Regulation of sarcoplasmic reticulum Ca2+reuptake in porcine airway smooth muscle

Abstract

Regulation of intracellular Ca2+concentration ([Ca2+]i) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca2+release and reuptake. The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is key to replenishment of SR Ca2+stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca2+indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca2+]iresponses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca2+]itransients to 1 μM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca2+]ioscillations (which were previously shown to result from repetitive SR Ca2+release/reuptake). However, when ACh-induced [Ca2+]ioscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca2+]itransients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca2+]ioscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca2+release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca2+reuptake, potentially through altered PLN phosphorylation.