COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression.
Journal: 2002/December - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
Two cyclooxygenase isozymes, COX-1 and -2, are known to catalyze the rate-limiting step of prostaglandin synthesis and are the targets of nonsteroidal antiinflammatory drugs. Here we describe a third distinct COX isozyme, COX-3, as well as two smaller COX-1-derived proteins (partial COX-1 or PCOX-1 proteins). COX-3 and one of the PCOX-1 proteins (PCOX-1a) are made from the COX-1 gene but retain intron 1 in their mRNAs. PCOX-1 proteins additionally contain an in-frame deletion of exons 5-8 of the COX-1 mRNA. COX-3 and PCOX mRNAs are expressed in canine cerebral cortex and in lesser amounts in other tissues analyzed. In human, COX-3 mRNA is expressed as an approximately 5.2-kb transcript and is most abundant in cerebral cortex and heart. Intron 1 is conserved in length and in sequence in mammalian COX-1 genes. This intron contains an ORF that introduces an insertion of 30-34 aa, depending on the mammalian species, into the hydrophobic signal peptide that directs COX-1 into the lumen of the endoplasmic reticulum and nuclear envelope. COX-3 and PCOX-1a are expressed efficiently in insect cells as membrane-bound proteins. The signal peptide is not cleaved from either protein and both proteins are glycosylated. COX-3, but not PCOX-1a, possesses glycosylation-dependent cyclooxygenase activity. Comparison of canine COX-3 activity with murine COX-1 and -2 demonstrates that this enzyme is selectively inhibited by analgesic/antipyretic drugs such as acetaminophen, phenacetin, antipyrine, and dipyrone, and is potently inhibited by some nonsteroidal antiinflammatory drugs. Thus, inhibition of COX-3 could represent a primary central mechanism by which these drugs decrease pain and possibly fever.
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Proc Natl Acad Sci U S A 99(21): 13926-13931

COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: Cloning, structure, and expression

Department of Chemistry and Biochemistry, E280 Benson Science Building, Brigham Young University, Provo, UT 84602
To whom correspondence should be addressed. E-mail: ude.uyb@snommis_nad.
Communicated by John Vane, William Harvey Foundation, London, United Kingdom
Communicated by John Vane, William Harvey Foundation, London, United Kingdom
Received 2002 Apr 17; Accepted 2002 Aug 5.

Abstract

Two cyclooxygenase isozymes, COX-1 and -2, are known to catalyze the rate-limiting step of prostaglandin synthesis and are the targets of nonsteroidal antiinflammatory drugs. Here we describe a third distinct COX isozyme, COX-3, as well as two smaller COX-1-derived proteins (partial COX-1 or PCOX-1 proteins). COX-3 and one of the PCOX-1 proteins (PCOX-1a) are made from the COX-1 gene but retain intron 1 in their mRNAs. PCOX-1 proteins additionally contain an in-frame deletion of exons 5–8 of the COX-1 mRNA. COX-3 and PCOX mRNAs are expressed in canine cerebral cortex and in lesser amounts in other tissues analyzed. In human, COX-3 mRNA is expressed as an ≈5.2-kb transcript and is most abundant in cerebral cortex and heart. Intron 1 is conserved in length and in sequence in mammalian COX-1 genes. This intron contains an ORF that introduces an insertion of 30–34 aa, depending on the mammalian species, into the hydrophobic signal peptide that directs COX-1 into the lumen of the endoplasmic reticulum and nuclear envelope. COX-3 and PCOX-1a are expressed efficiently in insect cells as membrane-bound proteins. The signal peptide is not cleaved from either protein and both proteins are glycosylated. COX-3, but not PCOX-1a, possesses glycosylation-dependent cyclooxygenase activity. Comparison of canine COX-3 activity with murine COX-1 and -2 demonstrates that this enzyme is selectively inhibited by analgesic/antipyretic drugs such as acetaminophen, phenacetin, antipyrine, and dipyrone, and is potently inhibited by some nonsteroidal antiinflammatory drugs. Thus, inhibition of COX-3 could represent a primary central mechanism by which these drugs decrease pain and possibly fever.

