Yu. A. Kovalitskaya

Characteristics of non-opioid β-endorphin receptor

Tritium-labeled selective agonist of non-opioid β-endorphin receptor, the decapeptide immunorphine ([2H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid β-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid β-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [2H]immunorphinereceptor complex was 2.4 ± 0.1 nM) and β-endorphin (Ki of the [2H]immunorphine specific binding was 2.9 ± 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro2]pentarphine (VKPFY) (Ki values were 0.0060 ± 0.0004, 2.7 ± 0.2, 2.6 ± 0.2, and 2.8 ± 0.2 nM, respectively) and is insensitive to naloxone and [Met5]enkephalin (Ki > 100 μM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [2H]immunorphine. Values of the specific binding of 8.4 nM [2H]immunorphine to rat adrenal, spleen, myocardium, and brain membranes were determined to be 1146.0 ± 44.7, 698.6 ± 28.1, 279.1 ± 15.4, and 172.2 ± 1.8 fmol/mg protein, respectively. Unlabeled β-endorphin, pentarphine, [Pro2]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [2H]immunorphine to membranes from these organs. No specific binding of [2H]immunorphine to rat liver, lung, kidney, and intestine membranes was found.

Immunostimulating effect of the synthetic peptide octarphin corresponding to β-endorphin fragment 12–19

We have synthesized the peptide TPLVTLFK corresponding to β-endorphin fragment 12–19 (dubbed octarphin) and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The octarphin peptide was labeled with tritium (specific activity 28 Ci/mol), and its binding to murine peritoneal macrophages was studied. [3H]Octarphin was found to bind to macrophages with high affinity (Kd = 2.3 ± 0.2 nM) and specificity. The specific binding of [3H]octarphin was inhibited by unlabeled β-endorphin and the selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (Ki = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled nalox-one, α-endorphin, γ-endorphin, or [Met5]enkephalin (Ki > 10 μM). Inhibitory activity of unlabeled octarphin analogs was more than 100 times lower than that of unlabeled octarphin. Octarphin was shown to stimulate activity of murine immuno-competent cells in vitro and in vivo: at concentration of 1–10 nM it enhanced the adhesion and spreading of peritoneal macrophages as well as their ability to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro; the peptide administered intraperitoneally at a dose of 20 μg/animal on day 7, 3, and 1 prior to isolation of cells increased activity of peritoneal macrophages as well as spleen T- and B-lymphocytes.

Elucidation and Characteristics of Non-opioid β-Endorphin Receptors in Rat Adrenal Cortex

Abstractβ-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid β-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid β-endorphin receptors on rat adrenal cortex membranes (Kd1 = 39.6 ± 2.0 nM, Bmax1 = 40.7 ± 2.3 pmol/mg protein; Kd2 = 0.25 ± 0.01 μM, Bmax2 = 187.8 ± 9.4 pmol/mg protein). β-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 ± 9.2 nM); naloxone, [Met5]enkephalin, and α- and γ-endorphins tested in parallel were inactive. Immunorphin at concentrations of 10–9-10–6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, while intramuscular injection of immunorphin at doses of 10-100 μg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.

Synthetic Peptide VKGFY and Its Cyclic Analog Stimulate Macrophage Bactericidal Activity through Non-Opioid β-Endorphin Receptors

We synthesized linear and cyclic pentapeptides corresponding to the sequence 369-373 of human immunoglobulin G heavy chain—VKGFY (referred to as pentarphin and cyclopentarphin, respectively). The effect of pentarphin and cyclopentarphin on phagocytosis of Salmonella typhimurium virulent 415 strain bacteria by mouse peritoneal macrophages in vitro was studied. Control experiments showed that macrophages actively captured these bacteria, but did not digest them: the captured microbes were viable and continued to proliferate inside the phagocytes; within 12 h all macrophage monolayer was destroyed (incomplete phagocytosis). If 1 nM pentarphin or cyclopentarphin was added to the cultivation medium, macrophage bactericidal activity was significantly increased and they digested all captured microorganisms within 6 h (complete phagocytosis). To study the receptor binding properties of pentarphin and cyclopentarphin we prepared 125I-labeled pentarphin (179 Ci/mmol specific activity). The binding of 125I-labeled pentarphin to mouse peritoneal macrophages was highaffinity (Kd = 3.6 ± 0.3 nM) and saturable. Studies on binding specificity revealed that this binding was insensitive to naloxone and [Met5]enkephalin, but completely inhibited by unlabeled cyclopentarphin (Ki = 2.6 ± 0.3 nM), immunorphin (Ki = 3.2 ± 0.3 nM), and β-endorphin (Ki = 2.8 ± 0.2 nM). Thus, the effects of pentarphin and cyclopentarphin on macrophages are mediated by naloxone-insensitive receptors common for pentarphin, cyclopentarphin, immunorphin, and β-endorphin.

