Effect of proinflammatory cytokines on regulation of sarcoplasmic reticulum Ca2+ reuptake in human airway smooth muscle.
Airway inflammation leads to increased intracellular Ca(2+) ([Ca(2+)](i)) levels in airway smooth muscle (ASM) cells. Sarcoplasmic reticulum Ca(2+) release and reuptake are key components of ASM [Ca(2+)](i) regulation. Ca(2+) reuptake occurs via sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA) and is regulated by the inhibitory protein phospholamban (PLB) in many cell types. In human ASM, we tested the hypothesis that inflammation increases PLB, thus inhibiting SERCA function, and leading to maintained [Ca(2+)](i) levels. Surprisingly, we found that human ASM does not express PLB protein (although mRNA is detectable). Overnight exposure to the proinflammatory cytokines TNFalpha and IL-13 did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (via CaMKII) occurs in human ASM. In fura-2-loaded human ASM cells, we found that the CaMKII antagonist KN-93 significantly slowed the rate of fall of [Ca(2+)](i) transients induced by ACh or bradykinin (in zero extracellular Ca(2+)), suggesting a role for CaMKII-mediated SERCA regulation. SERCA expression was decreased by cytokine exposure, and the rate of fall of [Ca(2+)](i) transients was slowed in cells exposed to TNFalpha and IL-13. Cytokine effects on Ca(2+) reuptake were unaffected by additional exposure to KN-93. These data indicate that in human ASM, SERCA is regulated by mechanisms such as CaMKII and that airway inflammation maintains [Ca(2+)](i) levels by decreasing SERCA expression and slowing Ca(2+) reuptake.
Sathish
Venkatachalem
V
Departments of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
Thompson
Michael A
MA
Bailey
Jeffrey P
JP
Pabelick
Christina M
CM
Prakash
Y S
YS
Sieck
Gary C
GC
D000109:Acetylcholine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology*; D018793:Interleukin-13 / Q000494:pharmacology*; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D012137:Respiratory System / Q000201:enzymology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D000099211:Phospholamban
D000109:Acetylcholine; D000255:Adenosine Triphosphate; D001920:Bradykinin; D002118:Calcium; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D018836:Inflammation Mediators; D018793:Interleukin-13; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D014409:Tumor Necrosis Factor-alpha; D000099211:Phospholamban; C506946:ATP2A2 protein, human