Glycosylation interference on RhoA activation: focus on G-CSF.

Glycosylation of cytokines appears to be responsible for several differences in their activity, and focusing on G-CSF, several divergences between the non-glycosylated G-CSF, Filgrastim, and the glycosylated G-CSF, Lenograstim, have been reported. To verify the role of G-CSF glycosylation in mediating these differences we tested in vitro the effects on the RhoA activation of the different G-CSFs, including deglycosylated Lenograstim. The results showed that Filgrastim induced sustained-RhoA activation while Lenograstim did not do so. Deglycosylated Lenograstim mimicked Filgrastim, resulting in RhoA hyper-activation. These in vitro findings demonstrate that the glycosylation of G-CSF plays a crucial role in RhoA activation.

Cardioprotection by ouabain and digoxin in perfused rat hearts.

This work was aimed at determining the cardioprotective effect of digitalis glycosides in rat heart, and to relate it with Na, K-ATPase inhibition and ERK1/2 activation. Isolated working rat hearts were perfused in the presence of ouabain or digoxin, which were used at concentrations ranging from 10 to 10 M. The hearts were then subjected to 30 minutes of global normothermic ischemia followed by 120 minutes of retrograde reperfusion; irreversible tissue injury was determined on the basis of triphenyltetrazolium chloride staining. Significant cardioprotection was observed with 10 M and 10 M ouabain (ischemic injury averaged 7.0 +/- 3.5% and 8.3 +/- 0.6% versus 37.3 +/- 2.0% in controls, P < 0.01 in each case). Hearts treated with digoxin showed decreased ischemic injury at 10 M and 10 M (18.0 +/- 1.5% and 14.2 +/- 1.0%, P < 0.01 versus control in both cases). In parallel experiments, ERK2 phosphorylation was increased by 10 to 10 M ouabain, while ERK1 and ERK2 phosphorylation was increased by 10 to 10 M digoxin. The cardioprotective effect was not related to Na, K-ATPase inhibition, since Rbuptake was not significantly different between control and treated hearts.

Erythrocyte sodium pump stimulation by ouabain and an endogenous ouabain-like factor.

Cardiac glycosides inhibit the sodium pump. However, some studies suggest that nanomolar ouabain concentrations can stimulate the activity of the sodium pump. In this study, using the Na(+)/K(+)-ATPase of human erythrocytes, we compared the effect of digoxin, ouabain and an ouabain like-factor (OLF), on (86)Rb uptake. Ouabain concentrations below 10(-9) M significantly stimulate Rb(+) uptake, and the maximal increase above base-line values is 18 +/- 5% at 10(-10) M ouabain. No stimulation is observed in the same conditions by digoxin. OLF behaved like ouabain, producing an activation of Rb(+) flux at concentrations lower than 10(-9) M ouabain equivalents (14 +/- 3% at 10(-10) M). Western blot analysis revealed the presence of both alpha(1) and alpha(3) pump isoforms in human erythrocytes. Our data confirm the analogies between OLF and ouabain and suggest that Na(+)/K(+)-ATPase activation may be related to the alpha(3) isoform. In addition, we investigated whether ouabain at different concentrations was effective in altering the intracellular calcium concentration of erythrocytes. We found that ouabain at concentration lower than 10(-9) M did not affect this homeostasis.

Production of ouabain-like factor in normal and ischemic rat heart.

Endogenous ouabain-like factor (OLF) has been detected in mammalian plasma, adrenal gland, and hypothalamus. We investigate whether cardiac tissue may also produce OLF. HPLC chromatographic separation of cardiac extracts showed that RIA-determined OLF activity coincided with the elution profile of exogenous ouabain and with the ability to inhibit 86Rb uptake in human erythrocytes. OLF activity was remarkably higher in excised hearts (3.94 +/- 0.84 pmol/g wet weight by RIA) than in rat blood (0.05 +/- 0.02 pmol/ml). Similar values were obtained in perfused working hearts, without significant changes over time from 5 to 30 minutes of aerobic perfusion. Significant OLF release in the perfusion buffer was also observed (0.54 +/- 0.05 pmoles over 30 minutes). In hearts subjected to 15 minutes of aerobic perfusion followed by 15 minutes of global myocardial ischemia OLF concentration was remarkably increased (8.59 +/- 1.13 versus 4.58 +/- 0.57 pmol/g wet weight by RIA, P < 0.01; an increase after ischemia was confirmed by the assay of 86Rb uptake). Our findings suggest that the rat heart is able to produce OLF, and that its concentration increases during ischemia. Myocardial OLF might modulate the Na/K-ATPase, producing relevant effects on ionic homeostasis and/or gene transcription.