pathway was not permeable to Sr²⁺, and was only partially blocked by 75 µM 2-APB (56 ± 9.6 % n = 3) or 1 µM La³⁺ (48 ± 10.8 %, n = 3). Calcium entry induced by classical agonists (bradykinin, carbachol, histamine, ATP) was not permeable to Sr²⁺, and was only partially blocked by 1 µM La³⁺ (50.3 ± 6.7 %, n = 5), but was abolished in the presence of 75 µM 2-APB. TRP channels are believed to mediate several types of voltage-independent calcium entry, with TRP 1 and 4 commonly associated with SOC, and TRP 3 and 6 with receptor-operated calcium entry. We used RT-PCR to detect which htrp genes were expressed in these cultured HASMCs. Both htrp4 and -6 were expressed in all HASMCs studied, whereas htrp3 was only expressed in some cell lines (3 of 12), and then only in passages later than 5. htrp1 gene expression was only present in ‘leaky’ cells that showed a basal Ca²⁺ influx. There was no detectable expression of htrp2, -5 or -7 in any cells. OAG, which may directly activate TRP3 and -6, was able to induce calcium influx (n = 9), as did the DAG kinase inhibitor R59022. In conclusion, we have found three calcium influx pathways in cultured HASMCs, which can be differentiated in terms of their permeability to Sr²⁺, and block by 2-APB and sub-micromolar lanthanides. Our results allow us to speculate that in these cultured cells TRP4 may underlie SOC, whereas TRP1, when present, may be constitutively active and underlie the basal influx. The data, whilst in no way conclusive, would be consistent with agonist-induced calcium entry being mediated via TRP6.

This work was supported by The Wellcome Trust (062554) and MRC (C.G.M.).