NaCl and fluid secretion by the intestine of the teleost Fundulus heteroclitus: involvement of CFTR.
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Sections of posterior intestine of the euryhaline killifish Fundulus heteroclitus adapted to sea water were stimulated by the calcium ionophore ionomycin (1 micromol l(-1)) in combination with agents to elevate intracellular cyclic AMP levels, 0.5 mmol l(-1) dibutyryl-cyclic AMP (db-cAMP) with 0.1 mmol l(-1) 3-isobutyl-1-methylxanthine (IBMX). Intestinal bag preparations from recently fed animals (but not from overnight unfed animals) changed from fluid absorption (+18.9+/-8.30 microl cm(-2) h(-1), N=8) in the untreated control period to net fluid secretion after stimulation (-7.43+/-1.30 microl cm(-2) h(-1), N=8, P<0.01; means +/- S.E.M.), indicative of the capacity of teleost intestine to undergo secretion. Posterior intestinal pieces mounted in vitro in Ussing-style membrane chambers showed net Cl(-) uptake (+2.245+/-0.633 microequiv cm(-2) h(-1), N=7) that turned to net secretion following stimulation by ionomycin + db-cAMP + IBMX (-3.809+/-1.22 microequiv cm(-2) h(-1), N=7, P<0.01). Mucosal application of the anion channel blocker 1 mmol l(-1) diphenylamine-2-carboxylate (DPC) after ionomycin + db-cAMP + IBMX treatment significantly reduced serosal-to-mucosal unidirectional Cl(-) flux (P<0.001), net Cl(-) flux (P<0.05), short-circuit current (I(sc), P<0.001) and tissue conductance (G(t), P<0.001), while 0.1 mmol l(-1) 4,4'-diisothiocyano-2,2'-stilbene-disulphonic acid (DIDS, a blocker of anion exchange) was without effect. Stimulation by db-cAMP + IBMX (no ionomycin) significantly increased unidirectional fluxes, I(sc) and G(t) but did not produce net Cl(-) secretion. Ionomycin alone produced a transient increase in I(sc) but had no effect on G(t) and caused no significant changes in unidirectional or net Cl(-) fluxes. Addition of db-cAMP + IBMX after ionomycin treatment produced net secretion of Cl(-) and large increases in unidirectional fluxes and G(t). Cystic fibrosis transmembrane conductance regulator (CFTR) was immunocytochemically localized with a monoclonal mouse antibody to the carboxy terminus and found to be present in the cytoplasm and basolateral membranes of all enterocytes and in the brush-border membrane of some cells, whereas NKCC immunofluorescence, demonstrating the presence of the Na(+)/K(+)/2Cl(-) cotransporter, was present in the cytoplasm and brush-border membrane. We conclude that the teleost intestine is capable of salt and fluid secretion only if intracellular Ca(2+) and cyclic AMP pathways are stimulated together and that this secretion appears to involve activation of CFTR ion channels in the apical membrane of a subpopulation of enterocytes. (+info)
Redistribution of immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water.
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Cellular distribution of cystic fibrosis transmembrane conductance regulator (CFTR) immunofluorescence was detected by monoclonal antibody directed to the C terminus of killifish CFTR (kfCFTR) in chloride cells of fresh water (FW) adapted fish and animals transferred to sea water (SW) for 24h, 48h and 14+ days. Confocal microscopy allowed localization within mitochondria-rich (MR) cells to be determined as superficial (i.e. in the apical membrane) or deeper within the cytoplasm of the cells. In FW, 90 % of MR cells had diffuse kfCFTR immunofluorescence in the central part of the cytosol, with only 8.1 % having apical kfCFTR, which was 6.6+/-0.54 microm below the microridges of surrounding pavement cells. Curiously, FW but not SW pavement cells also had positive immunofluorescence to kfCFTR. After 24h in SW, a time when kfCFTR expression is elevated, a condensed punctate immunofluorescence appeared among 18.8 % of MR cells, 13.4+/-0.66 microm (mean +/- S.E.M.) below the surface of the cells. By 48h, a majority (76.3 %) of MR cells had punctate kfCFTR distribution and the distance from the surface was less (7.8+/-0.2 microm), a distribution approaching the SW-acclimated condition (i.e. all MR cells showing kfCFTR immunofluorescence, 6.1+/-0.04 microm below the surface). In contrast, NKCC immunofluorescence was condensed and localized in lateral parts of MR cell complexes in FW animals and then redistributed to the whole basal cytoplasm after acclimation to SW. CFTR, the anion channel responsible for Cl(-) secretion in marine teleosts, redistributes in MR cells during SW acclimation by condensation of a diffuse distribution below the apical crypt, followed by translocation and insertion in the apical membrane. NKCC, the cotransporter that translocates Cl(-) across the basolateral membrane, moves from an eccentric cytosolic location in FW to a diffuse basolateral localization in SW chloride cells. (+info)
Short- and long-term influences of heavy metals on anionic drug efflux from renal proximal tubule.
