Involvement of calpain-calpastatin in cigarette smoke-induced inhibition of lung endothelial nitric oxide synthase.
We reported that cigarette smoke extract (CSE) causes decreases in the activity and expression of endothelial nitric oxide synthase (eNOS) and calpain activity in pulmonary artery endothelial cells (PAECs). Calpains are a family of calcium-dependent endopeptidases, and their specific endogenous inhibitor is calpastatin. In this study, we evaluated the role of calpain-calpastatin in CSE-induced decrease in eNOS gene expression. PAEC were incubated with 5-10% CSE for 2-24 h. eNOS gene transcription rate, eNOS messenger ribonucleic acid (mRNA) half-life, and the activity and protein contents of calpain and calpastatin were measured. Incubation of PAEC with CSE caused significant decreases in eNOS gene transcription and calpain activity and an increase in calpastatin protein content. eNOS mRNA half-life was not significantly altered by CSE. To investigate whether CSE-induced inhibition of eNOS gene expression is caused by decreased calpain activity due to an increase in calpastatin protein content, we cloned calpastatin gene from PAEC and constructed adenovirus vectors containing calpastatin. Overexpression of calpastatin mimics the inhibitory effects of CSE on calpain activity and on the activity, protein, and mRNA of eNOS. The cell-permeable calpain inhibitor, calpastatin peptide, inhibits acetylcholine-induced endothelium-dependent relaxation of the pulmonary artery. Incubation of PAEC with an antisense oligodeoxyribonucleotide of calpastatin prevented CSE-induced increases in calpastatin protein and CSE-induced decreases in calpain activity, eNOS gene transcription, activity and protein content of eNOS, and NO release. These results indicate that CSE-induced inhibition of eNOS expression in PAEC is caused by calpain inhibition due to an increase in calpastatin protein content.
Cui
Zhaoqiang
Z
Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Han
Zhaosheng
Z
Li
Zhaozhong
Z
Hu
Hanbo
H
Patel
Jawaharlal M
JM
Antony
Veena
V
Block
Edward R
ER
Su
Yunchao
Y
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D004727:Endothelium / Q000187:drug effects / Q000201:enzymology; D006207:Half-Life; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D012907:Smoking / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D014158:Transcription, Genetic
D002135:Calcium-Binding Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D010455:Peptides; D012333:RNA, Messenger; C033668:calpastatin; D052250:Nitric Oxide Synthase Type III; D002154:Calpain