Umberto Montali

Increased circulating levels of ouabain-like factor in patients with asymptomatic left ventricular dysfunction

Much evidence has been accumulated that human plasma contains digitalis‐like factor(s) with Na/K ATPase inhibitor properties. Increased concentrations of ouabain‐like factor (OLF) have been reported in patients with moderate to severe hypertension and in patients with overt congestive heart failure due to dilated cardiomyopathy.

Evidence for an endogenous ouabain-like immunoreactive factor in human newborn plasma coeluted with ouabain on HPLC.

The identification in human plasma of ouabain as an endogenous digitalis-like factor (EDLF) claimed by Hamlyn et al. has recently been contradicted by two studies which failed to demonstrate endogenous ouabain-like immunoreactivity in HPLC fractions in which exogenous ouabain was eluted. In this paper we report the results obtained on the cross-reactivity with antiouabain antibodies of an EDLF purified by us from human newborn cord plasma. We found that this EDLF coeluted with ouabain on HPLC and cross-reacted both with rabbit anti-ouabain antiserum and with the purified antibodies, which excluded possible interferences due to antibodies directed against non-ouabain portions of the immunogen. Similar but not identical slopes of the ouabain and EDLF displacements curves were observed in all competition ELISA experiments. The inhibitory effect of EDLF on erythrocyte 86Rb uptake was reversed by antiouabain antiserum and antibodies. The concentration of EDLF in newborn plasma, in the four different purifications studied ranged from 30 to 380 pM ouabain equivalents (o.e.) by ELISA and from 100 to 300 pM o.e. by 86Rb uptake. Our data thus support the existence, in human newborn plasma, of a factor with both biological and immunological ouabain-like properties, although not necessarily identical to ouabain.

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

Abstract: 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.

Selective inhibition of human erythrocyte Na+/K+ ATPase by cardiac glycosides and by a mammalian digitalis like factor

Na+/K+ATPase is a transport membrane protein which contains the functional receptor for digitalis compounds. In this work we compare the inhibition curves of Na+/K+ATPase measured by the inhibition of 86Rb uptake in human red blood cells by cardiac glycosides and by an endogenous digitalis like factor (EDLF) extracted from human newborn cord blood. The curves of Na+/K+TPase inhibition show a monophasic shape for ouabain, strophantidin, digitoxin, proscillaridin and EDLF whereas a biphasic shape for ouabagenin, digoxin, digoxigenin and digitoxigenin. All the drugs are potent inhibitors of erythrocyte Na+/K+ATPase with an IC50 ranging from 1.8 x 10(-9) M to 1.4 x 10(-11) M for the higher affinity binding site and from 1.8 x 10(-6) M to 5.5 x 10(-9) M for the lower affinity site. Digitoxigenin is the most active showing the higher active site at 1.4 x 10(-11) M. Ouabain and digoxin have higher affinity compared with their corresponding genins, while digitoxigenin shows a binding site with higher affinity than the respective cardiac glycosides. The increased affinity of the drugs to Na+/K+ATPase may be related to a lipophilic region in correspondence of the carbons 10, 9, 11, 12, 13 of the steroid nucleus, situated in the opposite side with respect of the C-OH-14. The comparison of the inhibition curves and the HPLC profile of newborn EDLF and of the investigated cardenolides suggest that EDLF may be a compound identical or very similar to ouabain.

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 10-8 to 10-5 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 10-7 M and 10-5 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 10-6 M and 10-5 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 10-7 to 10-5 M ouabain, while ERK1 and ERK2 phosphorylation was increased by 10-6 to 10-5 M digoxin. The cardioprotective effect was not related to Na+, K+-ATPase inhibition, since Rb+uptake was not significantly different between control and treated hearts.

