Recent evidence suggests that paracrine signaling agents, such as endothelin (ET), nitric oxide (NO), superoxide (O2-), and prostanoids can modulate mammalian renal function by affecting both hemodynamic and epithelial ionic transport pathways. Since these signaling pathways have been described in fish blood vessels, we hypothesized that they may control salt transport across the gill epithelium--the primary site of ion excretion in marine teleost fishes. We found that ET, the NO donors sodium nitroprusside and spermine NONOate, and the prostanoid PGE2 each can produce a concentration-dependent reduction in the short circuit current (Isc) across the isolated opercular epithelium of the killifish (Fundulus heteroclitus), the generally accepted model for the marine teleost gill epithelium. Sarafotoxin S6c was equipotent to ET-1, suggesting that ETB receptors are involved. Incubation with NG-nitro-L-arginine methyl ester (L-NAME) or indomethacin reduced the effect of subsequent addition of SRXS6c by 17 and 89%, respectively, suggesting the presence of an ET to NO and PGE axis. The effects of l-NAME and indomethacin were not additive, but the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) reduced the effect of SRXS6c by 34% and preincubation with l-NAME, indomethacin, and TEMPOL reduced the SRXS6c response to zero. This suggests a direct role for O2- in this axis. COX-2 appears to be the major enzyme involved in this axis because the specific COX-2 inhibitor NS-398 was twice as effective as the COX-1 inhibitor SC560 in inhibiting the SRXS6c effect. The Isc was stimulated by the EP2 agonist butaprost and inhibited by the EP(1,3) agonist sulprostone, suggesting both stimulatory and inhibitory PGE receptors in this tissue. Carbaprostacyclin (PGI2 analog), thromboxane A2, PGF(2alpha), and PGD2 did not affect the Isc. Our data are the first to suggest the importance of an ET-stimulated and NO-, O2(-)-, and PGE2-mediated signaling axis that can modify active extrusion of NaCl across the killifish opercular epithelium and, by inference, the marine teleost gill epithelium.
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.
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