Endotoxin induction of nitric oxide synthase and cyclooxygenase-2 in equine alveolar macrophages.
OBJECTIVE
To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses.


METHODS
Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively.


RESULTS
Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent. Conclusion-Lipopolysaccharide induces COX-2 and iNOS in equine macrophages.


CONCLUSIONS
The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses.
Hammond
R A
RA
Department of Biochemical Pharmacology, St Bartholomew's and the Royal London School of Medicine and Dentistry, United Kingdom.
Hannon
R
R
Frean
S P
SP
Armstrong
S J
SJ
Flower
R J
RJ
Bryant
C E
CE
D000818:Animals; D001483:Base Sequence; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism; D005260:Female; D006736:Horses / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*; D011863:Radioimmunoassay / Q000662:veterinary; D016415:Sequence Alignment; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D007527:Isoenzymes; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D014409:Tumor Necrosis Factor-alpha; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone