A new member of the cyclooxygenase (COX) family, COX-3, has been identified and characterized in canine tissues (Chandrasekharan et al., 2002). It has been suggested that COX-3 is the long sought target of acetaminophen since isolated COX-1 and COX-2 are minimally inhibited by this drug. COX-3 mRNA is identical to COX-1 mRNA except that the former retains all or part of intron 1. We have shown that COX-3 mRNA is relatively abundant in cultured cerebral endothelium (Kis et al., 2003) as well as in freshly harvested cerebral microvessels (Kis et al., 2004) of rat and that these preparations are unusually sensitive to the inhibition of prostaglandin synthesis by acetaminophen. For example, elevated levels of prostaglandin production in LPS-treated cultured rat endothelial cells are reduced by administration of clinically given doses of acetaminophen. The purposes of this study are: 1) To determine whether the entire intron-1 is inserted into the COX-3 mRNA in rat. 2) To determine the complete sequence of the rat COX-3 mRNA and to predict the amino acid sequence of the protein likely to be produced. 3) To transfect COS-7 cells with a rat COX-3 mRNA construct to determine whether it will lead to synthesis of the predicted protein. 4) To examine the COX activity of the predicted protein by challenging stable transfected COS-7 cells with arachidonic acid. COS-7 cells normally have a low level of endogenous prostaglandin production. We used RT-PCR and sequencing of the PCR product from total and mRNA from cultured endothelial cells. We used primary cultures of rat cerebral endothelial cells as well as freshly harvested cerebral microvessels because they show abundant expression of COX-3 mRNA. For transient transfection COS-7 cells were cultured in 24-well plates and were transfected with a COX-3 p3XFLAG-CMV-14 vector and used antibodies against the FLAG fusion protein in western blotting and immunostaining. Our results indicate that rat COX-3 mRNA from cerebral endothelium retains the entire intron-1 from the parent COX-1 gene. Retention of intron-1 in rat, composed of 98 base pairs, shifts the reading frame for COX-3 mRNA away from homology with COX-1 mRNA, and leads to a smaller protein unlike COX-1 with a predicted amino acid sequence of 127 amino acids and molecular weight of ~13 kDa. Transfection of COS-7 cells with the rat COX-3 mRNA resulted in detection of the predicted protein on western blotting, and localization of the protein around the cell nucleus. Our experiments also showed that the protein encoded by rat COX-3 does not have COX activity, since exogenous arachidonic acid failed to substantially increase prostaglandin production. Because this protein has a completely different amino acid sequence than COX-1 and COX-2 and we could not detect COX activity of this protein, we named it as Cyclooxygenase Variant Protein (COVAP). While COVAP does not directly convert arachidonic acid to prostaglandins, we cannot exclude the possibility that there is an interaction between acetaminophen and COVAP which inhibits the activity of the other COXs. It is also possible that COX-3 mRNA, due to its similarity to COX-1 mRNA, plays an important role in modulating COX-1 expression in endothelium.

Chandrasekharan et al., Proc Natl Acad Sci USA 99:13926, 2002
Kis et al., J Cerebral Blood Flow Metab 23:1287, 2003
Kis et al., Mol Brain Res, in press, 2004