Trypanosoma cruzi epimastigotes: regulation of myo-inositol transport by effectors of protein kinases A and C.

Inositol is the precursor for most Trypanosoma cruzi surface molecules, including phosphoinositides, glycosylinositolphospholipids and glycosylphosphatidylinositol anchors. As the parasite is an inositol auxotroph, the inositol transport system might be a potential target for new trypanocide drugs, as some of its properties are different from its mammalian counterpart. Here, we investigated the modulation exerted by effectors of PKA and PKC on this transport system to comply with the parasite physiology. Pre-incubation of the cells with either dibutyryl-cyclic AMP (25 microM) or forskolin (30 microM) decreased the myo-inositol uptake by half, this effect being reversed by KT5720 (PKA inhibitor). Conversely, pre-incubation of the cells with PMA (2.8 microg/ml) or serum (5%) had a approximately 50% stimulation in myo-inositol uptake, being this effect reversed by staurosporine (0.5 microM) or sphingosine (10 microM). These results allow us to conclude that the myo-inositol transport system in T. cruzi epimastigotes is inhibited by PKA and stimulated by PKC effectors.

Protein kinase C-mediated inhibition of renal Ca2+ ATPase by physiological concentrations of angiotensin II is reversed by AT1- and AT2-receptor antagonists.

Angiotensin II (Ang II) increases the cytosolic Ca2+ concentration in different cell types. In this study, we investigate the effect of Ang II on the Ca2+ ATPase of purified basolateral membranes of kidney proximal tubules. This enzyme pumps Ca2+ out of the cytosol in a reaction coupled to ATP hydrolysis, and it is responsible for the fine-tuned regulation of cytosolic Ca2+ activity. Ca2+-ATPase activity is inhibited by picomolar concentrations of Ang II, with maximal inhibition being attained at approximately 50% of the control values. The presence of raising concentrations (10(-11) to 10(-7) M) of losartan (an AT1-receptor antagonist) or PD123319 (an AT2-receptor antagonist) gradually reverts inhibition by Ang II. Both the phospholipase C (PLC) inhibitor U-73122 (10(-6) M) and the inhibitor of protein kinase C (PKC) staurosporine (10(-7) M) prevent inhibition of the Ca2+ pump by Ang II. Incubation of the previously isolated membranes with a PKC activator-the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (10(-8) M)-mimics the inhibition found with Ang II, and the effects of the compounds are not additive. Taken as a whole, these results indicate the Ang II inhibits Ca2+-ATPase by activation of a PKC system present in primed state in these membranes after binding of the hormone to losartan- and PD123319-sensitive receptors coupled to a PLC. Therefore, inhibition of the basolateral membrane Ca2+-ATPase by kinase-mediated phosphorylation appears to be one of the pathways by which Ang II promotes an increase in the cytosolic Ca2+ concentration of proximal tubule cells.

Sphingosine-1-phosphate formation activates phosphatidylinositol-4 kinase in basolateral membranes from kidney cells: crosstalk in cell signaling through sphingolipids and phospholipids.

Sphingosine-1-phosphate (S1P) and phosphatidylinositol-4 phosphate [PtdIns(4)P] are important second messengers in various cellular processes. Here, we show that S1P and PtdIns(4)P are formed in purified basolateral membranes (BLM) derived from kidney proximal tubules, indicating the presence of a plasma membrane associated SPK (BLM-SPK) and phosphatidylinositol-4 kinase (PI-4K). We observed that S1P synthesis is linear with time, dependent on protein concentration, and saturable in the presence of increasing concentrations of sphingosine. Different from the observations on cytosolic SPKs, the formation of S1P by BLM-SPK is Mg(2+)-independent and insensitive to the classical inhibitor of the cytosolic SPKs, DL-threo-dihydrosphingosine. With sphingosine as substrate, the enzyme shows cooperative kinetics (n = 3.4) with a K(0.5) value of 0.12 mM, suggesting that BLM-SPK is different from the previously characterized cytosolic SPK. The formation of PtdIns(4)P markedly inhibits BLM-SPK activity. Conversely, a strong activation of PtdIns(4)P synthesis by the formation of S1P is observed. Taken together, these results indicate that (i) basolateral membranes from kidney cells harbor a SPK activity that potentially regulates renal epithelium function, and (ii) the formation of S1P mediated by SPK enhances PI-4K activity, while PtdIns(4)P in turn inhibits SPK, suggesting an interplay between these lipid signaling molecules. These findings suggest the possibility of crosstalk between sphingolipids and glycerolipids, which might be involved in the regulation of transepithelial fluxes across the BLM of kidney cells.

