Study of degradation pathways of Amadori compounds obtained by glycation of opioid pentapeptide and related smaller fragments: stability,reactions, and spectroscopic properties

Reactions between biological amines and reducing sugars (the Maillard reaction) are among the most important of the chemical and oxidative changes occurring in biological systems that contribute to the formation of a complex family of rearranged and dehydrated covalent adducts that have been implicated in the pathogenesis of human diseases. In this study, chemistry of the Maillard reactions was studied in four model systems containing fructosamines (Amadori compounds) obtained from the endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu), leucine-enkephalin methyl ester, structurally related tripeptide (Tyr-Gly-Gly), or from amino acid (Tyr). The degradation of model compounds as well as their ability to develop Maillard fluorescence was investigated under oxidative conditions in methanol and phosphate buffer pH 7.4 at two different temperatures (37 and 70 degrees C). At 37 degrees C, glycated leucine-enkephalin degraded slowly in methanol (t(1/2) approximately 13 days) and phosphate buffer (t(1/2) approximately 9 days), producing a parent peptide compound as a major product throughout a three-week incubation period. Whereas fluorescence slowly increased over time at 37 degrees C, incubations off all studied Amadori compounds at 70 degrees C resulted in a rapid appearance of a brown color and sharp increase in AGE (advanced glycation end products)-associated fluorescence (excitation 320 nm/emmision 420 nm) as well as in distinctly higher amounts of fragmentation products. The obtained data indicated that the shorter the peptide chain the more degradation products were formed. These studies have also helped to identify a new chemical transformation of the peptide backbone in the Maillard reaction that lead to beta-scission of N-terminal tyrosine side chain and p-hydroxybenzaldehyde formation under both aqueous and nonaqueous conditions.     

Isolation and structure identification of a morphine-like peptide "enkephalin" in bovine brain.

The ability of bovine brain extracts to compete in a selective fashion for opiate receptor binding is attributable to a small peptide. The substance has been purified to homogeneity and identified as comprising two penta-peptides HTyrGlyGlyPheLeuOH (Leucine-enkephalin) and HTyrGlyGlyPheMetOH (methionine enkephalin). Bovine brain contains 4 times as much leucine-enkephalin as methionine-enkephalin in contrast to pig brain in which these ratios are reversed. Competition for opiate receptor binding by leucine-enkephalin is reduced more by sodium and enhanced more by manganese than is the case for methionine-enkephalin, suggesting that leucine-enkephalin may be a “purer” agonist than methionine-enkephalin.     

Peptidergic nerves in human dental pulp. An immunocytochemical study

The peptidergic innervation of human dental pulp was studied with indirect immunofluorescence and immunoperoxidase techniques. Pulpal nerve fibres displaying immunoreactivity for cholecystokinin, calcitonin gene-related peptide, C-terminal flanking peptide of neuropeptide tyrosine, leucine-enkephalin, methionine-enkephalin, neuropeptide K, neuropeptide tyrosine, peptide with N-terminal histidine and C-terminal isoleucine, somatostatin-28, substance P and vasoactive intestinal polypeptide were observed. Immunoreactive axon varicosities were detectable within radicular and coronal nerve trunks and within the nerve plexus of Raschkow in the para-odontoblastic region. Many peptidergic nerve fibres were observed in association with blood vessels of various sizes. Substance P- and calcitonin-gene-related peptide-immunoreactive axons were visible in the odontoblastic layer. The occurrence of VIP- and PHI-immunoreactive fibres lends support to the hypothesis that human tooth may be supplied by parasympathetic nerves. The immunocytochemical results here shown provide a morphological basis to previous experimental studies concerning the possible roles of neuropeptides in nociception mechanisms, control of the blood flow and modulation of the inflammatory response in dental tissues.     

The effect of glycation on the chemical and enzymatic stability of the endogenous opioid peptide, leucine-enkephalin, and related fragments

Nonenzymatic glycation is a posttranslational modification of peptides and proteins by sugars, which, after a cascade of reactions, leads to the formation of a complex family of irreversibly changed adducts implicated in the pathogenesis of human diseases. The stability of the Amadori compounds, the last reversible intermediates, determines the further course of the reaction. To provide information concerning the fate of glycated opioid peptides introduced into human circulation, the enzymatic (80% human serum) and chemical (phosphate buffer) stability of three Amadori compounds related to the endogenous opioid pentapeptide, leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu), and to its N-terminal fragments: N-(1-deoxy-D-fructos-1-yl)-l-tyrosyl-glycyl-glycyl-L-phenylalanyl-L-leucine, N-(1-deoxy-D-fructos-1-yl)-L-tyrosyl-glycyl-glycine, and N-(1-deoxy-D-fructos-1-yl)-L-tyrosine were investigated. The results obtained in human serum indicate that N-terminal glycation of leucine-enkephalin significantly enhances proteolytic stability. While leucine-enkephalin itself was rapidly degraded (t1/2 = 14.8 min), the glycated-derivative was slowly converted (t1/2 = 14 h) to the corresponding Amadori /compound of Tyr-Gly-Gly and Phe-Leu. In phosphate buffer, the rate of hydrolysis of the Amadori compounds depends on the structure and length of the peptide moiety as well as on the concentration of the phosphate buffer. The hydrolysis patterns for the Amadori compounds in phosphate buffer and in human serum were not the same and appear to be specific for each substrate.     

