Modulating effect of opioid peptides on hemopoiesis in stress

The influence of leu-enkephalin and dalargin on the blood system was studied during immobilization stress in mice. The early transmitted reactions of the peripheral blood were shown to decrease upon single drug infusions after immobilization. At later terms the activation of bone marrow hematopoiesis was not registered in mice receiving opioid peptides in contrast to the control animals. It correlates with drug-induced decrease in the mitotic activity of bone-marrow cells. Suppressive effect of opioids on hematopoiesis during stress was connected with their decreasing effect on corticosteroid level in the animal plasma. The latter can suggest indirect influence of enkephalins on bone marrow hematopoiesis in immobilization stress.     

Comparative effect of opioid receptor ligands on cell division in the epithelium of the tongue in white rats

Beta-endorphin, leu-enkephalin, dalargin and naloxone influences on cell division have been studied in tongue epithelium of white rats. The preparations were administered at a dose of 0.1 ml per 100 g body weight as a 2.10(-9) M solution. Cell division was studied 24 hours after administration. beta-endorphin, leu-enkephalin, dalargin and naloxone caused a 1.5-1.7-fold increase in the number of DNA-synthesizing nuclei, which was accompanied by an adequate rise in mitotic index in experiments with dalargin.     

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.     

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.     

Immunomodulating action of neuropeptides in in vitro systems

The work presents the data on the immunostimulating properties of neuropeptides. As revealed in this study, the leu-enkephalin level in blood sera (taken from 55 patients) inversely correlates with the intensity of the proliferative response of lymphocytes to phytohemagglutinin. In in vitro systems dalargin promotes the increase or decrease of the proliferative response of lymphocytes to phytohemagglutinin, depending on the proliferative activity of cells in response to this mitogen, and also leads to an increase in the number of rosette-forming cells. Leu-enkephalin in doses of 100, 10, 1, 0.1 micrograms/ml and dalargin in a dose of 0.1 microgram/ml inhibit the migration of leukocytes.     

Serotonin excess in the brain modifies the effect of beta-endorphin and dalargin on the processes of learning and memory]

In experiments on outbred female rats the influence was studied and compared of two representatives of endogenous opioids beta-endorphine and the analogue of leu-enkephalin dalargin on the processes of learning and memory in normal conditions and at the change of functional state of serotoninergic system of the brain. Parallel, the influence was studied of neuropeptides on the content of serotonin (5-OT) and its metabolite--5-oxyindolacetic acid in various areas of the brain in control and at the 5-OT redundancy. Conditioned reflexes (CRs) were used of two-way avoidance and defensive CRs. It has been established that administration of neuropeptides to intact animals influences in different directions the elaboration of the CR of two-way avoidance and maze defensive CR, but also worsens their preservation. Redundancy of 5-OT in the brain modifies behavioural effects of beta-endorphine and dalargin manifested in appearance of new effect and elimination and change of direction of the effects observed in the intact animals. Redundancy of 5-OT in the brain changes metabolic effects of beta-endorphine and particularly of dalargin. The obtained data testify to a dependence of the effects of beta-endorphine and dalargin on the functional state of 5-OT-ergic system.     

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 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.     

Functioning of the pituitary-adrenal system during the vestibulo-vegetative syndrome and administration of dalargin and nalorphine

Changes in ACTH, cortisol, beta-endorphin have been investigated during vestibulo-vegetative syndrome (VVS) and injections of dalargin (leu-enkephalin analog) and nalorphine (agonist-antagonist of opioid receptors) in 9 volunteers with low level vestibulo-vegetative stability. Cumulative coriolis acceleration test during rotations on a special chair was used for VVS modelling. Dalargin (1-4 mg), nalorphine (5 mg) and placebo (NaCl solution) were injected intravenously 5-15 min before rotation. A significant increase in ACTH, cortisol and beta-endorphin plasma levels has been observed. Mean positive linear correlation (r greater than +0.6) between ACTH and beta-endorphin and ACTH and cortisol was noted immediately after the test only when dalargin was injected. It is suggested that in VVS there develops a hormonal conflict, i. e. an adequate hormonal release is disturbed.     

