Action of opioid peptides on nerve tissue growth and regeneration processes in the rat

Using organotypic cultures of the sympathetic ganglia and spinal cord from rats, studies have been made of the effect of opioid peptides on the development of the nervous tissue. It was found that endogenous opioid peptides (leu- and met-enkephalins, beta-endorphin) within the concentrations investigated (10(-9)-10(-10) M), stimulate the growth of neurites, affect the rate of migration and proliferation of the glial and fibroblast-like cells. The effect was observed at the 2nd--5th days of cultivation, depending on the object investigated. Naloxone, a blockator of the opiate receptors, does not abolish the stimulating effect of the opioid peptides. Using clonal line of fibroblast-like cells L6, it was shown that leu-enkephalin decreases the sensitivity to contact inhibition of growth. On the basis of the data obtained, it is suggested that endogenous opioid peptides act as non-specific factors of growth regulation in the development and regeneration of the nervous tissue. Taking into account the role of endorphins in the activity of noci-antinociceptive system possible significance of these compounds in post-injury reparation is discussed.     

Influence of opioid peptides on nerve tissue in vitro

The influence of opioid peptides (gamma- and beta-endorphins, leu- and met-enkephalins, as well as certain synthetic analogs of enkephalin) was investigated on organotypic cultures of rat spinal and sympathetic ganglia. The cellular composition and size of the zone of growth were evaluated on the basis of intravital observations and an analysis of the specimen obtained using the method of impregnation, according to Holmes and the detection of catecholamines with glyoxylic acid. It was established that under the action of all the investigated substances that possess high affinity for opiate receptors, growth of the neurites from an explant was enhanced, and the number of glial and fibroblastoid cells in the growth zone was increased. The effect was observed most distinctly on a model of sympathetic ganglia. The tested compounds exhibited a significant growth-stimulating effect in the range of concentrations 10^–8–10^–14 M. The maximum size of the growth zone of the explants of the sympathetic ganglia in the case of a mean effective concentration of the peptides 10^–10 M by the third to fifth day of culturing was approximately 2–2.5 times this value in the control. The reaction was similar to the response of the nerve cells to nerve growth factor, used as a standard. Thus, the opioid peptides exhibit a pronounced growth effect on the structures of the nerve tissue under conditions of culture. It is suggested that this group of compounds, together with its currently well-known functions, may play a definite role in processes of the development and regenera-of nerve tissue.Institute of Experimental Cardiology, All-Union Cardiologic Science Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 550–557, July–August, 1985.     

Influence of captopril on the in vitro and in vivo effects of leu- and met-enkephalins

Captopril (10(-6) g/ml) enhanced and prolonged the inhibitory effect of leu- and met-enkephalins on the contractions of the isolated guinea-pig ileum section induced by electrical stimulation. In mice, the drug (25 mg/kg, subcutaneously) increased the degree and duration of the analgetic effect of enkephalins. It is concluded that the analgetic effect of captopril is related to the influence on the activity of the endogenous antinociceptive system.     

Effect of ethanol on the concentration of enkephalins in the brain of rats with different levels of alcoholic motivation

Radioimmunoassay was used to measure the content of leu- and met-enkephalins in white random-bred rats divided into groups according to the duration of ethanol anesthesia and the levels of 15% ethanol consumption under the conditions of free choice. The concentration of neuropeptides was determined in the cortex of the large hemispheres, striatum, thalamus, and medulla oblongata. The short-sleeping animals manifested elevated concentration of leu-enkephalin in the cortex and that of met-enkephalin in the striatum, medulla oblongata, and thalamus. Prolonged alcoholization under the conditions of free choice led, in the much-drinking animals, to decreased concentration of leu- and met-enkephalins in the striatum, thalamus and medulla oblongata and to increased concentration of leu-enkephalin in the cortex. The importance of leu- and met-enkephalins in the pathogenesis of chronic experimental alcoholism in rats with different alcoholic motivation is considered.     

Effects of opioid peptides and naloxone on tissue from the central and peripheral nervous system in culture

A study was made of the effects of opioid peptides (leu-enkephalin and dalargin AE-1, its synthetic analog) and of naloxone, an opiate receptor blocker, on organotypic cultures of spinal cord and spinal ganglia cells. The cellular composition and size of explant outgrowth was estimated according to in vitam morphological observations. It was found that all the opioid peptides tested at concentrations of 10^–9-10^–10M exercise a clear-cut growth-promoting effect on cultures from the spinal cord as well as those from the peripheral nervous system [4, 5]. Naloxone at a concentration of 10^–5-10^–6 M does not block peptide action, but itself stimulates growth. It was also proved that opioid peptides act as trophic factors for spinal ganglia nerve cells, increasing their survival in culture. Endorphins can thus serve as growth factors for tissues of the peripheral as well as the central nervous system. The likely processes lying at the root of the growth-promoting and trophic effects of endorphins on nerve tissue are discussed.Institute of Experimental Cardiology of the All-Union Cardiological Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 227–233, March–April, 1986.     

Effect of steroids and antisteroids on human meningioma cells in primary culture

Human meningiomas are rich in progestin receptors but virtually devoid of oestrogen receptors. We have studied the hormonal sensitivity of meningioma cells in vitro during 8 days of primary culture in the presence of different steroids and antisteroids. On day 8 the thymidine labelling index (TLI) was determined as a measure of cell growth. To date 30 cultures have been established from 39 tissue specimens. 13 cultures had a TLI below 1.0 and their growth were not affected by hormones. The TLI of the other 17 cultures was 3.0 +/- 1.7 (mean +/- SD; range 1.2-7.7). Following culture in the presence of 1 and 10 nM progesterone TLI was 83 +/- 28% (n = 9) and 61 +/- 29% (n = 3) of that of the control cultures respectively. Although in individual cultures occasional differences were found, the overall values are not statistically different from 100. Similarly, 1 nM of oestradiol and testosterone had no effect on the TLI (n = 3). Tamoxifen at 1 nM increased the TLI to 138% in one culture and decreased it to 66% of the control in another. The antiprogestin mifepristone (RU 486) in concentrations of 0.1, 1.0, 10, 100 and 1000 nM decreased the TLI to 72 +/- 30; 54 +/- 20; 55 +/- 20; 59 +/- 18 and 65 +/- 10 respectively (n = 6-15; P less than 0.05 vs control). It is concluded that although a growth promoting effect of progestins on meningioma could not be shown, the therapeutic possibilities of antiprogestins warrant further investigation.     

Simultaneous extraction of beta-endorphin and leu- and met-enkephalins from human and rat plasma

A simple, rapid and reliable procedure is described to simultaneously concentrate and purify beta-endorphin, leu- and met-enkephalins from small volumes of human and rat plasma before radioimmunoassay is performed. It uses C18 Sep-Pak reverse phase cartridges. The effectiveness of different protease inhibitors in preventing degradation of opiates by plasma and different solvent systems for eluting opiates is also evaluated.     

Comparison of the neuro- and immunomodulator properties of low-molecular neuropeptides

A study was made of the effect of the low-molecular neuropeptides, leu- and met-enkephalins, thyroliberin (TRH), the C-end tripeptides, gastrin (MAF) and oxytocin (MIF) on the content of biogenic monoamines and their metabolites and on the production of humoral antibodies to sheep red blood cells. The action of the peptides enumerated was compared to that of the peptide immunostimulant, tuftsin. All the peptides (upon intraventricular administration) with the exception of tuftsin affect the content of brain biogenic monoamines or their metabolites. Moreover, upon intravenous injection the neuropeptides under study except met-enkephalin exert a modulating action on the immune response pattern and intensity Leu-enkephalin, MIF and MAF have immunostimulant activity similar to tuftsin. TRH given in high doses (100 and 150 mg/kg) provokes almost a two-fold decrease in the antibody titer. This peptide has an immunosuppressant effect when administered both intravenously and intracisternally. It is suggested that neuro- and immunomodulator effects have much in common at the level of cell receptors.     

The growth-stimulating action of the preparation Balis-2 on sympathetic ganglia in culture

The effect of Balis-2 drug on the growth and differentiation of nervous tissue was studied on organotypic culture of the sympathetic ganglia. It was established that this agent is able to stimulate fiber outgrowth from explant and increase mean value of the maximal magnitude of the zone of the growth by concentration 0.001% and 0.0001%. It was found that Balis-2 drug is able to increase the intensity of reaction by 1.8-2 times. It is suggested that Balis-2 drug can be used as a new neurotropic agent.     

Coculture of human endothelium and smooth muscle cells: functional heterogeneity of endothelial cells from zones with different susceptibility to atherosclerosis

Using co-culture technique and 3H-thymidine radioautography we have studied effects of human aortic endothelial cells (EC), isolated separately from zones of low (LP) and high (HP) probability of atherosclerosis of grossly normal and atherosclerotic aortas, on 3H-thymidine incorporation by human intimal smooth muscle cells (SMCs). It was found that EC activity depended on a zone of probability, from which the cells were isolated, and on the degree of atherosclerotic lesion. The first-passage ECs from grossly normal LP zones inhibited 3H-thymidine incorporation, compared to control, incubated without Ecs, SMCs (63.5 +/- 27.5%). Cells from HP zones of the same vessels were less active or stimulated SMC proliferation (99.4 +/- 42.9%). EC cultures obtained from both LP and HP zones of atherosclerotic vessels had, as a rule, no effect or increased 3H-thymidine incorporation by SMCs (100.3 +/- 19.8 and 124.1 +/- 20.1%). In contrast to morphologically heterogeneous primary and first-passage cultures obtained from high seeding density, EC monolayers obtained with a split 1:10 were composed predominantly of small mononuclear cells. These cultures effectively inhibited SMC DNA synthesis independently of a zone of probability and a degree of atherosclerotic lesion of aorta (60.4 +/- 10.0 and 51.5 +/- 12.7%). The obtained data suggest that EC morphological heterogeneity is accompanied by functional changes of cells and may be involved in atherosclerotic plaque formation.     

Effect of substance P on nerve fiber regeneration in tissue culture

Explants of the ganglion trigeminale from chick embryos (PNS) and of the hippocampus from fetal rats (CNS) were cultivated in maximow chambers with growth medium or maintanance medium. Varied concentrations of substance P (SP . 3 CH3COOH . 4 H2O) were added. 1. The effect of substance P (SP) is related to concentration. In the presence of 10(-7)M SP in the growth medium and of 10(-4)M SP in the maintanance medium the cultivation of PNS cultures indicates positive results. These doses are suitable. 2. Within the first 24 hours in vitro SP stimulates the index of area in PNS cultures. The index of characterizes the relation of the outgrowth zone to the explant. In CNS cultures a significant difference of this effect was not observed. 3. The index of growth of nerve fibers may compare the test cultures with the control cultures. SP significantly increases the index of fiber growth in PNS cultures. A stimulation of CNS cultures was observed, significance was not found. 4. From the beginning of the cultivation with SP up to 48 hours in vitro the growth of nerve fibers significantly increases in the treated cultures in comparison with the control cultures. After this time the growth of nerve fibers decreased and a morphological conformity of test cultures and controls was observed. 5. The role of SP is discussed in specific activity on PNS tissue in vitro. The reactive neurons may be from the medio dorsal group of cells of the sensible ganglion.     

Transforming growth factor-beta enhances calcitonin-induced cyclic AMP production and the number of calcitonin receptors in long-term cultures of human umbilical cord blood monocytes in the presence of 1,25-dihydroxycholecalciferol.

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide, abundant in bone, that regulates both proliferation and differentiation of a wide variety of cells, but its role in osteoclast differentiation remains controversial. We have recently shown that long-term cultures of human cord blood monocytes, in the presence of 1,25 dihydroxycholecalciferol (1,25-(OH)2D3), give rise to cells that express two markers of the osteoclast phenotype, namely, the vitronectin receptor (VNR) and the calcitonin receptor (CTR). TGF-beta enhanced the proportion of cells expressing the VNR. In the present study, we investigated the effect of TGF-beta on the expression of CTR in cord blood monocytes cultured during 3 weeks in the presence of 1,25-(OH)2D3. When added within the first 2 weeks of culture, TGF-beta (500 pg/ml) significantly decreased the cell protein content. TGF-beta alone did not stimulate basal cAMP production. The 10 nM-sCT-stimulated cAMP production was enhanced by increasing TGF-beta concentrations from 50 pg/ml to 1,000 pg/ml: for 500 pg/ml TGF-beta, it was 294 +/- 28% vs. 140 +/- 25% for control cultures (p less than 0.01). The sCT dose-response curves showed a higher cAMP production from 10(-9) M to 10(-7) M of sCT in the presence of 500 pg/ml TGF-beta than in control cultures. The increase was 325 +/- 36% in the presence of TGF-beta and 195 +/- 13% in the absence of TGF-beta, for 10(-7) M sCT (p less than 0.01). This effect of TGF-beta on cAMP production was not observed either when it was added to monocyte cultures the last day or 2 hours before the end of the culture or in MCF7, a human breast cancer cell line that expresses CTR. [125I]-sCT binding studies performed on confluent cells showed similar Kd in control and TGF-beta-treated cells. By contrast, the CTR number was significantly increased in the presence of TGF-beta: 6.1 +/- 2 x 10(4) receptors per cell in control cultures and 28.8 +/- 8.1 x 10(4) receptors per cell in TGF-beta-treated cultures (p less than 0.05). It is thus suggested that TGF-beta increases the number of CTR of these cells that have other features of preosteoclasts. The role of this cytokine on the process of osteoclast differentiation and in bone resorption is thus emphasized.     

Inability of dimethylthiourea to limit tissue necrosis during acute myocardial infarction in rabbits.

This study examined the effect of treatment with dimethylthiourea (DMTU), a highly cell-permeable scavenger of hydroxyl radicals, on tissue necrosis in rabbit hearts during myocardial ischemia and reperfusion. Sixty-two rabbits underwent 45 minutes of coronary occlusion with, or without, coronary reperfusion for 3 hours. A saline vehicle, or DMTU (500 mg/kg intravenously [iv]) was administered over 45 minutes starting either 10 minutes before or 10 minutes after coronary occlusion, or 10 minutes before coronary reperfusion. Anatomic risk zone size was assessed using microsphere autoradiography, and the area of necrosis was determined using tetrazolium staining. Cardiac hemodynamics and risk zone size were similar for all treatment groups. No differences were observed in the extent of tissue necrosis (normalized to risk zone size) for saline- and DMTU-treated rabbits subjected to 45 minutes (61.2 +/- 23.1% vs. 70.6 +/- 16.5%) or 225 minutes (82.8 +/- 5.4% vs. 78.3 +/- 5.9%) of permanent coronary occlusion without reperfusion. Similarly, tissue necrosis in rabbits with 45 minutes coronary occlusion followed by 3 hours reperfusion was not significantly reduced when DMTU was administered either 10 minutes before coronary occlusion, 10 minutes after coronary occlusion, or 10 minutes before coronary reperfusion (67.0 +/- 9.9%; 57.6 +/- 10.6%; 68.3 +/- 13.3%) compared to saline-treated controls (76.6 +/- 10.5%). These results demonstrate that the hydroxyl radical scavenger DMTU does not appear to influence the progression of myocyte injury in this experimental model of acute myocardial infarction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of vanadate on (Na+-K+)-stimulated ATPase activity in the brain of rats of different ages

Experiments were carried out on infant rats aged five days and on adult rats (of both sexes) to investigate vanadate inhibition of (Na+-K+)ATPase activity in various parts of the brain. Vanadate was administered in 10(-5), 10(-7), 10(-8), 10(-9) and 10(-10) mol/l concentration. The enzyme activity and the effect of vanadate were studied in the tissue of the cerebral cortex, subcortical formations and the medulla oblongata. It was demonstrated that an inhibitory effect of vanadate on ouabain-sensitive ATPase could be determined in the brain of very young rats, i.e. in the immature nervous tissue. It was further demonstrated that the inhibitory effect of vanadate (in low concentrations) was significantly more potent in the nervous tissue of adult rats than in the CNS tissue of 5-day-old animals. Lastly, attention is drawn to certain differences in the sensitivity of ouabain-sensitive ATPase to the action of vanadate indifferent parts of the CNS in both the given age groups.     

Variables controlling somatomedin production by cultured human fibroblasts

Somatomedin is secreted by multiple types of cultured cells including human fibroblasts. Since the control of somatomedin (Sm) production by fibroblasts may be an important regulatory step in cell division, we undertook studies to define the variables in tissue culture experimental design that may have significant effects on the secretion of Sm. Cell density was an important parameter in determining basal Sm production rates. Cultures plated at 2.5 X 10(4) cells/well produced 0.38 +/- 0.06 U/ml/10(5) cells whereas an increase in culture density to 6.2 X 10(4) cells/well was associated with a decrease in Sm production per cell to 0.23 +/- 0.04 U/ml/10(5) cells (P less than .01). Cultures stimulated by platelet-derived growth factor (PDGF) showed a similar reduction in Sm production with increasing cell density. Epidermal growth factor was stimulatory (5.4-fold increase) in low-density cultures but had no effect when high-density cultures were used. If the experiments were initiated between 2 and 4 days after the last media change there were no significant differences in basal or PDGF-stimulated Sm concentrations. Between days 5 and 9 however, there was a progressive increase in the basal Sm production rate. The duration of incubation was an important variable since Sm production increased during the first 12 h in noncycling cells and showed an accelerated increase between 4 and 8 h in cycling cells. Cells between the eighth and 12th passage had similar basal Sm production (0.22 +/- 0.04 U/ml/10(5) cells) rates; cells between the 19th and 20th passages had significantly higher basal Sm production rates (0.41 +/- 0.05 U/ml/10(5) cells) (P less than .01). These results suggest that several variables, particularly cell density and passage number, are critical variables when quantitating the effect of hormones and growth factors on Sm production by cultured fibroblasts.     

Amyloid-beta1-42 treatment does not have a specific effect on cholinergic neurons in in vitro basal forebrain neuronal cultures of rat

The neurotoxic effect of amyloid-beta peptide (1-42) was investigated in cultures of neuronal tissue derived from the basal forebrain of embryonic rat. The axonal varicosities of the cholinergic cells were revealed by vesicular acetylcholine transporter staining, and the axonal varicosities in general by synaptophysin immunohistochemistry. The results demonstrate that the treatment of in vitro neuronal cultures with 20 microM amyloid-beta peptide (1-42) for 2 days on day 5, 12 or 15 exerted a neurotoxic effect on both the cholinergic and the non-cholinergic neurons. In the same cultures, the absolute number of synaptophysin-positive axon varicosities was reduced to greater extent (control: 203 +/- 37/field vs treated: 101 +/- 16/field) than the number of vesicular acetylcholine transporter-immunoreactive (control: 48 +/- 4/field vs treated: 0/field) structures. It is concluded that amyloid-beta peptide (1-42) does not have a specific effect only on the cholinergic neurons, but affects non-cholinergic neurons as well.     

Drug evaluations using neuronal networks cultured on microelectrode arrays

We used spontaneously active neuronal networks derived from dissociated embryonic murine spinal cord and auditory cortex and grown on substrate-integrated thin-film microelectrodes to determine characteristic responses to the cannabinoid agonists anandamide (AN) and methanandamide (MA). AN and MA reversibly inhibited spike and burst production in both tissue types. Responses of 21 cultures ranging in age from 23 to 111 days in vitro (d.i.v.) showed high intra- and inter-culture reproducibility at all ages. However, responses were tissue and substance-dependent. AN and MA were equipotent in cortical cultures and terminated bursting and spiking at 2.5 +/- 0.9 microM (n = 10). Spinal cultures were shut-off by 1.3 +/- 0.7 microM (n = 15) AN, but required 5.8 +/- 1.2 microM MA for activity cessation. MA, but not AN, demonstrated a biphasic influence: excitation at 0.25-3.5 microM and suppression at 4-7.1 microM. Palmitoylethanolamide, a related lipophilic molecule with no reported binding to the CBI receptor (to which AN and MA bind in the central nervous system), did not affect network activity at concentrations up to 6.5 microM. Irreversible cessation of activity was observed after 30 min applications of AN or MA at > 7 microM.     

Influence of fitness on the integrated neuroendocrine response to aerobic exercise until exhaustion

A group of trained and sedentary men performed an incremental graded exercise-test to exhaustion in order to assess the organic response of the two main stress-activated systems: the sympathetic nervous system with its endocrine component (the adrenal medulla), and the hypothalamic-pituitary-adrenal (HPA) axis. Maximal plasma concentrations of ACTH, cortisol and endogenous opioids (beta-endorphins) were obtained at the end of the exercise-test in the trained group. Thus ACTH increased from basal value of 21.25 +/- 2.5 pg/ml to 88.78 +/- 11.8 pg/ml at the end of the exercise (p<0.01); cortisol, from 16.56 microg/dl +/- 4.94 microg/dl to 23.80 +/- 4.57 microg/dl in min 15 of the recovery period (p<0.001); and beta-endorphin from 21.80 +/- 8.33 pmol/ml to 64.36 +/- 9.8 pmol/ml in min 3 of the recovery period (p<0.05). Catecholamine levels were increased from initial values at the end of the effort test in both control and trained groups. Control subjects exhibited a higher responsiveness compared to trained and showed superior intrinsic stimulation of the sympathetic nervous system. These results reveal a different response according to fitness in a physical stress situation.     

Opiate regulation of IL-1beta and TNF-alpha in cultured human articular chondrocytes

In order to investigate if beta-endorphins anti-inflammatory effect in cartilage-damaging states is mediated via tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), we examined its influence on these two cytokines in vitro. Human articular chondrocytes were obtained from patients undergoing total knee arthroplasty and stimulated with beta-endorphin (60-6000 ng/ml). Protein levels of TNF-alpha and IL-1 beta were measured by ELISA in supernatants from articular chondrocyte cultures. beta-Endorphin significantly increased the levels of IL-1 beta for all concentrations used after 15 min incubation, and when stimulated with 600 and 6000 ng/ml after 24 h incubation. The opioid-induced increase in IL-1 beta was blocked by naltrexone in the group tested. TNF-alpha expression was also significantly stimulated by 60 and 600 ng/ml beta-endorphin after 15 min, an effect blocked by naltrexone in the group tested. These findings indicate that the mechanism of beta-endorphins anti-inflammatory influence in cartilage-damaging states is not apparently mediated via these two cytokines modulation.