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It has been reported that canrenone, which is used in hypertensive therapy as an antialdosteronic drug, may also act as a blocker of ouabain effects. Several studies suggest that human plasma contains an endogenous ouabainlike factor similar to ouabain, which may be increased in hypertension, in pregnancy, and in the neonatal state. This study evaluated 1 the effect of canrenone on NaK ATPase in relation to ouabain in human placental membranes and erythrocytes by 3Houabain binding assay 2 the capacity of canrenone 10M to reverse the inhibition of NaKATPase by ouabain and by ouabainlike factor from umbilical cord plasma in human erythrocytes employing a 86Rb uptake assay. Increasing concentrations of canrenone 0–350M partially competed with 3Houabain binding in placental membrane 40 and erythrocytes 60. Scatchard plot from radioreceptor assay in placental membrane showed that ouabain and canrenone compete for the same binding site. In erythrocytes, canrenone completely reversed the inhibition caused by ouabain 5 × 109M and ouabainlike factor 2 × 109M ouabain equivalents. A reduction of inhibition of about 50 was observed with ouabain and ouabainlike factor respectively at a concentration of 5 × 108M and 2 × 108M ouabain equivalents. Our results thus provide evidence that canrenone, at therapeutical concentrations, is a partial competitive agonist of ouabain and of ouabainlike factor in human placental membranes and erythrocytes.

A hereditary defect in the outward ouabain-resistant, furosemide-sensitive (ORFS) Na+/K+ cotransport has recently been reported by Garay and Meyer in erythrocytes of essential hypertensive patients and some of their normotensive offsprings. Since Na+/K+ cotransport in erythrocytes operates bidirectionally, the same defect might be detectable in the ORFS component of K+ influx rate. To test this hypothesis outward and inward Na+/K+ cotransport measurements were performed in erythrocytes of hypertensives and normotensive controls. Our preliminary results confirm the observation of a reduced outward cotransport in essential hypertension and also suggest that inward cotransport may be reduced in essential hypertension.

1. To investigate endogenous cardiac glycoside-like compounds in plasma and their ability to inhibit the sodium pump, digoxin-like immunoreactivity [digoxin-like immunoreactive substance(s), DL1S] and 86Rb uptake by erythrocytes were measured in plasma extracts from normal adults, hypertensive adults and neonates. 2. DLIS levels in neonate plasma extracts were significantly higher than those found for normotensive or hypertensive adults. No difference was observed between normotensive and hypertensive subjects. DLIS was significantly increased when boiled plasma was extracted. 3. Extracts of boiled neonate and adult plasma inhibited 86Rb uptake. Instead, when boiling was omitted, no detectable inhibition was found in extracts of plasma from normotensive or hypertensive adult subjects. When present, the inhibition resulted from a depression of the ouabain-sensitive (sodium-pump-mediated) component, and, for the boiled neonate plasma only, also of the ouabain-resistant component. When the data from the different groups were pooled, a statistically significant inverse relationship between DLIS and erythrocyte 86Rb uptake was observed. Furthermore, in a subgroup of samples in which determinations were made before and after boiling in the same samples, an inverse correlation was found between changes in DLIS and changes in ouabain-sensitive (but not ouabain-resistant) 86Rb uptake. 4. Plasma extracts incubated with albumin at a physiological concentration significantly decreased (by approximately 20%) the inhibition of 86Rb uptake observed. 5. These findings support the existence of one or more endogenous compounds which both bind to antidigoxin antibodies and inhibit transmembrane cation transport. Part of this inhibition may, however, not involve the sodium pump. Furthermore, this chemically unidentified substance(s) may be bound to plasma proteins which partly reduce its action in vivo.

An unique endogeneous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (α1, α2and α3) of the alpha subunits of Na+/K+-ATPase in ratEDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral α3isoform and a steep dose-response curve with the same apparent potency for rat α2and α1isoforms of high (10-7M) and low affinity (3 × 10-5M) for ouabain. These results indicate that the Na+/K+-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain

AbstractAfter 6 and 12 months of ripening, samples of Asiago d’Allevowere analyzed for quality traits. Cheeses were produced during 3 periods using milk from cows fed a total mixed ration (TMR, May) or grazing on alpine pasture (AG) in early (July) and late (Sept.) summer. Data were submitted to ANOVA considering ripening, milk production period and farm as main effects, and whole cheese weight as covariate. During ripening, pH of AG-cheese was significantly lower than that of TMR-cheese; crude fat and protein significantly increased. According to period, July-samples showed the significantly lowest value of dry matter (DM), maybe due to a lower crude fat con-tent; however, variability in skimming method could have altered proximate composition. No texture differences were found, although increasing weight of whole cheese significantly reduced max shear force as result of a lower DM content. Lightness (L*) and yellowness (b*) significantly decreased during ripening. AG feeding system caused a lower L* and higher b* than TMR one, probably as a consequence of a different amount of milk pigments. Cheese varied also within AG season: Sept.-samples showed the lowest L* value and the highest b*.

AbstractA simple and rapid method based on the in vitrobovine lymphocyte proliferation was developed to assess the immunomodulatory activity of natural compounds extracted from plant and foods. The proliferation of lymphocytes was measured by the MTT [3-(4, 5- dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay. This test was used to measure the immuno-enhancing activity of 2 plant extracts and 2 samples of fermented milk after 48 h of incubation. To evaluate the effect of the cow physiological phase on the assay response, the tests were repeated using lymphocytes taken from non-pregnant dry, pregnant dry and post partum cows. The assay was not affected by the cow physiological phase and the results obtained were comparable. Both plant extracts enhanced lymphocyte growth, in particular the extract 2 was slightly more potent than extract 1. The fermentation of milk with proteolytic starter cultures, such as FAIR E-63 and LA 2, can generate peptidic compounds from milk protein with potential immunostimulating activity. These samples tested in cell culture inhibited lymphocyte proliferation because they showed an acidic pH of 4.5. However, this bioassay was enough sensitive to detect biological activity of different compounds also at low concentrations.