Effects of Non-Opiate Analogue of Leu-Enkephalin on the Ion Currents, Number of Nucleoli, and p53 Expression in Isolated Cardiomyocytes of Albino Rats.

Acute exposure of isolated ventricular cardiomyocytes to non-opiate analogue of leu-enkephalin (NALE peptide: Phe-D-Ala-Gly-Phe-Leu-Arg) in a concentration of 100 µg/liter and 6-h incubation in NALE solution did not significantly change ATP-dependent K+ current, L-type Ca2+ current, p53 protein expression, and number of nucleoli in the cardiomyocyte nuclei. Incubation of cardiomyocytes with NALE (100 µg/liter) in combination with NOP receptor blocker J-113397 (1 mg/liter) was followed by an increase in Ca2+ L-type current and the number of p53+ cells. The exposure of cardiomyocytes to NALE in a concentration 1000 µg/liter induced similar changes in the studied parameters (increase in Ca2+ L-type current and number of p53+ cardiomyocytes); an increase in the mean number of nucleoli was also observed. Our findings suggest that NALE peptide has direct effect on cardiomyocytes and NOP receptors are involved in this effect.

Effects of Neonatal Administration of Non-Opiate Analogues of Leu-Enkephalin to Heart Tissue Homeostasis of Prepubertal Albino Rats Exposed to Hypoxia.

Hypobaric hypoxia (pO2 65 mm Hg, duration 4 h) induced a significant increase in the number of cardiomyocytes expressing р53, beclin-1, endothelial NO synthase and accumulation and degranulation of mast cells in the epicardium in hearts of prepubertal female rats (age 45-47 days); the number of cardiomyocytes with nucleoli decreased, while the number of single-nucleolar cardiomyocytes increased after this exposure. Five-fold administration of non-opiate analogue of leu-enkephalin (NALE peptide: Phe-D-Ala-Gly-Phe-Leu-Arg; 100 µg/kg) during the neonatal period reduced the severity of the post-hypoxic changes in the heart. Neonatal administration of NALE (100 µg/kg) against the background of NO synthase blockade with L-NAME (50 mg/kg) did not abolish the cardioprotective effects of the peptide. A similar correction of posthypoxic changes in the heart was observed after neonatal administration of original peptide G (Phe-D-Ala-Gly-Phe-Leu-Gly; 100 µg/kg). Thus, NO synthase-NO system and C-terminal amino acid Arg in the molecule of non-opiate analogue of leu-enkephalin are not required for the cardioprotective effects of peptides. Non-opiate leu-enkephalin analogs, peptides NALE and G, can be considered as promising substances for increasing heart resistance to hypoxia during later age periods.

Role of Amino Acid Arginine and Nitric Oxide in Mechanisms of Cytoprotective Effect of Non-Opiate Leu-Enkephalin Analogue In Vitro.

Incubation of primary culture of pulmonary fibroblasts with non-opiate analogue of leuenkephalin (NALE; Phe-D-Ala-Gly-Phe-Leu-Arg, 0.1 µM) reduced generation of superoxide anion-radical (by 20.7%) and decreased the number of p53+ cells (by 40.2%) induced by exposure to H2O2 (60 µM). The cytoprotective effect of NALE was potentiated by NO synthase inhibitor L-NAME (1 mM): the number of p53+ cells decreased by 65.3% and morphometric parameters of the cell nuclei and nucleoli were improved. Incubation of pulmonary fibroblasts culture with peptide G (Phe-D-Ala-Gly-Phe-Leu-Gly, 0.1 µM) also significantly reduced the damaging effect of H2O2: the number of p53+ cells decreased by 73.5%, the area of cell nuclei returned to normal, and generation of superoxide anion-radical decreased by 18.4%. These results indicate that C-terminal amino acid Arg and activation of NO synthase are not involved in the direct cytoprotective effect of NALE.

[Influence of monosodium glutamate consumption by albino rats during pregnancy and lactation on their offspring].

