Abstract
A splice variant of cyclooxygenase-1 (COX-1), COX-1b (previously termed as COX-3), has been identified in canine tissues as an acetaminophen-sensitive isoform, but the sequence of COX-1b mRNA and the encoded protein are not known in rats. We cloned and sequenced rat COX-1b mRNA from cerebral endothelial cells. Sequence analysis indicated that the 98-base pair intron-1 of COX-1 gene remains unprocessed in the COX-1b mRNA, causing a frameshift mutation and a 127-amino acid open reading frame with no sequence similarity with known cyclooxygenases. Transient and permanent transfection of COS-7 cells with a vector containing the rat COX-1b cDNA resulted in synthesis of a protein of the expected size. We generated an affinity-purified polyclonal antibody against the rat COX-1b protein. Western blot analysis of rat tissues using this antibody demonstrated the likely existence of rat COX-1b protein in vivo with the highest expression in heart, kidney, and neuronal tissues. Our results on both stable and on transiently transfected COS-7 cells suggest that rat COX-1b does not have cyclooxygenase activity and does not have any effect on the inhibition of prostaglandin production by acetaminophen. Because this protein has a completely different amino acid sequence than COX-1 and COX-2 and it does not have cyclooxygenase activity, we suggest a name cyclooxygenase variant protein to distinguish it from the known prostaglandin-synthesizing cyclooxygenase isoforms.
Footnotes
-
This work was supported by National Institutes of Health Grants HL-30260, HL-66074, HL-65380, and DK-62372 and AHA Bugher Foundation Award 0270114N (to D.W.B.).
-
doi:10.1124/jpet.104.079533.
-
ABBREVIATIONS: COX, cyclooxygenase; bp, base pair; CEC, cerebral endothelial cell; DMEM, Dulbecco's modified Eagle's medium; RT-PCR, reverse transcription-polymerase chain reaction; PCOX, partial cyclooxygenase; PCR, polymerase chain reaction; PGE2, prostaglandin E2; ELISA, enzyme-linked immunosorbent assay; COVAP, cyclooxygenase variant protein.
-
↵1 These authors contributed equally to this work.
- Received October 19, 2004.
- Accepted January 12, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|