Normal human bone marrow suppressor cells and in liver cirrhosis

Suppressor properties of bone marrow cells were studied in healthy donors and patients with hepatocirrhosis using the technique registrating the activity of bone marrow B-suppressors by the inhibition of xenogenic target cell proliferation. The activity of bone marrow suppressor cells in patients with various types of hepatocirrhosis was reduced as compared to healthy subjects. In addition, the in vitro spontaneous proliferation level of bone marrow cells in hepatocirrhosis was considerably higher than that of healthy donors. This fact can be possibly attributed to the decline in the number of bone marrow B-suppressors or inhibition of their functional activity in hepatocirrhosis. Peripheral blood lymphocytes of these patients, like the lymphocytes of healthy donors, showed practically no suppressive effect in vitro.     

Interleukine-17-induced inhibitory effect on late stage murine erythroid bone marrow progenitors

Recent studies have shown that the T cell-derived cytokine, interleukin-17 (IL-17), stimulates hematopoiesis, specifically granulopoiesis inducing expansion of committed and immature progenitors in bone marrow. Our previous results pointed to its role in erythropoiesis too, demonstrating significant stimulation of BFU-E and suppression of CFU-E growth in the bone marrow from normal mice. As different sensitivities of erythroid and myeloid progenitor cells to nitric oxide (NO) were found, we considered the possibility that the observed effects of IL-17 were mediated by NO. The effects of recombinant mouse IL-17, NO donor (sodium nitroprusside - SNP) and two NO synthases inhibitors (L-NAME and aminoguanidine) on erythroid progenitor cells growth, as well as the ability of IL-17 to induce nitric oxide production in murine bone marrow cells, were examined. In addition, we tested whether the inhibition of CFU-E colony formation by IL-17 could be corrected by erythropoietin (Epo), the principal regulator of erythropoiesis. We demonstrated that IL-17 can stimulate low level production of NO in murine bone marrow cells. Exogenously added NO inhibited CFU-E colony formation, whereas both L-NAME and aminoguanidine reversed the CFU-E suppression by IL-17 in a dose-dependent manner. The inhibition of CFU-E by IL-17 was also corrected by exposure to higher levels of Epo. The data obtained demonstrated that at least some of the IL-17 effects in bone marrow related to the inhibition of CFU-E, were mediated by NO generation. The fact that Epo also overcomes the inhibitory effect of IL-17 on CFU-E suggests the need for further research on their mutual relationship and co-signalling.     

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.     

Cimetidine induced pancytopenia. Effect on human CFU-MIX colony formation

We describe a 26 year-old male with a pancytopenia possibly due to cimetidine. Using progenitor cell culture techniques we investigated the mechanism of this bone marrow toxicity. Our results show a cimetidine dose-dependent inhibition of normal human CFU-GM colony formation as described by Fitchen and Koeffler in 1980. No differences in growth inhibition were found between the patients' recovery marrow and the controls. Toxicity on normal human CFU-MIX colony formation was, however, far more pronounced. At concentrations as low as 5 micrograms/ml the numbers of CFU-MIX colonies were decreased by almost 20% and more than 30% in cultures of two normal bone marrow samples. A significant decrease in CFU-MIX colony size was measured even at therapeutic levels (0.5 micrograms/ml). No obvious decrease in CFU-GM colony size was noticed at low concentrations. Experiments with T-cell- and monocyte-depleted bone marrow samples gave similar results: a pronounced inhibition of the CFU-MIX colony formation at low concentrations of cimetidine whereas the CFU-GM formation was less affected. It is therefore very unlikely that Accessory cells play part in the cimetidine induced CFU-MIX inhibition. Our results suggest the existence of H2 histamine receptors on human CFU-MIX (= multipotent progenitor cell). Blocking these receptors prevents the multipotent progenitor cell from going into the DNA-synthesis phase of the cell cycle.     

Simian immunodeficiency virus inhibits bone marrow hematopoietic progenitor cell growth.