The consequences of dietary intake of significant amounts of monosodium glutamate (MSG) are the excess body weight; structural and functional disorders of the central nervous system, liver, kidneys. We have not found information about the influence of excessive using of the MSG by a woman during pregnancy and lactation on the fetus and infants. The aim of the study was the experimental evaluation of the MSG consumption consequences during pregnancy and lactation to the offspring health. Material and methods. The offspring of 3-month old pregnant female white Wistar rats, who received 1% MSG solution (200 mg per kg of body weight per day) ad libitum as the source of liquid during the pregnancy and lactation, have been studied (MSG group). The control group included offspring of pregnant female rats that received water as the source of liquid. In 25-day-old offspring histological examination and morphometry of the nucleo-nucleolar apparatus of neurons in the neocortex of the proper parietal lobe, cardiomyocytes of the subendocardial zones of the left and right ventricles have been performed. Gravimetry have been also carried out (body weight and weight of brain, heart, liver, kidney, thymus and spleen); mitotic activity of anterior corneal epithelium has been evaluated, the state of erythrocyte membranes have been analyzed by the method of acid erythrograms; behavioral tests "Open field", "The elevated plus-maze test", "Hanging on a horizontal wire" have been performed. Results. MSG consumption during pregnancy and lactation led to an increase of brain (by 19.1%) and kidneys (by 7.8%) relative masses; masses of thymus and spleen were decreased. Significant decrease of locomotor activity and increase of time of hanging in "horizontal wire test" were registered. A histological study showed an increase in the number of nucleoli in the neurons of the V layer of the neocortex of the proper parietal lobe (control - 1.56±0.09; MSG group - 1.81±0.07, р=0.03); decrease of the nucleolar parameters of cardiomyocytes; increase of mitotic activity of anterior corneal epithelium (control - 4.021±0.612‰; MSG group - 6.985±0.889‰, р=0.019). A decrease of the resistance of erythrocyte membranes to acid hemolysis was also registered. Conclusion. The results obtained indicate the effect of oral consumption of MSG food additive during pregnancy and lactation on the organism of the offspring.

Cytoprotective Effect of Echinochrome A in Primary Culture of Pulmonary Fibroblasts from Albino Rats under Conditions of Oxidative Stress.

We studied the effect of echinochrome A on the primary culture of pulmonary fibroblasts under normal conditions and under oxidative stress. Exposure to echinochrome A (100 µM, 6 h) did not affect the production of superoxide radical by fibroblasts, area of their nuclei, and number of nucleoli, but reduced the total area of nucleolar organizer regions against the background of stable proliferative activity of the culture. Two-hour oxidative stress induced by hydrogen peroxide solution (60 µM) increased the generation of superoxide radical, decreased DNA-synthetic activity of fibroblasts, area of fibroblast nuclei, and total area of nucleolar organizer regions. Preliminary administration of echinochrome A significantly attenuated the damaging effect of oxidative stress: the intensity of production of superoxide radicals decreased, DNA-synthetic activity and nucleus area of fibroblasts partially recovered; normalization of the total area of nucleoli was accompanied by an increase in their number.

Hepatoprotective Effect of Neonatal Administration of Non-Opioid Leu-Enkephalin Analogue in Adult Albino Rats Subjected to Antenatal Hypoxia.

We studied the effects of neonatal administration of non-opioid leu-enkephalin analogue peptide NALE) on the morphological parameters of the liver and redox status of 60-dayold albino rats subjected to antenatal hypoxia. In animals subjected to antenatal hypoxia, a decrease body weight and reduction of the area of hepatocytes and total area of their nucleoli were observed; these changes were accompanied by activation of free-radical processes and impairment of antioxidant defense in the liver and blood serum. Intraperitoneal administration of NALE (100 µg/kg, daily) during postnatal days 2-6 normalized body weight, hepatocyte area, and total area of their nucleoli, significantly reduced the intensity of ROS generation, and improved antioxidant protection in the liver and blood serum in 60-day-old animals subjected to antenatal hypoxia.

Delayed Effects of Neonatal Administration of Non-Opioid Analog of Leu-Enkephalin on Cerebral Consequences of Antenal Hypoxia.

In 60-day-old male rats after antenatal hypoxia, the body weight and the absolute weight of the cerebral hemispheres were significantly lower than in the progeny of intact animals. Analysis of brain sections stained with silver nitrate revealed reduced number of nucleoli in neocortical layer II and hippocampal CA1 neurons and smaller area of neuronal nuclei in neocortical layer V and total area of nucleoli in neurons of all studied zones. The animals demonstrated increased locomotor activity in the elevated plus-maze test. Chemiluminescent analysis of brain homogenates revealed the presence of oxidative stress at the organ level. Neonatal administration of non-opioid analog of leu-enkephalin (peptide NALE) after antenatal hypoxia normalized body weight, neutralized morphometric changes in the nucleoli and nuclei of neurons in the neocortex and hippocampus, and improved oxidative status of the brain. In 30- and 60-day-old male rats subjected to antenatal hypoxia and receiving peptide NALE during the neonatal period, behavioral responses were partially normalized. Non-opioid analog of leu-enkephalin can be a promising drug for correction of cerebral consequences of antenatal hypoxia.

Correction of Negative Effect of Antenatal Hypoxia on Liver Tissue Homeostasis in Newborn Albino Rats with Opioid Peptides.

We studied the possibility of correction of the negative effects of antenatal hypoxia on the liver tissue homeostasis in 7-day-old albino rats by administration of opioid peptides in a dose of 100 µg/kg on postnatal days 2-6. Administration of mixed µ/δ-opioid receptor agonist Dalargin neutralized deviations of gravimetric indicators, parameters of proliferative activity, and activity of the nucleolar apparatus of hepatocytes. Administration of the non-opiate Leu-enkephalin analogue did not normalize gravimetric parameters and nucleolar apparatus parameters, however, it significantly increased the pool of proliferating hepatocytes. Both peptides significantly reduced the intensity of free radical oxidation, improved antioxidant antiradical defense and resistance to peroxidation in the liver tissue of animals subjected to antenatal hypoxia.

Cytoprotective Effect of Peptide Sedatin, an Agonist of µ/δ-Opioid Receptors, on Primary Culture of Pulmonary Fibroblasts of Albino Rats under Conditions of Oxidative Stress.

We studied the effects of a synthetic analogue of dermorphin peptide sedatin on DNA synthesis, nucleolar apparatus, and parameters of free radical oxidation in the primary culture of pulmonary fibroblasts under conditions of oxidative stress. Oxidative stress significantly enhanced production of superoxide anion radical in the culture, sufficiently inhibited DNA synthesis in fibroblasts, and reduced the size of cell nuclei and parameters of the nucleolar apparatus. Sedatin prevented accumulation of free radical oxidation products and changes in karyometry parameters induced by oxidative stress. The peptide completely eliminated changes in the parameters of fibroblast nucleolar apparatus and abolished the inhibitory effect of oxidative stress on the number of DNA-synthesizing cells. Pretreatment with non-selective opioid receptor antagonist naloxone hydrochloride partially abolished the effects of sedatin in the primary culture of pulmonary fibroblasts.

Effect of Proline-Containing Oligopeptides PGP and RGP on Proliferative and Protein-Synthesizing Activity of Cultured Pulmonary Fibroblasts under Conditions of Oxidative Stress.

We studied the effect of glyprolines Pro-Gly-Pro (PGP) and Arg-Gly-Pro (RGP) on the primary culture of pulmonary fibroblasts from newborn albino rats under normal conditions and during oxidative stress. Under physiological conditions, the peptides had no effect on parameters of cell division. Hydrogen peroxide induced intensive oxidative stress accompanied by suppression of protein-synthesizing function. When hydrogen peroxide was added to the culture containing the test peptides, correction of the oxidative status was observed accompanied by activation of DNA-synthesizing activity and inhibition of lucigenin-dependent chemiluminescence.

Effects of antenatal hypoxia on tissue homeostasis in the myocardium of albino rats: early and delayed consequences.

Body weight, absolute and relative heart weight, DNA-synthetic activity of cardiomyocytes, and number of nucleoli in cardiomyocyte nuclei were reduced in newborn albino rats exposed to antennal hypoxia. All these changes developed against the background of oxidative stress. In mature animals, the absolute and relative heart weights were decreased, but the ratio of mononuclear cardiomyocytes in both ventricles was higher than in the control. These changes were accompanied by an increase in the percent of collagen fibers in the myocardial stroma. In the right ventricle, an increase in the cardiomyocyte length to width ratio and a decrease in the number of nucleoli were found. The observed changes can probably induce heart function disturbances under pathological conditions.

Effects of antenatal hypoxia on the parameters of tissue homeostasis in the liver of albino rats.

We studied age-dependent dynamics of some liver parameters in albino rats after antenatal hypoxia. A decrease in body weight, absolute liver weight in newborn and mature rats, and a reduction of relative weight of the liver in mature animals were observed under these conditions. It was found that antenatal hypoxia promotes a decrease in DNA-synthetic activity of hepatocytes, intensifies free radical processes in the liver of newborn rats, and reduces the number of nucleoli in hepatocytes. In mature animals, antenatal hypoxia was followed by a decrease in hepatocyte sizes and area of nucleoli, an increase in the number of binuclear cells relative to mononuclear cells, and a decrease in the number of nucleoli in hepatocyte nuclei. These data can reflect impairment of tissue homeostasis in the liver of mammals exposed to antenatal hypoxia.

