The role of chondroitin sulfate chains of urinary trypsin inhibitor in inhibition of LPS-induced increase of cytosolic free Ca2+ in HL60 cells and HUVEC cells.
Preincubation of HL60 cells and HUVEC cells with urinary trypsin inhibitor (UTI) inhibited increase of cytosolic free Ca2+ induced by LPS. In contrast, an increase of cytosolic free Ca2+ induced by LPS was not inhibited by deglycosylated UTI, UTI treated with monoclonal antibody of chondroitin sulfate. 45Ca2+ binding showed that UTI binds 45Ca2+ dose-dependently. Scatchard plot analysis showed that UTI has two binding sites for Ca2+, a high affinity binding site (Kd=15 microM) and a low affinity site (Kd=150 microM), and that UTI has more than 70 Ca2+ binding sites per molecule. The Ca2+ binding capacity of deglycosylated UTI and UTI treated with monoclonal antibody of chondroitin sulfate was markedly depressed. Furthermore, UTI forms multi-polymers in the presence of Ca2+ as demonstrated by gel filtration and agarose gel electrophoresis. These results suggest that UTI is a physiological Ca2+ chelator on the cells and that the action is due to chondroitin sulfate chains of UTI.
Kanayama
N
N
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, 3600 Handa-cho, 431-31, Japan.
Maehara
K
K
Suzuki
M
M
Fujise
Y
Y
Terao
T
T
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000378:metabolism; D002850:Chromatography, Gel; D004730:Endothelium, Vascular / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000494:pharmacology*
D006023:Glycoproteins; D008070:Lipopolysaccharides; D014361:Trypsin Inhibitors; D002809:Chondroitin Sulfates; C028665:urinastatin; D002118:Calcium