[Synthetic peptide KKRR corresponding to the human ACTH fragment 15-18 is an antagonist of the ACTH receptor].

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [3H]ACTH (11–24) and [3H]ACTH (15–18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [3H]ACTH-(11–24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (Kd 1.8 ± 0.1 nM). Twenty nine fragments of ACTH (11–24) were synthesized, and their ability to inhibit the specific binding of [3H]ACTH (11–24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15–18) (KKRR) (Ki 2.3 ± 0.2 nM), whose [3H] labeled derivative binds to rat adrenocortical membranes (Kd 2.1 ± 0.1 nM) with a high affinity. The specific binding of [3H]ACTH-(15–18) was inhibited by 100% by unlabeled ACTH (11–24) (Ki 2.0 ± 0.1 nM). ACTH (15–18) in the concentration range of 1–1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.

Nonopioid effect of β-endorphin

This review presents the generalized literature data and the results of our own research of the nonopioid effect of β-endorphin, an opioid neuropeptide interacting not only with opioid but also with nonopioid (insensitive to the opioid antagonist naloxone) receptors. The roles of the hormone and its receptors in regulation of the immune, nervous, and endocrine systems are discussed. The effect of neuromediator on the immune system mediated by both opioid and nonopioid receptors is considered in detail. The data on distribution and function of the nonopioid β-endorphin receptor in human and animal organisms are presented. All available data on the characteristics of the nonopioid β-endorphin receptor obtained by means of radioligand analysis are given. The discussed information is supposed to extend our conceptions of the role of β-endorphin in mammals and to be of extensive use in medicine and pharmacology.

Phenotypic Manifestation of Gene Expression Encoding Xyloglucanase from Penicillium canescens in Transgenic Aspen Plants

Plant xyloglucans play an important role in the processes of cell wall extension, determine their mechanical properties, thus affecting growth and morphology of individual cells and whole organs. Being one of the main components of hemicellulose, xyloglucans play a particular physiological role in woody plants. To study xyloglucan physiological role, transgenic aspen (Populus tremula L.) plants with a recombinant sp-Xeg gene from the fungus Penicillium canescens were produced. Constitutive expression of this gene in the heterologous surrounding was confirmed by RT-PCR method. The analysis of protein extracts from the leaves of greenhouse-grown plants and microshoots grown in vitro showed activation of xylogluconase in transgenic lines. The strongest activation (1.6-fold) was observed in the leaf extracts (clone PtXVXeg1b) and in vitro microshoots (clone PtXVXeg1c). In transgenic plants, the relative content of pentosans in the wood was declined. In control plants (Pt genotype), it was equal to 148 mg/g dry wt, whereas in tested clones (PtXVXeg1a, PtXVXeg1b, and PtXVXeg1c), it varied from 100 to 140 mg/g dry wt. The strongest decrease (by 31%) in the content of pentosans was observed for the line PtXVXeg1c; the content was equal to 102.1 ± 1.5 mg/g dry wt. A comparative analysis of leaf morphology revealed an increase in the length of petiole and a decrease in the length of the main vein in transgenic lines. In control plants, the ratio of the petiole length to the length of the main vein was equal to 0.49, whereas in transgenic plants, it varied from 0.51 to 0.66. A significant increase of this index was observed in 12 from 14 transgenic lines.

Stimulation of the Viability of Early Mouse Embryos Cultured in Vitro by β-Endorphin-Like Peptides

The growth of a mammalian embryo is accompanied by complex interactions between the embryo and the mother, as well as between the embryo cells themselves. Peptide growth factors play an important role in cell division, differentiation, and morphogenesis during mammalian embryogenesis. Several families of growth factors of various origins influence preimplantation of mammalian embryos. These are insulin-like (IGF), epidermal (EGF), and platelet-derived (PDGF) growth factors, as well as transforming growth factor b (TGF-b), fibroblast growth factor (FGF), and leukemia-inhibiting factor (LIF) [1]. The growth of the blastocyst, which is the final stage of preimplantation, proved to depend on EGF and LIF [2]. These two factors promote the formation of a favorable conditions for blastocyst implantation and the subsequent growth of the embryo. β -Endorphin can be assigned to the peptide growth factors that regulate embryo growth [3]. Follicular cells around the oocyte are known to secrete β -endorphin. This seems to influence oocyte maturation and ovulation [4]; the fact that the endometrium cells secrete β -endorphin [5] suggests that this substance has an effect on the interaction between the embryo and the mother. The in vitro sensitivity of growing preimplantation mouse embryos to the agonists and antagonists of opioid receptors suggests that the latter are present on the embryos and bind β -endorphin [6]. In the 1980s, the fragment 364–377 of the heavy chain of immunoglobulin G (IgG) similar to the central part of the β -endorphin molecule has been identified in extracts of the human placenta [7]. In the Pushchino Branch of the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Institute of Biochemistry of the Russian Academy of Sciences, β -endorphin-like peptides were synthesized. These are immunorphin (SLTCLVKGFY), which is the fragment 364–373 of the heavy chain of human IgG, and its fragments: pentarphin (VKGFY), the immunorphin 6–10 fragment; cyclopentarphin, cyclo(VKGFY); and cyclodipentarphin, cyclo(VKGFYVKGFY). These substances were shown to be selective antagonists of non-opioid (naloxon-insensitive) β -endorphin receptors that were detected on cells of the rat immune system and on membranes from the rat myocardium, adrenal glands, spleen, and brain [8, 9]. We have showed previously that immunorphin at a concentration of 10 –7 I stimulated early growth of mouse embryos in vitro [10]. Therefore, immunorphin may be considered a β -endorphin-like nonspecific growth factor. In this study, the effects of the β -endorphin-like peptides pentarphin ( 10 –7 I), cyclopentarphin ( 10 –7 I), and cyclodipentarphin 10 –6 I) on the growth of two-, four-, and eight-cell mouse embryos were studied in vitro. In the control group, none of the above peptides were added to the growing embryos. The specificity of the peptide effect was confirmed in experiments on the effect of a “cocktail” of amino acids constituting these peptides (Val, Lys, Gly, Phe, and Tyr; 10 –7 M) on mouse embryo preimplantation. In total, 344 and 215 embryos were used in the experiment and control, respectively.

Expression of xyloglucanase sp-Xeg gene from Penicillium canescens accelerates growth and rooting in transgenic aspen plants

The properties of transgenic aspen (Populus tremula) clones carrying the recombinant gene of xyloglucanase sp-Xeg from Penicillium canescens have been analyzed. Complex modifications were revealed both in the composition of the wood and in the plant phenotype. Biometric analysis showed that shoot dimensions increased by 24.8%, 25% and 26% in the PtXIV-Xeg1a, PtXVXeg1a and PtXVXeg1b lines, respectively. The number of internodes in some transgenic clones also increased. Modifications in rhizogenesis have been shown for the first time in the plants with the recombinant gene of xyloglucanase: in vitro rooting efficiency exceeded the control value in 13 out of 25 lines. Maximum rooting efficiency was observed in the PtXVXeg1a line (3.2-fold higher than in the control). A reliable increase in the root system mass (by 20% to 52%) under greenhouse conditions was observed for 8 out of 25 clones. A lower pentosan content in the wood was shown for all lines. The data on xyloglucanase activity and pentosan content generally correlated with phenotypic modifications.

Investigation of beta-endorphin reception in preimplantation development of a mouse embryo in vitro

The effect of β-endorphin on 2-, 4-, and 8-cell embryo development in vitro was studied. It is shown that the hormone has no effect on a 2-cell embryo development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number of blastocyst formations increases in the presence of 0.1 μM β-endorphin in embryo cultured medium, and the number of blastocysts with abnormal structure decreases. The effect of the hormone on the change of intracellular concentration of Ca2+ ions in 2-, 4-, and 8-cell mouse embryos has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase and phospholipase activity blockers, and naloxone on the change of intracellular concentration of Ca2+ ions in the early mouse embryo in the presence of β-endorphin has also been studied. It is shown that 2-cell embryos have opioid and nonopioid β-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopioid β-endorphin receptors. It is also shown that the effect of β-endorphin in the early mouse embryo through nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.