Abstract
Plasma membrane Ca(2+) influx, especially store-operated Ca(2+) entry triggered by sarcoplasmic reticulum (SR) Ca(2+) release, is a key component of intracellular calcium concentration ([Ca(2+)]i) regulation in airway smooth muscle (ASM). Agonist-induced Ca(2+) oscillations in ASM that involve both influx and SR mechanisms have been previously demonstrated. In nonexcitable cells, [Ca(2+)]i oscillations involve Ca(2+) influx via arachidonic acid (AA) -stimulated channels, which show similarities to store-operated Ca(2+) entry, although their molecular identity remains undetermined. Little is known about AA-regulated Ca(2+) channels or their regulation in ASM. In enzymatically dissociated human ASM cells loaded with the Ca(2+) indicator, fura-2, AA (1-10 μM) triggered [Ca(2+)]i oscillations that were inhibited by removal of extracellular Ca(2+). Other fatty acids, such as the diacylglycerol analog, 1-oleoyl-2-acetyl-SN-glycerol, oleic acid, and palmitic acid (10 μM each), failed to elicit similar [Ca(2+)]i responses. Preincubation with LaCl3 (1 μM or 1 mM) inhibited AA-induced oscillations. Inhibition of receptor-operated channels (SKF96,365 [10 μM]), lipoxygenase (zileuton [10 μM]), or cyclooxygenase (indomethacin [10 μM]) did not affect oscillation parameters. Inhibition of SR Ca(2+) release (ryanodine [10 μM] or inositol 1,4,5-trisphosphate receptor inhibitor, xestospongin C [1 μM]) decreased [Ca(2+)]i oscillation frequency and amplitude. Small interfering RNA against caveolin-1, stromal interaction molecule 1, or Orai3 (20 nM each) reduced the frequency and amplitude of AA-induced [Ca(2+)]i oscillations. In ASM cells derived from individuals with asthma, AA increased oscillation amplitude, but not frequency. These results are highly suggestive of a novel AA-mediated Ca(2+)-regulatory mechanism in human ASM, reminiscent of agonist-induced oscillations. Given the role of AA in ASM intracellular signaling, especially with inflammation, AA-regulated Ca(2+) channels could potentially contribute to increased [Ca(2+)]i in diseases such asthma.