Cyclooxygenase cloning in dogfish shark, Squalus acanthias, and its role in rectal gland Cl secretion.
The present studies were carried out with the aims to determine the cDNA sequence for cyclooxygenase (COX) in an elasmobranch species and to study its role in regulation of chloride secretion in the perfused shark rectal gland (SRG). With the use of long primers (43 bp) derived from regions of homology between zebrafish and rainbow trout COX-2 genes, a 600-bp product was amplified from SRG and was found to be almost equally homologous to mammalian COX-1 and COX-2 (65%). The full-length cDNA sequence was obtained by 5'-RACE and by analyzing an EST clone generated by the EST Project of the Mt. Desert Island Biological Laboratory Marine DNA Sequencing Center. The longest open reading frame encodes a 593-amino acid protein that has 68 and 64% homology to mammalian COX-1 and COX-2, respectively. The gene and its protein product is designated as shark COX (sCOX). The key residues in the active site (Try(385), His(388), and Ser(530)) are conserved between the shark and mammalian COX. sCOX contains Val(523) that has been shown to be a key residue determining the sensitivity to COX-2-specific inhibitors including NS-398. The mRNA of sCOX, detected by RT-PCR, was found in all tissues tested, including rectal gland, kidney, spleen, gill, liver, brain, and heart, but not in fin. In the perfused SRG, vasoactive intestinal peptide (VIP) at 5 nM induced rapid and marked Cl(-) secretion (basal: <250 microeq x h(-1) x g(-1); peak response: 3,108 +/- 479 microeq x h(-1) x g(-1)). In the presence of 50 microM NS-398, both the peak response (2,131 +/- 307 microeq x h(-1) x g(-1)) and the sustained response to VIP were significantly reduced. When NS-398 was removed, there was a prompt recovery of chloride secretion to control values. In conclusion, we have cloned the first COX in an elasmobranch species (sCOX) and shown that sCOX inhibition suppresses VIP-stimulated chloride secretion in the perfused SRG.
Yang
T
T
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Forrest
S J
SJ
Stine
N
N
Endo
Y
Y
Pasumarthy
A
A
Castrop
H
H
Aller
S
S
Forrest
J N
JN
Schnermann
J
J
Briggs
J
J
D000595:Amino Acid Sequence; D001003:Anal Canal / Q000201:enzymology* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002712:Chlorides / Q000378:metabolism*; D003001:Cloning, Molecular; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary; D015232:Dinoprostone / Q000096:biosynthesis; D004284:Dogfish / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D020098:Natriuretic Peptide, C-Type / Q000494:pharmacology; D009578:Nitrobenzenes / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D012007:Rectum / Q000201:enzymology / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D002712:Chlorides; D016861:Cyclooxygenase Inhibitors; D018076:DNA, Complementary; D009578:Nitrobenzenes; D012333:RNA, Messenger; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D020098:Natriuretic Peptide, C-Type; D014660:Vasoactive Intestinal Peptide; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone