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This Article Full Text Full Text (PDF) All Versions of this Article: 294/4/L787    most recent 00461.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Sathish, V. Articles by Sieck, G. C. PubMed PubMed Citation Articles by Sathish, V. Articles by Sieck, G. C.

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