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LETTERS TO THE EDITOR
Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina
Received August 29, 2005; accepted September 19, 2005.
). According to published data so far, this splice variant of COX-1, which we and others prefer to call COX-1b rather than COX-3 because it is not a unique gene product, has no role in prostaglandin production except perhaps in dog. Furthermore, it seems that the target of acetaminophen in most situations is COX-2 (Graham and Scott, 2005; Kis et al., 2005b).
Our primary concern, which prompted us to write the review, was that unchallenged findings and speculations were leading to inappropriate conclusions concerning the physiological role of COX-3, especially in humans but also in rodents. Our review attempted to make the COX-3 issues clearer and address such issues as whether acetaminophen is a selective COX-3 inhibitor and whether COX-3 potentially plays a role in the thermoregulation in rodents and humans. We believe that the statements in our review are accurate, fair, and balanced. In addition, we have no financial conflict of interest in this research area. In their lengthy letter, Simmons et al. (2005) have taken many of our comments out of context and provided their own interpretation and justification. Rather than address these issues individually, we encourage interested scientists to carefully evaluate the original publications in this area, as well as our review and their letter, to make their own judgment concerning our statements and conclusions. Nonetheless, to illustrate the complexity and confusion involved in the COX-3 studies, it may prove helpful for us to directly address one of the primary points made by Simmons et al. (2005) in their letter.
In the original study by Chandrasekharan et al. (2002), the authors state that the antibody against canine COX-3 was made using peptide sequences corresponding to the first 12 amino acids from both human and mouse. These individual peptides were synthesized and coupled to keyhole limpet hemocyanin, and a 50:50 mixture was injected into New Zealand White rabbits (Chandrasekharan et al., 2002). A theoretical concern of this unusual approach is that the sequence homologies among the first 12 amino acids for COX-3 between mouse or human and canine are approximately 50%; thus, the polyclonal antibodies based upon sequences for human and/or mouse cannot be expected to recognize canine COX-3 in the Western blots. The approach to COX-3 antibody production and its methodological description were so unusual and confusing that even Dr. Simmons misreported it in a subsequent review (Simmons, 2003): "immunoblots were performed using an antibody directed toward the sequence encoded by the 5' end of intron-11 [sic] in human. Proteins of 65-, 53-, and 50-kDA were detected with this antibody in human aorta (Chandrasekharan et al., 2002)." No reference was made to the use of the mouse peptide in antibody production. In addition to the confusion surrounding the antibody production, the sequence used and referred to as the first 12 amino acids of "mouse (MSREFDPEAPRNC) COX-3" (Chandrasekharan et al., 2002) shows only about 50% homology to the respective sequence of the actual mouse COX-3 (MSRESDPSGAPT) (Kis et al., 2005a); however, it shows complete homology to the first 12 amino acids of the canine COX-3 (MSREFDPEAPRN) (Simmons et al., 2004). Thus, it is not surprising that the antibody was effective on canine tissues! Moreover, the reported canine COX-3 sequence (Simmons et al., 2004) can be found in GenBank as canine COX-1, deposited by Simmons and colleagues (accession no. NM_001003023). Therefore, our criticism is not "unsubstantiated by any data," and the readers have several good reasons to question their results using this antibody (Chandrasekharan et al., 2002).
In contrast to Simmons et al. (2005), we and others think that the name COX-3 should be reserved to a yet undiscovered COX isoform, and we also reject the concept that acetaminophen is a selective COX-3 inhibitor. Simmons's COX-3 should be added to the long list of COX splice variants with unknown function with the name of COX-1b or COX-1v. The broader context of our review is our continuing concern that despite considerable investments of time and effort by investigators, many subject areas such as this one suffer from a lack of critical assessment and discussion of study details, results, and conclusions.
Footnotes
ABBREVIATIONS: COX, cyclooxygenase.
References
Chandrasekharan NV, Dai H, Roos KLT, Evanson NK, Tomsik J, Elton TS, and Simmons DL (2002) COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure and expression. Proc Natl Acad Sci USA 99: 13926–13931.
Graham GG and Scott KF (2005) Mechanism of action of paracetamol. Am J Ther 12: 46–55.[CrossRef][Medline]
Kis B, Snipes JA, and Busija DW (2005a) Acetaminophen and the COX-3 puzzle: sorting out facts, fictions and uncertainties. J Pharmacol Exp Ther 315: 1–7.
Kis B, Snipes JA, Simandle SA, and Busija DW (2005b) Acetaminophen-sensitive prostaglandin production in rat cerebral endothelial cells. Am J Physiol Regul Integr Comp Physiol 288: R897–R902.
Simmons DL (2003) Variants of cyclooxygenase-1 and their roles in medicine. Thromb Res 110: 265–268.[CrossRef][Medline]
Simmons DL, Botting RM, and Hla T (2004) Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol Rev 56: 387–437.
Simmons DL, Chandrasekharan NV, Dai H, Roos KL, and Tomsik J (2005) Comments on "Acetaminophen and the Cyclooxygenase-3 Puzzle: Sorting out Facts, Fictions, and Uncertainties." J Pharmacol Exp Ther 315: 1412–1414.
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