Agonist-induced production of inositol phosphates in human airway smooth muscle cells in culture

Abstract

Smooth muscle cells (SMC) from human bronchi were isolated by elastase treatment, subcultured, and characterized by their positive reaction with a monoclonal antibody against α-smooth muscle actin (αSMA). In each cell line tested, at least 95% of the cells were positively stained. The functional properties of these cells were examined by measuring the metabolism of inositol phosphates (IPs). For that purpose, cells were incubated for 3 days before reaching confluency in the presence of myo-[ 3H]inositol in order to label the phosphoinositide pool, and the various [ 3H]IPs were separated by HPLC on a SAX column with a phosphate gradient. IP 1 isomers were separated in three peaks; IP 2, IP 3, IP 4, IP 5 and IP 6 (phytic acid) were each eluted as single peaks. The identity of the [ 3H]peaks was verified with corresponding [ 3H]IP standards. The accumulation of [ 3H]IPs was measured by incubating cells up to 30 min in the presence of 10 mM LiCl, with or without a bronchoconstrictor agent (carbachol, histamine, PGF 2α). Histamine, 10 −4 M, elicited a four times larger IP accumulation than carbachol, 10 −4 M, and than PGF 2α, 5 10 −5 M. Dose-response curves were established for histamine and carbachol in the range 10 −7−10 −4 M. At 10 −7 M, carbachol was more effective than histamine in stimulating the IP metabolism. Atropine blocked the response to carbachol, and diphenhydramine inhibited the effect of histamine, indicating the specificity of the response to the agonists. These results indicate that cultured human bronchial SMC are a suitable preparation for studying physiological aspects of membrane transduction in the airways.