Made available in DSpace on 2017-07-10T14:57:34Z (GMT). No. of bitstreams: 1 Jardel_ Nimet.pdf: 1924881 bytes, checksum: 4755b41b1458253b217679d76b6eeead (MD5) Previous issue date: 2016-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Abstract: This study evaluated the use of gill and liver of Astyanax bifasciatus as histological biomarkers for biomonitoring of streams with different uses and land occupation. The fish were collected by electric fishing technique, in six streams classified as afforested, rural and urban, in the basin of the lower Iguaçu River, west region of Paraná State, in December 2014. It was tested the hypothesis that in streams that are influenced by effluents from urban and rural areas occur greater number and histopathological severity in gill and liver of A. Bifasciatus. In this context, the objective of this study was to correlate the presence and degree of histopathology of gill and liver with the environmental variables among streams with different uses and land occupation. The environments considered afforested showed higher values of dissolved Oxygen (DO) and lower conductivity (CO) when compared to the rural and urban environments. The gills of fish from the afforested streams indicated normal functioning of the organ, while in the rural and urban streams, the histopathology in the gills suggested light to moderate damage to the organ. In relation to the liver, the histopathology indicated light to moderate damage in rural streams and moderate to severe damage in urban streams. When considering the abiotic variables and frequencies of histopathology of gill and liver, the afforested streams were separated from rural and urban areas. The histopathology in gills and livers were significantly more severe, corroborating our hypothesis. It was possible to distinguish streams with different uses and land occupation, therefore, these histopatologias showed favorable biomarkers for studies of biomonitoring / Resumo: Este estudo avaliou o uso de brânquia e fígado de Astyanax bifasciatus como biomarcadores histológicos para biomonitoramento de riachos com diferentes usos e ocupação do solo. Os peixes foram coletados por meio da técnica de pesca elétrica, em seis riachos classificados em florestados, rurais e urbanos, na bacia do baixo Rio Iguaçu, região Oeste do Estado do Paraná, em dezembro de 2014. Testou-se a hipótese de que em riachos que sofrem influências de efluentes oriundos de áreas rurais e urbanas ocorrem maior número e severidade histopatológicas em brânquia e fígado de A. bifasciatus. Nesse contexto, este estudo teve como objetivo correlacionar a presença e o grau de histopatologias de brânquia e fígado com as variáveis ambientais entre riachos com diferentes usos e ocupação do solo. Os ambientes considerados florestados apresentaram maiores valores de Oxigênio dissolvido (OD) e menores de condutividade (CO) quando comparados com os ambientes rurais e urbanos. As brânquias dos peixes dos riachos florestados indicaram funcionamento normal do órgão, enquanto que nos riachos rurais e urbanos, as histopatologias nas brânquias sugeriram danos leves para moderados ao órgão. Em relação ao fígado as histopatologias indicaram danos leves para moderados nos riachos rurais e danos moderados para severos nos riachos urbanos. Ao considerar as variáveis abióticas e as frequências das histopatologias de brânquia e fígado, os riachos florestados foram separados dos rurais e urbanos. As histopatologias nas brânquias e fígados foram significativamente mais severas, corroborando a nossa hipótese. Foi possível distinguir os riachos com diferentes usos e ocupação do solo, portanto, essas histopatologias se mostraram biomarcadores favoráveis para estudos de biomonitoramento

