The presence and activity of SP-D in porcine coronary endothelial cells depend on Akt/PI3K, Erk and nitric oxide and decrease after multiple passaging.
Surfactant protein D (SP-D) mediates clearance of microorganisms and modulates inflammation in response to cytotoxic stimulation. It is present in various epithelia, but also in vascular smooth muscle and endothelial cells. Experiments were designed to determine whether or not SP-D is present in porcine coronary arterial endothelial cells and if so, to investigate the molecular mechanisms underlying this presence. The expression of SP-D, NO synthase, Akt 1/2 and Erk 1/2 proteins was determined in cultures at passages 1 (#1) and 4 (#4). SP-D in primary cells existed in three isoforms (37-38 kDa and 50 kDa). The 37-38 kDa SP-D forms were the dominant isoforms in the porcine endothelium and were prominent at #1 but partially lost at #4. Tumor necrosis factor-alpha (TNF-alpha) significantly augmented the level of SP-D expression at #1 but not at #4. The basal level of 37-38 kDa SP-D isoforms at #1 was reduced by L-NAME, wortmannin and PD 98059. The low basal expression at #4 could be increased by DETA NONOate (donor of NO) or insulin (activator of PI(3)K/Akt). The presence of nitric oxide synthase was reduced while that of Akt 1/2 and Erk 1/2 was increased at #4. In cells both at passages 1 and 4, TNF-alpha downregulated NO synthase and up-regulated p-Erk 1/2 protein. The present findings demonstrate the presence of SP-D in endothelial cells which is NO-, PI(3)K/Akt- and Erk-dependent. They suggest a protective role of SP-D in these cells.
Lee
Mary Y K
MY
Department of Pharmacology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, L2-48, Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong.
Sørensen
Grith L
GL
Holmskov
Uffe
U
Vanhoutte
Paul M
PM
D000818:Animals; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism; D046508:Culture Techniques; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D037663:Pulmonary Surfactant-Associated Protein D / Q000235:genetics / Q000502:physiology*; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D020033:Protein Isoforms; D037663:Pulmonary Surfactant-Associated Protein D; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases