Elucidation and characteristics of non-opioid beta-endorphin receptors in rat adrenal cortex.

beta-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of non-opioid beta-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 micro M, Bmax2 = 187.8 +/- 9.4 pmol/mg protein). beta-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 +/- 9.2 nM); naloxone, [Met5]enkephalin, and alpha- and gamma-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 micro g/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.

Characteristics of non-opioid beta-endorphin receptor.

Tritium-labeled selective agonist of non-opioid beta-endorphin receptor, the decapeptide immunorphine ([3H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid beta-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [3H]immunorphine-receptor complex was 2.4 +/- 0.1 nM) and beta-endorphin (Ki of the [3H]immunorphine specific binding was 2.9 +/- 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro3]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 microM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [3H]immunorphine. Values of the specific binding of 8.4 nM [3H]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 beta-endorphin, pentarphine, [Pro3]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [3H]immunorphine to membranes from these organs. No specific binding of [3H]immunorphine to rat liver, lung, kidney, and intestine membranes was found.

Macrophage-stimulating peptides VKGFY and cyclo(VKGFY) act through nonopioid beta-endorphin receptors.

We have synthesized two peptides, VKGFY and cyclo(VKGFY) (referred to as pentarphin (PNT) and cyclopentarphin (cPNT), respectively), and found that both peptides at 1 nM concentration increased the adhesion and spreading of murine peritoneal macrophages as well as their bactericidal activity in vitro, as shown by phagocytosis of Salmonella typhimurium virulent strain 415. PNT administered intraperitoneally at dose 20 microg/mouse on day 7, 3, and 1 prior to the isolation of macrophages also enhanced the macrophage adhesion and spreading. The receptor binding characteristics of PNT and cPNT were examined using 125I-labeled PNT. The binding of labeled PNT to peritoneal macrophages was high-affinity (K(d)=3.6 nM) and saturable. It was not inhibited by naloxone (NAL) or [Met(5)]enkephalin ([Met(5)]ENK) but completely inhibited by unlabeled cPNT (K(i)=2.6 nM), immunorphin (IMN, decapeptide SLTCLVKGFY, corresponding to the IgG heavy-chain sequence 364-373) (K(i)=3.2 nM) or beta-endorphin (beta-END) (K(i)=2.8 nM). Thus, the effects of PNT and cPNT on macrophages are mediated by NAL-insensitive receptors common for PNT, cPNT, IMN, and beta-END.

Synthetic peptide VKGFY and its cyclic analog stimulate macrophage bactericidal activity through non-opioid beta-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 strainbacteria 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 (125)I-labeled pentarphin (179 Ci/mmol specific activity). The binding of (125)I-labeled pentarphin to mouse peritoneal macrophages was high-affinity (K(d) = 3.6 +/- 0.3 nM) and saturable. Studies on binding specificity revealed that this binding was insensitive to naloxone and [Met(5)]enkephalin, but completely inhibited by unlabeled cyclopentarphin (K(i) = 2.6 +/- 0.3 nM), immunorphin (K(i) = 3.2 +/- 0.3 nM), and beta-endorphin (K(i) = 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 beta-endorphin.

beta-Endorphin-like peptide SLTCLVKGFY is a selective agonist of nonopioid beta-endorphin receptor.

It has been found that beta-endorphin (beta-END) and a synthetic beta-END-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin, IMN) corresponding to the sequence 364-373 of human IgG heavy chain stimulate Con A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met(5)]enkephalin ([Met(5)]ENK) and an antagonist of opioid receptors naloxone (NAL) tested in parallel were not active. The stimulating effect of beta-END and IMN on T lymphocyte proliferation was not inhibited by NAL. Studies on receptor binding of (125)I-labeled IMN to T lymphocytes revealed that it binds with high affinity to NAL-insensitive binding sites (K(d) = 7.0 +/- 0.3 nM). Unlabeled beta-END completely inhibited the specific binding of (125)I-labeled IMN to NAL-insensitive binding sites on T lymphocytes (K(i) = 1.1 +/- 0.2 nM). Thus, beta-END and IMN bind to common NAL-insensitive binding sites on T lymphocytes and enhance Con A-induced proliferation of these cells.

Effect of synthetic beta-endorphin-like peptide immunorphin on human T lymphocytes.

Beta-endorphin and the synthetic beta-endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met(5)]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of beta-endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of (125)I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (K(d) = 7.0 +/- 0.3 nM). Unlabeled immunorphin completely inhibits (125)I-labeled beta-endorphin specific binding to naloxone-insensitive receptors on T lymphocytes (K(i) = 0.6 +/- 0.1 nM). Thus, beta-endorphin and immunorphin interact with common naloxone-insensitive receptors on T lymphocytes.

[Interaction of synthetic decapeptide SLTCLVKGFY with human T lymphocytes]. / Vzaimodeistvie sinteticheskogo dekapeptida SLTCLVKGFY s T-limfotsitami cheloveka.

The synthetic peptide SLTCLVKGFY, corresponding to the 364-373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [125I] beta-endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (Ki 0.6 nM). The fragments 3-10, 4-10, 5-10, and 6-10 of Immunorphin also inhibited the binding (Ki 2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and, therefore, are not opioid. The Kd values of the specific binding of 125I-labeled Immunorphin and its 6-10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.

Synthetic beta-endorphin-like peptide immunorphin binds to non-opioid receptors for beta-endorphin on T lymphocytes.

The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [125I]beta-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K(i) = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 1.0 nM) possessed the ability to inhibit specific binding of [125I]beta-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. K(d) values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively.

[Hormone-like activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1]. / Gormonopodobnye svoistva sinteticheskogo dekapeptida, sootvetstvuiushchego adrenokortikotropinopodobnoi posledovatel'nosti immunoglobulina G1 cheloveka.

The antiproliferative and immunosuppressive in vitro effects of immunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11-20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10(-11)-10(-7) M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strain Salmonella typhimurium 415. By using a 125I-labeled "addressing" fragment of ACTH ¿[125I]ACTH-(13-24)¿, we showed that MT-4 cells express specific receptors for ACTH (Kd 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [125I]ACTH-(13-24) to these receptors with Ki1 of 0.38 and Ki2 of 0.34 nM, respectively. Specific receptors for ACTH (Kd 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to complete with labeled ACTH-(13-24) for binding to these receptors (Ki = 1.8 nM) and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.

Study of immunosuppressive activity of a synthetic decapeptide corresponding to an ACTH-like sequence of human immunoglobulin G1.

The synthetic ACTH-like decapeptide H-Val-Lys-Lys-Pro-Gly- Ser-Ser-Val-Lys-Val-OH, corresponding to amino acid residues 11-20 of the variable part of the human IgG1 heavy chain (referred to as immunocortin) was found to have an immunosuppressive effect on cells in vitro: it inhibits blast transformation of mouse thymocytes and reduces spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against the virulent bacterial strain Salmonella typhimurium 415. Tritium-labeled immunocortin binds with high affinity to ACTH receptors on thymocytes and macrophages (Kd 2. 1 and 2.5 nM, respectively) and activates adenylate cyclase in these cells. Thus, the interaction of immunocortin with the target cell includes the following main steps: binding to the receptor, activation of adenylate cyclase, and elevation of the intracellular content of cAMP.