Journal
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
Volume 294, Issue 4, Pages L787-L796Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajplung.00461.2007
Keywords
sarco(endo)plasmic reticulum calcium-ATPase; phospholamban; calmodulin; calmodulin kinase
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Funding
- NCRR NIH HHS [1 UL1 RR-024150-01] Funding Source: Medline
- NHLBI NIH HHS [R01 HL074309, HL-74309] Funding Source: Medline
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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 mu M ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W7 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 W-7 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.
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