Os filamentos branquiais da lagosta Americana, Homarus americanus, foram dissociados em solução salina fisiológica e os diversos tipos celulares separados em gradiente de 30, 40, 50 e 80% de sacarose através de centrifugação de alta velocidade. As células provenientes de cada solução de sacarose foram incubadas em 65Zn2+ visando caracterizar a tomada de zinco para cada tipo celular. A caracterização do acúmulo de zinco em cada célula foi investigada na presença e ausência de 10mM de cálcio (CaCl2), variadas concentrações de NaCl e pH, presença de 100 µM de verapamil, nifedipina (inibidores de canais de Ca2+) e ionóforo de cálcio, A23187. O influxo de 65Zn2+ foi descrito pela cinética de Michaelis-Menten nas concentrações de zinco variando de 1-1000 µM.. O cálcio externo não afetou o transporte de Zn para as células presentes no gradiente de 30% a 50% de sacarose, mas atuou como estimulador para as células em gradiente de 80% de sacarose. O cálcio reduziu o Km e a velocidade máxima de transporte (Vmax) para as células de 30% de sacarose, enquanto duplicou aparentemente a velocidade máxima de transporte para as células provenientes do meio em 80% de sacarose. Os resultados sugerem que o cálcio, sódio e prótons entram nas células branquiais através de um canal para cátion com ampla especificidade. Diferenças observadas no transporte de zinco em relação aos diferentes tipos de células aparentemente estão relacionadas com as diferentes taxas de afinidade de cada transportador em cada tipo celular. O transporte de 65Zn2+ também foi estudado em filamentos branquiais isolados e intactos, demonstrando propriedades de transporte muito parecidas com as observadas pelas células em gradiente de 80% de sacarose. Os resultados sugerem que a tomada de Zn se dá por processo de transporte na membrana apical das brânquias. Um modelo experimental para o transporte de Zn em células de brânquias de lagostas é apresentado. / Gill filaments of the American lobster, Homarus americanus, were dissociated in a physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in 65Zn2+ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell was investigated in the presence and absence of 10 mM calcium (CaCl2), variable NaCl concentrations and pH values, and 100 µM verapamil, nifedipine (calcium channel inhibitors), and the calcium ionophore, A23187. 65Zn2+ influxes were hyperbolic functions of zinc concentration (1-1000 µM) and followed the Michaelis-Menten kinetics. External calcium was neutral to cells from 30% to 50% sucrose, but stimulatory for cells from 80% sucrose. However, calcium reduced both apparent zinc binding affinity (Km) and maximal transport velocity (Jmax) for 30% sucrose cells, while doubling the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by way of an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were partially inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. 65Zn2+ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomena. A working model for zinc transport by lobster gill cells is presented.

Compared with the free atmosphere, the aquatic environment is oxygen poor. As a result many secondarily aquatic organisms have adaptations that allow them to continue to use the atmosphere, directly or indirectly, to supply their oxygen requirements. This thesis examines how diving insects use bubbles of air collected at the surface of the water as oxygen reserves, gills and flotation devices, and how an aquatic angiosperm channels convective flows of air from its emergent leaves to its submerged organs. 1. Backswimmers (Anisops spp.) begin a dive positively buoyant, but rapidly enter a protracted period of near neutral buoyancy. A bubble of air held on the insect’s abdomen shrinks as respiration consumes its oxygen, while at the same time highly soluble carbon dioxide dissolves into the surrounding water. The reduced air volume confers neutral buoyancy. In response to low oxygen partial pressure (PO2) in the bubble, oxygen is released from large haemoglobin cells in the abdomen. The haemoglobin’s sensitivity to falling PO2 maintains the oxygen tension between 5.1 and 2.0 kPa. This stabilises the volume and buoyancy of the bubble. During a dive the haemoglobin and air-store supply 0.25 and 0.26 μL of oxygen, respectively. 2. The oxygen affinity of backswimmer haemoglobin determines the stability of the neutrally buoyant phase as well as its ability to satisfy the insect’s respiration. An oxygen equilibrium curve (OEC) determined in vivo has a highly sigmoid shape and an oxygen affinity of 3.9 kPa. In comparison with OEC made in vitro, the in vivo measurements show increased cooperativity and oxygen affinity, consistent with the presence of cationic effectors. Models strongly support the accuracy of the in vivo OEC method. 