Effect of HMG-CoA-reductase inhibitor on DNA synthesis and free radical oxidation in the gastric mucosa under normal conditions and during indometacin-induced ulcerative process in the stomach of albino mice.

We studied the effect of simvastatin (24 mg/kg per os for 30 days) on DNA synthesis ((3)H-thymidine autoradiography) and free radical oxidation (chemiluminescent method) in the gastric mucosa of albino mice under normal conditions and in ulcerative process induced by single indometacin administration. Simvastatin treatment activated free radical oxidation, which was seen from enhanced chemiluminescence in the mucosa homogenate (by 1.7-4.6 times). Administration of indometacin against the background of simvastatin treatment potentiated local oxidative stress and inhibited DNA synthesis. Under these conditions, the area of ulcerative lesion in the gastric mucosa increased by 3.0 times.

Effects of analogues of hydra peptide morphogen on DNA synthesis in the myocardium of newborn albino rats.

DNA-synthetic activity of myocardial cells was studied by (3)H-thymidine autoradiography in newborn albino rats after intraperitoneal injection of hydra peptide morphogen and its analogues. Administration of hydra peptide morphogen stimulated proliferative activity in the myocardium. Short analogues of hydra peptide morphogen, 6C and 3C peptides, produced a similar effect. Administration of arginine-containing analogue of hydra peptide morphogen significantly reduced the number of DNA-synthesizing nuclei in the ventricular myocardium of newborn albino rats. The role of the structure of the peptide molecule in the realization of the morphogenetic effects of hydra peptide morphogen is discussed.

[Effect of intrauterine hypoxia upon newborn albino rat tooth germ cells anabolic activity].

The aim of this study was to examine the intrauterine hypoxia influence on dental hard tissue development. Pregnant rats were exposed in hypoxic environments between day 14 and 19 of pregnancy. The study was performed on 36 newborn albino rats. Analysis of nucleolar organizator parameters were performed in enameloblasts, odontoblasts and saliva gland epitheliocytes. Data obtained demonstrated that intrauterine hypoxia decreased nucleolar organizator quantity in enameloblasts of tooth germ.

[Antenatal hypoxia influence upon osteogenesis processes].

It was described the results of antenaltal hypoxia's influence upon osteogenesis processes. Reliable changes of the ossification nodus of the lower jaw, that in turn, influenced the frequency of occurrence of dentition anomalies were determined.

[Regulatory peptides influence on hypoxia induced tooth germ pathology of new born albino rats].

Intrauterine hypoxia decreased the nucleolar organizator quantity in enameloblasts of newborn albino rats tooth germ. Influence the leu-encepgalin synthetic analog dalargin and non-opiate synthetic leu-encephalin on newborn rats from 2 to 6 day abolished the intrauterine hypoxia affect. The main mechanism of peptide antioxidant effect may be NO system stimulation.

Gastroprotective effect of dalargin in gastropathy due to treatment with nonsteroid antiinflammatory drugs.

We studied the effect of dalargin and its analogue [Dala]2-leu-enkephalin on the gastric mucosa in indomethacin-receiving animals. Indomethacin treatment was followed by severe injury to the gastric mucosa, decrease in proliferative activity of the epithelium, and stimulation of free-radical processes in gastric tissues. Dalargin significantly decreased the area of erosive and ulcerative lesions, had a normalizing effect on proliferation of epithelial cells, and reduced the degree of oxidative stress. Administration of [Dala]2-leu-enkephalin did not improve the state of the gastric mucosa.

Effect of beta-casomorphin-7 on DNA synthesis in cell populations of newborn albino rats.

We studied the effect of beta-casomorphin-7 on DNA synthesis in cell populations of newborn albino rats. Intraperitoneal administration of a beta-casein fragment heptapeptide beta-casomorphin-7 (1 mg/kg, 1 or 5 injections) activated proliferative processes in the myocardium and ectodermal and endodermal epithelium of newborn rats.

Effect of dalargin on DNA synthesis in the gastric mucosa of albino rats.

3H-thymidine autoradiography and chemiluminescence study demonstrated pronounced effect of dalargin on the state of the gastric mucosa in albino rats. Dalargin stimulated DNA synthesis in the epithelium of the gastric mucosa and increased buffer capacity of its antiradical and antioxidant systems. Dalargin analogue not containing arginine ([D-Ala2]-leu-enkephalin) had little effect on these parameters. NO synthase inhibitor L-NAME abolished the effects of dalargin on DNA synthesis in the gastric mucosa. Our results suggest that the NO system plays an important role in the effect of dalargin on the gastric mucosa.