Yury A. Zolotarev

Binding of synthetic fragments of β-endorphin to nonopioid β-endorphin receptor

Selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin (SLTCLVKGFY) was labeled with tritium (the specific activity of 24 Ci/mmol). [3H]Immunorphin was found to bind to nonopioid β‐endorphin receptor of mouse peritoneal macrophages (Kd = 2.0 ± 0.1 nM). The [3H]immunorphin specific binding with macrophages was inhibited by unlabeled β‐endorphin (Ki = 2.9 ± 0.2 nM) and was not inhibited by unlabeled naloxone, α‐endorphin, γ‐endorphin and [Met5]enkephalin (Ki > 10 µM). Thirty fragments of β‐endorphin have been synthesized and their ability to inhibit the [3H]immunorphin specific binding to macrophages was studied. Unlabeled fragment 12–19 (TPLVTLFK, the authors name of the peptide octarphin) was found to be the shortest peptide possessing practically the same inhibitory activity as β‐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 β‐endorphin (Ki = 2.4 ± 0.2 and 2.7 ± 0.2 nM, respectively). Copyright

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

Two selective agonists of nonopioid β‐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 [3H]octarphin specific binding to the myocardial membranes was inhibited by unlabeled β‐endorphin (Ki = 1.9 ± 0.2 nM) and immunorphin (Ki = 2.2 ± 0.3 nM). The [3H]immunorphin specific binding with the membranes was inhibited by unlabeled β‐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, [Met5]enkephalin and [Leu5]enkephalin. Thus, β‐endorphin, immunorphin and octarphin bind to the common high‐affinity naloxone‐insensitive receptor of the rat myocardial membranes. Copyright

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 β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (Kd = 2.6 ± 0.2 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met5]enkephalin, and [Leu5]enkephalin, as well. The [3H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (Ki = 2.4 ± 0.2 nM) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (Ki = 2.9 ± 0.2 nM). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [3H]immunorphin with membranes (Ki = 2.8 ± 0.2 nM). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright

Interaction of synthetic peptide octarphin with rat myocardium membranes

A selective agonist of non-opioid β-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 β-endorphin receptor of rat myocardium membranes before EMI: Kd1 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 (Kd2 = 13.3 ± 0.4 nM), and in 48 h its almost complete restoration (Kd4 = 2.2 ± 0.3 nM). The results indicate participation of non-opioid β-endorphin receptor in the regulation of myocardial activity.

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

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12-19 of β-endorphin, a selective agonist of nonopioid β-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 β-endorphin may be involved in the regulation of the activity of the pituitary and adrenal glands.

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

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of nonopioid β‐endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [3H]Octarphin was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from rat adrenal cortex (Kd = 35.7 ± 2.3 nM, Bmax = 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, [Met5]enkephalin, and [Leu5]enkephalin as well. At the same time, the [3H]octarphin‐specific binding with adrenal cortex membranes was inhibited by unlabeled β‐endorphin (Ki = 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 β‐endorphin. Copyright

Comparative study of the neuroprotective and nootropic activities of the carboxylate and amide forms of the HLDF-6 peptide in animal models of Alzheimer’s disease

A comparative study of the neuroprotective and nootropic activities of two pharmaceutical substances, the HLDF-6 peptide (HLDF-6-OH) and its amide form (HLDF-6-NH2), was conducted. The study was performed in male rats using two models of a neurodegenerative disorder. Cognitive deficit in rats was induced by injection of the beta-amyloid fragment 25–35 (βA 25–35) into the giant-cell nucleus basalis of Meynert or by coinjection of βA 25–35 and ibotenic acid into the hippocampus. To evaluate cognitive functions in animals, three tests were used: the novel object recognition test, the conditioned passive avoidance task and the Morris maze. Comparative analysis of the data demonstrated that the neuroprotective activity of HLDF-6-NH2, evaluated by improvement of cognitive functions in animals, surpassed that of the native HLDF-6-OH peptide. The greater cognitive/ behavioral effects can be attributed to improved kinetic properties of the amide form of the peptide, such as the character of biodegradation and the half-life time. The effects of HLDF-6-NH2 are comparable to, or exceed, those of the reference compounds. Importantly, HLDF-6-NH2 exerts its effects at much lower doses than the reference compounds.

Interaction of the synthetic peptide octarphin with rat adrenal cortex membranes

The synthetic peptide octarphin (TPLVTLFK, fragment 12–19 of β-endorphin), a selective agonist of the nonopioid β-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 [3H]octarphin specifically binds to the membranes with high affinity: Kd1 = 36.3 ± 2.5 nM, Bmax1 = 41.0 ± 3.8 pmol/mg protein. The specific binding of [3H]octarphin to the membranes was inhibited by unlabeled β-endorphin (Ki = 33.9 ± 3.6 nM) and the agonist of the non-opioid receptor decapeptide immunorphin (Ki = 36.8 ± 3.3 nM). Unlabeled naloxone, [Leu5]- and [Met5]enkephalins, α- and γ-endorphins, and corticotropin were inactive (Ki > 1 μM). Both cold and heat shocks decreased the binding affinity: Kd2 = 55.6 ± 4.2 nM and Kd3 = 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 β-endorphin receptor of adrenal cortex membranes.

α 1 -Thymosin, α 2 -interferon, and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The 125I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (Kd = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α2-interferon (IFN-α2), α1-thymosin, (TM-α1), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α1 and the 131–135 sequence of human IFN-α2 (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (Ki > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α1, IFN-α2, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.

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.