3. It has long been assumed that a bubble of air held over the spiracles of an insect enables the uptake of oxygen from the surrounding water and thus acts as a ‘gas gill’. Oxygen diffuses into a bubble of air when its PO2 is lower than the surrounding water, but a coincident higher nitrogen partial pressure causes it to dissolve. Several models have been produced to describe the gas exchange process, but all are based on untested assumptions of gill parameters. Measurements of gas gill volume and PO2 made on water bugs (Agraptocorixa eurynome) demonstrate that both drop quickly at the beginning of a dive, but PO2 reaches a stable level while the gas gill continues to dissolve. The importance of ventilation in maintaining an acceptable rate of oxygen consumption is also shown. 4. Interconnected gas spaces within the leaves, stems and rhizomes are a common feature of many emergent aquatic plants. Pressurised air from the leaves and culms of these plants ventilate these lacunae, flowing back to the atmosphere through efflux points. Unlike most aquatic plants, which have simple interconnected pith spaces, sacred lotus, Nelumbo nucifera, possess discrete gas canals which only interconnect where a leaf grows from the rhizome. Silicone casts and pneumatic tests of the gas canals reveal a complex repeating pattern of interconnections which channel air from specific regions of the leaf blade to the rhizome and out through efflux points on adjacent leaves. 5. Lotus, Nelumbo nucifera, possess in the centre of their leaves a specialised efflux organ which connects the gas canals in the leaves and stems with the atmosphere through the apertures of large stomata. Measurements made on excised lotus leaves and in situ reveal that the large stomata act as exhaust valves, opening and closing in a diurnal pattern to regulate the flow of pressurised gas from the leaf lamina and gas canals. This behaviour is shown to regulate gas flow rate and direction. The aquatic environment offers similar respiratory challenges to both plants and insects. While the oxygen uptake and transport mechanisms evolved by these groups are markedly different, they all function according to the same physical laws. Diving insects are separated from the atmosphere while underwater, forcing them to rely on oxygen either carried with them from the surface or extracted from the surrounding water. Emergent aquatic plants have permanent access to atmospheric oxygen, but must transport it long distances from their aerial leaves and stems to their roots and rhizomes. This thesis examines the uptake and storage of oxygen by diving insects and the gas transport system of the sacred lotus. / Thesis(Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

<p>During the smoltification the Atlantic salmon (Salmo salar) develop different adaptations to survive in oceanic environment. One of the most important adaptations is the ability to excrete the surplus of salt through the gills. The excretion is controlled by an enzyme called Na+/K+-ATPase which is produced in an α-subunit by two gene isoforms called α1a and α1b. Enzyme activity is increasing during the smoltification process and is a strong indicator for that the process is taking place. The aim of this study was to investigate a landlocked strain of Atlantic salmon and see how the enzyme activity is developing in comparison with the gene expression of the mRNA that is coded for the enzyme. The study was carried out between March and May in the hatchery in Brattfors, Värmland. Fish were sampled at four occasions. The enzyme activity was compared between two groups of salmon where one group had full ration of food, 100% and the other group had a 15% food ration. The enzyme activity for the 100% group was then compared with the gene expression from the same group. The hypothesis was that food availability should effect smoltification and that the 15% group would have a faster increase in activity compared with the 100% group. There should also be some correlation between enzyme activity and gene expression. Na+/K+-ATPase enzyme activity showed no major differences between the groups except for a significant difference at the last sampling. Both groups had a large increase in activity from the second to the third sampling with a peak on 3.16 µmol ADP/mg/h at most. This was followed by a drop in activity at the last sampling date. The gene expression showed a fast increase of the α1b gene over the study with drop in the last sampling and the α1a gene had a constant increase from the first to the last sampling. The comparison with enzyme activity and gene expression showed a weak correlation. Compared with studies done on anadromous salmon and the land locked salmon in this study had a different development in gene expression. This could be explained that the different life strategies play an important role how the genes are expressed.