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

• Published in: American journal of physiology. Lung cellular and molecular physiology 2008-04, Vol.294 (4), p.L787-L796
• Main Authors: Sathish, Venkatachalem, Leblebici, Figen, Kip, Sertac N., Thompson, Michael A., Pabelick, Christina M., Prakash, Y. S., Sieck, Gary C.
• Format: Article
• Language: English
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.00461.2007

Regulation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca 2+ release and reuptake. The sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) is key to replenishment of SR Ca 2+ 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 Ca 2+ indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca 2+ ] i responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca 2+ ] 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 [Ca 2+ ] i oscillations (which were previously shown to result from repetitive SR Ca 2+ release/reuptake). However, when ACh-induced [Ca 2+ ] i oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca 2+ ] i transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca 2+ ] i oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca 2+ release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca 2+ reuptake, potentially through altered PLN phosphorylation.