Neuroimmunomodulation with enkephalins: in vitro enhancement of natural killer cell activity in peripheral blood lymphocytes from cancer patients

To initiate investigations into the effects of enkephalins on immune function in cancer patients, the effect of methionine-enkephalin and leucine-enkephalin on natural killer (NK) cell activity in isolated peripheral blood lymphocytes from cancer patients was investigated. Incubation of lymphocytes with either enkephalin resulted in significant increases in NK cell activity. At effector:target cell ratios of 100:1, 33:1 and 11:1 leucine-enkephalin significantly (p less than 0.05) enhanced NK activity at dilutions of 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml. Similar results were obtained with methionine-enkephalin with the exception that the 10(-6) dilution gave insignificant changes at both the 33:1 and 11:1 cell ratios. The results indicate a difference in dose response to both enkephalins between lymphocytes from cancer patients and normal volunteers.     

Isolation and structure identification of a morphine-like peptide "enkephalin" in bovine brain.

The ability of bovine brain extracts to compete in a selective fashion for opiate receptor binding is attributable to a small peptide. The substance has been purified to homogeneity and identified as comprising two penta-peptides HTyrGlyGlyPheLeuOH (Leucine-enkephalin) and HTyrGlyGlyPheMetOH (methionine enkephalin). Bovine brain contains 4 times as much leucine-enkephalin as methionine-enkephalin in contrast to pig brain in which these ratios are reversed. Competition for opiate receptor binding by leucine-enkephalin is reduced more by sodium and enhanced more by manganese than is the case for methionine-enkephalin, suggesting that leucine-enkephalin may be a “purer” agonist than methionine-enkephalin.     

Enkephalin-binding protein in human blood

An enkephalin-binding protein was found in human plasma and serum. The protein was partially purified by DEAE-cellulose column chromatography. The binding of [3H]leucine-enkephalin to this protein was competitively inhibited by unlabeled leucine- and methionine-enkephalin and various peptide hormones such as beta-endorphin and glucagon, but not by Leu-enkephalin-amide. The fact that amide derivatives of leucine-enkephalin and methionine-enkephalin did not inhibit the binding suggests that c-terminuses of enkephalins might have an important part in binding the protein. From these results, physiological roles of the enkephalin-binding protein are discussed.     

Fluctuations in cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monophosphate during the mitotic cycle of the acellular slime mould Physarum polycephalum.

Cyclic adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic guanosine 3′:5′-monophosphate (cyclic GMP) have been determined at half-hourly intervals throughout the mitotic cycle of Physarum polycephalum. Cyclic AMP was constant at 1pmole/mg protein throughout except for a transient peak of 17pmoles/mg protein in the last quarter of G2. Cyclic GMP was more variable (2–4pmole/mg protein) rising to 9.5pmole/mg protein during the 3 hour S period and to 7pmole/mg protein during the last hour of G2. The significance of these changes is discussed.     

Reduced mechanical activity of perfused rat heart following morphine or enkephalin peptides administration

In the isolated and perfused rat heart, the addition of morphine, methionine-enkephalin or leucine-enkephalin to the coronary perfusate, significantly reduces the mechanical activity by negatively affecting both the heart rate and the developed tension. These effects are dose dependent and maximally evident with leucine-enkephalin. Furthermore all the opioids strongly reduce the activity of isoproterenol-stimulated hearts. The suggestion is made that opioid peptides directly influence the cardiac mechanical activity possibly by interacting with membrane-receptor systems.     

Inhibitory action of opioid peptides on ouabain-sensitive Na+-K+ and Ca2+-dependent ATPase activities in bovine cardiac sarcolemma

The present study demonstrates that morphine (10(-6) and 10(-5) M), methionine-enkephalin or leucine-enkephalin (10(-10), 10(-8), and 10(-6) M) were able to inhibit significantly, in a dose-dependent manner, both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. The inhibitory action of these opioids on the two ATPases was not antagonized by preincubation with naloxone (10(-6) M). Naloxone alone (10(-8), 10(-6) and 10(-5) M) did not affect both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. Heat-denatured methionine-enkephalin (10(-6) M) or leucine-enkephalin (10(-6) M) also unaffected both the ATPases. The possibility is also discussed that opioid peptides may regulate myocardial contractility by modulating the movement of ions across the heart sarcolemma.     

