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 microM 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+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 microM isoproterenol or 100 microM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 microM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 microM 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., beta-agonists vs. nitric oxide).
Highlights
In addition to these mechanisms, we recently demonstrated the existence of controlled Ca2ϩ influx in airway smooth muscle (ASM) [2, 22] that occurs via store-operated Ca2ϩ channels [SOCC; termed capacitative Ca2ϩ entry [24, 29, 30]] in response to sarcoplasmic reticulum (SR) Ca2ϩ depletion, allowing for replenishment of intracellular Ca2ϩ stores [2, 22]
Whereas in our previous work on SOCC we demonstrated that Ca2ϩ influx occurs following SR Ca2ϩ depletion either with the sarco(endo)plasmic reticulum Ca2ϩ-ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) or with caffeine, it must be noted that caffeine depletes only ryanodine-sensitive SR Ca2ϩ stores
We demonstrated that cAMP activation via salbutamol, a 2-agonist, inhibits ACh-induced [Ca2ϩ]i oscillations in ASM that occur via repetitive SR Ca2ϩ release and reuptake [28]
Summary
CAMP cross-activation of protein kinase G contributed to SOCC inhibition These data demonstrate that a Ni2ϩ/La3ϩ-sensitive Ca2ϩ influx in ASM triggered by SR Ca2ϩ 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., -agonists vs nitric oxide). We demonstrated that salbutamol, a 2-agonist [28], as well as NO donors [26] inhibit ACh-induced [Ca2ϩ]i oscillations in ASM that occur via repetitive SR Ca2ϩ release and reuptake Both cAMP and cGMP influence the plasma membrane via membrane hyperpolarization [9, 15] and inhibition of Ca2ϩ influx via L-type Ca2ϩ channels [16, 17]. We examined the effect of cAMP and cGMP on SOCC-mediated Ca2ϩ influx in porcine ASM cells
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