</p> / <p>Under smoltifieringen utvecklar atlantlaxen (Salmo salar) olika anpassningar för att överleva i havsmiljö. En av de vikigaste anpassningarna är att utsöndra överskott av salt via gällarna. Exkretionen är kontrollerad av ett enzym som heter Na+/K+-ATPas som produceras i en α-subenhet av två isoformer av gener som heter α1a och α1b. Enzym aktiviteten ökar under smoltifieringen och är en stark indikator på att processen sker. Målet med denna studie var att undersöka en sjövandrande stam av atlantlax och se hur enzymaktiviteten utvecklas i jämförelse med gen expressionen av mRNA som kodar för produktionen av enzymet. Studien genomfördes vid fiskodlingen i Brattfors, Värmland där prover togs vid fyra tillfällen. Enzymaktiviteten jämfördes mellan två grupper av lax där en grupp fick full matranson 100 % och en grupp fick 15 % matranson. Senare jämfördes enzymaktiviteten för 100 % gruppen med gen expressionen inom samma grupp. Hypotesen var att tillgängligheten på mat skulle påverka smoltifieringen och att 15 % gruppen skulle ha en snabbare ökning i jämförelse med 100 % gruppen. Det skulle också vara en viss korrelation mellan enzymaktivitet och gen expression. Na+/K+-ATPas enzym aktiviteten visade inga större skillnader mellan grupperna förutom vid sista provtagningen. Båda grupperna hade en stor ökning från den andra till den tredje provtagningen med den högsta aktiviteten på 3.16 µmol ADP/mg/h. Detta följdes av ett fall i aktivitet vid sista provtagningen. Gen expressionen visade en snabb ökning av α1b genen över studien med en nedgång vid sista provtagningen och α1a hade en konstant men mindre ökning från första till sista provtagningen. Jämförelsen mellan enzymaktivitet och gen expression visade på en svag korrelation. Det fanns en skillnad i gen expression mellan studier gjorda på anadrom lax och sjövandrande lax i denna studie. Detta kan förklaras av att de olika livsstrategierna spelar en betydande roll i hur generna uttrycks.</p>

<p>A gill filament-based assay for the cytochrome P450 1A (CYP1A)-catalysed activity ethoxyresorufin <i>O</i>-deethylase (EROD) was developed in rainbow trout (<i>Oncorhynchus mykiss</i>) and applied to Atlantic salmon (<i>Salmo salar</i>), Arctic charr (<i>Salvelinus alpinus</i>), Atlantic cod (<i>Gadus morhua</i>), saithe (<i>Pollachius virens</i>), and spotted wolffish (<i>Anarhichas minor</i>). Exposure to waterborne β-naphthoflavone (βNF; 10^-6 M) induced branchial EROD activity in all species but the spotted wolffish. In rainbow trout exposed to low concentrations of benzo[a]pyrene (BaP; 10^-9 M) and the textile dye indigo (10^-8 M) the gills responded more rapidly than the liver to BaP, and indigo induced branchial but not hepatic EROD activity.</p><p>A CYP1A-dependent BaP adduct formation was shown in gills of fish exposed to waterborne ³H-BaP, i.e. the adduct formation was enhanced by βNF and blocked by ellipticine (CYP1A inhibitor). The predominant location for BaP adducts was the secondary lamellae (most exposed part of the gill filament), whereas the CYP1A enzyme was also present in the primary lamellae of the gill filament. Hence, in addition to the cell-specific expression of CYP1A an important determinant for the localisation of adducts seemed to be the bioavailability of BaP. This idea is supported by the fact that the CYP1A enzyme was induced only in secondary lamellae by BaP (10^-7 M) and indigo (10^-6 M), whereas it was induced in both primary and secondary lamellae by 3,3´,4,4´,5-pentachlorobiphenyl (10^-8 M). Apparently, readily metabolised inducers (BaP and indigo) are biotransformed in the secondary lamellae.</p><p>My results show that gill filament EROD activity is a sensitive biomarker of exposure to waterborne dioxin-like pollutants, and that the assay has potential for use in monitoring. Furthermore, the results suggest that readily metabolised dioxin-like compounds absorbed via the gills may undergo first-pass metabolism in the gill cells and therefore remain undetected by monitoring of EROD activity in the liver.</p>

