Interaction of Cholera Toxin B-subunit with Human T-lymphocytes.

In this work, 125I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (Kd = 3.3 nM) was determined. The binding of the 125I-labeled CT-B was inhibited by unlabeled interferon-α2 (IFN-α2), thymosin-α1 (TM-α1), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α1 and 131-135 of IFN-α2 (Ki 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (Ki > 1 µM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.

Binding of Synthetic LKEKK Peptide to Human T-Lymphocytes.

The synthetic peptide LKEKK corresponding to sequence 16-20 of human thymosin-α1 and 131-135 of human interferon-α2 was labeled with tritium to specific activity 28 Ci/mol. The [3H]LKEKK bound with high affinity (Kd = 3.7 ± 0.3 nM) to donor blood T-lymphocytes. Treatment of cells with trypsin or proteinase K did not abolish [3H]LKEKK binding, suggesting the non-protein nature of the peptide receptor. The binding was inhibited by thymosin-α1, interferon-α2, and cholera toxin B subunit (Ki = 2.0 ± 0.3, 2.2 ± 0.2, and 3.6 ± 0.3 nM, respectively). Using [3H]LKEKK, we demonstrated the existence of a non-protein receptor common for thymosin-α1, interferon-α2, and cholera toxin B-subunit on donor blood T-lymphocytes.

[Octarphin--Nonopioid Peptide of the Opioid Origin].

The data on the properties and mechanism of action of the peptide octarphin (TPLVTLFK, the fragment 12-19 of ß-endorphin)--a selective agonist of nonopioid (insensitive to the action of the opioid antagonist naloxone) ß-endorphin receptor found on n immune cells (peritoneal macrophages, T and B lymphocytes of spleen and blood), endocrine (adrenal cortex, hypothalamus), cardiovascular (cardiomyocytes) systems are analyzed and systematized. Binding to the receptor octarphin increases increases the mitogen-induced pro- liferation of human and mouse T and B lymphocytes in vitro, activates murine peritoneal macrophages in vitro and in vivo, stimulates growth of human T-lymphoblast cell lines Jurkat and MT-4, inhibits adenylate cyclase activity of rat adrenal cortex membranes and suppresses the secretion of glucocorticoids from the adrenal gland into the blood. It was shown that in a concentration range of 1-1000 nM the peptide increases the activity of inducible NO-synthase (iNOS), and the content of NO and cGMP in lipopolysaccharide-activated murine peritoneal macrophages. Taking into account that NO acts as a primary activator of soluble guanylate cyclase (sGC), it can be assumed that the activating effect of octarphin on macrophages is realized in the following way: increase in th iNOS expression --> increase in the NO production --> increase in the sGC activity --> increase in intracellular levels of cGMP.

The second life of antibodies.

Antibodies (immunoglobulins, Ig) are used by the immune system to identify and neutralize foreign objects and are responsible for antigen-binding and effector functions. Immunoglobulin G (IgG) is the major serum immunoglobulin of a healthy human (~75% of the total Ig fraction). The discovery in 1970 of the endogenous tetrapeptide tuftsin (Thr-Lys-Pro-Arg, fragment 289-292 of the C(H2)-domain of the heavy (H) chain of IgG), possessing both immunostimulatory and neurotrophic activities, was an impetus for the search for new biologically active peptides of immunoglobulin origin. As a result, fragments of the H-chain of IgG produced as a result of enzymatic cleavage of IgG within the antigen-antibody complex were discovered, synthesized, and studied. These fragments include rigin (341-344), immunorphin (364-373), immunocortin (11-20), and peptide p24 (335-358) and its fragments. In this review the properties of these peptides and their role in regulating the immune response are analyzed.

Interaction of synthetic peptide octarphin with human blood lymphocytes.

The synthetic peptide octarphin (TPLVTLFK, fragment 12-19 of ß-endorphin), a selective agonist of nonopioid ß-endorphin receptor, was prepared with specific activity 28 Ci/mmol. The binding of [3H]octarphin to T and B lymphocytes isolated from the blood of donors was studied. It was found that [3H]octarphin binds both to T and B cells with high affinity: Kd = 3.0 ± 0.2 and 3.2 ± 0.3 nM, respectively. The specific binding of [3H]octarphin to T and B lymphocytes was competitively inhibited by unlabeled ß-endorphin (Ki = 1.9 ± 0.2 and 2.2 ± 0.3 nM, respectively) and was not inhibited by unlabeled naloxone, [Met(5)]enkephalin, [Leu(5)]enkephalin, α-endorphin, and γ-endorphin. Thus, T and B lymphocytes of human blood possess a nonopioid ß-endorphin receptor whose binding is provided by the fragment 12-19 (the octarphin sequence).