Euglena gracilis as a model for the study of Cu2+ and Zn2+ toxicity and accumulation in eukaryotic cells.

We have observed the effect of copper and zinc on the biology of Euglena gracilis. The cells displayed different sensitivities to these metals, as the apparent LC50 for Cu2+ was 0.22 mM, and for Zn2+ it was 0.88 mM. While Zn2+ was able to increase cell proliferation even at 0.1 mM, the minimal CuCl2 concentration tested (0.02 mM) was sufficient to impair cell division. Higher concentrations of these metals not only inhibited cell division in a concentration-dependent manner, but also interfered with the metabolism of E. gracilis. A higher accumulation of proteins and lipids per cell was observed at the DI50 concentration for metal-treated cells. These results suggest that the test concentration of both metals leads to a failure in completing cell division. Ultrastructural analysis indicated a chloroplast disorganization in copper-treated cells, as well as the presence of electron dense granules with different shapes and sizes inside vacuoles. Microanalysis of these granules indicated an accumulation of copper, thus suggesting a detoxification role played by the vacuoles. These results indicate that E. gracilis is an efficient biological model for the study of metal poisoning in eukaryotic cells. They also indicate that copper and zinc (copper being more poisonous) had an overall toxic effect on E. gracilis and that part of the effect can be ascribed to defects in the structure of chloroplast membranes.

Inositol metabolism in Trypanosoma cruzi: potential target for chemotherapy against Chagas' disease

Chagas' disease is a debilitating and often fatal disease caused by the protozoan parasite Trypanosoma cruzi. The great majority of surface molecules in trypanosomes are either inositol-containing phospholipids or glycoproteins that are anchored into the plasma membrane by glycosylphosphatidylinositol anchors. The polyalcohol myo-inositol is the precursor for the biosynthesis of these molecules. In this brief review, recent findings on some aspects of the molecular and cellular fate of inositol in T. cruzi life cycle are discussed and identified some points that could be targets for the development of parasite-specific therapeutic agents.

Effect of cAMP on macromolecule synthesis in the pathogenic protozoa Trypanosoma cruzi

A síntese de macromoléculas de T. cruzi em cultura foi monitorada usando traçadores radioativos. Células de diferentes dias em cultura mostraram uma incorporaçäo preferencial de precursores comco se seguez: 1 dia para (3H)-timidina; 3 dias para (3H)-uridina e 4 dias para (3H)-leucina. Estudos autoradiográficos mostraram que (3H)-leucina. Estudos autoradiográficos mostraram que (3H)-timidina foi incorporada no DNA de ambos, cinetoplasto e núcleo, nesta ordem. Alteraçöes no conteúdo intracelular de cAMP seja por adiçäo de dibutiril-cAMP ou por estimulaçäo de adenilciclase por isoproterenol, causav am inibiçäo na síntese de DNA, RNA e proteínas. A adiçäo destes agentes que elevam o conteúdo intracelular de cAMP em culturas de T.cruzi provocou inibiçäo de crescimento, com resultado da síntese macromolecular imperfeita. Foi observada uma discriminaçäo entre os estereoisômeros de isoproterenol, sendo a configuraçäo L, a mais ativa