A gill filament-based assay for the cytochrome P450 1A (CYP1A)-catalysed activity ethoxyresorufin O-deethylase (EROD) was developed in rainbow trout (Oncorhynchus mykiss) and applied to Atlantic salmon (Salmo salar), Arctic charr (Salvelinus alpinus), Atlantic cod (Gadus morhua), saithe (Pollachius virens), and spotted wolffish (Anarhichas minor). Exposure to waterborne β-naphthoflavone (βNF; 10-6 M) induced branchial EROD activity in all species but the spotted wolffish. In rainbow trout exposed to low concentrations of benzo[a]pyrene (BaP; 10-9 M) and the textile dye indigo (10-8 M) the gills responded more rapidly than the liver to BaP, and indigo induced branchial but not hepatic EROD activity. A CYP1A-dependent BaP adduct formation was shown in gills of fish exposed to waterborne 3H-BaP, i.e. the adduct formation was enhanced by βNF and blocked by ellipticine (CYP1A inhibitor). The predominant location for BaP adducts was the secondary lamellae (most exposed part of the gill filament), whereas the CYP1A enzyme was also present in the primary lamellae of the gill filament. Hence, in addition to the cell-specific expression of CYP1A an important determinant for the localisation of adducts seemed to be the bioavailability of BaP. This idea is supported by the fact that the CYP1A enzyme was induced only in secondary lamellae by BaP (10-7 M) and indigo (10-6 M), whereas it was induced in both primary and secondary lamellae by 3,3´,4,4´,5-pentachlorobiphenyl (10-8 M). Apparently, readily metabolised inducers (BaP and indigo) are biotransformed in the secondary lamellae. My results show that gill filament EROD activity is a sensitive biomarker of exposure to waterborne dioxin-like pollutants, and that the assay has potential for use in monitoring. Furthermore, the results suggest that readily metabolised dioxin-like compounds absorbed via the gills may undergo first-pass metabolism in the gill cells and therefore remain undetected by monitoring of EROD activity in the liver.

During the smoltification the Atlantic salmon (Salmo salar) develop different adaptations to survive in oceanic environment. One of the most important adaptations is the ability to excrete the surplus of salt through the gills. The excretion is controlled by an enzyme called Na+/K+-ATPase which is produced in an α-subunit by two gene isoforms called α1a and α1b. Enzyme activity is increasing during the smoltification process and is a strong indicator for that the process is taking place. The aim of this study was to investigate a landlocked strain of Atlantic salmon and see how the enzyme activity is developing in comparison with the gene expression of the mRNA that is coded for the enzyme. The study was carried out between March and May in the hatchery in Brattfors, Värmland. Fish were sampled at four occasions. The enzyme activity was compared between two groups of salmon where one group had full ration of food, 100% and the other group had a 15% food ration. The enzyme activity for the 100% group was then compared with the gene expression from the same group. The hypothesis was that food availability should effect smoltification and that the 15% group would have a faster increase in activity compared with the 100% group. There should also be some correlation between enzyme activity and gene expression. Na+/K+-ATPase enzyme activity showed no major differences between the groups except for a significant difference at the last sampling. Both groups had a large increase in activity from the second to the third sampling with a peak on 3.16 µmol ADP/mg/h at most. This was followed by a drop in activity at the last sampling date. The gene expression showed a fast increase of the α1b gene over the study with drop in the last sampling and the α1a gene had a constant increase from the first to the last sampling. The comparison with enzyme activity and gene expression showed a weak correlation. Compared with studies done on anadromous salmon and the land