Synthetic peptide octarphin (TPLVTLFK), a selective agonist of nonopioid ß-endorphin receptor, stimulates nitric oxide synthesis in macrophages.

Synthetic peptide octarphin (TPLVTLFK, a selective agonist of nonopioid ß-endorphin receptor) was able to activate in a dose-dependent manner murine macrophages to express nitric oxide (NO) synthase and to produce NO. Octarphin required lipopolysacharide for the optimal induction of NO production. Octarphin-dependent NO production was sensitive to inhibition by dexamethasone and the NO synthase specific inhibitor NG-monomethyl-L-arginine. In the concentration range of 1-1000 nM, octarphin increased the cyclic 3',5'-guanosine monophosphate (cGMP) content in macrophages stimulated with lipopolysacharide. The effect was dependent on the peptide concentration and was maximal at a concentration of 100 nM. Thus, octarphin stimulates both NO and cGMP production in macrophages.

Interaction of synthetic peptide octarphin (TPLVTLFK) with human blood lymphocytes.

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12-19 of ß-endorphin, a selective agonist of non-opioid ß-endorphin receptor, was labeled with tritium to specific activity of 29 Ci/mmol. The analysis of [(3) H]octarphin binding to human T and B lymphocytes separated from normal human blood revealed the existence of one type of high-affinity binding sites (receptors): Kd 3.0 and 3.2 nM, respectively. Besides unlabeled octarphin, unlabeled ß-endorphin possessed the ability to inhibit the specific binding of [(3) H]octarphin to Т and B lymphocytes (Ki 1.9 and 2.2 nМ, respectively). Tests of the specificity of the receptors revealed that they are not sensitive to naloxone, α-endorphin, γ-endorphin, [Met(5) ]enkephalin, and [Leu(5) ]enkephalin. Thus, both T and B lymphocytes from normal human blood express non-opioid receptor for ß-endorphin. Binding of the hormone to the receptor provides a fragment 12-19.

C-Terminal Region of Caveolin-3 Contains a Stretch of Amino Acid Residues Capable of Diminishing Symptoms of Experimental Autoimmune Encephalomyelitis but Not Rheumatoid Arthritis Modeled in Rats.

A short synthetic peptide from the C-terminal part of the caveolin-3 structure was tested for experimental autoimmune encephalomyelitis (EAE) treatment in rats. The structure-function similarity established between the novel synthetic peptide of pCav3 and the well-known immunomodulator immunocortin determined pCav3's ability to reduce EAE symptoms in Dark Agouti (DA) rats injected with pCav3 (500 µg/kg). pCav3 was found to interfere with the proliferation of lymphocytes extracted from the LNs of DA rats primed with homogenate injection, with IC50 = 0.42 µM (2.35 mcg/mL). pCav3 affected EAE in a very similar manner as immunocortin. The high degree of homology between the amino acid sequences of pCav3 and immunocortin corresponded well with the therapeutic activities of both peptides, as demonstrated on EAE. The latter peptide, possessing a homologous structure to pCav3, was also tested on EAE to explore whether there were structural restrictions between these peptides implied by the MHC-involved cell machinery. Consequently, immunocortin was further examined with a different autoimmune disease model, collagen-induced arthritis (CIA), established in Sprague-Dawley rats. CIA was established using an intentionally different genetic platform than EAE. Based on the results, it was concluded that the effectiveness of pCav3 and immunocortin peptides in EAE rat model was almost identical, but differed in the rat model of rheumatoid arthritis; thus, efficacy may be sensitive to the MHC type of animals used to establish the autoimmune disease model.

Detection of nonopioid ß-endorphin receptor in the rat myocardium.

Two selective agonists of nonopioid ß-endorphin receptor, synthetic peptides TPLVTLFK (octarphin) and SLTCLVKGFY (immunorphin), were labeled with tritium to specific activity of 29 and 25 Ci/mmol, respectively. Both labeled peptides were found to bind to high-affinity naloxone-insensitive binding sites on the membranes isolated from the rat myocardium (Kd = 2.0 ± 0.2 and 2.5 ± 0.3 nM, respectively). The [(3)H]octarphin specific binding to the myocardial membranes was inhibited by unlabeled ß-endorphin (Ki = 1.9 ± 0.2 nM) and immunorphin (Ki = 2.2 ± 0.3 nM). The [(3)H]immunorphin specific binding with the membranes was inhibited by unlabeled ß-endorphin (Ki = 2.3 ± 0.3 nM) and octarphin (Ki = 2.4 ± 0.3 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α-endorphin, γ-endorphin, [Met(5)]enkephalin and [Leu(5)]enkephalin. Thus, ß-endorphin, immunorphin and octarphin bind to the common high-affinity naloxone-insensitive receptor of the rat myocardial membranes.

Synthetic peptide TPLVTLFK (octarphin) reduces the corticosterone production by rat adrenal cortex through nonopioid ß-endorphin receptor.