Effects of opioid peptides on thyrotropin-releasing hormone release from the rat caecum in vitro

Effects of opioid peptides (beta-endorphin, dynorphin (1-13). alpha-neoendorphin, beta-neoendorphin, leucine-enkephalin, methionine-enkephalin) on the release of thyrotropin-releasing hormone (TRH) from the rat caecum were studied in vitro. The rat caecum was incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium). The amount of TRH release from the rat caecum into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat caecum was inhibited significantly in a dose-related manner with the addition of opioid peptides. The inhibitory effects of opioid peptides on ir-TRH release from the rat caecum were blocked with an addition of naloxone. The elution profile of acid-methanol-extracts of rat caecum on Sephadex G-10 was identical to that of synthetic TRH. The findings suggest that opioid peptides inhibit TRH release from the rat caecum in vitro.     

Crystal structure of methionine-enkephalin

The crystal structure of methionine-enkephalin has been determined by X-ray crystallography. There are two independent pentapeptides in the asymmetric unit and both display extended backbone conformations with their side chains arranged alternately below and above the backbone plane. The two molecules form a hydrogen-bonded head-to-tail dimer similar in conformation to one dimeric pair of leucine-enkephalin molecules in a previously reported crystal structure.     

阿片肽对骨髓未成熟B细胞的免疫学效应

 已经证明一些细胞象脾淋巴细胞、血T细胞、自然杀伤细胞和血小板存在有阿片受体。本文在骨髓细胞上的研究提示未成熟B细胞表面也存在有这类神经肽的受体,通过阿片肤类结合到细胞上可以观察到不同的效应。试验结果显示M-脑啡肽、L-脑啡肽和α-内啡肽对骨髓未成熟B细胞抗体生成反应的抑制分别为76.6％、68.6％和67.7％。相反,β-内啡肽增进抗体反应达78.0％。M-脑啡肽和两种内啡肽在未成熟B细胞上的效应能够被拮抗剂纳络酮所逆转,提示反应涉及阿片肽受体机制。     

Synenkephalin in bovine and human spinal cord

Summary Synenkephalin, which comprises 70 residues at the aminoterminal of proenkephalin, was studied with immunocytochemical methods in the human and bovine spinal cord. Immunoreactive fibers had the same general distribution as methionine-enkephalin, but not as leucine-enkephalin fibers. They were found in all spinal layers and were most numerous in lamina II (outer zone) and V–VI (lateral portion). Synenkephalin immunoreactivity was overall less dense than that of the enkephalins. These results suggest that proenkephalin is the precursor protein also in enkephalinergic neurons of the human spinal cord.     

Localization of biotransformational enzymes along the crypt-villus axis of the rat intestine. Evaluation of two cell isolation procedures

Rat intestinal epithelial cells were isolated by EDTA-chelation, combined with gentle shaking (modified Weiser procedure) or with strong longitudinal vibration (Harrison/Webster procedure). Both methods yield large numbers of viable cells and are relatively easy to use. Electronmicroscopical and biochemical data indicate that cell fractions from different levels of the villous region can be obtained only by the modified Weiser procedure. When strong mechanical forces are involved (Harrison-Webster procedure) the villus epithelium is released according to an all-or-nothing process. The biotransformational capacity of cell fractions, obtained from different levels of the villi by the modified Weiser procedure, was investigated. It was shown that the rate of metabolism of 7-ethoxycoumarin and 1-naphthol was substantially higher in lower villous cells than in cells isolated from the upper villous region. O-Deethylation of 7-ethoxycoumarin decreases from 145 +/- 13 pmole/min mg cell protein (72 +/- 4% conjugated) in lower villous cells to 62 +/- 12 pmole/min mg cell protein (37 +/- 6% conjugated) in tip cells. Glucuronidation of 1-naphthol decreased from 495 +/- 23 pmole/min mg cell protein (lower villous cells) to 137 +/- 13 pmole/min mg cell protein (tip cells).     