Peptidergic nerves in human dental pulp

Summary The peptidergic innervation of human dental pulp was studied with indirect immunofluorescence and immunoperoxidase techniques. Pulpal nerve fibres displaying immunoreactivity for cholecystokinin, calcitonin gene-related peptide, C-terminal flanking peptide of neuropeptide tyrosine, leucine-enkephalin, methionine-enkephalin, neuropeptide K, neuropeptide tyrosine, peptide with N-terminal histidine and C-terminal isoleucine, somatostatin-28, substance P and vasoactive intestinal polypeptide were observed. Immunoreactive axon varicosities were detectable within radicular and coronal nerve trunks and within the nerve plexus of Raschkow in the para-odontoblastic region. Many peptidergic nerve fibres were observed in association with blood vessels of various sizes. Substance P- and calcitonin-gene-related peptide-immunoreactive axons were visible in the odontoblastic layer. The occurrence of VIP- and PHI-immunoreactive fibres lends support to the hypothesis that human tooth may be supplied by parasympathetic nerves. The immunocytochemical results here shown provide a morphological basis to previous experimental studies concerning the possible roles of neuropeptides in nociception mechanisms, control of the blood flow and modulation of the inflammatory response in dental tissues.     

Chromatographic analysis of the enkephalin immunoreactivity in the nucleus locus coeruleus of the cat after local colchicine administration

Cell bodies immunoreactive for methionine- and leucine-enkephalin are found in the area of the locus coeruleus (dorsolateral pons) of the cat after injection of colchicine in the ascending projections of the nucleus. Using radioimmunoassay procedures, it is shown that colchicine induces a significant increase in methionine- and leucine-enkephalin-immunoreactive material in this area of the brain. High pressure liquid chromatography analysis demonstrated that the immunoreactive materials were authentic methionine- and leucine-enkephalin. The methionine- and leucine-enkephalin patterns were identical in the colchicine injected and non-injected sides of the dorsolateral pons. It is suggested that, in this area of the brain, colchicine (i) does not significantly modify the processing of proenkephalin to form the pentapeptides methionine- and leucine-enkephalin, and (ii) does not induce the appearance of new substances reactive to the enkephalin antisera employed.     

Synthesis of hexose-related imidazolidinones: Novel glycation products in the Maillard reaction

Carbohydrate-peptide esters which mimic the reactivity of sugar 6-phosphates in nonenzymatic glycations were used as model compounds for the study of the Maillard reaction in vitro. We found that intramolecular cyclization of the monosaccharide ester in which the sugar moiety (D-glucose or D-galactose) is linked, through the C-6 hydroxy group, to the C-terminal carboxy group of the endogenous opioid pentapeptide leucine-enkephalin, in methanol as the solvent, resulted in the formation of imidazolidinone diastereoisomers having cis or trans relative geometry of the substituents at the imidazolidinone ring moiety. The diastereoisomeric imidazolidinones were separated and each transformed by hydrolysis into the corresponding D-gluco- and D-galacto-related imidazolidinone products of leucine-enkephalin. Along with the previous evidence that, from the same sugar-peptide esters by changing the reaction conditions Amadori rearrangement products could be obtained [Horvat et al. (1998) J Chem Soc Perkin Trans 1:99–13], the presented results point to the possibility that similar carbohydrate-related imidazolidinones may also be generated in the early stage of the Maillard reaction in vivo.     

Enkephalin-binding protein in human blood

An enkephalin-binding protein was found in human plasma and serum. The protein was partially purified by DEAE-cellulose column chromatography. The binding of [3H]leucine-enkephalin to this protein was competitively inhibited by unlabeled leucine- and methionine-enkephalin and various peptide hormones such as beta-endorphin and glucagon, but not by Leu-enkephalin-amide. The fact that amide derivatives of leucine-enkephalin and methionine-enkephalin did not inhibit the binding suggests that c-terminuses of enkephalins might have an important part in binding the protein. From these results, physiological roles of the enkephalin-binding protein are discussed.     

