Mechanisms of Interleukin-4 Effects on Calcium Signaling in Airway Smooth Muscle Cells

Abstract

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 M), or deguelin (100 nM). Calcium transients in response to carbachol (10 M) were significantly decreased to 0.34 Ϯ 0.10 of control after 20-min treatment with IL-4 but were 1.10 Ϯ 0.26 and 1.08 Ϯ 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 M), an inhibitor of SR calcium uptake, decreased carbachol-stimulated transients within 4 min to 0.83 Ϯ 0.08 of control (n ϭ 6). However, for cells treated with IL-4 (50 ng/ml) plus CPA, transients decreased significantly more, to only 0.51 Ϯ 0.05 (n ϭ 6; p Ͻ 0.05). Longer exposures to IL-4 and a higher concentration of CPA (30 M) 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.T helper-2 cytokines, including IL-4 and the closely related cytokine IL-13, have important and complex effects on airway inflammation and airway cells (Brusselle et al

IL‐4 inhibits calcium transients in bovine trachealis cells by a ryanodine receptor dependent mechanism

“…Theoretically, IL‐4 could decrease calcium in the SR by inhibiting calcium uptake via SERCA and/or by stimulating the release of calcium from the SR. In a prior study of these same cells, IL‐4 inhibited calcium transients in response to carbachol, even in the presence of a high concentration of CPA (30 μM), an inhibitor of SERCA (22). This finding suggested that IL‐4 did not decrease SR calcium content by inhibiting calcium uptake and, therefore, must be increasing the rate of release of calcium from the SR.…”

“…A prior study showed that the phosphatidylinositol 3‐kinase (PI3K) antagonists wortmannin and deguelin both inhibited the effect of IL‐4 on calcium transients (22). Because the current experiments showed that IL‐4 causes a net loss of SR calcium by mechanisms dependent on RyR calcium release channels, the simplest explanation or model for our findings is that IL‐4 stimulates signaling pathways involving PI3K to increase the release of calcium through RyR, thereby causing a net loss of calcium from the SR.…”

“…These observations by others are important because these same concentrations of IL‐4 have been shown to elicit effects in other non‐hematopoietic cell types and IL‐4 is increased in the bronchial lavage fluid of subjects with asthma (20). Evidence also suggests that IL‐4 modulates calcium signaling in airway smooth muscle (21, 22). In single, bovine trachealis cells, IL‐4 (50 ng/ml) had only small effects on cytosolic calcium concentrations ([Ca 2+ ]i) by itself, but yet, over 20 min time, significantly inhibited the magnitudes of both carbachol‐ and caffeine‐stimulated calcium transients (21).…”

“…Because subsequent results showed that IL‐4 does inhibit calcium mobilization by ionomycin, a finding consistent with store depletion, a second goal was to determine the mechanism. Importantly, a prior study showed that an effect of IL‐4 on calcium transients could still be observed in the presence of cyclopiazonic acid (CPA), a selective inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) responsible for uptake of calcium into the SR (1, 22, 26). This observation suggested that IL‐4 does not deplete SR calcium by inhibiting SERCA activity, but, instead, increases the rate of calcium release to cause a net loss of calcium from the SR. That study also showed that the IP3R antagonist xestospongin C did not inhibit the effect of IL‐4, which suggests that RyR and cADPR signaling might be involved in IL‐4 inhibition of calcium transients.…”

Airway Smooth Muscle in Bronchial Tone, Inflammation, and Remodeling

“…Despite such progress, the mechanisms that underlie spontaneous tone in inflammatory bronchoconstriction and alterations in the phenotype of smooth muscle cells that accompany airway inflammation remain unknown. In asthma, evidence suggests that intrinsic alterations in ASM excitation-contraction coupling exist as compared with tissue from healthy subjects (18)(19)(20)(21)(22). Greater focus on the role of intracellular calcium release channels, the importance of the mechanisms of calcium sensitization, and changes of expression of ion channels, receptors, and second messengers is needed in physiologically relevant preparations and in human tissue.…”