TY - JOUR
T1 - Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle
AU - Sathish, Venkatachalem
AU - Leblebici, Figen
AU - Kip, Sertac N.
AU - Thompson, Michael A.
AU - Pabelick, Christina M.
AU - Prakash, Y. S.
AU - Sieck, Gary C.
PY - 2008/4
Y1 - 2008/4
N2 - 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.
AB - 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.
KW - Calmodulin
KW - Calmodulin kinase
KW - Phospholamban
KW - Sarco(endo)plasmic reticulum calcium-ATPase
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U2 - 10.1152/ajplung.00461.2007
DO - 10.1152/ajplung.00461.2007
M3 - Article
C2 - 18245264
AN - SCOPUS:42049094613
SN - 1040-0605
VL - 294
SP - L787-L796
JO - American Journal of Physiology - Lung Cellular and Molecular Physiology
JF - American Journal of Physiology - Lung Cellular and Molecular Physiology
IS - 4
ER -