Molecular mechanisms of antioxidant action of dalargin on the liver in experimental cholestasis]

Changes of antioxidant activity of dalargin in the liver after naloxone (100 micrograms/kg) administration were examined in experiment on 144 rats with cholestasis. It was found that dalargin inhibited the activity of xanthine oxidase by 32-37% in different time periods after the injection. Dalargin and naloxone, when used in combination, had no effect on the enzyme activity. Glutathione-S-transferase activity rose by 38.0% and 21.8% on hour 1 and 3 after the injection, respectively, while simultaneous injection of dalargin and naloxone induced no changes in the enzyme activity after 1 hour, though decreased it by 36.8% and 26.4% on hour 3 and 5, respectively. Dalargin inhibited lipid peroxidation by 29-35%, simultaneous injection of dalargin and naloxone raised lipid peroxidation by 109.2%, 80.7% and 25.7% after 1, 3 and 5 hours, respectively. Dalargin injection elucidated a marked tendency to lowering of blood release of the liver-specific enzymes histidase and urokaninase in line with enhancement of their activity in the liver. A combined injection of dalargin and naloxone promoted high release of histidase and urokaninase in blood and did not change histidase activity in the liver in all cases. Urokanidase activity elevated in 5 hours. It was noticed that dalargin raised leu-enkephalin levels in the liver 3.5-fold 1 h after the injection. The reduced dalargin antioxidant effect coupled with naloxone pretreatment demonstrated indirect action of the neuropeptide on the liver via neuron receptors of the liver.     

Proof of stimulating effect of dalargin of the process of cell division through opiate receptors

The role of opiate receptors on cell division in corneal epithelium during administration of dalargin was analysed. Naloxone injection/200 micrograms/kg/decreased MI two times, DNA-synthesis 1.4 times over 24 hours. Naloxone prevented dalargin effect on cell proliferation. Another testimonies of dalargin opiate-binding mitogenic effect were the results of the study with dalargin analogues. They are agonists of opiate receptors too. These drugs, as well as dalargin, in a dose 10 micrograms/kg increased DNA-synthesis 1.5 times, MI and MIK 2.2 times. It turned out, that the administration of another two analogues of dalargin, which are not ligands of opiate receptors, probably do not cause an adequate increase of DNA-synthesis and mitotic index.     

Effect of dalargin on the processes of memory and learning in the rat

In experiments on rats, the influence was studied of dalargin on the elaboration and preservation of various homogeneous and heterogeneous conditioned reflexes (CRs) elaborated in single and multiple pairings. The effect of dalargin on the processes of learning and memory was compared with the action of the peptide on the activity of hypothalamic neurones. Administration of dalargin delayed the elaboration of maze defensive CRs and practically did not affect the elaboration of two-way avoidance. The preservation of CR also deteriorated under the influence of dalargin. Administration of dalargin 10 min before the CRs testing did not prevent their reproduction. When using CRs elaborated in a single pairing, dalargin disturbed the preservation of the drinking CR and improved that of passive avoidance CR. Dalargin in this dose affected the emotional state of animals in the open field and did not significantly affect their motor activity. Dalargin suppressed impulse activity in 17 out of 22 tested neurones of the lateral hypothalamus, with maximum effect in 20-50 min after its administration. The obtained data show that the character of dalargin action on the elaboration of CR and mainly on its consolidation, depends on the character of the elaborated CR and is probably due to great extent to the effect of the peptide on the brain emotional mechanisms.     

The use of 1H-NMR-spectroscopy for the study of peptide degradation by angiotensin-converting enzyme

A new method for continuous registration of enzymatic hydrolysis of peptides involving 1H-NMR spectroscopy was developed. The advantages of the method were demonstrated, using dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg) hydrolysis catalyzed by human kidney angiotensin-converting enzyme as an example. It was shown that the maximal activity of the enzyme towards dalargin is observed at pH 7.8; Km is 0.35 mM. The enzyme is inhibited by the substrate (Kd = 0.55 mM). Cl- do not influence the catalytic activity of the enzyme with respect to dalargin. The stereospecificity of the angiotensin-converting enzyme towards dalargin diasteriomers was studied.     