Microcompartmentation of cAMP in wild-type and memory-mutant dunce strains of Drosophila melanogaster

The "enzyme-probe" method [Solti M, Friedrich P: Eur J Biochem 95:551, 1979] has been applied to characterize the cyclic AMP pool in wild-type Canton-S and memory-mutant dunceM11 strains of Drosophila melanogaster. The kinetics of cyclic AMP breakdown in whole fly homogenates by endogenous cyclic nucleotide phosphodiesterase(s) indicate that the cyclic AMP pool is divided into free and bound fractions. The bound fraction in Canton-S and dunceM11 is 0.5 and 1.5 pmole/mg fly, respectively. Considering the total cyclic AMP content of the two strains, 1.6 and 10 pmole/mg fly, respectively, we conclude that the bulk of excess cyclic AMP in the mutant is free nucleotide.     

Degradation of enkephalins: the search for an enkephalinase

Summary The opioid peptides methionine-enkephalin and leucine-enkephalin appear to exert their biological effects through a receptor mediated mechanism. There appears to be three potential mechanisms for enkephalin degradation which could serve to control enkephalin levels in the vicinity of enkephalin receptors. These are, 1) cleavage of the tyrosyl-glycine bond by aminopeptidases, 2) cleavage of the glycyl-glycine bond by a dipeptidyl aminopeptidase, and 3) cleavage of the glycyl-phenylalanine bond by a dipeptidyl carboxypeptidase. In this review the biochemical properties of these potential enkephalinases are described, and the evidence for each acting as an enkephalinase is reviewed.     

The monosynaptic connection: the modulating effect of opioid peptides on the plasticity of presynaptic neurons and identified synapses]

Study of opioid peptides (leucine-enkephalin and methionine-enkephalin) action on plastic properties of the system of monosynaptically connected neurones LPa7--LPa3, PPa3 and LPa8--LPa3, PPa3 was conducted in the snail brain. It has been shown that all three links in the system studied (presynaptic neurone, postsynaptic neurone and synapse) manifest one and the same type of plasticity--habituation to rhythmic stimulation. Enkephalins have a modulating action on plastic properties of the presynaptic neurone and synapse: they retard the habituation of the presynaptic neurone to intracellular stimulation and retard the development of habituation at synaptic level. However, changes in the character of postsynaptic response in the presence of enkephalins are not a direct consequence of their influence on plastic properties of the presynaptic neurone. Besides, enkephalines reduce the effectiveness of synaptic transmission in the given system: they reduce EPSP duration in the postsynaptic neurone.     

抗 P 物质单克隆抗体的特性研究

观察了细胞株(BTP-1)所分泌的抗 P 物质单克隆抗体与17种 P 物质类似物的交叉反应。该单克隆抗体所针对的是 P 物质的羧基末端,与羧基端的甲硫氨酸残基有较大的关系,而和P 物质类似物的拮抗作用的强弱无相关性。与9种小肽(亮氨酸脑啡肽、甲硫氨酸脑啡肽,内啡肽、皮啡肽、血管紧张素Ⅰ、Ⅱ、缓激肽,催产素、胰高血糖素)均无交叉反应,显示该单克隆抗体有较好的特异性。经免疫组织化学试验,中脑以下部位的脊髓后角Ⅱ层、黑质、三叉神经脊束核、脚间核等末梢纤维和第Ⅱ脑室腹侧灰质、被盖外侧核、中缝核等胞体核周质染成棕色。BTP-1属于大鼠 IgG 型单克隆抗体。描述了微载体大量培养杂交瘤和制备单克隆抗体的技术,并讨论了大量制备的可能性。     

Are opioid peptides co-localized with vasopressin or oxytocin in the neural lobe of the rat?

