Regulation of sarcoplasmic reticulum Ca^sup 2+^ reuptake in porcine airway smooth muscle

Regulation of intracellular Ca... (...) in airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca... release and reuptake via sarco-endoplasmic reticulum Ca... ATPase (SERCA). We examined regulation of SERCA in porcine ASM, hypothesizing that the regulatory protein phospholamban (PLN) and calmodulin (CaM)-CaM kinase (CaMKII) pathway play a role. In porcine ASM microsomes, we examined expression and extent of PLN phosphorylation following pharmacological inhibition of CaM (W7) vs. CaMKII (KN62/KN93), and found that PLN is phosphorylated by CaMKII. Using enzymatically-dissociated single ASM cells loaded with the Ca... indicator fluo-3 and imaged using fluorescence microscopy, the effects of PLN siRNA, W7 and KN62 on ... responses to ACh and direct SR stimulation were measured. PLN siRNA slowed the rate of fall of ... transients, as did both W7 and KN62. The two inhibitors additionally slowed reuptake in the absence of PLN. Pre-exposure to W7 or KN62 did not prevent initiation of ACh-induced ... oscillations (previously shown to represent repetitive SR Ca... release/reuptake). However, once ACh-induced ... oscillations reached steady-state, subsequent exposure to W7 or KN62 decreased oscillation frequency and amplitude and slowed the fall time of ... transients, suggesting SERCA inhibition. Exposure to W7 completely abolished ongoing ACh-induced ... oscillations. Pre-exposure to W7 or KN62 did not affect SR Ca... release induced by caffeine, indicating that RyR channels were not directly inhibited. These data indicate that in porcine ASM, the CaM-CaMKII pathway regulates SR Ca... reuptake, potentially through altered PLN phosphorylation. (ProQuest: ... denotes formulae/symbols omitted.)