Vasopressin neuropeptides and acquisition of heroin and cocaine self-administration in rats

The effect of the vasopressin neuropeptide des-glycinamide (Arg8)-vasopressin (DGAVP) on reducing the acquisition of intravenous heroin self-administration in rats was analyzed. When rats reduced in body weight were allowed to self-administer heroin for 1 h per day in the presence of a fixed time, non contingent food delivery schedule, it appeared that heroin intake was related in an orderly way to the unit dose of heroin delivered. DGAVP decreased heroin intake during days 4 and 5 of acquisition, especially when a high dose of heroin was delivered. DGAVP decreased heroin intake more effectively when rats were tested without the food delivery schedule and for 6 h instead of 1 h sessions per day. Structure activity relationship studies revealed that the peptide (pGlu4, Cyt6)AVP-(4-8) was the shortest active sequence mimicking the effect of DGAVP and that this peptide was somewhat more potent than DGAVP in this respect. The peptide (pGlu4,Cyt6)AVP-(4-9) increased the heroin intake of the rats. DGAVP and (pGlu4,Cyt6)-AVP-(4-8) also decreased cocaine intake of body weight reduced rats given the opportunity to self-administer cocaine intravenously in daily 6 h sessions. It is concluded that vasopressin neuropeptides may decrease the reinforcing efficacy of heroin and cocaine during acquisition of drug self-administration rather than interact with nutritional and environmental factors influencing drug taking behavior.     

Changes in morphine self-administration after exposure to ionizing radiation: Evidence for the involvement of endorphins

Recent findings have implicated endogenous opiates in radiation-induced behavioral change. The present experiment further investigated this hypothesis by observing alterations in morphine self-administration after irradiation. Under the presumption that the release of endogenous opiates would decrease the need for exogenously supplied morphine, we hypothesized that after radiation exposure morphine-experienced mice would self-administer less of the opiate. C57BL/6J mice had continuous access to two drinking flasks which contained either water or morphine in saccharine water. Irradiated mice drank significantly less morphine than did sham-irradiated controls. This decrease was naloxone-reversible and could not be entirely attributed to a generalized radiogenic hypodipsia or taste aversion. These results are consistent with the hypothesis that radiation-induced behavioral changes may be due, in part, to the fluctuations of endogenous opiates.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Summary Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

Enkephalins and pituitary hormone release: modification of responsiveness to LHRH.

An intraperitoneal injection of leucine-enkephalin into rats stimulates gonadotropin and prolactin release. To elucidate the mechanism of this releasing property of leucine-enkephalin, rat hemipituitaries were incubated with either enkephalin alone or enkephalin in combination with OHRH. Enkephalin alone had no effect on LH or prolactin release in vitro but potentiated the LH response to LHRH. Neither leucine-enkephalin nor LHRH alone had an effect on GH release; however, when combined, a GH response to LHRH occurred. These results suggest that leucine-enkephalin can modify the pituitary responsiveness to certain hypothalamic releasing hormones by a direct pituitary action.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

Mapping of the cAMP-dependent phosphorylation sites on the acetylcholine receptor.

We have synthesized a tetradecapeptide corresponding to residues 354-367 of the delta-subunit of Torpedo acetylcholine receptor. This peptide contains the sequence Arg-Arg-Ser-Ser which has been proposed as the site for phosphorylation of the acetylcholine receptor (AChR) by an endogenous cAMP-dependent protein kinase. We have shown that the synthetic peptide can be phosphorylated by the catalytic subunit of bovine heart cAMP-dependent protein kinase. Antibodies elicited against peptide 354-367 were shown to cross-react with native AChR and to bind specifically to the delta- and gamma-subunit as detected by immunoblotting. Furthermore, antipeptide antibodies were shown to inhibit specifically the cAMP-dependent phosphorylation of both the delta- and gamma-subunits. This suggests that the phosphorylation sites in the delta- and gamma-subunits are highly cross-reactive, and is in agreement with the demonstration that an endogenous cAMP-dependent kinase phosphorylates these two subunits, probably on homologous sequences. Tryptic digestion of the delta-subunit isolated from phosphorylated AChR yields a single 25-kd phosphorylated fragment. Immunoblotting experiments allowed us to map peptide 354-367 within this phosphorylated fragment.     