Abstract

Acetaminophen is often categorized as a nonsteroidal antiinflammatory drug (NSAID), even though in clinical practice and in animal models it possesses little antiinflammatory activity (1). Like NSAIDs, however, acetaminophen inhibits pain and fever and is one of the world's most popular analgesic/antipyretic drugs. Despite acetaminophen's long use and popularity it lacks a clear mechanism of action. Flower and Vane showed that acetaminophen inhibited cyclooxygenase (COX) activity in dog brain homogenates more than in homogenates from spleen (2). This gave rise to the concept that variants of COX enzymes exist that are differentially sensitive to this drug and that acetaminophen acts centrally. Yet, even though two isozymes of COX are known, neither isozyme is sensitive to acetaminophen at therapeutic concentrations of the drug in whole cells or homogenates. Instead, COX-1 and -2 in homogenates frequently exhibit the paradoxical property of being stimulated by submillimolar concentrations of acetaminophen and inhibited by supermillimolar levels of the drug (1). This finding suggests that neither isozyme is a good candidate for the site of action of acetaminophen.

In analyzing COX-1 and -2 RNA expression in dog tissues, our laboratory observed that the cerebral cortex of dog brain contains two distinct RNAs that hybridized to a canine COX-1 cDNA. One RNA was ≈2.6 kb in size and the other was ≈1.9 kb in size, and analyses of these RNAs suggest that they encode previously uncharacterized COX-1-related proteins.

All assays were carried out in the presence of 30 μM arachidonic acid.

Acknowledgments

We thank and acknowledge the assistance of Marcus Rampton, John Clinger, Jenny Taylor, Joel Wilson, and Eme Ekpo for assistance with Western blots and radioimmunoassays. We thank Kenneth D. Westover for suggesting the acronym PCOX. We gratefully acknowledge the financial support of the National Institutes of Health (Grant AR46688), the Brigham Young University Cancer Research Center, the Brigham Young University Technology Transfer Office, the Office of Research and Creative Work, and the College of Physical and Mathematical Sciences.

Acknowledgments

Abbreviations

COXcyclooxygenase
NSAIDnonsteroidal antiinflammatory drug
PCOX-1partial COX-1
RTreverse transcription-coupled
Abbreviations

Footnotes

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AF535138","term_id":"23452498","term_text":"AF535138"}}AF535138 and {"type":"entrez-nucleotide","attrs":{"text":"AF535139","term_id":"23573465","term_text":"AF535139"}}AF535139).

See commentary on page 13371.

