Trypanosoma cruzi: correlation of muscle lesions with contractile properties in the acute phase of experimental infection in mice (Mus musculus).

Parasitism in skeletal muscles and myositis are commonly observed during experimental Trypanosoma cruzi infection. The effect of T. cruzi infection on contractile properties of skeletal muscles in consecutive periods of the acute infection in BALB/c mice was studied. Albarrada strain (clone 4) which was isolated in Mexico and has demonstrated a high level of blood parasitemia and parasitism in skeletal muscles was used. Isolated strips of rectus abdominis muscle were subjected to direct electrical field in vitro. Alternatively, plantaris muscles were stimulated in situ through the sciatic nerve. The peak amplitudes of a single twitch and tetanus contractions were considered to estimate the mechanical properties of muscles. Histopathological analysis was performed to correlate functional changes with the evolution of tissue parasitism and tissue injury. Contractile properties of muscles were significantly attenuated during acute T. cruzi infection. The percentage of damaged muscles rather than the character of tissue pathology affected their contractile properties significantly.

Infection by Trypanosoma cruzi enhances anion conductance in rat neonatal ventricular cardiomyocytes.

Recent studies on malaria-infected erythrocytes have shown increased anion channel activity in the host cell membrane, increasing the exchange of solutes between the cytoplasm and exterior. In the present work, we addressed the question of whether another intracellular protozoan parasite, Trypanosoma cruzi, alters membrane transport systems in the host cardiac cell. Neonatal rat cardiomyocytes were cultured and infected with T. cruzi in vitro. Ion currents were measured by patch-clamp technique in the whole-cell configuration. Two small-magnitude instantaneous anion currents, outward- and inward-rectifying, were recorded in all noninfected cardiomyocytes. In addition, ~10% of cardiomyocytes expressed a large anion-preferable, time-dependent current activated at positive membrane potentials. Hypotonic (230 mOsm) treatment resulted in the disappearance of the time-dependent current but provoked a dramatic increase of the instantaneous outward-rectifying one. Both instantaneous currents were suppressed by intracellular Mg(2+). T. cruzi infection did not provoke new anion currents in the host cells but caused an increase of the density of intrinsic swelling-activated outward current, up to twice in heavily infected cells. The occurrence of a time-dependent current dramatically increased in infected cells in the presence of Mg(2+) in the intracellular solution, from ~10 to ~80%, without a significant change of the current density. Our findings represent one further, besides the known Plasmodium falciparum, example of an intracellular parasite which upregulates the anionic currents expressed in the host cell.

K(bg) and Kv1.3 channels mediate potassium efflux in the early phase of apoptosis in Jurkat T lymphocytes.

Microelectrode ion flux estimation (MIFE) and patch-clamp techniques were combined for noninvasive K(+) flux measurements and recording of activities of the dominant K(+) channels in the early phases of apoptosis in Jurkat cells. Staurosporine (STS, 1 microM) evoked rapid (peaking around 15 min) transient K(+) efflux, which then gradually decreased. This transient K(+) efflux occurred concurrently with the transient increase of the K(+) background (K(bg)) TWIK-related spinal cord K(+) channel-like current density, followed by a drastic decrease and concomitant membrane depolarization. The Kv1.3 current density remained almost constant. Kv1.3 activation was not altered by STS, whereas the inactivation was shifted to more positive potentials. Contribution of K(bg) and Kv1.3 channels to the transient and posttransient STS-induced K(+) efflux components, respectively, was confirmed by the effects of bupivacaine, predominantly blocking K(bg) current, and the Kv1.3-specific blocker margatoxin. Channel-mediated K(+) efflux provoked a substantial cellular shrinkage and affected the activation of caspases.

TRESK-like potassium channels in leukemic T cells.