locked salmon in this study had a different development in gene expression. This could be explained that the different life strategies play an important role how the genes are expressed. / Under smoltifieringen utvecklar atlantlaxen (Salmo salar) olika anpassningar för att överleva i havsmiljö. En av de vikigaste anpassningarna är att utsöndra överskott av salt via gällarna. Exkretionen är kontrollerad av ett enzym som heter Na+/K+-ATPas som produceras i en α-subenhet av två isoformer av gener som heter α1a och α1b. Enzym aktiviteten ökar under smoltifieringen och är en stark indikator på att processen sker. Målet med denna studie var att undersöka en sjövandrande stam av atlantlax och se hur enzymaktiviteten utvecklas i jämförelse med gen expressionen av mRNA som kodar för produktionen av enzymet. Studien genomfördes vid fiskodlingen i Brattfors, Värmland där prover togs vid fyra tillfällen. Enzymaktiviteten jämfördes mellan två grupper av lax där en grupp fick full matranson 100 % och en grupp fick 15 % matranson. Senare jämfördes enzymaktiviteten för 100 % gruppen med gen expressionen inom samma grupp. Hypotesen var att tillgängligheten på mat skulle påverka smoltifieringen och att 15 % gruppen skulle ha en snabbare ökning i jämförelse med 100 % gruppen. Det skulle också vara en viss korrelation mellan enzymaktivitet och gen expression. Na+/K+-ATPas enzym aktiviteten visade inga större skillnader mellan grupperna förutom vid sista provtagningen. Båda grupperna hade en stor ökning från den andra till den tredje provtagningen med den högsta aktiviteten på 3.16 µmol ADP/mg/h. Detta följdes av ett fall i aktivitet vid sista provtagningen. Gen expressionen visade en snabb ökning av α1b genen över studien med en nedgång vid sista provtagningen och α1a hade en konstant men mindre ökning från första till sista provtagningen. Jämförelsen mellan enzymaktivitet och gen expression visade på en svag korrelation. Det fanns en skillnad i gen expression mellan studier gjorda på anadrom lax och sjövandrande lax i denna studie. Detta kan förklaras av att de olika livsstrategierna spelar en betydande roll i hur generna uttrycks.

Induction of the cytochrome P450(CYP)1A protein and the connected increase in 7-ethoxyresorufin O-deethylase (EROD) activity are common biomarkers in fish. Enhanced activity of this protein signals exposure to Ah-receptor agonists such as chlorinated dioxins, co-planar polychlorinated biphenyls (PCBs) and certain polycyclic aromatic hydrocarbons (PAHs). The EROD biomarker is commonly analyzed in liver microsomes. However, the gill is directly exposed to waterborne pollutants, and in this thesis the gill filament EROD assay was therefore evaluated as a monitoring tool for waterborne CYP1A inducers in fish. Originally developed in rainbow trout (Oncorhynchus mykiss), the assay was here applied in various limnic and marine species. Following exposure to low waterborne concentrations of the readily metabolized CYP1A inducers benzo(a)pyrene (BaP) and indigo, a strong EROD induction was observed in the gill but not in the liver. This likely reflected metabolic clearance of the inducers in gill and other extrahepatic tissues. The high sensitivity of the gill was confirmed in studies of fish caged in waters in urban and rural areas in Sweden where the gill consistently showed a more pronounced EROD induction compared with the liver and the kidney. Fish caged in the reference waters showed surprisingly strong gill EROD induction and CYP1A immunostaining. Consequently, there may be CYP1A inducers present in the aquatic environment that are not yet identified. The assay was further applied in Atlantic cod (Gadus morhua) as a biomarker of exposure to crude oil and produced water (PW) from oil fields in the North Sea. The assay was finally adapted to detect inhibiting compounds, and an imidazole, a triazole and a plant flavonoid turned out to be potent gill EROD inhibitors. The overall conclusion from the studies of this thesis is that the gill filament EROD assay is a practical and sensitive biomarker of exposure to waterborne CYP1A inducers in various fish species. The induction of gill EROD activity in fish also at the reference sites in the field studies calls for further studies on background contamination in Swedish waters.