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12-19 of ß-endorphin, a selective agonist of nonopioid ß-endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [(3)H]Octarphin was found to bind to high-affinity naloxone-insensitive binding sites on membranes isolated from rat adrenal cortex (K(d) = 35.7 ± 2.3 nM, B(max) = 41.0 ± 3.6 pmol/mg protein). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to α-endorphin, γ-endorphin, [Met(5) ]enkephalin, and [Leu(5) ]enkephalin as well. At the same time, the [(3) H]octarphin-specific binding with adrenal cortex membranes was inhibited by unlabeled ß-endorphin (K(i) = 32.9 ± 3.8 nM). Octarphin at concentrations of 10(-9) -10(-6) M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, whereas intranasal injection of octarphin at doses of 5 and 20 µg/rat was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, octarphin decreases the adrenal cortex functional activity through the high affinity binding to nonopioid receptor of ß-endorphin.

Binding of synthetic peptide TPLVTLFK to nonopioid beta-endorphin receptor on rat brain membranes.

The synthetic peptide TPLVTLFK corresponding to the sequence 12-19 of beta-endorphin (referred to as octarphin) was found to bind to high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K(d) = 2.6 +/- 0.2 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to alpha-endorphin, gamma-endorphin, [Met(5)]enkephalin, and [Leu(5)]enkephalin, as well. The [(3)H]octarphin specific binding with brain membranes was inhibited by unlabeled beta-endorphin (K(i) = 2.4 +/- 0.2 nM) and a selective agonist of nonopioid beta-endorphin receptor decapeptide immunorphin SLTCLVKGFY (K(i) = 2.9 +/- 0.2 nM). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [(3)H]immunorphin with membranes (K(i) = 2.8 +/- 0.2 nM). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of beta-endorphin on rat brain cortex membranes.

Binding of cholera toxin B subunit to intestinal epithelial cells.

We have prepared 125I-labeled cholera toxin B subunit (125I-labeled CT-B, a specific activity of 98Ci/mmol) and found that it binds to rat IEC-6 and human Caco-2 intestinal epithelial cells with high affinity (Kd 3.6 and 3.7nM, respectively). The binding of labeled protein was completely inhibited by unlabeled thymosin-α1 (TM-α1), interferon-α2 (IFN-α2), and the synthetic peptide LKEKK that corresponds to residues 16-20 in TM-α1 and 131-135 in IFN-α2, but was not inhibited by the synthetic peptide KKEKL with inverted amino acid sequence (Ki>10µM). Thus, TM-α1, IFN-α2, and the peptide: LKEKK bind with high affinity and specificity to the cholera toxin receptor on IEC-6 and Caco-2 cells. It was found that CT-B and the peptide: LKEKK at concentrations of 10-1000nM increased in a dose-dependent manner the nitric oxide production and the soluble guanylate cyclase activity in IEC-6 and Caco-2 cells.

Interaction of cholera toxin B subunit with T and B lymphocytes.

We have prepared 125I-labeled cholera toxin B subunit (125I-labeled CT-B, a specific activity of 98Ci/mmol) and found that its binding to T and B lymphocytes from the blood of healthy donors was high-affinity (Kd 2.8 and 3.0nM, respectively). The binding of labeled protein was completely inhibited by unlabeled thymosin-α1 (TM-α1), interferon-α2 (IFN-α2), and the synthetic peptide LKEKK that corresponds to residues 16-20 in TM-α1 and 131-135 in IFN-α2, but was not inhibited by the synthetic peptide KKEKL with inverted amino acid sequence (Ki>10µM). Thus, TM-α1, IFN-α2, and the peptide: LKEKK bind with high affinity and specificity to CT-B receptor on donor blood T and B lymphocytes. It was found that CT-B and the peptide: LKEKK at concentrations of 10-1000nM increased in a dose-dependent manner the soluble guanylate cyclase activity in T and B lymphocytes.

Synthetic peptide SLTCLVKGFY competes with beta-endorphin for naloxone-insensitive binding sites on rat brain membranes.

The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain and its synthetic fragment VKGFY were found to compete with 125I-labeled beta-endorphin for high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K(i)=1.18+/-0.09 and 1.58+/-0.11 nM, respectively). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to [Met(5)]enkephalin and [Leu(5)]enkephalin as well. The K(d) values characterizing the specific binding of 125I-labeled immunorphin and its fragment Val-Lys-Gly-Phe-Tyr to these binding sites were determined to be 2.93+/-0.27 nM and 3.17+/-0.29 nM, respectively.

Beta-endorphin-like peptide SLTCLVKGFY reduces the production of 11-oxycorticosteroids by rat adrenal cortex through nonopioid beta-endorphin receptors.

Beta-endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid beta-endorphin receptors on rat adrenal cortex membranes (Kd = 31.6 +/- 0.2 nM, Bmax = 37.4 +/- 2.2 pmol/mg protein). Immunorphin at concentrations of 10(-9) to 10(-6) M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10-100 microg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.

Synthetic peptide octarphin (TPLVTLFK) inhibits the activity of the hypothalamus-pituitary-adrenal axis through nonopioid ß-endorphin receptor.