(11/164)
We recently demonstrated in isolated killifish renal proximal tubules that two classes of nephrotoxicants, aminoglycoside antibiotics and radiocontrast agents, rapidly decrease transport mediated by multidrug resistance protein 2 (Mrp2) by causing endothelin (ET) release and signaling through an ET(B) receptor and protein kinase C (PKC). In the present study, we used killifish proximal tubules, fluorescein methotrexate, a fluorescent model substrate for Mrp2, and confocal microscopy to examine the effects of two heavy metal salts (CdCl(2) and HgCl(2)) on Mrp2 function. Three patterns of effects were seen. First, exposing tubules to 10 microM CdCl(2) or 100 nM HgCl(2) for 30 min reduced Mrp2-mediated transport. This reduction was abolished by the ET(B) receptor antagonist, RES-701-1, and by the PKC-selective inhibitor, bis-indolylmaleimide I; neither of these pharmacological tools by itself affected transport. As with aminoglycoside antibiotics and radiocontrast agents, the acute effects of 10 microM CdCl(2) or 100 nM HgCl(2) on transport were also blocked by nifedipine, suggesting that Ca(2+) also initiated cadmium and mercury action. Second, exposure to higher concentrations of CdCl(2) and HgCl(2) appeared to be toxic. Third, exposing tubules for 6 to 24 h to lower levels of CdCl(2) increased Mrp2-mediated transport and Mrp2 immunostaining at the luminal membrane of the proximal tubule cells. Together, these findings indicate that exposure of renal proximal tubules to heavy metals initially leads to reduced Mrp2 function but is followed by an induction in Mrp2-mediated transport after long-term exposure. (+info)
Cytochrome P4501A (CYP1A) in killifish (Fundulus heteroclitus): heritability of altered expression and relationship to survival in contaminated sediments.
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Previous research has shown that killifish (Fundulus heteroclitus) inhabiting a creosote-contaminated site on the Elizabeth River in Virginia exhibit little induction of cytochrome P4501A (CYP1A) protein expression and activity upon exposure to typical CYP1A-inducing chemicals. We characterized the CYP1A response of first, second, and third generation laboratory-raised offspring of feral Elizabeth River killifish to exposure to sediments from the contaminated site as well as the prototypical polycyclic aromatic hydrocarbon (PAH)-type CYP1A inducers beta-naphthoflavone (BNF) and 3-methylcholanthrene (3-MC). The Elizabeth River offspring's responses were compared to those of offspring of killifish from two reference sites (King's Creek, Virginia, and Russell Creek, North Carolina). As with feral Elizabeth River killifish, the first generation embryos and larvae were refractory to CYP1A induction. However, the response observed in 3-year-old first generation adults, as well as with second and third generation fish, was much closer to that observed in reference-site fish. We suggest that the pattern of altered CYP1A response in Elizabeth River killifish, while persistent and heritable for one generation, is mostly nongenetically based. Additionally, we investigated the hypothesis that low CYP1A activity (measured as in ovo EROD activity) would correlate to increased survival in Elizabeth River sediment pore water; this hypothesis was not supported by our results. (+info)
A low genomic number of recessive lethals in natural populations of bluefin killifish and zebrafish.