Interaction of the synthetic peptide octarphin with rat adrenal cortex membranes.

The synthetic peptide octarphin (TPLVTLFK, fragment 12-19 of ß-endorphin), a selective agonist of the non-opioid ß-endorphin receptor, was labeled with tritium yielding specific activity of 28 Ci/mmol. The binding of [3H]octarphin to rat adrenal cortex membranes was studied under normal conditions as well as after cold and heat shocks. It was found that under normal conditions [(3)H]octarphin specifically binds to the membranes with high affinity: K(d1) = 36.3 ± 2.5 nM, B(max1) = 41.0 ± 3.8 pmol/mg protein. The specific binding of [(3)H]octarphin to the membranes was inhibited by unlabeled ß-endorphin (K(i) = 33.9 ± 3.6 nM) and the agonist of the non-opioid receptor decapeptide immunorphin (K(i) = 36.8 ± 3.3 nM). Unlabeled naloxone, [Leu(5)]- and [Met(5)]enkephalins, α- and γ-endorphins, and corticotropin were inactive (K(i) > 1 µM). Both cold and heat shocks decreased the binding affinity: K(d2) = 55.6 ± 4.2 nM and K(d3) = 122.7 ± 5.6 nM, respectively. In both cases, the maximal binding capacity of the receptor did not change. Thus, even a short-term thermal shock significantly affects the sensitivity of the non-opioid ß-endorphin receptor of adrenal cortex membranes.

Interaction of synthetic peptide octarphin with rat myocardium membranes.

A selective agonist of non-opioid ß-endorphin receptor synthetic peptide octarphin (TPLVTLFK, specific activity 28 Ci/mmol) was prepared. The [3H]octarphin binding to rat myocardium membranes before and after experimental myocardial infarction (EMI) was studied. It was found that [3H]octarphin with high affinity and specificity binds to non-opioid ß-endorphin receptor of rat myocardium membranes before EMI: K(d1) value of the [3H]octarphin specific binding to membranes was 1.8 ± 0.2 nM. In 3 h after EMI a sharp lowering in affinity of the binding is observed (K(d2) = 13.3 ± 0.4 nM), and in 48 h its almost complete restoration (K(d4) = 2.2 ± 0.3 nM). The results indicate participation of non-opioid ß-endorphin receptor in the regulation of myocardial activity.

Immunostimulating effect of the synthetic peptide octarphin corresponding to ß-endorphin fragment 12-19.

We have synthesized the peptide TPLVTLFK corresponding to ß-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 (K(d) = 2.3 ± 0.2 nM) and specificity. The specific binding of [3H]octarphin was inhibited by unlabeled b-endorphin and the selective agonist of nonopioid b-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K(i) = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled naloxone, a-endorphin, γ-endorphin, or [Met(5)]enkephalin (K(i) > 10 mM). 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 immunocompetent 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.

Nonopioid effect of ß-endorphin.

This review presents the generalized literature data and the results of our own research of the nonopioid effect of ß-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 ß-endorphin receptor in human and animal organisms are presented. All available data on the characteristics of the nonopioid ß-endorphin receptor obtained by means of radioligand analysis are given. The discussed information is supposed to extend our conceptions of the role of ß-endorphin in mammals and to be of extensive use in medicine and pharmacology.

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.

Synthetic peptide immunocortin stimulates the production of 11-oxycorticosteroides by rat adrenal cortex through ACTH receptors.

Adrenocorticotropic hormone (ACTH)-like peptide immunocortin (IMC) VKKPGSSVKV, corresponding to the amino acid sequence 11-20 of the variable part of human immunoglobulin G (IgG) 1 heavy chain, at concentrations of 10(-9)-10(-6) I was found to increase the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunocortin at doses of 10-100 microg/kg was found to stimulate the secretion of 11-oxycorticosteroids (CS) from the adrenals to the bloodstream. Immunocortin was labeled with tritium to specific activity of 22 Ci/mmol. Receptor binding studies revealed that [(3)H]immunocortin ([(3)H]IMC) bound with high affinity and specificity to ACTH receptors on rat adrenal cortex membranes (K(d)=2.1+/-0.2 nM, B(max)=1.1+/-0.1 pmol/mg protein).

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.

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.

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.

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.

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.