Presence of digitalis-like factor in mammalian plasma

We attempted to purify a digitalis-like factor from volume expanded dog plasma using an inhibitory effect on the binding of [3H]ouabain to intact human erythrocytes to monitor digitalis-like activity. A highly polar [3H] ouabain displacing compound was purified to a high degree using a combination of chromatographic procedures including reverse phase and gel filtration high performance liquid chromatography. This compound, a reversible inhibitor of [3H]ouabain binding, closely resembles ouabain in its polarity and significantly increases during saline infusion. Its molecular weight was estimated to be 343Da. Moreover, similar compound was consistently detected in other mammalian plasma.     

Evidence for the existence of the same endogenous digitalis-like factor in several mammalian species

1. Endogenous digitalis-like activity was studied comparatively in four mammalian species: guinea pig, dog, cow and rat. 2. Water extracts were prepared from guinea pig, dog, cow and rat hearts and assayed by ouabain radioreceptorassay, digoxin radioimmunoassay and digitoxin radioimmunoassay. Extracts were further analysed by fractionation by gel permeation chromatography with Sephadex G-25. 3. A similar behaviour was observed with the four species in the three assays. Extracts displaced tritiated ouabain binding to its receptor and labeled digoxin analogue binding to antidigoxin antibodies in a competitive manner. Displacement of labeled digitoxin analogue to antidigitoxin antibodies did not follow Michaelis-Menten kinetics. IC50 ratios between assays were similar for the four species studied. 4. Extracts from the four species exhibited a similar pattern when fractionated with Sephadex G-25. Endogenous digoxin-like immunoreactivity eluted after the salts, suggesting that the active material is of a molecular weight of less than 1000. 5. Results suggest that a similar endogenous factor endowed with digitalis-like characteristics is present in all mammalian species.     

The endogenous digitalis-like factor

Intensive search into the presence of endogenous digitalis-like factor (EDLF) started shortly after identification of the alpha subunit of the Na,K,-ATPase as being receptor for digitalis glycosides. After years of skepticism, present data testify EDLF really exists. Most probably, the EDLF has chemical structure of either ouabain or of one of its isomers. It is secreted by the adrenal cortex, and, under conditions of stress, it's secretion is regulated differently from the secretion of both gluco- and mineralocorticoids. The physiological role of the EDLF has not been fully understood yet. In the newborn's kidneys, the inhibition of the Na,K-ATPase may assist to increase elimination of surplus sodum from the organism. In individuals of any age, the inhibitory influence of EDLF upon Na,K-ATPase in the arterial wall smooth muscle cells increases peripheral vascular resistance and thus, blood pressure. In the tissue culture, direct positive inotropic influences of EDLF upon rat cardiomyocytes was observed. However, the importance of positive inotropic effect of the EDLF upon the heart in clinical medicine remains to be elucidated. (Mol Cell Biochem 160/161: 111–115, 1996)     

Isolation of a urinary digitalis-like factor indistinguishable from digoxin

A digitalis-like factor has been purified to apparent homogeneity from human urine based on the inhibitory effect on [3H] ouabain binding to intact human erythrocytes. The purification scheme involved large scale adsorption followed by preparative, semipreparative and analytical high-performance liquid chromatography. The purified material showed a prominent digoxin-like immunoreactivity. The behaviour of the isolated substance was identical to that of authentic digoxin in three high-performance liquid chromatography and three thin-layer chromatography systems. Moreover, fast atom bombardment mass spectrum and proton nuclear magnetic resonance spectrum suggested that the purified material may be indistinguishable from digoxin.     

Evidence that mammalian lignans show endogenous digitalis-like activities

Enterolactone, a lignan that has been identified in biological samples from man and several mammals, shares with ascorbic acid and cardiac glycosides a gamma-butyrolactone. It displaces 3H-ouabain from its binding sites on cardiac digitalis receptor and inhibits, dose dependently, the Na+, K+-ATPase activity of human and guinea-pig heart. The time dependence of this inhibition resembles that of dihydroouabain, a cardiac glycoside in which the lactone ring does not contain conjugated double bonds. The active concentrations of enterolactone as inhibitor of Na+,K+-ATPase are in the 10(-4) M range and, at those concentrations, the cross-reactivity with antidigoxin antibodies is low. Lignans may contribute to the putative digitalis-like activity found in tissues, blood and urine of several mammals including man.     

Purification and characterization of human urine-derived digitalis-like factor

We were able to purify a digitalis-like factor to apparent homogeneity from human urine based on the inhibitory effect on [3H]-ouabain binding to intact human erythrocytes. This ouabain displacing compound closely resembles ouabain in its polarity, molecular weight, non-peptidic nature and mode of action except for its UV absorbance spectrum. This compound sharing many biological activities of ouabain may be the endogenous ligand for the Na+, K+-ATPase and serve as a specific regulator of the sodium pump.     

Endogenous digitalis-like factor as a stimulator of endothelin secretion from endothelial cells

Effects of human urine-derived endogenous digitalis-like factor (EDLF) and ouabain on endothelin (ET) secretion were examined in cultured endothelial cells. ET was secreted in a linear fashion over 5 hours from bovine pulmonary artery endothelium into serum-free medium. EDLF stimulated ET secretion in a dose-dependent manner. In contrast, ouabain did not affect ET secretion at the concentration of 10(-9)-10(-5) M. These results indicate that human urine-derived EDLF is distinct from plant-derived ouabain and act as a stimulator of ET secretion by endothelial cells.     