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12-19 of ß-endorphin, a selective agonist of nonopioid ß-endorphin receptor, was labeled with tritium to specific activity of 29 Ci/mmol. The analysis of [(3)H]octarphin binding to rat pituitary and adrenal cortex membranes revealed the existence of one type of binding sites (receptors): Kd 5.9 and 35.6 nM, respectively. Octarphin at concentrations of 1-1000 nМ was shown to inhibit the adenylate cyclase activity of rat adrenocortical membranes, while its intramuscular injection at doses of 10-100 µg/kg was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, the nonopioid receptor of ß-endorphin may be involved in the regulation of the activity of the pituitary and adrenal glands.

Binding of synthetic fragments of beta-endorphin to nonopioid beta-endorphin receptor.

Selective agonist of nonopioid beta-endorphin receptor decapeptide immunorphin (SLTCLVKGFY) was labeled with tritium (the specific activity of 24 Ci/mmol). [3H]Immunorphin was found to bind to nonopioid beta-endorphin receptor of mouse peritoneal macrophages (Kd = 2.0 +/- 0.1 nM). The [3H]immunorphin specific binding with macrophages was inhibited by unlabeled beta-endorphin (Ki = 2.9 +/- 0.2 nM) and was not inhibited by unlabeled naloxone, alpha-endorphin, gamma-endorphin and [Met5]enkephalin (Ki > 10 microM). Thirty fragments of beta-endorphin have been synthesized and their ability to inhibit the [3H]immunorphin specific binding to macrophages was studied. Unlabeled fragment 12-19 (TPLVTLFK, the author's name of the peptide octarphin) was found to be the shortest peptide possessing practically the same inhibitory activity as beta-endorphin (Ki = 3.1 +/- 0.3 nM). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol). [3H]Octarphin was found to bind to macrophages with high affinity (Kd = 2.3 +/- 0.2 nM). The specific binding of [3H]octarphin was inhibited by unlabeled immunorphin and beta-endorphin (Ki = 2.4 +/- 0.2 and 2.7 +/- 0.2 nM, respectively).

Interaction of ACTH synthetic fragments with rat adrenal cortex membranes.

Synthetic peptide, corresponding to the amino acid sequence 11-24 of human adrenocorticotropic hormone (ACTH), was labeled with tritium (specific activity of 22 Ci/mmol). [(3)H]ACTH (11-24) was found to bind to rat adrenal cortex membranes with high affinity and specificity (K(d) = 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) have been synthesized and their ability to inhibit the specific binding of [(3)H]ACTH (11-24) to adrenocortical membranes has been investigated. Unlabeled fragment ACTH 15-18 (KKRR) was found to replace in a concentration-dependent manner [(3)H]ACTH (11-24) in the receptor-ligand complex (K(i) = 2.3 +/- 0.2 nM). ACTH (15-18) was labeled with tritium (specific activity of 20 Ci/mmol). [(3)H]ACTH (15-18) was found to bind to rat adrenal cortex membranes with high affinity (K(d) = 2.1 +/- 0.1 nM). The specific binding of [(3)H]ACTH (15-18) was inhibited by unlabeled ACTH (11-24) (K(i) = 2.2 +/- 0.1 nM). ACTH (15-18) at the concentration range of 1-1000 nM did not affect the adenylate cyclase activity in adrenocortical membranes.

Effect of beta-endorphin and beta-endorphin-like peptide immunorphin on the growth of human leukemic cells in vitro.

The influence of beta-endorphin and immunorphin on human leukemic cell growth in vitro was studied. It was shown that both peptides increase the growth of T-lymphoblastoid cells in a dose-dependent manner. The effect of these peptides on the 3H-thymidine incorporation into T-lymphoblastoid cell line Jurkat was not reversed by the antagonist of opioid receptor naloxone. Interestingly, these peptides had no effect on B-lymphoblastoid and promyelocyte cell growth, however they enhance 3H-thymidine incorporation into myeloid cell lines.

The Stimulating Effect of the beta-Endorphin-Like Peptide Immunorphin on the Human T-Lymphoblastoid Cell Line Jurkat Is Mediated by a Non-Opioid Receptor for beta-Endorphin.

It was shown that beta-endorphin and the synthetic decapeptide SLTCLVKGFY that corresponds to the amino acid sequence 364-373 of the human IgG heavy chain (referred to as immunorphin) is able to stimulate growth of the human T-lymphoblastoid cell line Jurkat. The antagonist of opioid receptors naloxone did not inhibit the stimulating effect of the peptides. Studies on [(3)H]-immunorphin binding to Jurkat cell receptors have demonstrated that it binds with high affinity to naloxone-insensitive receptors (K(d) = 1.3 nM; n = 5.2 x 10(5)). Unlabeled beta-endorphin and the 6-10 fragment of immunorphin completely inhibited the labeled ligand specific binding to naloxone-insensitive receptors on T lymphocytes (K(i) = 1.4 x 10(-7) and 3.7 x 10(-5) M, respectively). Thus, beta-endorphin and immunorphin share the naloxone-insensitive receptors on human T-lymphoblastoid cell line Jurkat.