RÉSUMÉ
We examined gonads and thyroid glands of Gulf killifish (Fundulus grandis) 1yr after the Deepwater Horizon oil spill. F. grandis were trapped from two impacted sites in Barataria Bay (Bayou St. Denis, Bay Jimmy) and an un-impacted site in East Texas (Sabine Pass). The greatest number of F. grandis were collected at Sabine Pass. F. grandis collected at Bayou St. Denis were smaller and had smaller Fulton condition factor scores than fish collected at Sabine Pass. Sex ratios were biased roughly 2:1 in favor of females at Sabine Pass and Bayou St. Denis. Gonad-somatic index (GSI) in males from Sabine Pass was double that of fish from Bay Jimmy while germinal epithelium thickness of the testes was 2.7 fold smaller in males from the impacted site. GSI and oocyte diameters in females from Bayou St. Denis were significantly smaller than females from Bay Jimmy or the reference site. There were no differences in thyroid follicle cell height. While total polyaromatic hydrocarbons at the impacted sites were no different from the reference site, the impacted sites did have greater concentrations of benzo[a]pyrene in sediment pore water. The finding of smaller GSI and testicular germinal epithelium in males from an impacted site suggest that exposure to a combination of oil and dispersants may adversely impact testicular function.
Sujet(s)
Surveillance de l'environnement/méthodes , Fundulidae/croissance et développement , Gonades/effets des médicaments et des substances chimiques , Pollution pétrolière/effets indésirables , Glande thyroide/effets des médicaments et des substances chimiques , Polluants chimiques de l'eau/toxicité , Animaux , Baies (géographie)/composition chimique , Femelle , Gonades/anatomopathologie , Golfe du Mexique , Louisiane , Mâle , Pollution pétrolière/analyse , Glande thyroide/anatomopathologie , Polluants chimiques de l'eau/analyseRÉSUMÉ
We investigated stage-dependent changes in sensitivity of the thyroid gland to perchlorate during development of African clawed frog tadpoles (Xenopus laevis) in relation to non-thyroidal iodide transporting tissues. Perchlorate-induced increases in thyroid follicle cell size and colloid depletion were blunted when exposures began at Nieuwkoop-Faber (NF) stage 55 compared to when exposures began at NF stages 49 or 1-10. To determine if the development of other iodide transporting tissues may contribute to this difference we first examined which tissues expressed transcripts for the sodium dependent iodide symporter (NIS). RT-PCR analysis revealed that NIS was expressed in stomach and small intestine in addition to the thyroid gland of X. laevis tadpoles. NIS mRNA was not detected in lung, kidney, skin, gill, muscle, heart or liver. Perchlorate sensitive (125)I uptake was found in stomach, lung, kidney, gill, and small intestine but not muscle, liver, or heart. Perchlorate-sensitive (125)I uptake by stomach was 6-10 times greater than in any other non-thyroidal tissue in tadpoles. While NF stage 49 tadpoles exhibited perchlorate-sensitive uptake in stomach it was roughly 4-fold less than that observed in NF stage 55 tadpoles. Although abundance of NIS gene transcripts was greater in stomachs from NF stage 55 compared to NF stage 49 tadpoles this difference was not statistically significant. We conclude that gastric iodide uptake increases between NF stages 49 and 55, possibly due to post-translational changes in NIS glycosylation or trafficking within gastric mucosal cells. These developmental changes in gastric NIS gene expression may affect iodide availability to the thyroid gland.
Sujet(s)
Larve/métabolisme , Métamorphose biologique/génétique , Perchlorates/métabolisme , Symporteurs/génétique , Glande thyroide/métabolisme , Xenopus laevis/métabolisme , Animaux , Femelle , MâleRÉSUMÉ
Nucleotide sequence comparisons have identified a gene product in the genome database of African clawed frogs (Xenopus laevis) as a probable member of the solute carrier family of membrane transporters. To confirm its identity as a putative iodide transporter, we examined the function of this sequence after heterologous expression in mammalian cells. A green monkey kidney cell line transfected with the Xenopus nucleotide sequence had significantly greater (125)I uptake than sham-transfected control cells. The uptake in carrier-transfected cells was significantly inhibited in the presence of perchlorate, a competitive inhibitor of mammalian Na(+)/iodide symporter. Tissue distributions of the sequence were also consistent with a role in iodide uptake. The mRNA encoding the carrier was found to be expressed in the thyroid gland, stomach, and kidney of tadpoles from X. laevis, as well as the bullfrog Rana catesbeiana. The ovaries of adult X. laevis also were found to express the carrier. Phylogenetic analysis suggested that the putative X. laevis iodide transporter is orthologous to vertebrate Na(+)-dependent iodide symporters. We conclude that the amphibian sequence encodes a protein that is indeed a functional Na(+)/iodide symporter in X. laevis, as well as R. catesbeiana.
