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Hepatocyte transmembrane potential (Vm) behaves as an osmometer and varies with changes in extracellular osmotic pressure created by altering the NaCl concentration in the external medium (Howard, L.D. and Wondergem, R. (1987) J. Membr. Biol. 100, 53). We now have demonstrated similar effects on Vm by increasing external osmolality with added sucrose and not altering ionic strength. We also have demonstrated that hyperosmotic stress-induced depolarization of Vm results from changes in membrane K+ conductance, gK, rather than from changes in the K+ equilibrium potential. Vm and aki of hepatocytes in liver slices were measured by conventional and ion-sensitive microelectrodes, respectively. Cell water vols. were estimated by differences in wet and dry weights of liver slices after 10-min incubations. Effect of hyperosmotic medium on membrane transference number for K+, tk, was measured by effects on Vm of step-changes in external [K+]. Hepatocyte Vm decreased 34, 52 and 54% when tissue was superfused with medium made hyperosmotic with added sucrose (50, 100 and 150 mM). Correspondingly, aKi increased 10, 18 and 29% with this hyperosmotic stress of added sucrose. Tissue water of 2.92 ± 0.10 kg H2O/kg dry weight in control solution decreased to 2.60 ± 0.05, 2.25 ± 0.06 and 2.22 ± 0.05 kg H2O/kg dry weight with additions to medium of 50, 100 and 150 mM sucrose, respectively. Adding 50 mM sucrose to medium decreased tK from 0.20 ± 0.01 to 0.05 ± 0.01. Depolarization by 50% with hyperosmotic stress (100 mM sucrose) also occurred in Cl-free medium where Cl- was substituted with gluconate. We conclude that hepatocytes shrink during hyperosmotic stress, and the aKi increases. The accompanying decrease in Vm is opposite to that expected by an increase in aKi, and at least in part results from a concomitant decrease in gK. Changes in membrane Cl- conductance most likely do not contribute to osmotic stress-induced depolarization, since equivalent decreases in Vm occurred with added sucrose in cells depleted of Cl- by superfusing tissue with Cl-free medium.

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Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-08-10T17:18:30Z No. of bitstreams: 2 Tese - Leandro Nascimento da Silva Rodrigues - 2014.pdf: 2636013 bytes, checksum: 332020037e85516d843bbcbfc5e4da75 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-15T13:41:26Z (GMT) No. of bitstreams: 2 Tese - Leandro Nascimento da Silva Rodrigues - 2014.pdf: 2636013 bytes, checksum: 332020037e85516d843bbcbfc5e4da75 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-15T13:41:26Z (GMT). No. of bitstreams: 2 Tese - Leandro Nascimento da Silva Rodrigues - 2014.pdf: 2636013 bytes, checksum: 332020037e85516d843bbcbfc5e4da75 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-11-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The dimorphic fungus Paracoccidioides is the etiological agent of paracoccidioidomycosis, a systemic mycosis with high relevance for the public health in Brazil and other Latin American countries such as Colombia and Venezuela. Generally, microorganisms require responses to stress conditions to survive in response to environmental changes and pathogenic organisms, particularly, require an effective response even higher to react against host defences. Osmotic stress has been used as a model to study signal transduction and seems to cause many cellular adaptations, which include signal transduction pathways modification, protein expression alteration and cellular volume and size regulation. In this work we have evaluated the proteomic profile of yeast cells of Paracoccidioides sp. (Pb01) obtained in osmotic stress condition. Data describe an osmoadaptative response of this fungus when subjected to this treatment. Proteins involved in the synthesis of the cell wall components were modulated, evidencing a remodelling of the cell wall. In addition, it was also observed alterations on the energy metabolism, given that proteins of the pentose phosphate pathway were abundant while proteins of the glycolysis were less abundant under osmotic stress condition. In addition changes in amino acid metabolism were also observed; more clearly the degradation of amino acids such as leucine, isoleucine and valine was induced during osmotic stress. Hereupon, our study suggests that Paracoccidioides sp. (Pb01) present a vast osmoadaptative repertoire; comprising different proteins which act complementarily and that this response could be able to minimize the effects caused by osmotic stress. / O fungo dimórfico Paracoccidioides é o agente etiológico da paracoccidioidomicose, uma micose sistêmica com grande relevância na saúde pública no Brasil e em outros países da América Latina, como Colômbia e Venezuela. Microrganismos, em geral, requerem respostas às condições de estresse para sobreviver às mudanças ambientais e patógenos, em particular, necessitam de uma resposta efetiva ainda maior para reagir às defesas do hospedeiro. O estresse osmótico é usado como um modelo para estudos de transdução de sinais e parece causar muitas adaptações celulares, as quais incluem alterações nas vias de transdução de sinais, expressão de proteínas e regulação do volume e tamanho celulares. Neste trabalho foi avaliado o perfil proteômico das células leveduriformes de Paracoccidioides sp. (Pb01) obtidas sob condições de estresse osmótico. Os dados evidenciam uma resposta osmoadaptativa deste fungo, quando submetido a este tipo de estresse. Proteínas envolvidas na biossíntese de componentes de parede celular foram moduladas, evidenciando um remodelamento de parede. Também foram observadas prováveis alterações no metabolismo de energia, tendo em vista que proteínas da via das pentoses fosfato mostraram-se abundantes, enquanto proteínas da via glicolítica mostraram-se em menor abundância frente às condições de estresse osmótico. Adicionalmente alterações no metabolismo de aminoácidos também foram observadas; de forma mais evidente a degradação de aminoácidos como leucina, valina e isoleucina foi induzida durante o estresse osmótico. Neste sentido, nosso estudo sugere que Paracoccidioides sp. (Pb01) possui um amplo repertório osmoadaptativo, composto por diferentes proteínas que atuam de maneira complementar e que devem atuar promovendo a minimização dos efeitos causados pelo estresse osmótico.