Effect of opioid peptides on the lymphatic drainage of the pancreas in the rat and dog

The effect of dalargin and some other ligands of the opioid receptors on drainage function of the pancreatic lymphatic system was studied in rats and dogs. In rats, dalargin (30-1000 micrograms/kg, subcutaneously) accelerated the elimination of Evans blue from beneath the pancreatic capsule in a dose-related manner. The effect of dalargin was attenuated by naloxone. Specific agonists of mu-, delta- and sigma-opioid receptors had no dalargin-like activity. In dogs, dalargin (60-80 micrograms/kg, subcutaneously) after the administration of Evans blue into the pancreas increased its concentration in the truncus lymphaticus and slowed down its penetration into the blood. Thus, dalargin accelerates the elimination of Evans blue from the pancreas due to the intensification of lymphatic drainage. The effect of dalargin was mediated by subpopulation of opioid receptors with which their certain selective ligands have but slight interaction.     

Effect of dalargin,a synthetic leu-enkephalin analogue on synaptic transmission in the Lorenzini ampullae of rays

Effects of dalargin, a synthetic leu-enkephalin analogue and its antagonist naloxone on synaptic transmission in afferent synapses of ray electroreceptors were investigated using an isolated preparation of Lorenzini ampullae from Black sea rays. It was shown that dalargin (10^–6–10^–10 mole liter) both decreased background activity and evoked activity of an afferent fiber in a dose-dependent manner. Naloxone (10^–5 mole/liter) also inhibited afferent impulsation and completely blocked responses of the Lorenzini ampullae to dalargin application. L-glutamate-induced excitatory responses were reduced in the presence of dalargin. It is suggested that the modulatory action of dalargin on glutamatergic synaptic transmission in the Lorenzini ampullae is exerted via specific opiate receptors.Translated from Neirofiziologiya, Vol. 25, No. 1, pp. 18–21, January–February, 1993.     

Effects of dalargin on proliferative processes and vertical migration of cells of the corneal epithelium during stress

We studied the effect of synthetic analogue of opioid peptides dalargin on kinetics of cell population of corneal epithelium during stress. It was found out that dalargin introduction before stress decreased the level of pathological mitoses induced by the acute stress action in white rats early in the morning. Dalargin prevented the activation of the proliferative process after the repeated stress activation, made stress milder. The stress induced vertical migration of the cells of the corneal epithelium normalized by dalargin. We believe these results show that dalargin has a good protective effect.     

Effects of several regulatory peptides on the functional activity of the pancreas in acute experimental pancreatitis

The influence of regulatory peptides (somatostatin, calcitonin, and dalargin) on xanthine oxidase activity and lipid peroxidation level in pancreatic tissues as well as on the release of pancreatic enzymes (alpha-amylase, trypsin, lipase, and transamidinase) into blood was studied in 205 rats with experimental acute pancreatitis. Somatostatin and dalargin were shown to have obvious antioxidant effect seen by reduced xanthine oxidase activity and MDA level. All studied peptides stimulate reduced release of pancreatic enzymes. Particularly, reduction of dalargin and somatostatin is caused by inhibition of their synthesis as well as by pancreas protective effect of the peptides. Release of enzymes reduced by calcitonin is probably associated only with inhibition of secretory activity of the pancreas.     

Effect of the hexapeptide dalargin on ornithine decarboxylase activity in the duodenal mucosa of rats in experimental duodenal ulcer

The effect of an opioid antiulcerogenic hexapeptide dalargin on ornithine decarboxylase activity of duodenal mucosa has been studied in rats with experimental duodenal ulcers induced by cysteamine. The intraperitoneal injection of 12.5 micrograms/kg of dalargin inhibited ulcerogenesis and activated the enzyme. The effect of the peptide was antagonized by an opiate antagonist naloxone. 5000 micrograms/kg of dalargin failed to inhibit the ulcer formation or to activate ornithine decarboxylase. Since ornithine decarboxylase activation is a marker of intensified cell proliferation and tissue regeneration, our results suggest that the antiulcerogenic effect of dalargin is due to the enhancement of duodenal mucosa regeneration.     