Neurohumoral effects of orphanin FQ/nociceptin: relevance to cardiovascular and renal function

Orphanin FQ/Nociceptin (OFQ/N) is a peptide whose structure resembles that of the endogenous opioid peptides (endorphins). OFQ/N and its receptor are distributed in neural tissue and brain regions involved in the regulation of pituitary hormone release. Functional studies have shown that this peptide evokes a unique pattern of cardiovascular and renal excretory responses. This review will focus on the neural and humoral effects of OFQ/N and how this peptide may participate in the regulation of cardiovascular and renal function.     

Peptidergic transmission in the brain. III. Hippocampal inhibition by the amygdala.

The mechanism of arginine vasopressin (AVP) action in the rat hippocampus has been determined. The peptide activates inhibitory interneurons and constricts cerebral microvessels. In the whole animal, each of these direct actions has secondary consequences for the excitability of pyramidal cells. Recent studies have shown that a peptide similar to AVP mediates endogenous neurotransmission in the hippocampus. Here we report experiments showing that the endogenous peptide activates the same mechanisms as exogenously applied AVP. The endogenous AVP-like peptide has no effect on the presynaptic fiber volley, or on pure somatic and dendritic postsynaptic potentials. These results are taken to exclude presynaptic mechanisms as explanations for the peptide's action. The endogenously released peptide inhibits individual pyramidal cells in single unit recording and excites presumed interneurons, just as AVP itself is known to act. The endogenous peptide is released only by stimuli applied to a nucleus that contains immunoreactive AVP and projects to the hippocampus.     

Evidence for the occurrence of the opioid octapeptide dynorphin-(1–8) in the neurointermediate pituitary of rats

Evidence is provided for the existence of the opioid peptide dynorphin-(1–8) in the neurointermediate pituitary of rats. The octapeptide was isolated by immunoadsorption to antibodies directed against porcine dynorphin-(1–13) followed by a variety of chromatographic separation procedures. The identity of the purified material with dynorphin-(1–8) was indicated by the following criteria: comigration with synthetic dynorphin-(1–8) on gelfiltration chromatography and high-performance liquid chromatography systems and liberation of a peptide with the same chromatographic behavior as leucine-enkephalin after sequential cleavage with trypsin and carboxypeptidase B.Radioimmunological estimations revealed that dynorphin-(1–8) is a major dynorphin-related opioid peptide in the pituitary of rats.     

Participation of leu-enkephalin in regulating carbohydrate metabolism

The experiments have been performed on 166 white male rats with a mass of 180-220 g. It has been revealed that leucine-enkephalin and its synthetic analogs prevent an increase in glucose blood levels and a decrease in glycogen liver levels caused by adrenaline and parathyroid hormone. At the same time the enkephalins inhibit the secretory activity of pancreatic beta-cells. The mechanisms of opioid peptide effect on carbohydrate metabolism are discussed.     

Analysis of peptide affinity to major histocompatibility complex proteins for the two-step binding mechanism

A novel approach to the analysis of an equilibrium two-step peptide-protein binding is developed and applied to the experimental data. The first step of the process is the release of an endogenous peptide from a binding groove and the second is the binding of an added peptide. The method developed enables us to determine consequently the maximum protein occupancy level (protein-binding capacity), the dissociation constant of an endogenous peptide, and the dissociation constant of a binding (antigenic) peptide. It is shown and confirmed by experimental data that the value of an equilibrium dissociation constant of a binding peptide could be much less than the experimental value of ED(50) (concentration of added peptide required to bind half of the protein), but not equal to that commonly assumed for major histocompatibility complex (MHC)-peptide binding. The model considered gives a clear understanding of why some peptides may be good binders to MHC protein in vitro, but do not exhibit anticipated activity on the cellular level and vice versa.     

Identification of dipeptidyl peptidase III in human neutrophils

We have found activity of dipeptidyl peptidase (DPP) III, one of the most important enkephalin-degrading enzymes in the central nervous system, in human neutrophils. HPLC analysis of the peptide fragments produced by treatment of leucine-enkephalin with isolated neutrophils in the presence of inhibitors of other enkephalin-degrading enzymes revealed that the enzyme in human neutrophils cleaved dipeptides from the NH(2) terminus of leucine-enkephalin, suggesting the presence of DPPIII activity in human neutrophils. Using a specific synthesized substrate and proteinase inhibitors, it was found that the neutrophils have 19.2 +/- 3.6 microM/h/5 x 10(6) cells of beta-naphthylamine for the enzyme. It was also confirmed that spinorphin and tynorphin, both reported to inhibit the activities of enkephalin-degrading enzymes, had potent inhibitory activities (IC(50): 4.0 and 0.029 microg/ml, respectively) against the enzyme. The presence of DPPIII activity in human neutrophils suggests that the biologically active peptides which are associated with enkephalin play a physiological role in regulating enkephalin or inflammatory mechanisms in peripheral tissues.