Correlation between endogenous digoxin-like immunoreactivity and 3H-ouabain displacement on erythrocyte membranes in extracts of human plasma

The existence of endogenous cardiac glycoside-like compounds and their property of being recognized by anti-digoxin antibodies is still a matter of controversy. In order to investigate this problem, endogenous digoxin-like immunoreactivity (measured by RIA) and digitalis-like radioreceptor activity (measured by displacement of 3H-ouabain from erythrocyte membranes) were assessed in plasma extracts of normal adults, pregnant women and newborns. These three groups were chosen because of their known widely different levels of digoxin-like immunoreactivity. Compared to adults, newborns and pregnant women had significantly higher levels not only of immunoreactivity but also of displacement of 3H-ouabain binding, the latter being due, according to Scatchard analysis, to a decrease of the affinity of ouabain to its cellular receptor rather than to its maximal binding capacity. Furthermore, immunoreactivity and binding displacement correlated significantly. Our data indicate that one (or more) compounds with cardiac glycoside-like properties (both immunological and at the receptor level) are present in the plasma of newborns and pregnant women, and confirm the idea that radioimmunological methods may be useful in studying endogenous inhibitors of the sodium pump.

FURTHER EVIDENCE FOR AN ENDOGENOUS DIGITALIS-LIKE COMPOUND IN NEWBORN AND ADULT PLASMA DETECTED BY ANTI-OUABAIN ANTISERUM

It is widely but not unanimously accepted that one or more endogenous digitalis-like factors (EDLF) circulate in human plasma. In this paper we provide confirmatory evidence that newborn plasma contains a factor with immunological and biological properties similar to ouabain and demonstrate that this factor may be present also in the adult. In fact, we obtained in newborn and adult plasma extracts, identical HPLC elution profiles of ouabain-like immunoreactivity and 86Rb erythrocyte uptake inhibitory activity with a major peak corresponding to the retention time of ouabain. The fact that immunoreactivity and biological digitalis-like activity in the peak are due to an identical substance is strongly supported by the correlation between RIA and 86Rb uptake inhibitory values observed in the purified fractions. Finally, the strong correlation between immunoreactivity observed in plasma samples after simple SepPak C18 extraction and after additional HPLC suggests that less purified samples may be assayed for screening purposes. However, for a more quantitative assessment, this simple extraction method needs a subsequent HPLC purification for eliminating an overestimation of values due to cross-reacting impurities.

Endogenous Digitalis-like Activity in the Newborn

Elevated levels of endogenous digoxin-like immunoreactivity have been reported in the body fluids of premature and full-term infants as well as in term pregnancy, in the amniotic fluid, and in human milk. Several lines of evidence suggest that these factors could also have biological properties in common with digitalis: i.e., they could represent truly endogenous digitalis-like factor(s). In recent years we succeeded in partially purifying this factor from umbilical cord blood, which represents an easily available source of this factor. The inhibitory activity of this factor on 86Rb uptake could be neutralized by antidigoxin antibodies (Fab fragments) and provided, for the first time, direct evidence of an association between digoxin-like immunoreactivity and biological digitalis-like activity. In addition, these antibodies could be used for immunoaffine chromatography as a purification step before separation by high-performance liquid chromatography. Preliminary experiments suggest that this endogenous compound has both a tissue and an isoenzyme selectivity and is not a well-known steroid (testosterone, progesterone, 17-OH progesterone, cortisol, dehydroepiandrosterone sulfate, and estradiol).

Detection of digitalis compounds using a surface plasmon resonance-based biosensor

An automated surface plasmon resonance-based biosensor system has been used to detect endogenous and exogenous digitalis-like factors (EDLF) in the pmolar range in real time. EDLF was purified from umbilical cord blood. EDLF has been suggested to play a role in hypertension and in perinatal adaptation. Highly specific polyclonal anti-ouabain antibodies showed a high affinity binding capacity for ouabain, ouabagenin and strophantidin with an IC50 value of 5 x 10(-10) M, 7.0 x 10(-10) M and 2 x 10(-8) M, respectively. EDLF cross-reacted with antibodies and its concentration in plasma at IC50 was around 50 pmol ouabain equivalent. This study shows the potential usefulness of the biosensor technology for biomolecular interaction analysis. The features of this technology (fully automated, measured in real time, sharpened response) offer several advantages compared with a traditional immunoassay like radioimmunoassay (RIA) in the detection of digitalis compounds in human fluids.

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.