COX2 in a euryhaline teleost,Fundulus heteroclitus: primary sequence, distribution, localization, and potential function in gills during salinity acclimation

Abstract

SUMMARY In the kidneys of mammals, cyclooxygenase type 2 (COX2) is expressed in medullary interstitial cells, the macula densa and epithelial cells of the cortical thick ascending limb where it generates prostaglandins that regulate hormone secretion, inhibit ion transport, and support cell survival during salt loading and dehydration. In teleosts, the gills are in direct contact with an aquatic environment and are the dominant site of osmoregulation. During transfers between salinities, specialized cells in the gills (chloride cells) rapidly regulate NaCl secretion for systemic osmoregulation while they simultaneously are exposed to acute osmotic shock. This study was conducted to determine if COX2 is expressed in the gills, and if so, to evaluate its function in cellular and systemic osmoregulation. Degenerate primers, reverse transcription–PCR and rapid amplification of cDNA ends were used to deduce the complete cDNA sequence of a putative COX2 enzyme from the gills of the euryhaline killifish (Fundulus heteroclitus). The 2738 base pair cDNA includes a coding region for a 610 amino acid protein that is over 70%identical to mammalian COX2. A purified antibody generated against a conserved region of mouse COX2 labeled chloride cells, suggesting that the enzyme may control NaCl secretion as an autocrine agent. Real-time PCR was then used to demonstrate that mRNA expression of the COX2 homologue was threefold greater in gills from chronic seawater killifish than in gills from chronic freshwater killifish. Expression of Na+/K+/2Cl–cotransporter and the cystic fibrosis transmembrane conductance regulator were also greater in seawater, suggesting that chronic COX2 expression in the gills is regulated in parallel to the key ion transporters that mediate NaCl secretion. Real-time PCR was also used to demonstrate that acute transfer from seawater to freshwater and from freshwater to seawater led to rapid, transient inductions of COX2 expression. Together with previous physiological evidence,the present molecular and immunological data suggest that constitutive branchial COX2 expression is enhanced in seawater, where prostaglandins can regulate NaCl secretion in chloride cells. Our data also suggest that branchial COX2 expression may play a role in cell survival during acute osmotic shock.

Physiological and molecular mechanisms of osmoregulatory plasticity in killifish after seawater transfer

“…Our present results ( Fig. 2A), as well as those in previous studies (Singer et al, 1998;Scott et al, 2004a;Scott and Schulte, 2005;Choe et al, 2006;Shaw et al, 2007), suggest that increases in the expression of ion transporter genes contribute to this response. Na + /K + -ATPase α 1a , NKCC1 and CFTR mRNA levels in the gills increased 12 h to 3 days after transfer of killifish to seawater, coinciding with subsequent increases in Na + /K + -ATPase activity at 7 days (Fig.…”

“…While a fair amount is known about transcriptional regulation of ion transport in the gills of killifish transferred to seawater (Singer et al, 1998;Scott et al, 2004a;Scott and Schulte, 2005;Choe et al, 2006;Shaw et al, 2007), much less is known about the opercular epithelium. Gene expression differs greatly between these tissues after transfer of intact killifish to freshwater (Scott et al, 2005a).…”

Paracellular pathway remodeling enhances sodium secretion by teleost fish in hypersaline environments

“…Na + ,K + -ATPase, which is responsible for producing the Na + transmembrane gradient that drives NKCC operation, has been cloned from F. heteroclitus (Semple et al, 2002). There is complex regulation of Na + ,K + -ATPase at the protein and mRNA expression levels associated with adaptation to seawater (Choe et al, 2006;Mancera and McCormick, 2000;McCormick et al, 2009;Scott and Schulte, 2005). Tilapia (Oreochromis mossambicus) in hypersaline conditions significantly shift their gill proteome, featuring increased expression of mitochondrial proteins and the stress protein NDRG1 (Kültz et al, 2013).…”

Urotensin II and its receptor in the killifish gill: regulators of NaCl extrusion

“…Much like endothelin, urotensin signals through a NO/prostanoid pathway to inhibit ion transport. When the killifish is in SW, this signaling pathway is thought to act as a brake on salt extrusion, helping the fish maintain ion homeostasis (Choe et al, 2006;Hyndman and Evans, 2007). It is unclear, however, whether there is cross-talk between UII and endothelin and these signaling pathways.…”