Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle.
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 microM 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.
Sathish
Venkatachalem
V
Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Leblebici
Figen
F
Kip
Sertac N
SN
Thompson
Michael A
MA
Pabelick
Christina M
CM
Prakash
Y S
YS
Sieck
Gary C
GC
D000109:Acetylcholine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics; D006706:Homeostasis; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D012143:Respiratory Physiological Phenomena; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000502:physiology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D013552:Swine; D014132:Trachea / Q000187:drug effects / Q000502:physiology*; D000099211:Phospholamban
D000109:Acetylcholine; D000255:Adenosine Triphosphate; D002118:Calcium; D002135:Calcium-Binding Proteins; D034741:RNA, Small Interfering; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban