Characterization of [Ca2+]i responses in primary vultures of mouse cardiomyocytes induced by Trypanosoma cruzi Trypomastigotes

Trypanosoma cruzi , the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 μM) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC

The stimulatory effect of angiotensin II on Na+-ATPase activity involves sequential activation of phospholipases and sustained PKC activity

AbstractAngiotensin II (Ang II) stimulates the proximal tubule Na+-ATPase through the AT1 receptor/phosphoinositide phospholipase Cβ (PI-PLCβ)/protein kinase C (PKC) pathway. However, this pathway alone does not explain the sustained effect of Ang II on Na+-ATPase activity for 30 min. The aim of the present work was to elucidate the molecular mechanisms involved in the sustained effect of Ang II on Na+-ATPase activity. Ang II induced fast and correlated activation of Na+-ATPase and PKC activities with the maximal effect (115%) observed at 1 min and sustained for 30 min, indicating a pivotal role of PKC in the modulation of Na+-ATPase by Ang II. We observed that the sustained activation of PKC by Ang II depended on the sequential activation of phospholipase D and Ca2+-insensitive phospholipase A2, forming phosphatidic acid and lysophosphatidic acid, respectively. The results indicate that PKC could be the final target and an integrator molecule of different signaling pathways triggered by Ang II, which could explain the sustained activation of Na+-ATPase by Ang II

Hydrochlorothiazide Potentiates Contractile Activity of Mouse Cavernosal Smooth Muscle

Introduction: Hydrochlorothiazide has a negative influence on penile erection but little is known about the mechanism(s) involved. Aims: To characterize the effects of this diuretic on mouse corpus cavernosum (CC) smooth muscle in vitro and ex vivo. Methods: CC strips of C57BL/6 mice (12–16 weeks old) were mounted in organ baths containing Krebs-Henseleit solution and tissue reactivity was evaluated. Expression of genes encoding diuretic targets and enzymes involved in penile erection were evaluated by polymerase chain reaction. Main Outcome Measures: Stimulation-response curves to phenylephrine (10 nmol/L–100 μmol/L) or to electrical field stimulation (1–32 Hz) were constructed, with or without hydrochlorothiazide. Strips of CC from mice after long-term hydrochlorothiazide treatment (6 mg/kg/day for 4 weeks) with or without amiloride (0.6 mg/kg/day for 4 weeks) in vivo also were studied. Nitric oxide and Rho-kinase pathways were evaluated. Results: Hydrochlorothiazide (100 μmol/L) increased the maximum response to phenylephrine by 64% in vitro. This effect was unaffected by the addition of indomethacin (5 μmol/L) but was abolished by N(ω)-nitro-L-arginine methyl ester (100 μmol/L). Hydrochlorothiazide (100 μmol/L) potentiated electrical field stimulation-induced contraction in vitro, but not ex vivo. Long-term treatment with hydrochlorothiazide increased the maximum response to phenylephrine by 60% and resulted in a plasma concentration of 500 ± 180 nmol/L. Amiloride (100μmol/L) caused rightward shifts in concentration-response curves to phenylephrine in vitro. Long-term treatment with hydrochlorothiazide plus amiloride did not significantly increase the maximum response to phenylephrine (+13%). Reverse transcriptase polymerase chain reaction did not detect the NaCl cotransporter in mouse CC. Hydrochlorothiazide did not change Rho-kinase activity, whereas amiloride decreased it in vitro and ex vivo (approximately 18% and 24% respectively). A 40% decrease in Rock1 expression also was observed after long-term treatment with hydrochlorothiazide plus amiloride. Conclusion: Hydrochlorothiazide potentiates contraction of smooth muscle from mouse CC. These findings could explain why diuretics such as hydrochlorothiazide are associated with erectile dysfunction

Trypanosoma cruzi-cardiomyocytes: New Contributions Regarding a Better Understanding of this Interaction

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75% inhibition in the T. cruzi-cardiomyocytes invasion

Involvement of the G(i/o)/cGMP/PKG pathway in the AT(2)-mediated inhibition of outer cortex proximal tubule Na(+)-ATPase by Ang-(1–7)

The molecular mechanisms involved in the Ang-(1–7) [angiotensin-(1–7)] effect on sodium renal excretion remain to be determined. In a previous study, we showed that Ang-(1–7) has a biphasic effect on the proximal tubule Na(+)-ATPase activity, with the stimulatory effect mediated by the AT(1) receptor. In the present study, we investigated the molecular mechanisms involved in the inhibition of the Na(+)-ATPase by Ang-(1–7). All experiments were carried out in the presence of 0.1 nM losartan to block the AT(1) receptor-mediated stimulation. In this condition, Ang-(1–7) at 0.1 nM inhibited the Na(+)-ATPase activity of the proximal tubule by 54%. This effect was reversed by 10 nM PD123319, a specific antagonist of the AT(2) receptor, and by 1 μM GDP[β-S] (guanosine 5′-[β-thio]diphosphate), an inhibitor of G protein. Ang-(1–7) at 0.1 M induced [(35)S]GTP[S] (guanosine 5′-[γ-[(35)S]thio]triphosphate) binding and 1 μg/ml pertussis toxin, an inhibitor of G(i/o) protein, reversed the Ang-(1–7) effect. Furthermore, it was observed that the inhibitory effect of Ang-(1–7) on the Na(+)-ATPase activity was completely reversed by 0.1 μM LY83583, an inhibitor of guanylate cyclase, and by 2 μM KT5823, a PKG (protein kinase G) inhibitor, and was mimicked by 10 nM d-cGMP (dibutyryl cGMP). Ang-(1–7) increased the PKG activity by 152% and this effect was abolished by 10 nM PD123319 and 0.1 μM LY83583. Taken together, these data indicate that Ang-(1–7) inhibits the proximal tubule Na(+)-ATPase by interaction with the AT(2) receptor that subsequently activates the G(i/o) protein/cGMP/PKG pathway