Effect of dalargin on the behavior of rats subjected to intrauterine ethanol exposure

Influence was studied of dalargin--synthetic analogue of enkephalin administered in early postnatal period to young rats with intrauterine ethanol injection, on their behaviour and conditioned activity. Parameters were established of spontaneous motor activity, behaviour in the open field, acquisition and preservation of conditioned reflexes of passive and active avoidance in experimental and intact rats, injected with dalargin. A reduction of sensitivity to corasol convulsive effect was observed under the influence of dalargin.     

Radioimmunologic and radioreceptor studies of the pharmacokinetics of the enkephalin analog dalargin in man

Pharmacokinetics of dalargin, an opioid hexapeptide, was investigated on 7 males by two approaches. Dalargin radioimmunoassay was performed using a highly specific antiserum reacting only with the whole molecule. In radioreceptor assay lyophilized rat brain membranes containing opiate receptors were used. 2-6 min after intravenous introduction of 1-10 mg dalargin, immunoreactive dalargin blood concentration was lower than 0.5 ng/ml. The results of radioreceptor assay were presented as a biexponential curve with a fast main phase of activity changes (90%, characteristic time 1.5-5.0 min) and a slow "clearance" phase (10% of the substance, characteristic time 85-200 min). Prolonged presence of receptor-active substances in the blood can be attributed to the products of dalargin degeneration, namely its N-terminal penta- and tetrapeptides.     

The effect of the synthetic leu-enkephalin analog dalargin on the proliferative activity of glioma C6 cells and on the intensity of their DNA synthesis]

Effect of dalargin, an opioid peptide (a synthetic analogue of Leu-enkephalin), on proliferation and intensity of DNA synthesis of C6 glioma cells was studied. Specific conditions of cultivation were selected, with a low control value of proliferation, which permitted to assess growth-stimulating effect of the peptide. Growth curves were plotted to assess peptide activity, which demonstrated that reaction was a many-phase process: a significant increase in cell number under peptide effect was observed only at the beginning of the logarithm phase and at the beginning of the prestationary phase of the growth curve. Cell number increased on average by 25-27% in the presence of dalargin as compared to control. Reaction of glioma DNA synthesis to dalargin also demonstrates complexity of the process: the peptide changes DNA synthesis, but as a rule, this process has a three-phase character and is not directly associated with the duration of cultivation in the presence of dalargin. Effect of naloxone, an opiate receptor blocker, was analysed to assess the receptor mechanism. It was found that reaction for naloxone and for combined effect of naloxone and dalargin was not the same.     

Effects of opioid receptor ligands on DNA synthesis and histamine contents in the gastric mucosa and blood of white rats

Using autoradiographic study with 3H-thymidine and spectrofluorometric method the authors studied the influence of opioid receptors' ligands on the DNA synthesis in the stomach epithelium histamine content and the blood. Leu-encephalin, B-endorphin, dalargin, napoxone were administered intraperitoneally to male rats. The dose of injection was 0.1 ml per 100 g body weight. It was observed that B-endorphin and dalargin 1.5-1.6 fold increased the quantity of DNA-synthesised nuclei in the epithelium of fundal stomach section. Leu-encephalin and dalargin increased the histamine concentration in the stomach, at the same time dalargin caused a rapid decrease of histamine concentration in the blood. Naloxone also decreased histamine concentration in the stomach. The obtained results are being discussed in connection with dalargin therapy of ulcerous diseases.     

The normalizing effect of dalargin on the glucocorticoid and opioid levels of the blood in CBA and C57Bl/6 mice undergoing footshock stress]

Radioreceptor investigations showed that it is impossible to interpret changes in the activity of opioid receptor ligands of mu and delta types in plasma of mice under footshock stress (FSS) as activation or depletion of opioid system (OS). There occurred qualitative changes characterized by interstrain differences (ID). These are also typical for dynamics of development of FSS effects on blood corticosterone levels. Administration of dalargin diminishes qualitative and quantitative changes in OS function at different periods after FSS preventing early rise of corticosterone levels after the exposure. In spite of common features in dalargin action on opioid and steroid metabolism in mice of both strains, ID are also present.     

