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Cyclooxygenase expression in canine platelets and Madin-Darby canine kidney cells

Patricia A. Kay-Mugford DVM, MSc1,2, Sally J. Benn MSc3, Jonathan LaMarre DVM, PhD4, and Peter D. Conlon DVM, PhD5
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  • 1 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Ontario, Canada N1G 2W1.
  • | 2 Present address is Schering-Plough Animal Health Corporation, Bldg U-23 Mailstop 4800, 1095 Morris Avenue, Union, NJ 07083-1982.
  • | 3 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Ontario, Canada N1G 2W1.
  • | 4 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Ontario, Canada N1G 2W1.
  • | 5 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Ontario, Canada N1G 2W1.

Abstract

Objective—To examine cyclooxygenase (COX) expression in canine platelets and Madin-Darby canine kidney (MDCK) cells in culture.

Sample Population—Canine platelets and MDCK cells.

Procedure—Total RNA was recovered from isolated canine platelets and MDCK cells. Northern blot analysis and reverse transcription-polymerase chain reaction (RTPCR), using complementary DNA probes and primers designed from the human COX sequences, were used to determine COX-1 and -2 (cyclooxygenase isoforms 1 and 2) messenger RNA (mRNA) expression.

Results—Following northern blot analysis, canine platelets were found to express only the 2.8-kb COX- 1 transcript; COX-2 was not detected. Canine MDCK cells expressed the 4.5-kb COX-2 transcript, in addition to the 2.8-kb COX-1 transcript. A single DNA band of 270 base pairs was identified following gel electrophoresis of the product obtained from RT-PCR of mRNA from canine platelets. Sequencing revealed that this PCR product was 90% homologous to a portion of the human COX-1 gene (Genbank M59979).

Conclusions and Clinical Relevance—Detection of COX-1 by RT-PCR of RNA obtained from canine platelets is a novel finding. The 90% homology of the PCR product with the human sequence suggests strong conservation between the canine and human COX-1 gene. Cloning and sequencing of the canine gene will be required to fully characterize homologous regions. Because of the importance of COX in the inflammatory process and as a potential target of currently available nonsteroidal anti-inflammatory drugs (NSAID), a better understanding of canine COX may improve our ability to use NSAID appropriately, achieve efficacy, and avoid potential adverse drug effects in dogs. (Am J Vet Res 2000;61:1512–1516)

Abstract

Objective—To examine cyclooxygenase (COX) expression in canine platelets and Madin-Darby canine kidney (MDCK) cells in culture.

Sample Population—Canine platelets and MDCK cells.

Procedure—Total RNA was recovered from isolated canine platelets and MDCK cells. Northern blot analysis and reverse transcription-polymerase chain reaction (RTPCR), using complementary DNA probes and primers designed from the human COX sequences, were used to determine COX-1 and -2 (cyclooxygenase isoforms 1 and 2) messenger RNA (mRNA) expression.

Results—Following northern blot analysis, canine platelets were found to express only the 2.8-kb COX- 1 transcript; COX-2 was not detected. Canine MDCK cells expressed the 4.5-kb COX-2 transcript, in addition to the 2.8-kb COX-1 transcript. A single DNA band of 270 base pairs was identified following gel electrophoresis of the product obtained from RT-PCR of mRNA from canine platelets. Sequencing revealed that this PCR product was 90% homologous to a portion of the human COX-1 gene (Genbank M59979).

Conclusions and Clinical Relevance—Detection of COX-1 by RT-PCR of RNA obtained from canine platelets is a novel finding. The 90% homology of the PCR product with the human sequence suggests strong conservation between the canine and human COX-1 gene. Cloning and sequencing of the canine gene will be required to fully characterize homologous regions. Because of the importance of COX in the inflammatory process and as a potential target of currently available nonsteroidal anti-inflammatory drugs (NSAID), a better understanding of canine COX may improve our ability to use NSAID appropriately, achieve efficacy, and avoid potential adverse drug effects in dogs. (Am J Vet Res 2000;61:1512–1516)