Summary The content of vasopressin, oxytocin, neurophysin, leucine-enkephalin, methionine-enkephalin, dynorphin-(1–13), and -neoendorphin in the rat neurohypophysis was measured after different periods of dehydration and after depolarisation of isolated neural lobes and of neurosecretory nerve endings. The rates at which the amount of neurohypophysial hormone and opioid peptides decreased, and the changes in the ratios between the amount of vasopressin or oxytocin and opioid peptide in the neurohypophysis after dehydration and in the incubation medium after depolarization in vitro cast some doubt on, and can be explained by mechanisms other than co-localisation of the different peptides.     

Regional heterogeneity of two high affinity binding sites for 3H-WB-4101 in mouse brain

Binding studies of the tritium labeled α-adrenergic antagonist WB-4101 to mouse brain homogenate at equilibrium, reveal two binding sites: a super high affinity site (0.25 nM, 0.070 pmole/mg. protein) and a high affinity site (3.0 nM, 0.19 pmole/ mg. protein). Kinetic experiments, which measure the rates of association and dissociation of ³H-WB-4101 further confirm the existence of two binding sites. Differences in the distribution of the two sites in five regions of mouse brain are described. In addition, we present evidence suggestive of a postsynaptic localization of both binding sites.     

25-Hydroxyvitamin D-1alpha-hydroxylase activity is diminished in human prostate cancer cells and is enhanced by gene transfer

The hormone 1alpha,25-dihydroxyvitamin D (1alpha,25(OH)(2)D) inhibits growth and induces differentiation of prostate cells. The enzyme responsible for 1alpha,25(OH)(2)D synthesis, 25-hydroxyvitamin D (25(OH)D)-1alpha-hydroxylase (1alpha-OHase), has been demonstrated in human prostate cells. We compared the levels of 1alpha-OHase activity in prostate cancer cell lines, LNCaP, DU145 and PC-3 and in primary cultures of normal, cancerous and benign prostatic hyperplasia (BPH) prostate cells. We observed a marked decrease in 1alpha-OHase activity in prostate cancer cells, including an undetectable level of activity in LNCaP cells. Transient or stable transfection of 1alpha-OHase cDNA into LNCaP cells increased 1alpha-OHase activity from undetectable to 4.95pmole/mg+/-0.69pmole/mg and 5.8pmole/mg+/-0.7pmole/mg protein per hour, respectively. In response to 25(OH)D, the prohormone of 1alpha,25(OH)(2)D, the transfected LNCaP cells showed a significant inhibition of 3H-thymidine incorporation (37%+/-6% and 56%+/-4% at 10(-8)M for transiently and stably transfected cells, respectively). These findings support an important autocrine role for 1alpha,25(OH)(2)D in the prostate and suggest that the re-introduction of the 1alpha-OHase gene to prostate cancer cells, in conjunction with the systemic administration of 25(OH)D, constitutes an endocrine form of gene therapy that may be less toxic than the systemic administration of 1alpha,25(OH)(2)D.     

Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary

In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH.     

Exposure to Exogenous Enkephalins Disrupts Reproductive Development in the Eastern Lubber Grasshopper,Romalea microptera (Insecta: Orthoptera)

Enkephalins play a major role in reproductive physiology in crustaceans; however their role in reproductive development in insects is largely unknown. We investigated the effect of exposure to exogenous leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), and the opioid antagonist naloxone on gonad development in the Eastern lubber grasshopper, Romalea microptera. Injection of either Leu-Enk or naloxone alone significantly increased the testicular index and testicular follicular diameter in males, and the ovarian index, oocyte length, and oocyte diameter in females. In contrast, injection of Met-Enk inhibited all measures of reproductive development in both sexes. Surprisingly, co-injection of naloxone with either enkephalin enhanced the effect associated with administration of the enkephalin alone. This study clearly demonstrates the ability of enkephalins to disrupt insect sexual development and also suggests the existence of conserved enkephaline-dependent regulatory mechanisms in insects and crustaceans.