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

Venkatachalem Sathish, Figen Leblebici, Sertac N. Kip, Michael A. Thompson, Christina M. Pabelick, Y. S. Prakash, Gary C. Sieck

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

Regulation of intracellular Ca2+ concentration ([Ca 2+]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 [Ca 2+]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.

Original languageEnglish (US)
Pages (from-to)L787-L796
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume294
Issue number4
DOIs
StatePublished - Apr 2008

Keywords

  • Calmodulin
  • Calmodulin kinase
  • Phospholamban
  • Sarco(endo)plasmic reticulum calcium-ATPase

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

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