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Despite the importance of selection against deleterious mutations in natural populations, reliable estimates of the genomic numbers of mutant alleles in wild populations are scarce. We found that, in wild-caught bluefin killifish Lucania goodei (Fundulidae) and wild-caught zebrafish Danio rerio (Cyprinidae), the average numbers of recessive lethal alleles per individual are 1.9 (95% confidence limits 1.3 to 2.6) and 1.4 (95% confidence limits 1.0 to 2.0), respectively. These results, together with data on several Drosophila species and on Xenopus laevis, show that phylogenetically distant animals with different genome sizes and numbers of genes carry similar numbers of lethal mutations. (+info)
Lighting environment predicts the relative abundance of male colour morphs in bluefin killifish (Lucania goodei) populations.
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Animal communication occurs when an animal emits a signal, the signal is transmitted through the environment, and then detected by the receiver. The environment in which signalling occurs should govern the efficacy of this process. In this study, I examine the relationship of lighting environment (light transmission and tree cover), location and the relative abundances of male colour morphs across seven drainages and 30 populations in the bluefin killifish, Lucania goodei. I found that males with blue anal fins were more common in populations with low transmission of ultraviolet (UV) and blue wavelengths. By contrast, males with red anal fins (and to a lesser extent, males with yellow anal fins) were more common in populations with high transmission of UV and blue wavelengths. High UV-blue light transmission should create a blue visual background and may make blue males less conspicuous and red males more conspicuous to conspecifics. Colour contrast with the visual background may be more important than total brightness of the colour pattern. These results indicate that natural selection for effective intraspecific communication drives the relative abundance of male colour morphs in different lighting habitats. (+info)
Strong positive selection and habitat-specific amino acid substitution patterns in MHC from an estuarine fish under intense pollution stress.
(15/164)
Population-level studies using the major histocompatibility complex (Mhc) have linked specific alleles with specific diseases, but data requirements are high and the power to detect disease association is low. A novel use of Mhc population surveys involves mapping allelic substitutions onto the inferred structural molecular model to show functional differentiation related to local selective pressures. In the estuarine fish Fundulus heteroclitus, populations experiencing strong differences in antigenic challenges show significant differences in amino acid substitution patterns that are reflected as variation in the structural location of changes between populations. Fish from a population genetically adapted to severe chemical pollution also show novel patterns of DNA substitution at a highly variable Mhc class II B locus including strong signals of positive selection at inferred antigen-binding sites and population-specific signatures of amino acid substitution. Heavily parasitized fish from an extreme PCB-contaminated (U.S. Environmental Protection Agency Superfund) site show enhanced population-specific substitutions in the a-helix portion of the inferred antigen-binding region. In contrast, fish from an unpolluted site show a significantly different pattern focused on the first strand of the B-pleated sheet. Whether Mhc population profile differences represent the direct effects of chemical toxicants or indirect parasite-mediated selection, the result is a composite habitat-specific signature of strong selection and evolution affecting the genetic repertoire of the major histocompatibility complex. (+info)
Cystic fibrosis transmembrane conductance regulator in teleost fish.
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The gills and intestinal epithelia of teleost fish express cystic fibrosis transmembrane conductance regulator (CFTR), and utilize this low conductance anion channel in the apical membrane for ion secretion in seawater gill and in the basolateral membrane for ion absorption in freshwater gill. Similarly, in the intestine CFTR is present in the basolateral membrane for intestinal absorption and also in the apical membrane of secreting intestine. The expression of CFTR and the directed trafficking of the protein to the apical or basolateral membrane is salinity-dependent. The CFTR gene has been cloned and sequenced from several teleost species and although all the major elements in the human gene are present, including two nucleotide binding domains that are common to all ATP binding cassette (ABC) transporters, the sequences are divergent compared to shark or human. In euryhaline fish adapting to seawater, CFTR, localized immunocytochemically, redistributes slowly from a basolateral location to the apical membrane while ion secretory capacity increases. The facility with which teleosts regulate CFTR expression and activation during salinity adaptation make this system an appealing model for the expression and trafficking operation of this labile gene product. (+info)