Suppression of ATP diphosphohydrolase/CD39 in human vascular endothelial cells.
Vascular ATP diphosphohydrolase/CD39 is an endothelial cell membrane protein with both ecto-ATPase and ecto-ADPase activities. Suppression of constitutive CD39 expression may result in elevated concentrations of ATP and ADP at the vascular interface that could predispose to thrombosis and inflammation. To study the effects of suppression of CD39 synthesis, stable 25-base antisense chimeric oligonucleotides targeting sequences at the 5' region of CD39 were designed. Transfection of these stable oligomers into cultured human endothelial cells resulted in dramatic decreases in levels of CD39 mRNA transcripts. Following transfection with antisense oligonucleotides, total ADPase activity fell from 26.0 +/- 3.1 in control cultures to 9.5 +/- 3.4 nmol of P(i) min(-1) (mg of protein)(-1) (p < 0.005); suppression of CD39 protein expression was also observed by Western blotting. Decreases in ATP diphosphohydrolase activity were associated with increases in concentrations of extracellular purine nucleotides released following stimulation of endothelial cells. Rates of initial hydrolysis of extracellular ATP released from purinergic agonist-stimulated endothelial cells decreased from 17.9 +/- 5.0 to 4.8 +/- 0.5 pmol min(-1) per 10(6) cells (p < 0.005) in antisense transfected cells. Therefore, CD39 regulates extracellular ATP concentrations and may be an important modulator of purinergic receptor activity in vascular endothelial cells.
Imai
M
M
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Kaczmarek
E
E
Koziak
K
K
Sévigny
J
J
Goepfert
C
C
Guckelberger
O
O
Csizmadia
E
E
Schulte Am Esch
J
J
Robson
S C
SC
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000378:metabolism; D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics; D001081:Apyrase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D001483:Base Sequence; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000378:metabolism / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D014471:Umbilical Veins
D015703:Antigens, CD; D016376:Oligonucleotides, Antisense; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D001081:Apyrase; C067869:CD39 antigen