Purification from conditioned medium and chemical identification of a factor that inhibits somatic embryogenesis in carrot

Somatic embryogenesis is strongly inhibited in cultures of carrot (Daucus carota L.) cells when the cell density is high. The inhibition is caused by factors that are released by cells into the medium of such cultures. In this study, we purified and identified one of the inhibitory factors found in the medium of high-cell-density cultures of carrot cells. The inhibitory factor with the strongest apparent activity was purified by fractionation with ethylacetate, chromatography on an octadecylsilyl (ODS) silica gel-column and HPLC. The inhibitory factor had a single peak of absorbance at 280 nm and was identified as 4-hydroxybenzyl alcohol by mass spectrometry and 1H- and 13C-NMR spectroscopy. Authentic 4-hydroxybenzyl alcohol strongly inhibited the formation of somatic embryos at a concentration equal to that in high-cell-density cultures. These results suggest that 4-hydroxybenzyl alcohol is a major factor that accumulates in high-cell-density cultures of carrot cells and inhibits somatic embryogenesis.     

Effect of dalargin on the activity of cytochrome oxidase and glutamate dehydrogenase in rat spinal cord dissociated cultured neurons

Dissociated cultured neurons from the rat embryo spinal cord were grown for six days in the presence of dalargin, the synthetic analog of leu-enkephalin. Then the activities of two enzymes of energy metabolism, cytochrome oxidase (CO) and glutamate dehydrogenase (GDH), were studied in these neurons using quantitative cytochemical technique. Dalargin, which possesses the properties of nerve growth factor, enhanced the nerve cell growth and increased the activity of the above enzymes, with GDH activity being increased more significantly. According to the classical standpoint, increased GDH activity under conditions of acute energy deflciency favors the invoivement of some amino acids in a citric acid cycle for subsequent reproduction. One can suggest, in this relation, that the increased energy production caused by the enhanced nerve cell growth in the presence of dalargin was partially compensated by the amino acid splitting. The results allow us to suggest that the effect of dalargin (growth factor) on the nerve cells is similar to the effects of the extremal factors, and requires additional energy to be supplied.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 95–99, March–June, 1996.     

Effect of a stable analog of leu-enkephalin, dalargin, on the cell division of the corneal epithelium in white rats

The effect of Leu-enkephalin analog--dalargin--on the corneal epithelium proliferation has been studied in white rats. 10 microliter dalargin per 1 kg body weight were administered intraperitoneally at 8 a.m. The mitotic index (MI), DNA synthesis cell index and label intensity (LI) were determined every 4 hours over a 24-hour period. The results obtained demonstrate that dalargin stimulates DNA synthesis in cells throughout the entire period of action. MI increased only 4, 8, 12 hours after dalargin administration. Mean daily DNA synthesis cell index and MI increased 2.1-fold and 3.1-fold, respectively after dalargin administration. It is suggested that dalargin activates the cell division processes by speeding up mitosis, shortening the premitotic period, accelerating the speed of the DNA synthesis and increasing cell proliferation pool.     

Role of opioid peptides in the neurotrophic control of the acetylcholine-sensitive membrane of rat skeletal muscle fibers

The influence of opiate peptides on the development of hypersensitivity to acetylcholine has been revealed in muscle fibers membrane after denervation of rat diaphragm muscle. The addition of 1 X 10(-8) M beta-endorphin or dalargin to the culture medium prevented the appearance of extra-junctional acetylcholine sensitivity. The peptide containing only three amino acids and identical to the initial dalargin region did not possess the same effect. Moreover, if this peptide was present in the culture medium in the concentration higher than that of dalargin, the effect of dalargin was blocked. It is suggested that the neurotrophic regulation of acetylcholine-sensitive membrane properties of skeletal muscle fibers is accomplished with opiate neurogenic peptide, e.g. beta-endorphin.     

Main ethanol metabolizing alcohol dehydrogenases (ADH I and ADH IV): biochemical functions and the physiological manifestation

The range of the biochemical reactions which can be catalyzed by ADH I and ADH IV is extremely wide. The most characterized functions of these enzymes are protection against excess endogenous acetaldehyde, products of lipid peroxidation, exogenous alcohols and some xenobiotics. It was found also that ADH I and ADH IV are important members of the enzyme system synthesizing retinoic acid (especially during embryogenesis). They can oxidize some steroids and participate in bioamine and prostaglandin metabolism but so far the extent of their contribution to the latter processes is under discussion. Recent data suggest a correlation between the activity of ADH I in some organs and fine physiological processes including behavior regulation and craving for alcohol in albino rats.