Sujet(s)
Protéines de transport membranaire/physiologie , Perchlorates/pharmacologie , Rana catesbeiana/métabolisme , Symporteurs/antagonistes et inhibiteurs , Symporteurs/métabolisme , Xenopus laevis/métabolisme , Animaux , Cellules COS , Chlorocebus aethiops , Femelle , Muqueuse gastrique/métabolisme , Radio-isotopes de l'iode/métabolisme , Rein/métabolisme , Mâle , Protéines de transport membranaire/génétique , Phylogenèse , ARN messager/métabolisme , Sodium/métabolisme , Glande thyroide/métabolisme , TransfectionRÉSUMÉ
Comparisons of the primary structures of the Na,K-ATPase alpha-isoforms reveal the existence of regions of structural divergence, suggesting that they are involved in unique functions. One of these regions is the isoform-specific region (ISR), located near the ATP binding site in the major cytoplasmic loop. To evaluate its importance, we constructed mutants of the rodent wild-type alpha1 and alpha3 isoforms in which the ISR was replaced with irrelevant sequences, i.e., the analogous region from the rat gastric H,K-ATPase catalytic subunit or a region from the human c-myc oncogene. Opossum kidney (OK) cells were transfected with wild-type rat alpha1, alpha3, or their corresponding chimeras and selected in ouabain. Introduction of either mutant produced ouabain-resistant colonies, consistent with functional expression of the chimeric protein and indicating that the ISR is not essential for overall Na,K-ATPase function. The introduced chimeras were then characterized enzymatically by measuring the relative rate of K(+) and Li(+) deocclusions. Results showed that exchanges of both alpha1 and alpha3 ISRs significantly modified the sensitivity for the enzyme to either K(+) or Li(+). Subsequent treatment of the cells with phorbol esters revealed an altered Na,K-ATPase transport in response to protein kinase C activation for the alpha1 chimeras. No changes were observed for the alpha3 isoform, suggesting that it is not sensitive to PKC regulation. These results demonstrated that the ISR plays an important role in ion deocclusion and in the response to PKC (only for the alpha1 isoform).
Sujet(s)
Protéine kinase C/métabolisme , Sodium-Potassium-Exchanging ATPase/métabolisme , Séquence d'acides aminés , Animaux , Humains , Cinétique , Modèles moléculaires , Données de séquences moléculaires , Potassium/métabolisme , Isoformes de protéines/génétique , Isoformes de protéines/métabolisme , Structure tertiaire des protéines , Protéines proto-oncogènes c-myc/génétique , Protéines proto-oncogènes c-myc/métabolisme , Rats , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/métabolisme , Alignement de séquences , Sodium-Potassium-Exchanging ATPase/génétique , TempératureSujet(s)
Sodium-Potassium-Exchanging ATPase/composition chimique , Séquence d'acides aminés , Substitution d'acide aminé , Animaux , Lignée cellulaire , Isoenzymes/composition chimique , Isoenzymes/métabolisme , Données de séquences moléculaires , Sous-unités de protéines/composition chimique , Sous-unités de protéines/métabolisme , Rats , Protéines recombinantes/composition chimique , Protéines recombinantes/métabolisme , Alignement de séquences , Similitude de séquences d'acides aminés , Sodium-Potassium-Exchanging ATPase/métabolismeRÉSUMÉ
Specific functions served by the various Na(+)-K(+)-ATPase alpha-isoforms are likely to originate in regions of structural divergence within their primary structures. The isoforms are nearly identical, with the exception of the NH(2) terminus and a 10-residue region near the center of each molecule (isoform-specific region; ISR). Although the NH(2) terminus has been clearly identified as a source of isoform functional diversity, other regions seem to be involved. We investigated whether the central ISR could also contribute to isoform variability. We constructed chimeric molecules in which the central ISRs of rat alpha(1)- and alpha(2)-isoforms were exchanged. After stable transfection into opossum kidney cells, the chimeras were characterized for two properties known to differ dramatically among the isoforms: their K(+) deocclusion pattern and their response to PKC activation. Comparisons with rat full-length alpha(1)- and alpha(2)-isoforms expressed under the same conditions suggest an involvement of the central ISR in the response to PKC but not in K(+) deocclusion.