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

Departments of ¹Physiology and Biomedical Engineering, and ²Anesthesiology, Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 6 November 2007 ; accepted in final form 28 January 2008

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⁺]_i responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca²⁺]_i transients 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⁺]_i oscillations (which were previously shown to result from repetitive SR Ca²⁺ release/reuptake). However, when ACh-induced [Ca²⁺]_i oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca²⁺]_i transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca²⁺]_i oscillations 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.

sarco(endo)plasmic reticulum calcium-ATPase; phospholamban; calmodulin; calmodulin kinase

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