The self-fertilizing mangrove rivulus, Kryptolebias marmoratus, is an amphibious fish capable of reversible gill remodelling when moving between aquatic and terrestrial environments. In this thesis I determined how plastic morphological and physiological respiratory traits were integrated during transitions between environments. In two isogenic lineages, I found that behaviour (increased emersion) of individual fish caused gill morphological changes (enlargement of the interlamellar cell mass (ILCM)) that reduced gill surface area. I also found that large ILCMs that formed after 7 d of air exposure increased both gill ventilation and critical oxygen tension (Pcrit) when fish returned to water. These results indicate that large ILCMs reduce aquatic respiratory function, and increased gill ventilation was unable to maintain oxygen uptake at extreme levels of hypoxia. Ultimately, this study highlights the trade-offs in gill structure and function during the transition between air and water, and demonstrates that differences in behaviour can generate morphological variation.

Os filamentos branquiais da lagosta Americana, Homarus americanus, foram dissociados em solução salina fisiológica e os diversos tipos celulares separados em gradiente de 30, 40, 50 e 80% de sacarose através de centrifugação de alta velocidade. As células provenientes de cada solução de sacarose foram incubadas em 65Zn2+ visando caracterizar a tomada de zinco para cada tipo celular. A caracterização do acúmulo de zinco em cada célula foi investigada na presença e ausência de 10mM de cálcio (CaCl2), variadas concentrações de NaCl e pH, presença de 100 µM de verapamil, nifedipina (inibidores de canais de Ca2+) e ionóforo de cálcio, A23187. O influxo de 65Zn2+ foi descrito pela cinética de Michaelis-Menten nas concentrações de zinco variando de 1-1000 µM.. O cálcio externo não afetou o transporte de Zn para as células presentes no gradiente de 30% a 50% de sacarose, mas atuou como estimulador para as células em gradiente de 80% de sacarose. O cálcio reduziu o Km e a velocidade máxima de transporte (Vmax) para as células de 30% de sacarose, enquanto duplicou aparentemente a velocidade máxima de transporte para as células provenientes do meio em 80% de sacarose. Os resultados sugerem que o cálcio, sódio e prótons entram nas células branquiais através de um canal para cátion com ampla especificidade. Diferenças observadas no transporte de zinco em relação aos diferentes tipos de células aparentemente estão relacionadas com as diferentes taxas de afinidade de cada transportador em cada tipo celular. O transporte de 65Zn2+ também foi estudado em filamentos branquiais isolados e intactos, demonstrando propriedades de transporte muito parecidas com as observadas pelas células em gradiente de 80% de sacarose. Os resultados sugerem que a tomada de Zn se dá por processo de transporte na membrana apical das brânquias. Um modelo experimental para o transporte de Zn em células de brânquias de lagostas é apresentado. / Gill filaments of the American lobster, Homarus americanus, were dissociated in a physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in 65Zn2+ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell was investigated in the presence and absence of 10 mM calcium (CaCl2), variable NaCl concentrations and pH values, and 100 µM verapamil, nifedipine (calcium channel inhibitors), and the calcium ionophore, A23187. 65Zn2+ influxes were hyperbolic functions of zinc concentration (1-1000 µM) and followed the Michaelis-Menten kinetics. External calcium was neutral to cells from 30% to 50% sucrose, but stimulatory for cells from 80% sucrose. However, calcium reduced both apparent zinc binding affinity (Km) and maximal transport velocity (Jmax) for 30% sucrose cells, while doubling the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by way of an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were partially inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. 65Zn2+ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomena. A working model for zinc transport by lobster gill cells is presented.