The pathogenic mechanisms underlying the depressed hematopoietic functions seen in human immunodeficiency virus-infected individuals were explored in rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVmac). Bone marrow hematopoietic progenitor cell colony formation, both granulocyte/macrophage (CFU-GM) and erythrocyte (BFU-E), was shown to be decreased in number in SIVmac-infected rhesus monkeys. SIVmac was readily isolated from bone marrow cells of infected monkeys and was shown to be harbored in macrophages rather than T lymphocytes. The in vitro infection of normal bone marrow cells by SIVmac inhibited colony formation. A striking in vivo correlation between increased SIVmac load in bone marrow cells and decreased hematopoietic progenitor cell colony growth was also shown. Finally, inhibition of SIVmac replication in bone marrow macrophages resulted in increased progenitor cell colony growth from bone marrow cells. These results suggest that the infection of bone marrow macrophages by the acquired immunodeficiency syndrome (AIDS) virus may contribute to depressed bone marrow hematopoietic progenitor cell growth. Moreover, inhibition of AIDS virus replication in these macrophages might induce significant improvement in hematopoietic function.     

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.     

TGF-β Inhibition Restores Terminal Osteoblast Differentiation to Suppress Myeloma Growth

Background

Multiple myeloma (MM) expands almost exclusively in the bone marrow and generates devastating bone lesions, in which bone formation is impaired and osteoclastic bone resorption is enhanced. TGF-β, a potent inhibitor of terminal osteoblast (OB) differentiation, is abundantly deposited in the bone matrix, and released and activated by the enhanced bone resorption in MM. The present study was therefore undertaken to clarify the role of TGF-β and its inhibition in bone formation and tumor growth in MM.

Methodology/Principal Findings

TGF-β suppressed OB differentiation from bone marrow stromal cells and MC3T3-E1 preosteoblastic cells, and also inhibited adipogenesis from C3H10T1/2 immature mesenchymal cells, suggesting differentiation arrest by TGF-β. Inhibitors for a TGF-β type I receptor kinase, SB431542 and Ki26894, potently enhanced OB differentiation from bone marrow stromal cells as well as MC3T3-E1 cells. The TGF-β inhibition was able to restore OB differentiation suppressed by MM cell conditioned medium as well as bone marrow plasma from MM patients. Interestingly, TGF-β inhibition expedited OB differentiation in parallel with suppression of MM cell growth. The anti-MM activity was elaborated exclusively by terminally differentiated OBs, which potentiated the cytotoxic effects of melphalan and dexamethasone on MM cells. Furthermore, TGF-β inhibition was able to suppress MM cell growth within the bone marrow while preventing bone destruction in MM-bearing animal models.

Conclusions/Significance

The present study demonstrates that TGF-β inhibition releases stromal cells from their differentiation arrest by MM and facilitates the formation of terminally differentiated OBs, and that terminally differentiated OBs inhibit MM cell growth and survival and enhance the susceptibility of MM cells to anti-MM agents to overcome the drug resistance mediated by stromal cells. Therefore, TGF-β appears to be an important therapeutic target in MM bone lesions.     

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.     

Ultrastructure of cardiomyocytes in the peri-infarct zone during the treatment of experimental myocardial infarct in rats using the hexapeptide dalargin

The experiments on white rats with induced myocardial infarction have studied the influence of dalargin on the infarction size and peri-infarction zone ultrastructure. 24 hours later the decrease in the infarction zone size was detected in rats who had received dalargin in a dose of 50 and 100 micrograms/kg. In the peri-infarction zone the increase in glycogen quantity, the lower degree of lipid infiltration, the increase in mitochondrial number and mitochondrial energy effectiveness coefficient were noted, as compared to control animals. Sarcolemma of cardiomyocytes from the peri-infarction zone in rats on dalargin was impermeable for colloidal lanthanum. The decrease in the infarction size under the effect of dalargin is explained by its influence on the survival of cardiomyocytes in the peri-infarction zone.     

Photosensitization of leukemic cells and normal bone marrow cells by 514 nm laser light and effects of laser light on migration inhibition and lymphokine response

In a model for ex-vivo purging of bone marrow grafts, leukemic cells and normal bone marrow cells were treated with merocyanine 540 and exposed to 514 nm laser light. With this treatment, 99.9999% of leukemic cells were killed while 55% of the normal bone marrow cells survived. The deleterious effects of laser light alone in the absence of photosensitizer were not observed as determined by cell viability, cell migration, and response of target cells to human migration inhibition factor. These results indicate that laser light induced photodynamic therapy can be useful for ex-vivo autologous bone marrow purging without regard to the deleterious effects of laser light alone.