ABSTRACT
Mammalian acetyl-CoA carboxylase (ACC) is present in two isoforms, alpha and beta, both of which catalyze formation of malonyl-CoA by fixing CO2 into acetyl-CoA. ACC-alpha is highly expressed in lipogenic tissues whereas ACC-beta is a predominant form in heart and skeletal muscle tissues. Even though the tissue-specific expression pattern of two ACC isoforms suggests that each form may have a distinct function, existence of two isoforms catalyzing the identical reaction in a same cell has been a puzzling question. As a first step to answer this question and to identify the possible role of ACC isoforms in myogenic differentiation, we have investigated in the present study whether the expression and the subcellular distribution of ACC isoforms in H9c2 cardiac myocyte change so that malonyl-CoA produced by each form may modulate fatty acid oxidation. We have observed that the expression levels of both ACC forms were correlated to the extent of myogenic differentiation and that they were present not only in cytoplasm but also in other subcellular compartment. Among the various tested compounds, short-term treatment of H9c2 myotubes with insulin or okadaic acid rapidly increased the cytosolic content of both ACC isoforms up to 2 folds without affecting the total cellular ACC content. Taken together, these observations suggest that both ACC isoforms may play a pivotal role in muscle differentiation and that they may translocate between cytoplasm and other subcellular compartment to achieve its specific goal under the various physiological conditions.
Subject(s)
Rats , Acetyl-CoA Carboxylase/metabolism , Acetyl-CoA Carboxylase/drug effects , Animals , Cell Differentiation/drug effects , Cell Line , Cell Membrane Permeability , Chromones/pharmacology , Cytosol/enzymology , Cytosol/drug effects , Digitonin/pharmacology , Immunoblotting , Insulin/pharmacology , Isoenzymes , Morpholines/pharmacology , Myocardium/cytology , Okadaic Acid/pharmacology , PhosphorylationABSTRACT
Cell viability requires the perfect functioning of the processes controlling ATP and Ca2+ homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with disruption of ATP and Ca2+ homeostasis. Therefore, the study of the mechanisms by which different T. cruzi stages regulate the intracellular Ca2+ distribution and the ATP supply to maintain cell viability could provide new insights into the physiology of these parasites. One important objective of these studies is the identification of possible metabolic differences between host and parasite that could be exploited for the rational design of new and more effective trypanocidal drugs
Subject(s)
Animals , Adenosine Triphosphate/metabolism , Calcium/metabolism , Homeostasis , Trypanosoma cruzi/metabolism , Digitonin/pharmacology , Homeostasis/drug effects , Energy Metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/growth & developmentABSTRACT
By using the fluorescent Ca2+ indicator fura 2, submicromolar levels of intracellular Ca2+ have been detected in Trypanosoma cruzi different stages. The intracellular transport mechanisms involved in maintaining Ca2+ homeostasis in T. cruzi have been characterized by measuring Ca2+ transport in digitonin-permeabilized cells. Two intracellular calcium transport systems have been detected. Ca2+ uptake by the mitochondria occurs by an electrophoretic mechanism, is inhibited by antimycin A, FCCP, and ruthenium red, and stimulated by respiratory substrates, phosphate and acetate. This pool has a high capacity and low affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.6-0.7 microM. Ca2+ uptake by the endoplasmic reticulum is inhibited by high concentrations of vanadate and anticalmodulin agents, and stimulated by ATP. This pool has a low capacity and a high affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.05-1.0 microM. In addition, calmodulin has been purified from T. cruzi epimastigotes and shown to stimulate the homologous plasma membrane Ca(2+)-ATPase and cyclic-AMP phosphodiesterase. The gene encoding this protein has been cloned and sequenced and shown to have a great homology to mammalian calmodulin. The role of the plasma membrane of T. cruzi in the regulation of [Ca2+]i has been studied using fura 2-loaded epimastigotes or plasma membrane vesicles prepared from epimastigotes. Plasma membrane vesicles transport Ca2+ in the presence of Mg2+ and have a high affinity, vanadate-sensitive (Ca(2+)-Mg2+)-ATPase with an apparent Km for free Ca2+ of 0.3 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Animals , Calcium/metabolism , Homeostasis , Trypanosoma cruzi/metabolism , Antimycin A/pharmacology , Biological Transport , Ca(2+) Mg(2+)-ATPase/drug effects , Ca(2+) Mg(2+)-ATPase/metabolism , Calmodulin/antagonists & inhibitors , Calmodulin/metabolism , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Cell Membrane Permeability/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Digitonin/pharmacology , Fura-2 , Homeostasis/drug effects , Imidazoles/pharmacology , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Membrane Potentials/drug effects , Mitochondria/drug effects , Ruthenium Red/pharmacology , Trifluoperazine/pharmacology , Trypanosoma cruzi/drug effects , Vanadates/pharmacologyABSTRACT
O estudo da açäo do esteviosídeo sobre a fisiologia celular é amplamente justificado em funçäo da utilizaçäo deste glicosídeo na dieta e nas indústrias farmacêuticas e de alimentos. Neste trabalho, analisaram-se os efeitos que soluçöes de esteviosídeo, nas concentraçöes finais de 3,3 x 10*-9M, 3,3 x 10*-4M, provocaram sobre a estabilidade das membranas dos diversos componentes do compartimento lisossômicos, isolados de fígados de camundongos. Os resultados indicaram que as soluçöes nas concentraçöes 3,3 x 10*-6M, 3,3 x 10*-5M e 3,3 x 10*-4M têm efeito estabilizador sobre os complexos de membranas de lisossomos hepáticos, enquanto que as soluçöes nas concentraçöes 3,3 x 10*-9M, 3,3 x 10*-8M e 3,3 x 10*-7M näo determinaram nenhum efeito estatisticamente significante que pudesse ser avaliado através da metodologia aqui empregada