Cyclic nucleotide regulation of store-operated Ca2+influx in airway smooth muscle

Abstract

Sarcoplasmic reticulum (SR) Ca2+release and plasma membrane Ca2+influx are key to intracellular Ca2+([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+depletion triggers influx via store-operated Ca2+channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+influx in enzymatically dissociated porcine ASM cells. SR Ca2+was depleted by 1 μM cyclopiazonic acid in 0 extracellular Ca2+([Ca2+]o), nifedipine, and KCl (preventing Ca2+influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]oand characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 μM isoproterenol or 100 μM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 μM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 μM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+influx was dependent on the extent of SR Ca2+depletion, sensitive to Ni2+and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+influx in ASM triggered by SR Ca2+depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., β-agonists vs. nitric oxide).