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1 Department of Physiology & Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota, United States
2 Department of Anesthesiology, Mayo Clinic College of Medicine, Rochester, Minnesota, United States
3 Department of Anesthesiology, Mayo Clinic College of Medicine, Rochester, Minnesota, United States; Department of Physiology & Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota, United States
4 Department of Physiology & Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota, United States; Department of Anesthesiology, Mayo Clinic College of Medicine, Rochester, Minnesota, United States
* To whom correspondence should be addressed. E-mail: sieck.gary{at}mayo.edu.
Regulation of intracellular Ca2+ ([Ca2+]i) in airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca2+ release and reuptake via sarco-endoplasmic reticulum Ca2+ 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 Ca2+ indicator fluo-3 and imaged using fluorescence microscopy, the effects of PLN siRNA, W7 and KN62 on [Ca2+]i responses to ACh and direct SR stimulation were measured. PLN siRNA slowed the rate of fall of [Ca2+]i 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 [Ca2+]i oscillations (previously shown to represent repetitive SR Ca2+ release/reuptake). However, once ACh-induced [Ca2+]i oscillations reached steady-state, subsequent exposure to W7 or KN62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca2+]i transients, suggesting SERCA inhibition. Exposure to W7 completely abolished ongoing ACh-induced [Ca2+]i oscillations. Pre-exposure to W7 or KN62 did not affect SR Ca2+ 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 Ca2+ reuptake, potentially through altered PLN phosphorylation.
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