LY294002, but not wortmannin, increases intracellular calcium and inhibits calcium transients in bovine and human airway smooth muscle cells

Abstract

To characterize the effect that a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002, has on cytosolic calcium concentrations ([Ca 2+] i), bovine airway smooth muscle cells (BASMC) and cultured human bronchial smooth muscle cells (HBSMC) were loaded with fura 2-AM, imaged as single cells and [Ca 2+] i measured ratiometrically. LY294002 (50 μM) increased [Ca 2+] i by 294±76 nM ( P<0.01, n=13) and 230±31 nM ( P<0.001, n=10) in BASMC and HBSMC, respectively, and increases occurred in the absence of extracellular calcium. In contrast, after pre-treatment with thapsigargin, LY294002 no longer increased [Ca 2+] i. This calcium mobilization by LY294002 was associated with a significant functional effect since LY294002 also inhibited calcium transients to carbachol (45±23 nM), caffeine (45±32 nM), and histamine (20±22 nM), with controls of 969±190, 946±156, and 490±28 nM, respectively. Wortmannin, a different PI3-kinase inhibitor, neither increased [Ca 2+] i nor inhibited transients. Also, LY294002 increased [Ca 2+] i in the presence of wortmannin, U-73122, and xestospongin C. We concluded that LY294002 increased [Ca 2+] i, at least in part, by mobilizing intracellular calcium stores and inhibited calcium transients. The effects of LY294002 on [Ca 2+] i were not dependent on wortmannin-sensitive PI3-kinases, phospholipase C, or inositol trisphosphate receptors (IP 3R). For BASMC and HBSMC, LY294002 has effects on calcium regulation that could be important to recognize when studying PI3-kinases.