In this study, we present patch-clamp characterization of the background potassium current in human lymphoma (Jurkat cells), generated by voltage-independent 16 pS channels with a high ( approximately 100-fold) K+/Na+ selectivity. Depending on the background K+ channels density, from few per cell up to approximately 1 open channel per microm2, resting membrane potential was in the range of -40 to -83 mV, approaching E (K) = -88 mV. The background K+ channels were insensitive to margotoxin (3 nM), apamine (3 nM), and clotrimazole (1 microM), high-affinity blockers of the lymphocyte Kv1.3, SKCa2, and IKCa1 channels. The current depended weakly on external pH. Arachidonic acid (20 microM) and Hg2+ (0.3-10 microM) suppressed background K+ current in Jurkat cells by 75-90%. Background K+ current was weakly sensitive to TEA+ (IC50 = 14 mM), and was efficiently suppressed by externally applied bupivacaine (IC50 = 5 microM), quinine (IC50 = 16 microM), and Ba2+ (2 mM). Our data, in particular strong inhibition by mercuric ions, suggest that background K+ currents expressed in Jurkat cells are mediated by TWIK-related spinal cord K+ (TRESK) channels belonging to the double-pore domain K+ channel family. The presence of human TRESK in the membrane protein fraction was confirmed by Western blot analysis.

Methyl-beta-cyclodextrin reversibly alters the gating of lipid rafts-associated Kv1.3 channels in Jurkat T lymphocytes.

The voltage-dependent Kv1.3 potassium channels mediate a variety of physiological functions in human T lymphocytes. These channels, along with their multiple regulatory components, are localized in cholesterol-enriched microdomains of plasma membrane (lipid rafts). In this study, patch-clamp technique was applied to explore the impact of the lipid-raft integrity on the Kv1.3 channel functional characteristics. T lymphoma Jurkat cells were treated for 1 h with cholesterol-binding oligosaccharide methyl-beta-cyclodextrin (MbetaCD) in 1 or 2 mM concentration, resulting in depletion of cholesterol by 63 +/- 5 or 75 +/- 4%, respectively. Treatment with 2 mM MbetaCD did not affect the cells viability but retarded the cell proliferation. The latter treatment caused a depolarizing shift of the Kv1.3 channel activation and inactivation by 11 and 6 mV, respectively, and more than twofold decrease in the steady-state activity at depolarizing potentials. Altogether, these changes underlie the depolarization of membrane potential, recorded in a current-clamp mode. The effects of MbetaCD were concentration- and time-dependent and reversible. Both development and recovery of the MbetaCD effects were completed within 1-2 h. Therefore, cholesterol depletion causes significant alterations in the Kv1.3 channel function, whereas T cells possess a potential to reverse these changes.

Pathologic changes in lungs caused by Mexican isolates of Trypanosoma cruzi in the acute phase of infection in mice.

Chagas' disease, which is caused by the protozoan parasite Trypanosoma cruzi, is a significant public health problem in the Americas. Its clinical presentation varies significantly in different geographic regions. Using experimental infection in mice, we studied the pathologic changes in lungs in the acute phase of the disease caused by three Mexican isolates of T. cruzi. Clusters of parasites and inflammatory reactions were found in the walls of conducting airways and pulmonary vessels. Inflammation was more intense in the small vessels. Although the parasites were not found in the alveolar walls, severe pathologic changes in these structure were observed and included alveolar wall thickening and inflammatory infiltration. Furthermore, serous liquid, fibrin fibers, hyaline membranes, and erythrocytes were found in the alveolar spaces. The pathomorphologic changes observed in the infected mice are consistent with pneumonitis.

Mechanism of luminal Ca2+ and Mg2+ action on the vacuolar slowly activating channels.

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca(2+)-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channel's closed conformations. Vacuolar Ca2+ and Mg2+ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a 'permeable blocker'. Vacuolar Ca(2+)-with a much higher affinity than Mg(2+)-slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca(2+)-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channel's closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca2+ at the vacuolar side. The specificity for Ca2+ compared to Mg2+ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca2+ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.

Potassium-selective channel in the red beet vacuolar membrane.

In higher plants the vacuolar K(+)-selective (VK) channel was identified solely in guard cells. This patch-clamp study describes a 40 pS homologue of the VK channel in Beta vulgaris taproot vacuoles. This voltage-independent channel is activated by submicromolar Ca(2+), and is ideally selective for K(+) over Cl(-) and Na(+).