Footnotes

References

  • 1. Botting R M. Clin Infect Dis. 2000;31:8202–8210.[PubMed]
  • 2. Flower R J, Vane J R. Nature (London) 1972;240:410–411.[PubMed]
  • 3. Sambrook J, Russell D Molecular Cloning: A Laboratory Manual. 3rd Ed. Plainview, NY: Cold Spring Harbor Lab. Press; 2001. [PubMed][Google Scholar]
  • 4. Simmons D L, Levy D B, Yannoni Y, Erikson R L. Proc Natl Acad Sci USA. 1989;86:1178–1182.
  • 5. Hla T. Prostaglandins. 1996;51:81–85.[PubMed]
  • 6. Simmons D L, Xie W, Chipman J G, Evett G E In: Prostaglandins, Leukotrienes, Lipoxins and PAF. Bailey J M, editor. New York: Plenum; 1991. pp. 67–78. [PubMed][Google Scholar]
  • 7. Salmon J A. Prostaglandins. 1978;15:383–397.[PubMed]
  • 8. O'Neill G P, Ford-Hutchinson A W. FEBS. 1993;330:156–160.[PubMed]
  • 9. Moyad M A. Semin Urol Oncol. 2001;19:280–293.[PubMed]
  • 10. Cohen S M, Shirai T, Steineck G. Scand J Urol Nephrol Suppl. 2000;205:105–115.[PubMed]
  • 11. Xie W, Chipman J G, Robertson D L, Erikson R L, Simmons D L. Proc Natl Acad Sci USA. 1991;88:2692–2696.
  • 12. Kennedy T A, Smith C J, Marnett L J. J Biol Chem. 1994;269:27357–27364.[PubMed]
  • 13. Matheson A J, Figgitt D P. Drugs. 2001;61:833–865.[PubMed]
  • 14. Riendeau D, Percival M D, Brideau C, Charleson S, Dube D, Ethier D, Falgueret J P, Friesen R W, Gordon R, Greig G, et al J Pharmacol Exp Ther. 2001;296:558–566.[PubMed][Google Scholar]
  • 15. Smith C J, Zhang Y, Koboldt C M, Muhammad J, Zweifel B S, Shaffer A, Talley J J, Masferrer J L, Seibert K, Isakson P C. Proc Natl Acad Sci USA. 1998;95:13313–13318.
  • 16. Kusuhara H, Matsuyuki H, Okumoto T. Prostaglandins Other Lipid Mediat. 1998;55:43–49.[PubMed]
  • 17. Goto K, Ochi H, Yasunaga Y, Matsuyuki H, Imayoshi T, Kusuhara H, Okumoto T. Prostaglandins Other Lipid Mediat. 1998;56:245–254.[PubMed]
  • 18. Ochi T, Motoyama Y, Goto T. Eur J Pharmacol. 2000;391:49–54.[PubMed]
  • 19. Ballou L R, Botting R M, Goorha S, Zhang J, Vane J R. Proc Natl Acad Sci USA. 2000;97:10272–10276.
  • 20. Martinez R V, Reval M, Campos M D, Terron J A, Dominguez R, Lopez-Munoz F J. J Pharm Pharmacol. 2002;54:405–412.[PubMed]
  • 21. Warner T D, Giuliano F, Vojnovic I, Bukasa A, Mitchell J A, Vane J R. Proc Natl Acad Sci USA. 1999;96:7563–7568.
  • 22. Buckley M M, Brogden R N. Drugs. 1990;39:86–109.[PubMed]
  • 23. Chopra B, Giblett S, Little J G, Donaldson L F, Tate S, Evans R J, Grubb B D. Eur J Neurosci. 2000;12:911–920.[PubMed]
  • 24. Li S, Wang Y, Matsumura K, Ballou L R, Morham S G, Blatteis C M. Brain Res. 1999;825:86–94.[PubMed]
  • 25. Li S, Ballou L R, Morham S G, Blatteis C M. Brain Res. 2001;910:163–173.[PubMed]
  • 26. Steiner A A, Li S, Llanos Q J, Blatteis C M. Neuroimmunomodulation. 2001;9:263–275.[PubMed]
  • 27. Dogan M D, Ataoglu H, Akarsu E S. Brain Res Bull. 2002;57:179–185.[PubMed]
  • 28. Schwartz J I, Chan C-C, Mukhopadhyay S, McBride K J, Jones T M, Adcock S, Moritz C, Hedges J, Grasing K, Dobratz D, et al Clin Pharmacol Ther. 1999;65:653–660.[PubMed][Google Scholar]
  • 29. Vigano A, Dalla Villa A, Cecchini I, Biasini G C, Principi N. Eur J Clin Pharmacol. 1986;31:359–361.[PubMed]
  • 30. Doughty R A, Giesecke L, Athreya B. Am J Dis Child. 1980;134:461–463.[PubMed]
  • 31. Gunsberg M, Bochner F, Graham G, Imhoff D, Parsons G, Cham B. Clin Pharmacol Ther. 1984;35:585–593.[PubMed]
  • 32. Toutain P L, Cester C C, Haak T, Laroute V. J Vet Pharmacol Ther. 2001;24:43–55.[PubMed]
  • 33. Cohen O, Zylber-Katz E, Caraco Y, Granit L, Levy M. Eur J Clin Pharmacol. 1998;54:549–553.[PubMed]
  • 34. Bovill J G. Eur J Anaesthesiol Suppl. 1997;15:9–15.[PubMed]
  • 35. Jurna I, Brune K. Pain. 1990;41:71–80.[PubMed]
  • 36. Plant M H, Laneuville O. Biochem J. 1999;344:677–685.
  • 37. Sanz A, Moreno J I, Castresana C. Plant Cell. 1998;10:1523–1537.
  • 38. Oliw E H, Su C, Sahlin M. Adv Exp Med Biol. 1999;469:679–685.[PubMed]
  • 39. Landino L M, Crews B C, Gierse J K, Hauser S D, Marnett L J. J Biol Chem. 1997;272:21565–21574.[PubMed]
  • 40. Ballif B A, Mincek N V, Barratt J T, Wilson M L, Simmons D L. Proc Natl Acad Sci USA. 1996;93:5544–5549.
  • 41. Liou J Y, Shyue S K, Tsai M J, Chung C L, Chu K Y, Wu K K. J Biol Chem. 2000;275:15314–15320.[PubMed]
  • 42. Simmons D L, Botting R M, Robertson P M, Madsen M L, Vane J R. Proc Natl Acad Sci USA. 1999;96:3275–3280.
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