Existence of different forms of adrenocorticotropin in rat hypothalamus

Adrenocorticotropin (ACTH)-like immunoreactivity and bioactivity were extracted from rat hypothalamus and fractionated by high pressure liquid chromatography. Analysis of the fractions either by radioimmunoassay or bioassay (corticosteroid production from rat adrenal cells) revealed several peaks of immunoreactivity and bioactivity. Only 20-25% of total ACTH-like immunoreactivity and bioactivity eluted with the same retention time as authentic ACTH 1-39. The results suggest that different forms of ACTH exist in rat hypothalamus.     

LRF producing cells of the mammalian hypothalamus

Summary The demonstration of perikarya of mediocellular neurones producing LRF, using indirect immunofluorescence on slides and anti synthetic LRF antibodies, requires both their activation and the inhibition of their axoplasmic transport. This fact suggests that LRF is present in an immunoreactive form, but usually in very low concentrations. Perikarya of neurons producing LRF are found principally in the preoptic and septal areas of the rat and decrease caudally, particularly beyond the retrochiasmatic area. Most of the axons coming from these perikarya are incorporated in the hypothalamoinfundibular tract and terminate around the capillaries of the primary portal plexus, particularly those of interealar plexus. Other axons (or axon collaterals) may be found in various areas (suprachiasmal crista, epithalamus, amygdala, mesencephalon) and form circuits recalling the “extrahypophyseal pathways” described for the magnocellular Gomori-positive neurons of the SON and PVN. These axons are probably concerned in intersegmental regulations involving “neurosecretory synapses”, particularly of the axosomatic type. The placement of stereotaxic lesions was used to determine the topography and direction of axoplasmic flow of the axons transporting LRF. The infundibular immunoreactive material, already discernible at the end of gestation in the foetus, shows considerable variations between birth and puberty, during the estrous cycle and under various other physiological or experimental conditions. The observations made under various experimental or physiological conditions suggest that, in the guinea-pig in particular, the greater part of the hypothalamic immunoreactive material is concentrated in the infundibular area. This area of accumulation is comparable to the distal neurohypophysis of the Gomori-positive neurosecretory system coming from the SON and PVN. This work was financed by the D.G.R.S.T. Contract No. 72-7-0375.     

Existence of carcinine, a histamine-related compound, in mammalian tissues

Carcinine (beta-alanylhistamine) was synthesized in vitro from histamine and beta-alanine. It was detected quantitatively using an HPLC method previously described for the quantification of the related compounds histamine, histidine, carnosine and 3-methylhistamine. Carcinine was identified in several tissue of the rat, guinea pig, mouse and human, and was then shown to be metabolically related in vivo to histamine, histidine, carnosine and 3-methylhistamine through radioisotopic labeling. The results demonstrate that carcinine may be concurrently quantitated using the same HPLC method as that used to measure histamine, histidine, carnosine and 3-methylhistamine. These findings suggest a role for carcinine in the carnosine-histidine-histamine metabolic pathway and in the mammalian physiologic response to stress.     

Morphological evidence of endogenous digitalis-like substance (EDLS) in the rat and macaque hypothalamus, using digoxin-immunohistochemistry

Morphological investigation of the endogenous digitals-like substance (EDLS) characterized as a natriuretic humoral agent has not been carried out to date; but EDLS can be detected indirectly by immunohistochemistry using cross-reactivity with digoxin-antibody. In this report, the brain and other organs of the rat and macaque were immunostained using commercially supplied digoxin-specific antibodies. The immunoreactive substances were mainly observed in the neurons of hypothalamic paraventricular and supraoptic nuclei, and varicose fibers in some areas of the hypothalamus and infundibulum. No immunopositive substances were detected in the pituitary anterior lobe, adrenal gland, or kidney of the rat.     

Age as a factor in 6-hydroxydopamine-induced plasticity in the hypothalamus

Summary The question of age as a possible factor influencing the regenerative response of catecholaminergic varicosities in the hypothalamus was investigated in the supraoptic commissure and the paraventricular, periventricular, and dorsomedial hypothalamic nuclei of rats that had received intraventricular injections of the neurotoxin 6-hydroxydopamine when they were (1) neonates, (2) young adults, or (3) senescent adults. After postneurotoxin survival for 4, 21, 56, or 180 days, the animals were perfused, and the hypothalamic tissue sections were cut and processed using a glyoxylic acid method for localizing catecholamines. Four days following neurotoxin administration, counts of fluorescent varicosities showed a significant loss of catecholamine varicosities in each of the four areas. Subsequently, at least partial restoration of numbers of catecholamine varicosities occurred in all hypothalamic areas in all three age groups. It is concluded that, following selective lesions induced by the neurotoxin 6-OH-DA, catecholamine varicosities were restored both in immature and mature groups. According to the evidence obtained experimentally, the rate of restoration was greater in the neonate group, whereas the percentage restoration attained varies according to the hypothalamic area studied and the age of the animal.     

Purification of a glucagon releasing factor from the rat hypothalamus

The purification of a peptide from the rat hypothalamus which is capable of stimulating the release of glucagon from the endocrine pancreas and therefore named glucagon releasing factor (GlRF), is described. Compositional analysis of the product obtained using well established techniques for peptide isolation revealed that GlRF appeared to consist of 30-31 amino acids. The significance of GlRF in the physiological regulation of glucagon secretion is yet to be determined but the existence of this very potent factor supports the concept of a hypothalamic-pancreatic neurohormonal link.