Nitric oxide causes apoptosis in pulmonary vascular smooth muscle cells.
Nitric oxide (NO), a product of certain cytokine-activated cells, affects rates of apoptosis, a mechanism of programmed cell death. We asked whether NO affected rates of apoptosis in pulmonary vascular cells. Using rat pulmonary artery smooth muscle cells, we studied direct effects of the NO donor SG-nitroso-acetyl-D,L-penicillamine (SNAP) and the effects of NO endogenously synthesized in response to bacterial lipopolysaccharide (LPS) and inflammatory cytokines interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha (a combination called cytomix for convenience). We determined apoptosis on the basis of light microscopy and the bromodeoxyuridine terminal deoxynucleotidyl transferase reaction (BrdUTdT). Both SNAP- and cytomix-induced synthesis of NO resulted in histologic evidence of apoptosis based upon fluorescence microscopy using propidium iodide. SNAP (10(-5) M) increased BrdUTdT-positive cells from 17.5 to 78.4% compared with basal medium alone, with the maximal response occurring at 15 h or exposure. Exposing cells to LPS and cytokines induced NO production (from 0.1 +/- 0.1 to 24.6 +/- 0.5 microM, P < 0.05) caused cytological changes consistent with apoptosis and led to an increase of increased BrdUTdT-positive cells from 11 to 41% at 12 h compared with basal medium alone. The competitive NO synthase inhibitor NG-monomethyl-L-arginine inhibited both NO synthesis and NO apoptosis, returning the proportion of BrdUTdT-positive cells (6%) to levels below control. L-Arginine (0.5 mM) restored percentages to those increase in response to endogenously synthesized NO, and NO is a potential mechanism of acute lung injury in response to inflammatory cytokines.
Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7210, USA.
J D
JD
Smith
S D
SD
McLean
D K
DK
Nakayama
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000502:physiology*; D001973:Bromodeoxyuridine / Q000032:analysis; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004253:DNA Nucleotidylexotransferase / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018836:Inflammation Mediators / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D009569:Nitric Oxide / Q000494:pharmacology* / Q000502:physiology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D011651:Pulmonary Artery / Q000737:chemistry / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D026423:S-Nitroso-N-Acetylpenicillamine
D017304:Annexin A5; D016207:Cytokines; D004791:Enzyme Inhibitors; D018836:Inflammation Mediators; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D004253:DNA Nucleotidylexotransferase; D001973:Bromodeoxyuridine; D010396:Penicillamine