Cyclooxygenase cloning in dogfish shark, Squalus acanthias, and its role in rectal gland Cl secretion

1 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; 3 Department of Medicine, Yale University, New Haven, Connecticut 06510; 4 Dartmouth College, Hanover, New Hampshire 03755; and 2 Mt. Desert Island Biological Laboratory, Salisbury Cove, Maine 04672 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: