Mechanisms of interleukin-4 effects on calcium signaling in airway smooth muscle cells.
In airway smooth muscle cells, interleukin (IL)-4 inhibited both carbachol- and caffeine-induced calcium mobilization from the sarcoplasmic reticulum (SR). Because of the known signaling pathways for IL-4 and importance of calcium uptake in maintaining SR calcium stores shared by agonists and caffeine, it was hypothesized that this rapid inhibitory effect might depend on phosphatidylinositol 3-kinase (PI3K) and on inhibition of calcium uptake by the SR. Enzyme-dispersed bovine trachealis cells were loaded with Fura-2/acetoxymethyl ester, and changes in cytosolic calcium were imaged in single cells. Cells were pretreated with inhibitors of PI3K, either wortmannin (100 nM), LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] (50 microM), or deguelin (100 nM). Calcium transients in response to carbachol (10 microM) were significantly decreased to 0.34 +/- 0.10 of control after 20-min treatment with IL-4 but were 1.10 +/- 0.26 and 1.08 +/- 0.23 when wortmannin or deguelin, respectively, was added along with IL-4. LY294002 alone had nonspecific effects on transients. In other experiments, cyclopiazonic acid (CPA) (5 microM), an inhibitor of SR calcium uptake, decreased carbachol-stimulated transients within 4 min to 0.83 +/- 0.08 of control (n = 6). However, for cells treated with IL-4 (50 ng/ml) plus CPA, transients decreased significantly more, to only 0.51 +/- 0.05 (n = 6; p < 0.05). Longer exposures to IL-4 and a higher concentration of CPA (30 microM) gave similar results. It was concluded that IL-4 did not inhibit transients in the presence of PI3K antagonists but that it did in the presence of CPA. This suggested that IL-4 inhibited calcium transients by mechanisms dependent upon a wortmannin-sensitive PI3K but not by inhibition of calcium uptake into the SR.
Ethier
Michael F
MF
Department of Medicine, LRB Room 319, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605, USA.
Cappelluti
Erika
E
Madison
J Mark
JM
D000818:Animals; D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002469:Cell Separation; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D066298:In Vitro Techniques; D015847:Interleukin-4 / Q000494:pharmacology*; D018721:Muscarinic Agonists / Q000494:pharmacology; D009130:Muscle, Smooth / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012137:Respiratory System / Q000166:cytology* / Q000187:drug effects; D012519:Sarcoplasmic Reticulum / Q000187:drug effects; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D000252:Calcium-Transporting ATPases; D002217:Carbachol; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D016257:Fura-2; D015847:Interleukin-4; D018721:Muscarinic Agonists; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases