Effect of canrenone on the digitalis site of Na+/K(+)-ATPase in human placental membranes and in erythrocytes.

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 ouabain-like 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 Na+/K(+)-ATPase in relation to ouabain in human placental membranes and erythrocytes by 3H-ouabain binding assay; (2) the capacity of canrenone (10 microM) to reverse the inhibition of Na+/K(+)-ATPase by ouabain and by ouabain-like factor (from umbilical cord plasma) in human erythrocytes employing a 86Rb uptake assay. Increasing concentrations of canrenone (0-350 microM) partially competed with 3H-ouabain 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 x 10(-9) M) and ouabain-like factor (2 x 10(-9) M ouabain equivalents). A reduction of inhibition of about 50% was observed with ouabain and ouabain-like factor respectively at a concentration of 5 x 10(-8) M and 2 x 10(-8) M (ouabain equivalents). Our results thus provide evidence that canrenone, at therapeutical concentrations, is a partial competitive agonist of ouabain and of ouabain-like factor in human placental membranes and erythrocytes.

Are serum inhibin concentrations new markers of placental tumours in the course of chemotherapy?

BACKGROUND: The study was conducted to evaluate whether the detection of serum molecular forms of inhibin (A and B) could be useful for the diagnosis, prognosis and follow-up of placental tumours. METHODS: A total of 17 patients with hydatidiform mole (n = 13), invasive mole (n = 1) or choriocarcinoma (n = 3) were studied; serum concentrations of inhibins A and B, human chorionic gonadotrophin (HCG) and its free beta subunit (HCGbeta) were measured before chemotherapy (after mole evacuation for eight patients) and also during the course of chemotherapy (for 10 patients). RESULTS: After evacuation or before chemotherapy for refractory disease, serum inhibin A and B concentrations were found to be increased in 10/17 and 4/17 patients, when HCG and HCGbeta were high in all patients. In 10 patients with a follow-up during treatment, nine had a high concentration of inhibin A which correlated with those of HCG and HCGbeta. Normalization of inhibin A was faster than that of HCG and HCGbeta for three and six patients respectively. There was no correlation between changes of inhibin B and HCGbeta concentrations. CONCLUSIONS: Our results suggest that inhibins A and B are not useful markers and that HCG determination still remains the most useful marker for diagnosis and follow-up of placental tumours.

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.

[Mesogastrectomy in the surgical treatment of gastric carcinoma. Experience with 61 cases]. / La mesogastrectomia nella chirurgia del carcinoma gastrico. Esperienza di 61 casi.

BACKGROUND: In gastric cancer surgery, to search for a technique to remove the entire posterior mesogastric region using a standardised operation using well defined methods and anatomic-embryological planes. METHODS: A concise description of the embryological evolution of the posterior mesogastrium allow the formation of the mesogastric fascia (and the supramesocolic fascia of the omentum--which is a continuation) to be documented. It is also clear that the mesogastric fascia is the embryological--anatomical equivalent of Treitz's fascia, pancreatic retro-head, and Toldt's retrocolic fascia, of which it is a structural continuation. Like Treitz's and Toldt's fascias, the mesogastric fascia also represents the surgical plane for the detachment of the region in question and allows maximum safety and radicality. By carrying out primary ligature of the arteries at the origin and the veins at the outlet, the entire posterior mesogastric region, with the relative lymph node stations, can be removed en bloc with maximum radicality and safety, and also in line with the principle of "no touch isolation". We used this technique to operate 61 cases, 17% of all cases of gastric carcinomas between 1973 and 1994. RESULTS: Mesogastrectomy was required in 87% of cases with carcinoma in a high localisation or widespread nature of the linitis plastica type. Only 23 cases (37%) were at pTNM II and III A stages. Thirty-eight cases (63%) were at stages III B and IV. In non-selected cases and those with severe associated pathologies and undergoing emergency surgery, and those cases that were extended beyond mesogastrectomy, morbidity was above all linked to pleural effusion. There were only 2 cases (3%) of operating mortality owing to two technical errors: an esophago-jejunal anastomotic dehiscence (the only case in the series, 1.6%) caused by esophageal cancer nests in the suture and a case of necrosis in the left hepatic region following the section of the left gastric artery at the origin despite the existence of a large hepatic collateral vessel. The results for stages II and III A were excellent: stage II, 100% survival at 5, 10 and 15 years; stage III A 88% survival at 5 years, 70% at 10 years, 55% at 15 years, but only two deaths from neoplasia at 2.7 and 4.6 years. The results for stages III B and IV are comparable to large series undergoing traditional forms of surgery. Postoperative conditions of nutrition and quality of life were good and patients resumed activities with the aid of constant chlorhydric-peptic replacement treatment and the total extraction of gastric mucosa. CONCLUSIONS: We believe that mesogastrectomy represents a real advance in both technical terms and results for stages II and III A; it is debatable for stages III B and IV, although individual cases who survived for more than 10 years were also reported. The case of a stage pT3N0M1 = IV pathology, with a single hepatic metastasis that increased until one year and then spontaneously resolved leaving the patient alive and in good health 20 years and 6 months after the operation is truly amazing.

[The single-stage surgery of colorectal neoplastic occlusion. The experience of 133 cases]. / La chirurgia in un tempo dell'occlusione neoplastica colon-rettale. Esperienza di 133 case.

BACKGROUND AND AIM: 133 cases of occluded colorectal neoplasms (14% of the entire series): 30 (23%) of the right colon, 103 (77%) of the left colon-rectum; 69 males (52%) and 64 females (48%); mean age 67.5 years old, range 33-91 years. pTNM: stage II, 28 cases (21%); stage III: 43 cases (32%); stage IV, 62 cases (47%). The aim of this study was to resolve the occlusive symptoms and to treat the neoplasm in a single operation. METHODS: In the 62 cases at stage IV, surgery was solely palliative: 49 (79%) derivations, 13 (21%) entero-enterostomies and 36 (58%) preternatural anus; 11 (18%) standard hemicolectomies, extended in two cases to hepatic resection, and 2 (3%) Hartmann's operations. In the 71 cases at stages II and III, surgery took the form of standard colic exeresis with primary ligature of the colonic vessels at source and at the outlet; 15 (21%) right colectomies, 50 (70%) left colectomies, extended in 6 cases (8%) to abdomino-perineal amputation; 6 segmentary colectomies, 3 (4%) of the transverse colon and 3 (4%) Hartmann's operations. The following aspects are essential in this single-stage surgery: urgency; massive dose antibiotic treatment limited to the pre- and perioperative stages; peritoneal cleansing using accurate, methodical, repeated and abundant lavage; perioperative colonic preparation using direct colotomic perioperative lavage or using a trans-buccoenteric access (using Grosz-Dennis tube); the peritonisation of the retroperitoneum with the omentum and the protection of the anastomosis using omental wrapping and active lavage and, for colorectal anastomosis, even using the 3-way tube, lavage and active aspiration, in a transanal trans- or sub-anastomotic position. Total parenteral feeding is useful for 6-7 days. RESULTS: In the 62 cases at stage IV, postoperative morbidity was 3 cases (6%): 3 suppurations of laparotomy, and mortality occurred in 10 cases (16%): one case of anastomotic disunion (pre-Gullino's tube), 3 cases of septic shock and 6 cardiorespiratory failures. Mean postoperative hospitalisation was 14 days. All these patients died owing to the spread of neoplasms within 1-40 months, mean 13 months. The worst results were obtained in entero-enterostomies: 1-9 months, mean 5 months. In the 71 cases at stages II and III, postoperative morbidity was 3 cases (4%): a small anastomotic filtration after right colectomy and 2 suppurations of the laparotomic incision; mortality amounted to 10 cases (14%): one case of septic shock, one of acute hepatitis, one intestinal infarction and one cardiac infarction, 3 pulmonary embolisms and 3 cardiorespiratory failures. Mean postoperative hospitalisation was 13 days, only 10 in cases of left colectomy with anastomosis protected by Gullino's tube. The long-term results were very good in these 71 patients: over 5-year survival of 50% (Kaplan-Meier). CONCLUSIONS: Using this courageous single-stage surgery, the results are optimal even at a distance, together with reduced surgical trauma and a shorter hospital stay.

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.

Inhibition of rat Na+/K+-ATPase isoforms by endogenous digitalis extracts from neonatal human plasma.

An unique endogenous 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 (alpha1, alpha2 and alpha3) of the alpha subunits of Na+/K+-ATPase in rat. EDLF 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 alpha3 isoform and a steep dose-response curve with the same apparent potency for rat alpha2 and alpha1 isoforms of high (10(-7) M) and low affinity (3 x 10(-5) M) 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.

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.

The stimulatory effect on human erythrocyte rubidium-86 uptake by anti-cardiac-glycoside antibodies.

Considerable, but as yet still controversial evidence indicates the presence, in mammalian tissues of endogenous digitalis-like factors (EDLFs) which inhibit cell membrane Na+, K(+)-adenosine triphosphatase (Na+, K(+)-ATPase) and which may cross-react with anti-digitalis antibodies. The aim of this study was to evaluate the effect of antibodies against cardiac glycosides on Na+, K(+)-ATPase in human erythrocytes. For this purpose, we measured the effect of antibodies against two different cardiac glycosides (anti-ouabain rabbit antiserum and anti-digoxin Fab fragments) on the activity of the Na+, K(+)-ATPase, as measured by erythrocyte rubidium-86 (86Rb) uptake, in subjects who had never come into contact with exogenous cardiac glycosides, and compared these results with the effect of two control rabbit sera: a normal serum and an antiserum to a non-related antigen. Anti-ouabain rabbit antiserum and anti-digoxin Fab fragments induced a significantly greater percentage change in 86Rb uptake in the erythrocytes than the two control sera (ANOVA followed by multiple comparison by the Games-Howell test). The average percentage change was +11.8 +/- 16.3% (n = 19) (mean +/- SD) for anti-ouabain antiserum +10.8 +/- 15.6% (n = 23) for anti-digoxin Fab fragments, -1.68 +/- 11.2% (n = 11) for anti-rhGM-CSF antiserum, and -5.8 +/- 11.7 (n = 10) for normal control serum. In a subgroup of ten subjects in whom the 3 antisera were tested simultaneously, the stimulation of erythrocyte 86Rb uptake induced by the two antidigitalis antibodies correlated significantly (r = 0.906, p = 0.001, n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin resistance and vasodilation in essential hypertension. Studies with adenosine.

Insulin-mediated vasodilation has been proposed as a determinant of in vivo insulin sensitivity. We tested whether sustained vasodilation with adenosine could overcome the muscle insulin resistance present in mildly overweight patients with essential hypertension. Using the forearm technique, we measured the response to a 40-min local intraarterial infusion of adenosine given under fasting conditions (n = 6) or superimposed on a euglycemic insulin clamp (n = 8). In the fasting state, adenosine-induced vasodilation (forearm blood flow from 2.6 +/- 0.6 to 6.0 +/- 1.2 ml min-1dl-1, P < 0.001) was associated with a 45% rise in muscle oxygen consumption (5.9 +/- 1.0 vs 8.6 +/- 1.7 mumol min-1dl-1, P < 0.05), and a doubling of forearm glucose uptake (0.47 +/- 0.15 to 1.01 +/- 0.28 mumol min-1dl-1, P < 0.05). The latter effect remained significant also when expressed as a ratio to concomitant oxygen balance (0.08 +/- 0.03 vs 0.13 +/- 0.04 mumol mumol-1, P < 0.05), whereas for all other metabolites (lactate, pyruvate, FFA, glycerol, citrate, and beta-hydroxybutyrate) this ratio remained unchanged. During euglycemic hyperinsulinemia, whole-body glucose disposal was stimulated (to 19 +/- 3 mumol min-1kg-1), but forearm blood flow did not increase significantly above baseline (2.9 +/- 0.2 vs 3.1 +/- 0.2 ml min-1dl-1, P = NS). Forearm oxygen balance increased (by 30%, P < 0.05) and forearm glucose uptake rose fourfold (from 0.5 to 2.3 mumol min-1dl-1, P < 0.05). Superimposing an adenosine infusion into one forearm resulted in a 100% increase in blood flow (from 2.9 +/- 0.2 to 6.1 +/- 0.9 ml min-1dl-1, P < 0.001); there was, however, no further stimulation of oxygen or glucose uptake compared with the control forearm. During the clamp, the ratio of glucose to oxygen uptake was similar in the control and in the infused forearms (0.27 +/- 0.11 and 0.23 +/- 0.09, respectively), and was not altered by adenosine (0.31 +/- 0.9 and 0.29 +/- 0.10). We conclude that in insulin-re15-76sistant patients with hypertension, adenosine-induced vasodilation recruits oxidative muscle tissues and exerts a modest, direct metabolic effect to promote muscle glucose uptake in the fasting state. Despite these effects, however, adenosine does not overcome muscle insulin resistance.

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).

Antidigoxin antibodies neutralize the effect of newborn endogenous digitalis-like factor on erythrocyte 86Rb uptake.

Increasing evidence indicates the existence of endogenous digitalis like factor(s) (EDLF). We recently reported on the partial purification of an EDLF from newborn (cord) blood which possesses both digoxin-like immunoreactivity and the ability to inhibit the cell membrane sodium pump measured as the inhibitory activity on erythrocyte 86Rb uptake. We here report that high affinity digoxin-binding antibodies (Fab fragments; Digibind, Burroughs Wellcome Co.) are capable of neutralizing the inhibitory activity on erythrocyte Rb uptake not only of digoxin but also of ouabain and of partially purified newborn EDLF. These results provide, to our knowledge for the first time, direct evidence that antidigitalis antibodies may cross-react with one or more circulating substances which share antigenic determinants with digoxin and ouabain and possess endogenous digitalis-like properties, strongly suggesting that these antibodies may be useful tools both for the assay of EDLF and for the study of its biological effects.

Comparison between endogenous digoxin-like immunoreactivity and 86Rb uptake by erythrocytes in extracts of human plasma.

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), DLIS] 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.

Acute hypotensive effect of calcium antagonists and endogenous digitalis-like immunoreactivity in human essential hypertension.

Evidence has been provided on the increased presence, in essential hypertension, of endogenous digitalis-like factor(s) [DLIS, digoxin-like immunoreactive substance(s)] able to cross-react with antidigoxin antibodies and to inhibit the membrane-bound sodium-potassium pump. An inhibition of the sodium pump could lead, in smooth muscle cells, to an increase of intracellular calcium ions and to an increase of total peripheral resistances. In this study the relation between plasma levels of DLIS and the acute hypotensive effect of a calcium antagonist (nifedipine) has been evaluated in a group of borderline to severe hypertensive patients and in a control group of normotensive subjects. The results obtained confirm that the hypotensive effect of nifedipine is related to pretreatment blood pressure and show, only in hypertensive patients, a significant relation of DLIS with both pretreatment blood pressure and blood pressure decrement induced by nifedipine. These findings are compatible with a possible role of DLIS in modulating cellular calcium handling.